Antibody molecules binding il-17a and il-17f and uses thereof

Abstract

The present disclosure relates to antibodies against IL-17A and IL-17F, or antigen-binding fragments thereof, derivatives comprising the antibodies or antigen-binding fragments thereof, and pharmaceutical compositions. In addition, the invention also relates to application of the antibody or the antigen binding fragment thereof in diagnosis and treatment of IL-17A and IL-17F related diseases.

Description

Antibody molecules binding to IL-17A and IL-17F and their applications Technical Field

[0001] The present disclosure belongs to the field of immunology, and more specifically, relates to antibodies or antigen-binding fragments thereof that simultaneously bind to interleukin 17A (IL-17A) and interleukin 17F (IL-17F), derivatives comprising the antibodies or antigen-binding fragments thereof, pharmaceutical compositions, and their use in treating diseases associated with IL-17A and IL-17F, such as autoimmune diseases or inflammatory diseases. Background Art

[0002] The interleukin-17 (IL-17) family consists of six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F. They can bind to their respective receptors, thereby mediating different physiological and pathological processes. Among them, the human IL-17A gene encodes a polypeptide consisting of 155 amino acids, including a 23-amino acid signal peptide and a 132-amino acid mature peptide chain. It forms a homodimer through disulfide bonds and has a molecular weight of 35kD. Studies have confirmed that IL-17A is an important cytokine, mainly secreted by cells such as Th17 cells and innate lymphoid cell subsets. It stimulates a variety of cell types such as fibroblasts, epithelial cells, synovial cells, and chondrocytes to secrete cytokines and chemokines, thereby playing an important role in inflammatory diseases. At the same time, IL-17A also has biological effects such as host defense and tissue repair (Nat. Immunol, 2019 Dec, 20(12): 1594-1602; Trends Immunol, 2017 May, 38(5): 310-322).

[0003] IL-17A has become an important target for the treatment of psoriasis, psoriatic arthritis, and various other autoimmune diseases. Inhibiting IL-17A has been clinically proven to be an effective therapeutic strategy. Two anti-IL-17A antibodies, secukinumab (Novartis) and ixekizumab (Eli Lily), have been approved for the treatment of patients with moderate-to-severe plaque psoriasis, psoriatic arthritis, and ankylosing spondylitis. Clinical trials have shown that secukinumab and ixekizumab have better clinical efficacy in psoriasis than established therapeutic drugs ustekinumab (anti-IL-12 / IL-23p40 monoclonal antibody) and adalimumab (anti-TNFα monoclonal antibody) (J Am Acad Dermatol, 2015 Sep, 73(3): 400-409; Br J Dermatol, 2020 Aug, 183(2): 294-302; Dermatol Ther (Heidelb), Jan, 12(1): 167-184).

[0004] IL-17F is the member of the IL-17 family that is most closely related to IL-17A in terms of sequence and biological properties. IL-17A and IL-17F are encoded at the same gene locus (6p12), share 55% protein sequence homology, and share the same receptors (IL-17RA and IL-17RC). IL-17A and IL-17F can each form a homodimer linked by a disulfide bond, or they can form a heterodimer, IL-17A & F (Nat Rev Immunol, 2009 Aug, 9(8): 556-567). IL-17A and IL-17F are upregulated in a variety of inflammatory tissues and can synergize with other proinflammatory cytokines to enhance the body's inflammatory response. Numerous studies have confirmed that abnormal expression of IL-17A and IL-17F is closely associated with a variety of autoimmune and inflammatory diseases, such as moderate to severe plaque psoriasis, hidradenitis, psoriatic arthritis, rheumatoid arthritis, ankylosing spondylitis, and asthma (Immunity, 2019 Apr 16; 50(4): 892-906; Nat Rev Rheumatol, 2018 Aug, 14(8): 453-466; Front Immunol, 2018 Aug 2, 9: 1682; Ann Rheum Dis, 2011 May; 70(5): 727-732). Given that IL-17A and IL-17F play similar roles in a variety of pathological processes, it is suggested that simultaneous neutralization of IL-17A and IL-17F may be more effective than inhibiting IL-17A alone.

[0005] Bimekizumab, a monoclonal antibody drug developed by UCB that simultaneously targets IL-17A and IL-17F, has entered the clinical phase III stage in the field of various autoimmune diseases. Among them, in multiple phase III clinical head-to-head studies for the treatment of moderate to severe plaque psoriasis, bimekizumab has been shown to be more effective than a variety of marketed drugs, including adalimumab, ustekinumab, and secukinumab, showing a higher level of skin lesion clearance. In these clinical studies, approximately 60% of patients treated with bimekizumab achieved complete skin lesion clearance at week 16, and the response lasted for one year, and the overall tolerability was good (N Engl J Med, 2021 Jul 8, 385(2): 142-152; N Engl J Med, 2021 Jul 8, 385(2): 130-141; Lancet, 2021 Feb 6, 397(10273): 487-498). The above results demonstrate the advantages of the strategy of simultaneously targeting IL-17A and IL-17F compared with other targets in the treatment of psoriasis.

[0006] Currently, there are no drugs targeting both IL-17A and IL-17F available in China. Therefore, developing monoclonal antibody drugs targeting IL-17A and IL-17F to enrich the clinical drug categories for related diseases and improve drug accessibility remains an urgent issue in this field.

[0007] SUMMARY OF THE INVENTION

[0008] The present disclosure provides an anti-IL-17A and IL-17F antibody or an antigen-binding fragment thereof, which specifically binds to IL-17A, IL-17F and / or IL-17A&F, and does not significantly bind to IL-17B, IL-17C, IL-17D and IL-17E.

[0009] In some embodiments, the IL-17A is a peptide having GenBank Accession No. NP_002181.1 (mRNA: NM_002190), and the IL-17F is a peptide having GenBank Accession No. NP_443104.1 (mRNA: NM_052872).

