A single-domain antibody against IL-6 and its uses

Abstract

The present invention belongs to the field of immunology and relates to a single-domain antibody against IL-6 and its use. The single-domain antibody is composed of a heavy chain, and the heavy chain includes a heavy chain CDR1 shown in any one of SEQ ID NO: 13-SEQ ID NO: 15, a heavy chain CDR2 shown in SEQ ID NO: 16, and a heavy chain CDR3 shown in any one of SEQ ID NO: 17-SEQ ID NO: 19. Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention uses biological genetic engineering technology to screen out a single-domain antibody specifically targeting IL-6, and the antibody has good affinity.

Description

Technical Field

[0001] The present invention relates to a single-domain antibody capable of specifically binding to IL-6 (hereinafter, abbreviated as "anti-IL-6 single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its pharmaceutical therapeutic use. Background Art

[0002] Interleukin-6 (IL-6) is a pleiotropic cytokine with a wide range of functions. IL-6 can regulate the growth and differentiation of various cells, has the functions of regulating immune responses, acute-phase responses, and hematopoiesis, and plays an important role in the body's anti-infection immune response. IL-6 is significantly altered in many diseases, and its dysregulation can cause many diseases, and its clinical manifestation is mainly an increase in the level of IL-6 during the onset. IL-6 is rapidly generated during the acute inflammatory response of internal and external injuries, surgical operations, stress responses, infections, brain death, tumor generation, and other conditions.

[0003] There are many target cells for the action of IL-6, including macrophages, hepatocytes, resting T cells, activated B cells, and plasma cells, etc.; its biological effects are also very complex, and it has been called B cell stimulatory factor 2, 26KD protein, B cell differentiation factor, hepatocyte stimulatory factor, etc. Its biological effects include: ① promoting the expression of IL-2r on the surface of T cells and enhancing the mitogenic effect of IL-1 and TNF on TH cells. ② As a hepatocyte stimulatory factor, it induces the synthesis of acute-phase response proteins during the acute inflammatory response caused by infection or trauma, and the increase in amyloid a and c-reactive protein is particularly obvious. ③ Promoting the proliferation, differentiation of B cells and the production of antibodies; the malignant B cells of multiple myeloma can both produce IL-6 and respond to IL-6, suggesting that IL-6 may act as an autocrine growth factor for these cells. ④ IL-6 can also effectively promote the cachexia induced by TNF and IL-1; promote glucocorticoid synthesis; stimulate osteoclast activity and keratinocyte growth; and can also promote the function of bone marrow hematopoiesis.

[0004] Studies have shown that the overproduction and signal transduction of IL-6 (especially the so-called trans-signaling) are involved in various diseases and disorders, such as sepsis, and various forms of cancer, such as multiple myeloma, renal cell carcinoma, plasma cell leukemia, lymphoma, B-lymphoproliferative disorders, and prostate cancer. Non-limiting examples of other diseases caused by excessive IL-6 production or signal transduction include osteoporosis, cachexia, psoriasis, mesangial proliferative glomerulonephritis, Kaposi's sarcoma, AIDS-related lymphoma, inflammatory diseases and disorders such as rheumatoid arthritis, systemic-onset juvenile idiopathic arthritis, hypergammaglobulinemia; regional enteritis, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, Castleman's disease, IgM γ-globulinopathy, cardiac myxoma, asthma (especially allergic asthma), and autoimmune insulin-dependent diabetes.

[0005] Single-domain antibodies are the new favorites in the antibody field. Due to their small molecular weight, bivalent, trivalent, or multispecific antibodies can be obtained through simple molecular cloning techniques. Due to their small molecular size, single-domain antibodies can achieve high yields in both prokaryotic expression systems (Escherichia coli) and eukaryotic expression systems (CHO cells, 293 cells, etc.). Currently, there is still a lack of single-domain antibody products that can effectively inhibit IL-6 in the prior art. Summary of the Invention

[0006] The object of the present invention is to provide a single-domain antibody that can specifically bind to IL-6 and its uses.

[0007] The first aspect of the present invention provides a single-domain antibody against IL-6, which is composed of a heavy chain, and the heavy chain includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are one of the following (1)-(4):

[0008] (1) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:16, CDR3 shown in SEQ ID NO:17;

[0009] (2) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:16, CDR3 shown in SEQ ID NO:19;

[0010] (3) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:16, CDR3 shown in SEQ ID NO:18;

[0011] (4) CDR1 shown in SEQ ID NO:15, CDR2 shown in SEQ ID NO:16, and CDR3 shown in SEQ ID NO:19.

[0012] The above CDR combinations (1)-(4) correspond to single-domain antibodies 3F7 (SEQ ID NO:4), 2C6 (SEQ ID NO:1), 2D7 (SEQ ID NO:2), and 2E10 (SEQ ID NO:3) in sequence.

[0013] All of the above sequences can be replaced with sequences having "at least 80% homology" with the sequence or sequences with only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0014] In one embodiment, in any one or more of the heavy-chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues can be replaced with their conservative amino acids respectively. Specifically, in the heavy-chain CDR1, 1 to 5 amino acid residues can be replaced with their conservative amino acids; in the heavy-chain CDR2, 1 to 5 amino acid residues can be replaced with their conservative amino acids; in the heavy-chain CDR3, 1 to 5 amino acid residues can be replaced with their conservative amino acids.

[0015] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies with exactly the same sequence have 100% homology.

[0016] In some embodiments, a sequence that differs from the foregoing sequence by only one or a few amino acid substitutions, for example, a sequence containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, can also achieve the object of the invention. These variant forms include (but are not limited to): deletion, insertion, and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called "conservative" amino acid substitutions. In the case of substitution, the substitution will preferably be a conservative amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids within the following groups (a)-(d) by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art know that the sequences of framework regions FR1-4 are not immutable, and the sequences of FR1-4 can adopt conservative sequence variants of the sequences disclosed in the present invention.

[0017] As used herein, the term "anti-IL-6 single domain antibody" of the present invention not only includes the complete single domain antibody, but also includes fragments, derivatives and analogs of the anti-IL-6 single domain antibody. As used herein, the terms "fragment", "derivative" and "analog" have the same meaning and all refer to polypeptides that substantially maintain the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusing a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or a secretion sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with an Fc tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0018] In a preferred embodiment, the heavy chain further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are respectively:

[0019] FR1 shown in SEQ ID NO:9 or a variant of FR1, the variant of FR1 containing at most 5 amino acid substitutions in the FR1;

[0020] FR2 shown in SEQ ID NO:10 or a variant of FR2, the variant of FR2 containing at most 5 amino acid substitutions in the FR2;

[0021] FR3 shown in SEQ ID NO:11 or a variant of FR3, the variant of FR3 containing at most 5 amino acid substitutions in the FR3;

[0022] FR4 shown in SEQ ID NO:12 or a variant of FR4, the variant of FR4 containing at most 5 amino acid substitutions in the FR4.

[0023] The second aspect of the present invention is to provide an amino acid sequence of an anti-IL-6 single domain antibody, the amino acid sequence of the single domain antibody being shown as any one of SEQ ID NOs: 1-4, or the single domain antibody having at least 80% sequence homology with the amino acid sequences of SEQ ID NOs: 1-4 and being capable of specifically binding to the IL-6 protein.

[0024] In one embodiment, the anti-IL-6 single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the amino acid sequences selected from SEQ ID NO: 1-4 or SEQ ID NO: 1-4, and is capable of specifically binding to the IL-6 protein.

[0025] The third aspect of the present invention is to provide an Fc fusion antibody or a humanized antibody of the anti-IL-6 single-domain antibody as described above.

[0026] The fourth aspect of the present invention is to provide a nucleotide molecule encoding the anti-IL-6 single-domain antibody as described above, the nucleotide sequence of which is shown in any one of SEQ ID NO: 5-8 respectively, or has at least 80% sequence homology with any one of SEQ ID NO: 5-8.

[0027] In one embodiment, the nucleic acid molecule encoding the anti-IL-6 single-domain antibody is selected from SEQ ID NO: 5-8 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with the nucleotide sequences selected from SEQ ID NO: 5-8, and the anti-IL-6 single-domain antibody encoded by it is capable of specifically binding to the IL-6 protein.

[0028] The fifth aspect of the present invention is to provide an expression vector, which contains a nucleotide molecule encoding an anti-IL-6 single-domain antibody, an Fc fusion antibody or a humanized antibody, and the nucleotide sequence of the anti-IL-6 single-domain antibody is shown in SEQ ID NO: 5-8 respectively or has at least 80% sequence homology with any one of SEQ ID NO: 5-8.