[0010] The present disclosure provides an anti-IL-17A and IL-17F antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof can specifically bind to IL-17A, IL-17F and / or IL-17A&F, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are:

[0011] (1) SEQ ID NO: 9, 10, 11, 12, 13, 14; or

[0012] (2) SEQ ID NO: 15, 16, 17, 18, 19, 20; or

[0013] (3) SEQ ID NO: 21, 22, 23, 24, 25, 26; or

[0014] (4) SEQ ID NO: 27, 28, 29, 30, 31, 32.

[0015] According to one aspect of the present disclosure, the anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have at least 80% to 100% sequence identity with the heavy chain variable region and the light chain variable region of any one of the following groups, respectively:

[0016] (1) SEQ ID NO: 1 and 2; or

[0017] (2) SEQ ID NOs: 3 and 4; or

[0018] (3) SEQ ID NO: 5 and 6; or

[0019] (4) SEQ ID NOs: 7 and 8; or

[0020] (5) SEQ ID NOs: 33 and 34.

[0021] In some preferred embodiments, the anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof described in the present disclosure are murine antibodies, chimeric antibodies, or humanized antibodies.

[0022] In some preferred embodiments, the anti-IL-17A and IL-17F antibodies are monoclonal antibodies.

[0023] In some preferred embodiments, the anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region comprises a native Fc or a modified variant Fc, and the Fc is derived from mouse or human.

[0024] In some preferred embodiments, the anti-IL-17A and IL-17F antibodies are full-length antibodies.

[0025] In some preferred embodiments, the anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof of the present disclosure are in the form of IgG1, IgG2, IgG3 or IgG4.

[0026] In some preferred embodiments, the antigen-binding fragments disclosed herein are Fab, Fv, scFv, F(ab')2, linear antibodies, or single domain antibodies.

[0027] In some embodiments, the present disclosure provides a conjugate formed by conjugating the aforementioned antibody or antigen-binding fragment thereof to a capture label or a detection label. The detection label includes but is not limited to a radionuclide, a luminescent substance (e.g., fluorescein), a colored substance, or an enzyme.

[0028] In some embodiments, the present disclosure provides a fusion protein, wherein one of the fused moieties comprises the anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof of the present disclosure.

[0029] In some embodiments, the present disclosure provides a bispecific antibody or multispecific antibody, wherein one antigen-binding domain of the bispecific antibody or multispecific antibody comprises the anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof of the present disclosure.

[0030] In some embodiments, the present disclosure provides a nucleic acid encoding any of the aforementioned antibodies or antigen-binding fragments thereof, and also provides a recombinant vector comprising the nucleic acid.

[0031] In some embodiments, the present disclosure provides a host cell comprising an expression vector as described herein or a nucleic acid encoding the antibody or antigen-binding fragment thereof. In some preferred embodiments, the host cell can be a prokaryotic cell, such as Escherichia coli, or a eukaryotic cell, such as yeast, or a mammalian cell, such as a CHO cell or HEK293 cell.

[0032] In some embodiments, the present disclosure provides a method for preparing the antibody or antigen-binding fragment thereof, comprising: culturing the host cell of the present disclosure under suitable conditions, and purifying the expression product from the cell.

[0033] In some embodiments, the present disclosure provides uses of the antibodies or antigen-binding fragments thereof for detecting, diagnosing, or treating diseases associated with the expression of IL-17A and IL-17F.

[0034] In some embodiments, the present disclosure provides a method for detecting IL-17A, IL-17F and / or IL-17A&F in a sample, comprising: contacting the sample with the aforementioned anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof; detecting formation of a complex between the anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof and IL-17A, IL-17F and / or IL-17A&F; optionally, the anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof is detectably labeled.

[0035] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof of the present disclosure, or an effective amount of a nucleic acid encoding the antibody or antigen-binding fragment thereof, or an effective amount of a recombinant vector containing the encoding nucleic acid, or an effective amount of a host cell containing the encoding nucleic acid, or an effective amount of a fusion protein of the present disclosure, or an effective amount of a bispecific antibody or multispecific antibody of the present disclosure. In some more preferred embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0036] In some specific embodiments, the pharmaceutical composition further comprises one or more additional therapeutic agents. The additional therapeutic agent is a pharmaceutical preparation that can be administered to a subject in combination with the pharmaceutical composition of the present disclosure.

[0037] In some embodiments, the present disclosure provides a method for treating a disease associated with expression of IL-17A and IL-17F in a subject, the method comprising administering a pharmaceutical composition of the present disclosure to a subject in need thereof. In some preferred embodiments, the disease is an autoimmune disease. In other preferred embodiments, the disease is an inflammatory disease. In some more preferred embodiments, the method further comprises administering to the subject an additional therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings further illustrate the novel features disclosed in this specification. The features and advantages disclosed in this specification can be better understood with reference to these drawings, but it should be understood that these drawings are only used to illustrate specific implementations of the principles disclosed herein and are not intended to limit the scope of the appended claims.

[0039] Figure 1 shows the binding of humanized antibody Z1 to human IL-17A and IL-17F. Figure 1A shows the binding activity of Z1 to human IL-17A, and Figure 1B shows the binding activity of Z1 to human IL-17F.

[0040] Figure 2 shows the binding of humanized antibody Z1 to other members of the IL-17 family.

[0041] Figure 3 shows the functional assay results of humanized antibody Z1 at the cellular level. Figure 3A shows the inhibition of IL-6 expression induced by IL-17A by Z1, Figure 3B shows the inhibition of IL-6 expression induced by IL-17F by Z1, and Figure 3C shows the inhibition of IL-6 expression induced by IL-17A and F by Z1.