[0029] In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (the nucleotide molecule encoding the anti-IL-6 single-domain antibody or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC1 by genetic engineering means), and other general expression vectors can also be selected as needed.

[0030] The sixth aspect of the present invention is to provide a host cell capable of expressing the anti-IL-6 single-domain antibody, Fc fusion antibody or humanized antibody as described above, or containing the expression vector as described above. The preferred host cells are bacterial cells, fungal cells or mammalian cells.

[0031] In another preferred embodiment, the host cells include prokaryotic cells or eukaryotic cells, including bacteria and fungi.

[0032] In another preferred embodiment, the host cell is selected from the group consisting of: Escherichia coli, yeast cells, mammalian cells, phages, or combinations thereof.

[0033] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of: Escherichia coli, Bacillus subtilis, Lactobacillus, Streptomyces, Proteus mirabilis, or combinations thereof.

[0034] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or combinations thereof.

[0035] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of: insect cells such as Spodoptera frugiperda, plant cells such as Nicotiana tabacum, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0036] In another preferred embodiment, the host cell is the suspension ExpiCHO-S cell.

[0037] In another preferred embodiment, the host cell is the suspension 293F cell.

[0038] The seventh aspect of the present invention is to provide a recombinant protein comprising the aforementioned anti-IL-6 single-domain antibody. The recombinant protein may be the single-domain antibody shown in SEQ ID NO: 1-4 as described above, or a single-domain antibody having at least 80% homology with SEQ ID NO: 1-4, or may also be a multi-epitope antibody, a multi-specific antibody, and a multivalent antibody; for example, the multi-epitope antibody may be composed of more than one sequence among SEQ ID NO: 1-4; the multivalent antibody may be composed of one of the sequences among SEQ ID NO: 1-4 repeated several times; the multi-specific antibody includes but is not limited to bispecific antibodies and trispecific antibodies; in addition, the recombinant protein may be a fragment, derivative, and analogue of the aforementioned antibody.

[0039] The eighth aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned anti-IL-6 single-domain antibody and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually determined according to the isoelectric point of the antibody (the pH of the aqueous carrier medium needs to deviate from the isoelectric point of the antibody and differ from the isoelectric point of the antibody by approximately 2).

[0040] The pharmaceutical composition of the present invention can be directly used to bind to the IL-6 protein molecule.

[0041] The pharmaceutical composition is used for treating diseases. In a preferred embodiment, the diseases are Castleman disease, multicentric Castleman disease, SARS-CoV-2 acute respiratory disease, smoldering multiple myeloma or multiple myeloma, leukemia.

[0042] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001 - 99 wt%, preferably 0.01 - 90 wt%, more preferably 0.1 - 80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solution, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be made into an injection form, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.

[0043] The ninth aspect of the present invention is to provide the use of the aforementioned anti-IL-6 single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a drug for treating diseases.

[0044] In a preferred embodiment, the diseases are Castleman disease, multicentric Castleman disease, SARS-CoV-2 acute respiratory disease, smoldering multiple myeloma or multiple myeloma, leukemia.

[0045] The present invention also provides a kit for detecting the level of IL-6, which contains the aforementioned anti-IL-6 single-domain antibody. In a preferred example of the present invention, the kit further includes a container, an instruction manual, a buffer, etc.

[0046] In a preferred embodiment, the kit includes an antibody that recognizes the IL-6 protein, a lysis medium for lysing the sample, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0047] In a preferred embodiment, the kit further contains a second antibody and an enzyme or fluorescent or radioactive label for detection, as well as a buffer.

[0048] In a preferred embodiment, the second antibody of the kit can be an antibody (as an anti-antibody) of the aforementioned anti-IL-6 single-domain antibody, and can be a single-domain antibody, a monoclonal antibody, a polyclonal antibody, or any other form of the antibody.

[0049] The present invention also provides a method for generating an anti-IL-6 single-domain antibody, including the steps:

[0050] (a) Under conditions suitable for generating single-domain antibodies, culture the host cell described in the sixth aspect of the present invention to obtain a culture containing the anti-IL-6 single-domain antibody;

[0051] (b) Isolate or recover the anti-IL-6 single-domain antibody from the culture;

[0052] (c) Optionally, purify and / or modify the anti-IL-6 single-domain antibody obtained in step (b).

[0053] Advantages

[0054] Compared with the prior art, the advantages of the present invention are as follows:

[0055] (1) The single-domain antibody of the present invention specifically targets the IL-6 protein with the correct spatial structure.

[0056] (2) For the single-domain antibody obtained in the present invention, the expression system has flexible selection. It can be expressed in both prokaryotic systems and eukaryotic systems such as yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the later production cost.

[0057] (3) For the single-domain antibody obtained in the present invention, the transformation of the multi-combination form of the antibody is simple. By simply concatenating through genetic engineering methods, multivalent and multispecific antibodies can be obtained, and its immune heterogeneity is very low. Without humanization modification, it will not produce a strong immune response.

[0058] (4) For the single-domain antibody obtained in the present invention, its affinity range is wider. Before affinity maturation, its affinity range can range from the nM level to the pM level, providing multiple choices for antibodies for different later uses. Description of the Drawings

[0059] To more clearly illustrate the technical solutions of the present application, the drawings required for use in the examples will be briefly introduced below. Obviously, for those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0060] Figure 1 It is the enrichment situation of the library for screening the anti-IL-6 antibody in Example 3;

[0061] Figure 2 It is the determination map of the antibody-antigen binding dose-effect curve (2C6, 2D7) in Example 12;

[0062] Figure 3 It is the determination map of the antibody-antigen binding dose-effect curve (2E10) in Example 12;

[0063] Figure 4 It is the graph for measuring the antibody-antigen binding dose-effect curve (3F7) in Example 12;

[0064] Figure 5 It is the graph for measuring the antibody-antigen binding dose-effect curve (Tab1, hIgG) in Example 12;

[0065] Figure 6 It is the graph of the detection result of the antibody neutralizing IL-6-induced TF1 cell proliferation in Example 13 (Tab1, hIgG);

[0066] Figure 7 It is the graph of the detection result of the antibody (eukaryotic sample) neutralizing IL-6-induced TF1 cell proliferation in Example 13 (2C6, 2D7);

[0067] Figure 8 It is the graph of the detection result of the antibody (eukaryotic sample) neutralizing IL-6-induced TF1 cell proliferation in Example 13 (2E10);

[0068] Figure 9 It is the graph of the detection result of the antibody (eukaryotic sample) neutralizing IL-6-induced TF1 cell proliferation in Example 13 (3F7). Detailed implementation manners

[0069] The present invention will be further described in detail below in conjunction with examples, so that those skilled in the art can implement it according to the description in the specification.

[0070] As used herein, "single-domain antibody" (sdAb, also called nanobody or VHH by the developer Ablynx) is well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies are antibodies having only a heavy chain (which naturally does not contain a light chain), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0071] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cows. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species (such as camels, dromedaries, llamas, and guanacos). Like intact antibodies, single-domain antibodies can selectively bind to specific antigens. A single-domain antibody can contain only the variable domain of the immunoglobulin chain, which has CDR1, CDR2, and CDR3 as well as framework regions.

[0072] As used herein, the term "Fc fusion antibody" refers to a protein produced by fusing the Fc segment of a target antibody with a functional protein molecule having biological activity using genetic engineering techniques.

[0073] The term "humanized antibody" refers to an antibody obtained by fusing the heavy-chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining regions (CDR1-3 sequences) of the target antibody into the variable region of a human antibody, or by mutating the amino acids of the target antibody according to the characteristics of the human antibody framework regions (FR1-4). Humanized antibodies can be prepared by synthetic methods or site-directed mutagenesis methods.

[0074] In the present invention, sequences with high homology to the sequences of CDR1-3 disclosed in the present invention can also yield single-domain antibodies against IL-6. In some embodiments, sequences having "at least 80% homology", "at least 85% homology", "at least 90% homology", "at least 95% homology", or "at least 98% homology" with the sequences in SEQ ID NO: 1-4 can all achieve the object of the invention.

[0075] In some embodiments, a sequence that differs from the sequences of SEQ ID NOs: 1-4 by only one or a few amino acid substitutions, e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, can also achieve the object of the invention. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider the so-called "conservative" amino acid substitutions. In the case of substitution, the substitution will preferably be a conservative amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids within the following groups (a)-(d) by another or a few amino acids within the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0076] This patent prepares the target protein and the truncated form of the target protein by genetic engineering techniques, and then immunizes Inner Mongolia Alxa Bactrian camels with the obtained antigen protein. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. By genetic engineering methods, the coding sequences of the variable regions of camel antibodies are recombined into a phage display vector, and specific antibodies against the antigen protein are screened out by phage display technology, and their ability to bind to the antigen is further detected.