[0042] Figure 4 shows the results of cell activity inhibition assays using humanized antibody Z1. Figure 4A shows the inhibition of IL-17A-induced SEAP release by Z1, Figure 4B shows the inhibition of IL-17F-induced SEAP release by Z1, and Figure 4C shows the inhibition of IL-17A&F-induced SEAP release by Z1.

[0043] FIG5 shows the results of in vivo activity testing of humanized antibody Z1. DETAILED DESCRIPTION

[0044] the term

[0045] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0046] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0047] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or the use of range descriptions are only for the purpose of brevity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, the description using ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly written out in this article. For example, the description of a range from 1 to 6 should be considered to specifically disclose subranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as specific numerical points within these ranges, such as 1, 2, 3, 4, 5, 6. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0048] When referring to a measurable value such as an amount, a temporal duration, etc., the term "about" is meant to include variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0049] As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

[0050] As used herein, the terms "IL-17A" and "IL-17F" refer to homodimeric molecules each formed by covalent bonds, and "IL-17A & F" refers to molecules existing in the form of heterodimers.

[0051] As used herein, the term "anti-IL-17A and IL-17F antibody" or "antibody that binds to IL-17A and IL-17F" refers to an antibody that can bind to IL-17A, IL-17F and / or IL-17A&F with sufficient affinity, that is, the antibody can recognize and bind to IL-17A, IL-17F, and IL-17A&F.

[0052] IL-17A, IL-17F, and IL-17A&F of human origin are represented as hIL-17A, hIL-17F, and hIL-17A&F, respectively. Therefore, expressions such as “anti-human IL-17A antibody,” “anti-human IL-17F antibody,” “anti-hIL-17A antibody,” “anti-hIL-17F antibody,” “anti-human IL-17A&F antibody,” “anti-hIL-17A&F antibody,” “antibody against hIL-17A,” “antibody against hIL-17F,” and “antibody against hIL-17A&F” specifically refer to antibodies that can bind to hIL-17A, hIL-17F, and / or hIL-17A&F with sufficient affinity so that the antibodies can be used as diagnostic agents and / or therapeutic agents targeting hIL-17A, hIL-17F, and / or hIL-17A&F.

[0053] Cynomolgus monkey-derived IL-17A is denoted as cyno IL-17A, and similarly, cynomolgus monkey-derived IL-17F is denoted as cyno IL-17F.

[0054] The term "antibody" as used herein typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have this structure. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). Each heavy chain comprises a heavy chain variable domain (VH) and a heavy chain constant domain (CH), also known as a heavy chain constant region (CH).

[0055] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be identified as one of two clearly distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0056] In the case of IgG, IgA, and IgD antibodies, the heavy chain constant region comprises three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a segment of variable length rich in proline and cysteine. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine ​​residues.

[0057] The term "variable region" or "variable domain" shows significant changes in the amino acid composition from one antibody to another, and is primarily responsible for antigen recognition and binding. The variable region of each light chain / heavy chain pair forms an antigen binding site, so that a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each contain three regions with extreme variability, referred to as hypervariable regions (HVRs), or more generally, referred to as complementary determining regions (CDRs). VH and VL each have four framework regions, FRs, represented by FR1, FR2, FR3, and FR4, respectively. Therefore, CDR and FR sequences typically appear in the following sequence of a heavy chain variable domain (VH) (or light chain variable domain (VL)): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)-FR4.

[0058] The term "Fc" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which region includes at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise indicated, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0059] As used herein, the types of "antibodies" in a broad sense may include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

[0060] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody or comprises an Fc region.

[0061] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced by a single cell clone that is directed against a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of these techniques.

[0062] The term "chimeric antibody" refers to a construct in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies. In a narrow sense, a chimeric antibody comprises all or most of the selected murine heavy and light chain variable regions operably linked to human light and heavy chain constant regions. The constant region sequences, or variants or derivatives thereof, can be operably associated with the disclosed heavy and light chain variable regions using standard molecular biology techniques to provide full-length anti-IL-17A and IL-17F antibodies that can be used per se or incorporated into the disclosed anti-IL-17A and IL-17F antibodies.

[0063] The term "humanized antibody" is a hybrid immunoglobulin containing the minimum sequence derived from a non-human immunoglobulin, immunoglobulin chain or its fragment. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residue from the CDR of the receptor is replaced by the residue from the CDR of a non-human species (donor antibody) with required specificity, affinity and performance, such as mouse, rat, rabbit or primate. In some cases, the framework region residues of the human immunoglobulin are replaced by corresponding non-human residues. In some cases, a "back mutation" can be introduced into a humanized antibody, wherein the residue in one or more FRs of the variable region of the receptor human antibody is replaced by the corresponding residue from the non-human species donor antibody. Such a back mutation can contribute to the appropriate three-dimensional configuration of one or more grafted CDRs and therefore improve affinity and antibody stability. Antibodies from various donor species can be used, and these donor species include but are not limited to mouse, rat, rabbit or non-human primate. In addition, a humanized antibody can be included in a receptor antibody or in a donor antibody without finding new residues, so as to further improve antibody performance.

[0064] It should be noted that the CDR and FR divisions of the monoclonal antibody variable regions disclosed herein are determined according to the Kabat definition. Other naming and numbering systems, such as Chothia, IMGT, or AHo, are also known to those skilled in the art. Therefore, humanized antibodies based on the monoclonal antibody sequences disclosed herein that contain one or more CDRs derived from any naming system are clearly within the scope of this disclosure.

[0065] The term "sequence identity" or "sequence similarity" or "sequence homology" refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence, after aligning the sequences (and introducing gaps, if necessary) to obtain maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed to determine percent amino acid sequence identity using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0066] The term "antibody fragment" comprises at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or its antigenic epitope, or a fusion protein product further derived from such a fragment, such as a single-chain antibody, an extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments (VL), variable heavy chain fragments (VH), Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific antibodies or multispecific antibodies formed by antibody fragments.