[0077] Now, the above technical solution will be disassembled and detailed, and described in the form of specific examples:

[0078] Example 1: Preparation of human IL-6 protein:

[0079] The human recombinant IL-6 protein used in this patent was obtained by the company's own expression and purification. The design scheme of the expression vector of the human recombinant IL-6 protein is as follows:

[0080] (1) Retrieve the coding sequence of IL-6 in NCBI, and its accession number is NP_000591.1. The accession number of the amino acid sequence encoded by this sequence is P05231.

[0081] (2) Clone the nucleotide sequence encoding the amino acids at positions 30 - 212 of the IL-6 protein (the amino acids at positions 30 - 212 are the extracellular domain of the IL-6 protein) into the vector pcDNA3.4 by gene synthesis. Sequence the constructed vector by Sanger sequencing, compare it with the original sequence, and after confirmation, extract a large amount of the recombinant plasmid, remove endotoxin, and transfect suspension 293F cells for the expression and purification of the target protein. The purity reaches more than 90%, meeting the requirements for animal immunization.

[0082] Example 2: Construction of a single-domain antibody library against IL-6 protein:

[0083] Mix 1 mg of the human recombinant IL-6 protein purified in Example 1 with an equal volume of Freund's complete adjuvant, and immunize a Bactrian camel from Alxa, Inner Mongolia once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations are carried out by mixing 1 mg of IL-6 protein with an equal volume of Freund's incomplete adjuvant for animal immunization. This immunization process is to intensively stimulate the camel to produce antibodies against the IL-6 protein.

[0084] After the animal immunization is completed, draw 150 mL of the camel's peripheral blood lymphocytes and extract the RNA of the cells. Synthesize cDNA using the extracted total RNA, and amplify VHH (variable heavy chain of antibody) using the cDNA as a template through nested PCR reaction.

[0085] Then, use restriction enzymes to digest the pMECS vector and the VHH fragment respectively, and then ligate the digested fragments and the vector. Transform the ligated fragments into competent cells TG1 by electroporation, construct a phage display library of IL-6 protein and determine the library capacity. The library capacity is approximately 1×10 9 , and at the same time, identify and detect the correct insertion rate of the library in the target fragment by colony PCR.

[0086] The results show that after PCR amplification of 30 colonies randomly selected from the library, 28 clones can amplify bands of the predicted size, and 2 clones amplify incorrect bands. Therefore, the correct insertion rate is 28÷30×100%≈93%.

[0087] Example 3: Screening of single-domain antibodies against IL-6 protein:

[0088] Take 200 μL of the recombinant TG1 cells in Example 2 and culture them in 2×TY medium. During this period, add 40 μL of helper phage VCSM13 to infect TG1 cells, and culture overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0089] Couple 500 μg of IL-6 protein diluted in 100 mM NaHCO3 at pH 8.3 onto an ELISA plate and place it at 4°C overnight. At the same time, set up a negative control well (medium control); the next day, add 200 μL of 3% skim milk and block at room temperature for 2 h; after blocking, add 100 μl of the amplified phage library (about 2×10 11 phage particles), and incubate at room temperature for 1 h; after incubating for 1 h, wash 15 times with PBS + 0.05% Tween-20 to wash away the unbound phage.

[0090] Dissociate the phage specifically bound to IL-6 protein with trypsin at a final concentration of 25 mg / mL, and infect Escherichia coli TG1 cells in the logarithmic growth phase. Culture at 37°C for 1 h, produce and collect the phage for the next round of screening. Repeat the same screening process for 1 round to gradually obtain enrichment.

[0091] When the enrichment factor reaches more than 10 times, the enrichment effect is as Figure 1 shown.

[0092] Figure 1 where P / N = the number of monoclonal bacteria grown after infecting TG1 bacteria with the phage eluted from the positive well in biopanning / the number of monoclonal bacteria grown after infecting TG1 bacteria with the phage eluted from the negative well. This parameter will gradually increase after enrichment; I / E = the total amount of phage added to the positive well in each round of biopanning / the total amount of phage eluted from the positive well in each round of biopanning. This parameter will gradually approach 1 after enrichment.