[0067] The term "Fab fragment" includes the heavy chain variable region and the light chain variable region, and also includes the constant region of the light chain and the first constant region CH1 of the heavy chain, which is a monovalent antibody fragment. The term "F(ab')2 fragment" includes two Fab fragments and the hinge region, which is a bivalent antibody fragment.

[0068] The term "Fd fragment" generally comprises the heavy chain variable region and the constant region CH1; the term "Fv fragment" contains the antibody heavy chain variable region and the light chain variable region, but no constant region, and is the smallest antibody fragment with a complete antigen binding site.

[0069] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0070] The term "fusion protein" refers to a larger molecule formed by linking different polypeptides / proteins through genetic recombination or chemical methods, and the connection may be made with or without a linker.

[0071] The term "multispecific antibody" refers to an antibody that is functionally linked (e.g., chemical coupling, genetic fusion, non-covalent binding, or other methods) to one or more other binding molecules to form a new antibody construct that binds to two or more different sites and / or targets. Among them, "bispecific antibodies" are more commonly used, which specifically refer to antibody constructs that are specific for two different antigens. Generally, bispecific antibodies or multispecific antibodies include at least two antigen-binding domains.

[0072] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or its antigen-binding fragment. In a broad sense, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, or an entire extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof, can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen or cell or preparation containing the antigen can be used to generate antibodies specific for the antigenic determinant. The antigen can be an isolated full-length protein, a cell surface protein (e.g., immunized with cells expressing at least a portion of the antigen on their surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein), or a protein construct (e.g., an Fc antigen). The antigen can be produced in genetically modified cells. Any of the aforementioned antigens can be used alone or in combination with one or more immunogenicity-enhancing adjuvants known in the art. The DNA encoding the antigen can be genomic or non-genomic (e.g., cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector can be used to transform cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

[0073] The term "epitope", also known as "antigenic determinant", refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are generally retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are generally lost after treatment with denaturing solvents. An epitope is generally composed of 3-15 amino acid residues. Methods for determining the epitope bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and the techniques described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0074] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer may be linear, cyclic, or branched, it may contain modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified, for example, by glycosylation, lipidation, acetylation, phosphorylation, methylation, or any other manipulation such as conjugation to a labeling component. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and the D or L optical isomers, as well as amino acid analogs and peptide mimetics. A polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by a specified nucleic acid sequence.

[0075] The term "amino acid modification" (or "modified amino acid") includes amino acid substitutions, insertions, and / or deletions in a polypeptide sequence. "Amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S32A refers to the replacement of serine at position 32 with alanine.

[0076] The sequence identity or homology of humanized antibody variable region and human receptor variable region can be measured as discussed herein, and when measured like this, will preferably share at least 60% or 65% sequence identity, more preferably at least 70%, 75%, 80%, 85% or 90% sequence identity, even more preferably at least 93%, 95%, 98% or 99% sequence identity. Preferably, the non-identical residue positions are different because of conservative amino acid replacements. " conservative substitution " is an amino acid replacement in which an amino acid residue is replaced by another amino acid residue with a side chain (R group) having similar chemical properties (for example, charge or hydrophobicity). In general, conservative amino acid replacements will not substantially change the functional properties of a protein. This area has defined families of amino acid residues with similar side chains. These families include amino acids containing basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR region or in the framework region of the disclosed antibodies can be replaced with other amino acid residues having similar side chains. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity can be adjusted upward to correct for the conservative nature of the substitution.

[0077] During the production of monoclonal antibodies, different physical and chemical factors can easily produce various post-translational modification (PTM) variants, such as glycosylation, oxidation, glycation, deamidation, isomerization, and terminal cyclization. These PTMs may cause changes in the physical and chemical properties of the antibody, change the interaction with the antibody Fc receptor, and affect the binding activity with the target antigen; the occurrence of some PTMs may even reduce antibody stability and cause immunogenicity (JARASCH et al., JOURNAL OF PHARMACEUTICAL SCIENCES, 2015). The negative effects of PTM sites can be eliminated by amino acid modification, such as conservative substitution. Amino acid substitutions in antibody CDRs for the purpose of modifying PTMs are also clearly within the scope of this disclosure.

[0078] The antibodies of the present disclosure may also include substitutions or modifications of the constant region (e.g., Fc), including but not limited to amino acid residue substitutions, mutations and / or modifications, which result in compounds with preferred characteristics including but not limited to altered pharmacokinetics, increased serum half-life, increased binding affinity, reduced immunogenicity, increased production, altered binding to Fc receptors (FcRs), enhanced or decreased ADCC or CDC, altered glycosylation and / or disulfide bonds, and modified binding specificity.

[0079] The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant for a specific antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, and flow cytometry.

[0080] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. When a "pharmaceutical composition" is present as a combination of separate formulations containing two or more different active ingredients, they can be administered simultaneously, sequentially, separately, or at intervals, with the goal of exerting the biological activities of the multiple active ingredients for combined treatment of a disease.

[0081] The term "pharmaceutical carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic agent is administered.

[0082] The term "effective amount" refers to a dosage of a pharmaceutical formulation of an antibody or antigen-binding fragment thereof of the present disclosure that produces the desired effect in a treated patient after administration to the patient in a single or multiple doses. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; the specific antibody administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0083] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0084] The term "transfection" as used herein refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

[0085] The term "stable transfection" or "stable transfection" refers to the introduction and integration of exogenous nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated the foreign DNA into its genomic DNA.

[0086] The term "isolated polynucleotide" or "isolated nucleic acid" refers to a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of this disclosure. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in a heterologous host cell or a purified (partially or substantially) polynucleotide in solution. An isolated polynucleotide includes a polynucleotide molecule contained in a cell that normally contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location. An isolated RNA molecule includes in vivo or in vitro RNA transcripts of the disclosure, as well as positive and negative strand forms and double-stranded forms.