[0093] Example 4: Screening of specific positive clones against IL-6 by phage enzyme-linked immunosorbent assay (ELISA):

[0094] The single-domain antibodies against IL-6 protein were screened for 2 rounds according to the screening method in Example 3 above. The phage enrichment factor against IL-6 protein reached more than 10. After the screening, 384 single colonies were selected from the obtained positive clones and inoculated into 96-well deep-well plates containing 2×TY medium with 100 μg / mL ampicillin, and a blank control was set. After culturing at 37 °C until the logarithmic phase, IPTG with a final concentration of 1 mM was added, and the culture was continued at 28 °C overnight.

[0095] Crude antibodies were obtained by osmotic lysis method; the IL-6 recombinant protein was separately released into 100 mM NaHCO3 at pH 8.3, and 100 μg of the protein was coated overnight at 4 °C in an ELISA plate. 100 μL of the obtained crude antibody solution was transferred to the ELISA plate with the antigen added and incubated at room temperature for 1 h; unbound antibodies were washed away with PBST, 100 μl of Mouse Anti-HA tag Antibody (HRP) (mouse anti-HA horseradish peroxidase-labeled antibody, Thermo Fisher) diluted 1:2000 was added, and the incubation was continued at room temperature for 1 h; unbound antibodies were washed away with PBST, the horseradish peroxidase chromogenic solution was added, and after reacting at 37 °C for 15 min, the stop solution was added, and the absorbance was read at a wavelength of 450 nm on an ELISA reader.

[0096] When the OD value of the sample well was more than 5 times that of the control well, it was determined as a positive clone well; the bacteria in the positive clone well were transferred and shaken in LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0097] According to the sequence alignment software VectorNTI, the gene sequences of each clone were analyzed. Clones with the same CDR1, CDR2, and CDR3 sequences were regarded as the same clone, while clones with different sequences were regarded as different clones. Finally, single-domain antibodies specifically targeting IL-6 protein were obtained (including single-domain antibodies 2C6, 2D7, 2E10, 3F7, and single-domain antibody clones 1C10, 1E5, 1E6, 2B9, 2F4, 3C2, 3C7, 3C8, 3C10, 3C12, 3G6, 4B12, 4D6, 1G8, 2C7 whose sequences are not shown).

[0098] The amino acid sequence of its antibody is in the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally, single-domain antibody protein is obtained.

[0099] The amino acid sequences of single-domain antibodies 2C6, 2D7, 2E10, and 3F7 are shown as SEQ ID NO:1-4 in sequence, and their nucleotide sequences are shown as SEQ ID NO:5-8 in sequence.

[0100] The CDR sequences of the 4 single-domain antibodies are shown in Table 1-3. The FR1 sequences of the 4 single-domain antibodies are as shown in SEQ ID NO:9, the FR2 sequences of the 4 single-domain antibodies are as shown in SEQ ID NO:10, the FR3 sequences of the 4 single-domain antibodies are as shown in SEQ ID NO:11, and the FR4 sequences of the 4 single-domain antibodies are as shown in SEQ ID NO:12.

[0101] Table 1 CDR1 sequences of 4 single-domain antibodies

[0102]

[0103] Table 2 CDR2 sequences of 4 single-domain antibodies

[0104]

[0105] Table 3 CDR3 sequences of 4 single-domain antibodies

[0106]

[0107] Example 5: Purification and expression of a specific single-domain antibody against IL-6 protein in the host bacterium Escherichia coli

[0108] The plasmids (pMECS-VHH) of different clones obtained by sequencing analysis in Example 4 were electrotransformed into Escherichia coli HB2151, and then spread on an LB + amp + glucose culture plate containing ampicillin and glucose, and cultured overnight at 37°C; single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin, and cultured overnight on a shaker at 37°C.

[0109] 1 mL of the overnight culture was inoculated into 330 mL of TB culture medium, and cultured on a shaker at 37°C. When the OD600nm value reached 0.6 - 0.9, 1 mM IPTG was added, and the culture was continued overnight on a shaker at 28°C; the cells were centrifuged to collect Escherichia coli, and the crude antibody solution was obtained by osmotic lysis.

[0110] The antibody was purified by nickel column affinity chromatography.

[0111] Example 6: Construction of a eukaryotic expression vector for the Fc fusion antibody of a single-domain antibody against IL-6

[0112] (1) Subclone the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened out in Example 4 was subjected to Sanger sequencing to obtain its nucleotide sequence;

[0113] (2) Synthesize the above nucleotide sequences (SEQ ID NO: 5 - 8 and the nucleotide sequences of other single - domain antibody clones with sequences not shown) into the vector RJK - V4 - hFC1 designed and modified by our company through sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of this vector is as described in Example 10;

[0114] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0115] (4) Sequentially sequence and identify the plasmid after extraction;

[0116] (5) Prepare the recombinant vector for subsequent eukaryotic cell transfection and expression after confirmation. Express the Fc protein of VHH by the method of Example 7 or 8 and purify the above antibody by the method of Example 9.

[0117] Example 7: Expression of a single - domain antibody against IL - 6 protein in suspension ExpiCHO - S cells

[0118] (1) Three days before transfection, passage and expand the culture of ExpiCHO - S 5 cells at a density of 2.5×10 TM / mL, transfer the calculated required cell volume to a 500 - mL shake flask containing fresh pre - warmed 120 mL (final volume) of ExpiCHO TM expression medium; make the cell concentration reach about 4×10 6 - 6×10 6 viable cells / mL;

[0119] (2) One day before transfection, dilute the ExpiCHO - S TM cells to a concentration of 3.5×10 6 viable cells / mL and culture the cells overnight;

[0120] (3) On the day of transfection, measure the cell density and the percentage of viable cells. The cell density before transfection should reach about 7×10 6 - 10×10 6 viable cells / mL;

[0121] (4) Dilute the cells to 6×10 TM with fresh pre - warmed ExpiCHO 6 expression medium at 37℃. Transfer the calculated required cell volume to a 500 - mL shake flask containing fresh pre - warmed 100 mL (final volume) of ExpiCHO TM expression medium;

[0122] (5) Gently invert and mix ExpiFectamine TM CHO reagent, dilute ExpiFectamine with 3.7 mL of OptiPRO TM medium TM CHO reagent, swirl or mix;

[0123] (6) Dilute plasmid DNA with 4 mL of chilled OptiPRO TM medium, swirl and mix;

[0124] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-IL-6 single domain antibody prepared in Example 6) complex at room temperature for 1 - 5 minutes, then gently add it to the prepared cell suspension, gently swirling the flask during the addition;

[0125] (8) Incubate the cells with shaking in a humidified air atmosphere containing 8% CO2 at 37°C;

[0126] (9) Add 600 μL of ExpiFectamine TM CHO Enhancer and 24 mL of ExpiCHO feed 1 day (18 - 22 hours) after transfection;

[0127] (10) Collect the supernatant approximately 8 days after transfection (when the cell viability is less than 70%).

[0128] Example 8: Expression of anti-IL-6 protein single domain antibody in suspension 293F cells

[0129] Recombinant single domain antibody expression experimental procedure (taking a 500 mL flask as an example):

[0130] (1) Three days before transfection, passage and expand 293F cells at 2.5×10 5 / mL, transfer the calculated required cell volume to a 500 mL flask containing 120 mL (final volume) of pre-warmed OPM-293CD05 Medium. Adjust the cell concentration to approximately 2×10 6 - 3×10 6 viable cells / mL.

[0131] (2) On the day of transfection, measure the cell density and the percentage of viable cells. The cell density should reach approximately 2×10 6 - 3×10 6 viable cells / mL before transfection.

[0132] (3) Dilute the cells with pre-warmed OPM-293CD05 Medium to 1×10 6cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed medium.

[0133] (4) Dilute the PEI (1 mg / mL) reagent with 4 mL of Opti-MEM medium, and mix well by swirling or pipetting up and down; dilute the plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-IL-6 single domain antibody prepared in Example 6) with 4 mL of Opti-MEM medium, mix well by swirling, and filter through a 0.22 μm filter tip. Incubate at room temperature for 5 min.

[0134] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15 - 20 minutes, then gently add it to the prepared cell suspension, swirling the shake flask gently during the addition.

[0135] (6) Culture the cells at 37 °C, 5% CO2, and 120 rpm on a shaker.

[0136] (7) Add 5 mL of OPM-CHOPFF05 feed at 24 h and 72 h after transfection.

[0137] (8) Collect the supernatant at about 7 days after transfection (when the cell viability is less than 70%).