[0087] The terms "nucleic acid molecule encoding," "coding DNA sequence," and "coding DNA" refer to the order of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0088] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found in, for example, Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. The antibodies or antigen-binding fragments of the present invention are engineered to have one or more human FR regions in addition to the non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website at http: / / imgt.cines.fr or from the Journal of Immunoglobulins (2001) ISBN: 012441351.

[0089] The engineered antibodies or antigen-binding fragments thereof disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes the antibodies can be purified and collected using conventional techniques. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0090] As used herein, the term "individual" or "subject" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0091] As used herein, the terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, the term "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance defects (e.g., transplant rejection).

[0092] The term "inflammation" as used herein refers to a basic pathological process that occurs when the body is stimulated and is mainly a defensive response. The term "autoimmune disease" refers to a disease in which the body's immune system malfunctions, causing the body to attack its own tissues. It has complex causes and symptoms vary depending on the type of disease and the organs affected. The inflammation and autoimmune diseases described in the present disclosure may include, but are not limited to, psoriasis, psoriatic arthritis, atopic dermatitis, hidradenitis suppurativa, systemic sclerosis, rheumatoid arthritis, systemic juvenile idiopathic arthritis (JIA), inflammatory bowel disease, systemic lupus erythematosus (SLE), asthma, chronic obstructive airway disease (COPD), ankylosing spondylitis and other spondyloarthritis.

[0093] As used herein, the term "treatment" refers to clinical intervention aimed at altering the course of a disease in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, ameliorating or relieving the condition, and alleviating or improving the prognosis.

[0094] The term "combination" as used herein refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. The therapies can be physical, such as radiotherapy, or chemical, such as administering a drug to a subject, and the drug also includes a combination drug. "Combination drug" refers to a combination of two or more pharmaceutical preparations, each of which has an active ingredient, that need to be used in combination when administered to a subject. The active ingredients can be mixed together to form a single dosing unit, or they can be independently administered as dosing units and used separately; when administered, different pharmaceutical preparations can be administered essentially synchronously, simultaneously or sequentially.

[0095] Example

[0096] The present disclosure is further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present disclosure only and are not intended to limit the scope of the present disclosure. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed according to conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0097] Example 1 Animal Immunization

[0098] 6-8 week old SD rats (Weitong Lihua, Animal Production License No.: SCXK (Beijing) 2019-0002) were selected for animal immunization. Each rat was injected with 30 μg of recombinant human IL-17A (hIL-17A) protein and 30 μg of recombinant human IL-17F (hIL-17F) protein, respectively. The first injection of hIL-17A and hIL-17F protein dose was 60 μg / rat, and the injection was once every week. The titer of the immune serum was tested by ELISA to determine the immune response of the animal. After 6 rounds of immunization, rats with higher serum titers were selected for euthanasia, and the spleen and lymph nodes were collected for the preparation of hybridoma cells.

[0099] Example 2 Preparation of anti-IL-17A and IL-17F mouse monoclonal antibodies

[0100] Lymph node and spleen cells from immunized rats were collected and mixed with Sp2 / 0 myeloma cells at a 1:1 ratio in electrofusion solution. Electrofusion (BTX) was performed according to the electrofusion protocol. The fused cells were resuspended in HAT medium (DMEM + 20% FBS + 1× HAT), mixed thoroughly to prepare a cell suspension, and then transferred to a 96-well plate. The fused cells were cultured in a 37°C, 5% CO2 incubator for 12 days. Hybridoma cells were initially screened using ELISA. Positive clones that bound to both human IL-17A (hIL-17A) and human IL-17A & F (hIL-17A & F) antigens were identified and then transferred to a 24-well plate for culture. Three days later, the hybridoma supernatant was again assayed by ELISA, and positive clones that bound to hIL-17A, hIL-17F, hIL-17A&F, and cynomolgus macaque IL-17A (cyno IL-17A) were selected. These positive clones were then subcloned using a semi-solid culture method to obtain stable, single hybridoma cells. The resulting monoclonal clones were further screened through binding activity assays and cell-based biological function assays, resulting in a total of 30 hybridoma cell lines with strong binding activity to hIL-17A, hIL-17F, and hIL-17A&F.

[0101] The selected hybridoma cell lines were expanded into culture flasks containing complete culture medium for antibody production. When the cell viability was approximately 30%, the cell supernatant was collected for purification. The cell culture supernatant was purified using a Protein A chromatography column (GE Healthcare). The concentration of the purified antibody was determined by absorbance at 280 nm, and the antibody purity was determined by SDS-PAGE (Invitrogen) and SEC-HPLC (Agilent 1260). Through combined activity assays and cell-level biological function assays, four hybridoma cell lines were finally selected for sequencing.

[0102] Conventional biological methods were used to extract total RNA from cells. Using the total RNA as a template, cDNA was synthesized by reverse transcription using the SuperScript III First-Strand Synthesis SuperMix Kit (Invitrogen). Using the cDNA as a template, amplification was performed using primers specific for the antibody constant region. The PCR products were separated by agarose gel electrophoresis, and the DNA fragments were purified and recovered. The amino acid sequences of the variable regions of the four murine monoclonal antibodies disclosed herein were obtained by nucleic acid sequencing and translation, as shown in Table 1.

[0103] Table 1 Amino acid sequences of the variable regions of four mouse monoclonal antibodies

[0104] Based on the variable region amino acid sequences disclosed in Table 1, the CDRs and FRs of the antibody variable regions were divided using the Kabat numbering convention. The six CDR sequences of each antibody are shown in Table 2 below. The numbers in brackets in Table 2 represent the sequence numbers, for example, (9) represents SEQ ID NO: 9.

[0105] Table 2 Amino acid sequences of the variable region CDRs of four mouse monoclonal antibodies

[0106] Example 3 Humanization of murine anti-IL-17A and IL-17F antibodies

[0107] The selected lead monoclonal antibody 1.103.12 was humanized: the constant domains of the parental (murine antibody) were replaced with human constant domains to prepare a chimeric antibody, and then humanized by selecting human antibody sequences based on the homology between the parental and human antibodies.

[0108] (1) Design of humanized antibodies

[0109] The sequence of the murine antibody 1.103.12 was aligned with human IgG germline sequences in the IMGT database. IGHV3-23*04 IGHJ1 and IGKV7-3*01 IGKJ2 were selected as templates for heavy and light chain CDR grafting, respectively. The heavy chain CDR regions of 1.103.12, namely HCDR1 (SEQ ID NO: 9), HCDR2 (SEQ ID NO: 10), and HCDR3 (SEQ ID NO: 11), were grafted into the framework regions of IGHV3-23*04 IGHJ1. The light chain CDR regions of 1.103.12, namely LCDR1 (SEQ ID NO: 12), LCDR2 (SEQ ID NO: 13), and LCDR3 (SEQ ID NO: 14), were grafted into the framework regions of IGKV7-3*01 IGKJ2. Various humanized variants were constructed by empirically backmutating specific framework region sites and combining backmutations.

[0110] (2) Expression and purification of humanized antibodies

[0111] The heavy and light chain genes of various humanized variants were synthesized and cloned into expression plasmids. The sequenced heavy and light chain expression plasmids were then co-transfected into 293F cells for expression and purification. The concentration of the purified antibodies was determined by absorbance at 280 nm, and the purity was assessed by SDS-PAGE and SEC-HPLC. After screening of the purified humanized variants, a molecule with superior activity was identified, designated Z1. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:33, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:34.

[0112] Example 4 Binding activity and selectivity of humanized antibody Z1

[0113] The ELISA method was selected to determine the binding activity of the humanized antibody Z1 obtained in Example 3 with human IL-17A, IL-17F and other IL-17 family members (IL-17B, IL-17C, IL-17D, IL-17E).

[0114] The experimental procedure is as follows: 100 μL / well of antigen (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E: 0.5 μg / mL; IL-17F: 2 μg / mL) was coated at 4°C overnight. Blocking was performed with 200 μL of blocking buffer (2% BSA in PBS) for 1 hour at room temperature. Wash three times with PBST, all washes performed on a BioTek automated plate washer. Add 100 μL of diluted antibody to each well, starting at 10 μg / mL or 80 nM, diluted 1:4. Incubate at room temperature for 2 hours. Wash three times with PBST. Add 100 μL of diluted anti-human HRP secondary antibody to each well and incubate at room temperature for 1 hour. Wash six times with PBST. Add 100 μL of TMB substrate to each well and incubate at room temperature for 5 minutes. The reaction was then terminated by adding 100 μL of stop solution to each well. The OD values ​​at a wavelength of 450 nm were read using an Envision microplate reader. The results are shown in Figures 1 and 2, respectively.

[0115] GraphPad Prism 8.0 software was used to calculate the half-maximal binding concentration (EC 50 ), EC of Z1 with IL-17A and IL-17F 50 The results show that humanized antibody Z1 has strong binding activity with both IL-17A and IL-17F, but has no significant binding activity with other members of the IL-17 family (IL-17B, IL-17C, IL-17D, IL-17E), indicating that the antibody obtained in the present disclosure has good selectivity.

[0116] Example 5 Affinity detection of humanized antibody Z1

[0117] The affinity of Z1 samples for hIL-17A, hIL-17F, hIL-17A & F, cyno IL-17A, and cyno IL-17F was determined using SPR technology (Biacore 8K). The experimental process is as follows:

[0118] Coupling of antibodies on CM5 chips: Activate the CM5 chip with a freshly prepared 1:1 mixture of 50 mM NHS: 200 mM EDC (NHS and EDC are from the amino coupling kit) at a flow rate of 10 μL / min for 420 s, then inject goat anti-human IgG Fc antibody (diluted in sodium acetate solution, pH = 5.0, concentration of 30 μg / mL) at a flow rate of 10 μL / min for 420 s, and then inject 1 M ethanolamine at a flow rate of 10 μL / min for 420 s to block excess active carboxyl groups on the chip.

[0119] Affinity testing procedure: Capture: Antibody Z1 was diluted in 1× HBS-EP running buffer and injected into the detection channel at 1 μg / mL at a flow rate of 30 μL / min. Antigens were serially diluted in 1× HBS-EP running buffer to the following concentrations: hIL-17A: 10 nM to 0.3125 nM; hIL-17F: 20 nM to 0.625 nM; hIL-17A & F: 5 nM to 0.3125 nM; cyno IL-17A: 10 nM to 0.3125 nM; cyno IL-17F: 20 nM to 0.625 nM. Detection: Antigens were sequentially flowed through the reference and experimental channels in increasing concentration. Binding was performed for 180 s and dissociation for 1200 s. The surface was regenerated with 50 mM HCl after each binding and dissociation step. Biacore Insight Evaluation software was used to fit a 1:1 binding model and calculate the antibody association rate constant, ka, dissociation rate constant, kd, and equilibrium dissociation constant, KD. The results are shown in Table 3. The data show that antibody Z1 has good affinity for hIL-17A, hIL-17F, hIL-17A & F, cyno IL-17A, and cyno IL-17F.

[0120] Table 3 Affinity of humanized antibody Z1

[0121] Example 6 In vitro functional experiments of humanized antibody Z1

[0122] (1) Cell-level biological activity assay: IL-6 Bioassay

[0123] Antibodies against IL-17A and IL-17F were used to inhibit the secretion of IL-6 by HFF-1 cells stimulated by IL-17A, IL-17F, and IL-17A&F to detect the biological activity of antibody Z1. The experimental procedure is as follows: HFF-1 cells in good growth condition were cultured at a rate of 1×10 4 Cells were seeded at a density of 100 cells / well in a 96-well cell culture plate and incubated overnight in an incubator. On the second day of the experiment, 50 μL of IL-17A (final concentration 50 ng / mL), IL-17F (final concentration 200 ng / mL), or IL-17A&F (final concentration 100 ng / mL) recombinant protein was added to each well of another 96-well plate. To the wells containing 50 μL of IL-17A, IL-17F, or IL-17A&F protein, 50 μL of diluted antibody Z1 was added to prepare a protein-antibody mixture. The antibody was diluted 1:3 to an initial concentration of 12 μg / mL, with 11 dilutions in total. The 96-well plate was incubated in a cell culture incubator for 1 hour. The plate containing HFF-1 cells was removed, the old medium was removed, and 100 μL of the protein-antibody mixture was added to each well. The cells were incubated in the cell culture incubator for a further 24 hours. The cell culture supernatant was collected and the IL-6 level in the cell supernatant was detected using a human interleukin-6 (IL-6) ELISA kit (Biolegend). The results are shown in FIG3 .

[0124] GraphPad Prism 8.0 software was used to fit the data of different antibody concentrations and corresponding IL-6 levels to a 4-parameter nonlinear logistic formula to calculate the half-maximal inhibitory concentration (IC50). 50 ) value, the IC values ​​of Z1 with IL-17A, IL-17F and IL-17A&F under this experimental condition 50 0.62nM, 1.41nM and 1.76nM respectively.

[0125] (2) Cell activity inhibition experiment: Reporter assay

[0126] Antibodies against IL-17A and IL-17 inhibited HEK-BliE stimulated by IL-17A, IL-17F and IL-17A&F TM The biological activity of antibody Z1 was detected by measuring the release of secretory embryonic alkaline phosphatase (SEAP) from IL-17 cells (InvivoGen). TM IL-17 cells were collected at 5×10 4Cells were seeded at a density of 10 cells / well in a 96-well plate and incubated in a cell culture incubator overnight. On the second day of the experiment, another 96-well plate was taken and 50 μL of IL-17A (final concentration of 50 ng / mL), IL-17F (final concentration of 200 ng / mL) or IL-17A&F (final concentration of 100 ng / mL) recombinant protein was added to each well. 50 μL of diluted antibody was added to the wells with 50 μL of IL-17A, IL-17F or IL-17A&F to prepare a protein-antibody mixture. The antibody was diluted at a ratio of 1:3, with an initial concentration of 10 μg / mL, for a total of 9 dilution concentrations. Incubate in a cell culture incubator at 37°C for 1 hour. TM The IL-17 cell culture plate was removed, and 100 μL of the protein-antibody mixture was added to each well. The cells were then incubated in a cell culture incubator for another 24 hours. The cell culture supernatant was collected and assayed for SEAP release using QUANTI-Blue Solution (InvivoGen). The results are shown in Figure 4.

[0127] GraphPad Prism 8.0 was used to fit and calculate the half-maximal inhibitory concentration (IC 50 ) value, the IC values ​​of Z1 with IL-17A, IL-17F and IL-17A&F under this experimental condition 50 2.05nM, 5.274nM and 3.27nM respectively.

[0128] Example 7 In vivo activity detection of humanized antibody Z1

[0129] Human IL-17A can bind to the mouse IL-17 receptor, thereby stimulating mice to express and secrete a variety of inflammatory factors and chemokines such as CXCL1 / KC. In order to verify that the antibody disclosed herein can block the binding of IL-17A to its receptor IL-17RA in vivo, thereby inhibiting the expression of chemokine CXCL1 / KC, an in vivo activity experiment in mice was implemented. The specific experimental process is as follows: C57BL / 6 female mice (Shanghai Nanmo, Animal License: SYXK (Shanghai) 2018-0002) were randomly divided into 8 groups and weighed, with 6 mice in each group. Among them, one group of mice was intraperitoneally injected with sterile PBS buffer, one group was injected with isotype control antibody IgG (3000μg / kg), and the remaining groups of mice were injected with different concentrations of antibody drug solutions of each group, including: antibody Z1 disclosed herein, reference antibody secukinumab, and the respective concentration gradients were 3000μg / kg, 300μg / kg, and 30μg / kg, respectively. Sixteen hours later, all groups except the PBS group received a subcutaneous injection of 150 μg / kg human IL-17A. Two hours later, blood samples were collected. Whole blood was allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm at 4°C for 10 minutes, and the supernatant serum was aspirated. Samples were aliquoted and stored at -80°C to avoid repeated freezing and thawing. CXCL1 / KC levels in mouse serum samples were measured using the Mouse CXCL1 / KC Quantikine ELISA Kit (R&D).

[0130] Data were analyzed using GraphPad Prism 8.0, with t-tests used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001. The data are shown in Figure 5. As shown, compared to the inactive isotype control antibody, Z1 significantly inhibited IL-17A-induced CXCL1 / KC expression in mice in a dose-dependent manner, demonstrating Z1's robust in vivo pharmacodynamic activity. Furthermore, the data in Figure 5 further demonstrate that at a dose of 300 μg / kg, Z1 vs. secukinumab exhibited 32% vs. 15% inhibition of CXCL1 / KC; at a dose of 3000 μg / kg, Z1 vs. secukinumab exhibited 51% vs. 39% inhibition. This indicates that Z1 exhibits slightly superior in vivo efficacy to the currently marketed reference antibody, secukinumab.

[0131] Example 8 In vivo PK study of humanized antibody Z1

[0132] In order to detect the pharmacokinetic parameters of the antibody Z1 disclosed herein in vivo, a single-dose intravenous PK experiment was carried out in crab-eating monkeys. The experimental process is as follows: Grouping: One day before administration, 4 animals that have passed the adaptive training (Kangshun Biology, Animal Production License No.: SCXK (Gui) 2018-0043) were selected and randomly divided into 2 groups according to the weight of the animals, namely test group one and test group two, with 2 animals in each group, half male and half female. Administration: Under sterile conditions, the test antibody Z1 and the reference antibody ixekizumab were diluted with sodium chloride injection and administered by a single intravenous push. The dosage was 3 mg / kg and the administration volume was 2 mL / kg. The first day was the first day of the experiment (D1). Blood sampling: Blood sampling sites were designed as follows: before administration (0 h), 5 min, 0.5 h, 2 h, 4 h, 6 h, 8 h, 24 h (D2), 48 h (D3), 96 h (D5), 168 h (D8), 240 h (D11), 336 h (D15), 504 h (D22), 672 h (D29), 840 h (D36), 1008 h (D43), and 1176 h (D50). Approximately 1 mL of blood was collected from a vein in one of the four extremities (avoiding the administration site). Whole blood was collected into a blood collection tube containing a coagulant and separating gel. After the blood clotted, it was centrifuged at 1800 g for 10 min. The collected serum was divided into two aliquots, labeled according to the blood sample identification method, and temporarily stored in a -80°C ultra-low temperature freezer to avoid repeated freezing and thawing. Sample determination: Quantitative analysis of blood drug concentrations was based on a quantitative ELISA method for coated human IL-17A. Z1 and ixekizumab monoclonal antibody samples were used as quantitative standards, and standard curves were prepared for the analysis of blood drug concentrations of different antibodies.

[0133] The half-lives of the two antibodies in cynomolgus monkeys were calculated using a first-order reaction model. The average half-life values ​​for each group are shown in Table 4. At 3 mg / kg intravenous administration, the half-life of Z1 was 17.2 days. Compared with the reference antibody, Z1 exhibited a longer half-life in cynomolgus monkeys.

[0134] Table 4 Half-life of humanized antibody Z1 in vivo

[0135] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

1. An anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof can specifically bind to IL-17A, IL-17F and / or IL-17A&F, and comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are: (1) SEQ ID NO: 9, 10, 11, 12, 13, 14; or (2) SEQ ID NO: 15, 16, 17, 18, 19, 20; or (3) SEQ ID NO: 21, 22, 23, 24, 25, 26; or (4) SEQ ID NO: 27, 28, 29, 30, 31, 32.

2. An anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region have at least 80% to 100% sequence identity with the heavy chain variable region and the light chain variable region of any one of the following groups, respectively: (1) SEQ ID NO: 1 and 2; or (2) SEQ ID NOs: 3 and 4; or (3) SEQ ID NO: 5 and 6; or (4) SEQ ID NOs: 7 and 8; or (5) SEQ ID NOs: 33 and 34.

3. The anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof according to claim 1 or 2, further comprising one or more of the following features: (1) It further comprises a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region comprises Fc; more preferably, Fc is derived from mouse or human; more preferably, the sequence of Fc is a natural or modified variant; (2) It is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody; (3) It is a monoclonal antibody; or a full-length antibody, or its antigen-binding fragment is Fab, Fv, scFv, F(ab')2, linear antibody, or single-domain antibody; (4) It is in the form of IgG1, IgG2, IgG3 or IgG4.

4. A conjugate, comprising any of the aforementioned anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof coupled to a capture label or a detection label, wherein the detection label comprises a radionuclide, a luminescent substance, a colored substance, or an enzyme.

5. A fusion protein, wherein one of the fused parts comprises any of the aforementioned anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof.

6. A bispecific antibody or multispecific antibody, wherein one of the antigen-binding domains comprises any of the aforementioned anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof.

7. A nucleic acid encoding any of the aforementioned anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof.

8. A recombinant vector comprising the nucleic acid of claim 7.

9. A host cell comprising the recombinant vector of claim 8 or the nucleic acid of claim 7.

10. The host cell according to claim 9, which is a prokaryotic cell, such as Escherichia coli; or a eukaryotic cell, such as yeast or a mammalian cell, such as a CHO cell or a HEK293 cell.

11. A method for preparing any of the aforementioned anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof, comprising: The host cell of claim 9 or 10 is cultured under suitable conditions, and the expression product is purified from the cell.

12. A method for detecting IL-17A, IL-17F and / or IL-17A&F in a sample, comprising: (1) contacting the sample with any of the aforementioned anti-IL-17A and IL-17F antibodies or antigen-binding fragments thereof; (2) detecting the formation of a complex between an anti-IL-17A and IL-17F antibody or an antigen-binding fragment thereof and IL-17A, IL-17F and / or IL-17A&F; optionally, the anti-IL-17A and IL-17F antibody or an antigen-binding fragment thereof is detectably labeled.

13. A pharmaceutical composition comprising an effective amount of the anti-IL-17A and IL-17F antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or an effective amount of the fusion protein according to claim 5, or an effective amount of the bispecific antibody or multispecific antibody according to claim 6, or an effective amount of the nucleic acid according to claim 7, or an effective amount of the recombinant vector according to claim 8, or an effective amount of the host cell according to claim 9 or 10.

14. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 13 or 14, further comprising one or more additional therapeutic agents.

16. A method for treating a disease associated with the expression of IL-17A and IL-17F in a subject, the method comprising administering the pharmaceutical composition of any one of claims 13 to 15 to a subject in need thereof.

17. The method of claim 16, wherein the disease is an autoimmune disease or an inflammatory disease.

18. The method of claim 16 or 17, further comprising administering to the subject an additional other therapeutic agent.

Images