[0138] Example 9: Purification of the single domain antibody against IL-6 protein

[0139] (1) Filter the protein expression supernatant obtained in Example 7 or 8 through a 0.45 μm disposable filter tip to remove insoluble impurities;

[0140] (2) Purify the above filtrate by affinity chromatography using a protein purifier. Utilize the ability of human Fc to bind to Protein A, and use agarose beads conjugated with Protein A for purification;

[0141] (3) Pass the filtrate through a Protein A pre-packed column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material;

[0142] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffer solutions;

[0143] (5) Elute the target protein bound to the column with a low-pH buffer solution;

[0144] (6) Immediately add the eluate to a Tris-HCl solution with a pH of 9.0 for neutralization;

[0145] (7) After dialysis of the neutralized protein solution above, perform SDS-PAGE analysis. After determining that the protein purity is above 95% and the concentration is above 0.5 mg / mL, store it at low temperature for later use.

[0146] Example 10: Construction of a eukaryotic expression vector for single-domain antibody

[0147] The target vector RJK-V4-hFC1 commonly mentioned for nanobodies is modified on the basis of the commercial vector pCDNA3.4 of the company invitrogen (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by fusing the Fc segment in the heavy chain coding sequence of human IgG1. That is, this vector contains the hinge region (Hinge), CH2, and CH3 regions of the IgG1 heavy chain. The specific modification scheme is as follows:

[0148] (1) Select the restriction enzyme cleavage sites XbaI and AgeI on pcDNA3.4;

[0149] (2) Introduce a multiple cloning site (MCS, Multiple Cloning Site) and a 6×His tag at the 5' end and 3' end of the Fc fragment coding sequence by overlapping PCR respectively;

[0150] (3) Amplify the above fragment by PCR using a pair of primers with XbaI and AgeI restriction enzyme cleavage sites respectively;

[0151] (4) Digest pcDNA3.4 and the recombinant DNA fragment in (3) with the restriction enzymes XbaI and AgeI respectively;

[0152] (5) Ligate the digested vector and the inserted fragment under the action of T4 ligase, then transform the ligation product into Escherichia coli, amplify, verify by sequencing, and obtain the recombinant plasmid.

[0153] The name with hFC1 refers to: the Fc fusion antibody obtained by eukaryotic expression after cloning the corresponding single-domain antibody sequence into RJK-V4-hFC1 (for example, 2C6-hFC1 is the Fc fusion antibody obtained by eukaryotic expression after cloning the nucleotide sequence corresponding to the single-domain antibody of 2C6 into RJK-V4-hFC1).

[0154] Example 11: Expression and purification of a tool antibody (Tab1) targeting human IL-6

[0155] In this article, Tab1 is Siltuximab.

[0156] The searched sequences were entrusted to General Biosystems (Anhui) Co., Ltd. for codon optimization in the mammalian cell expression system and cloned into the pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification, and the plasmid was extracted using a plasmid midiprep kit (MachereyNagel, Cat#740412.50). According to the addition of 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) per 100 mL of cells, transient expression was carried out in 293F cells (culture medium: FreeStyle293 Expression medium, Thermo, Cat#12338026 + F-68, Thermo, Cat#24040032) using PEI; 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added 6 - 24 h after transfection, and cultured at 8% CO2 and 130 rpm for about 7 - 8 days; when the cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column; after dialysis with PBS, the concentration was measured using Nanodrop, the purity was identified by SEC, and the binding ability was verified by indirect ELISA;

[0157] The Tab1 obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0158] Example 12: Determination of the antigen-binding dose-response curve of the antibody

[0159] This example was carried out using the standard enzyme-linked immunosorbent assay (ELISA) operation procedure.

[0160] (1) Coat 50 μL of 1 μg / mL IL-6 protein overnight at 4°C.

[0161] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0162] (3) Dilute VHH-hFc to 2 μg / mL, and then serially dilute the antibody 5-fold for a total of 8 concentration gradients. Here, VHH-hFc was obtained by purifying the anti-IL-6 protein single-domain antibody (which is fusion-expressed with Fc) prepared in Example 8 through Example 9. In addition, hIgG and Tab1 controls were also set respectively; Tab1 was prepared in Example 11;

[0163] (4) Wash the plate; add 50 μL of the single-domain antibody diluted in step (3), in duplicate, and incubate at 37°C for 1 h.

[0164] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37 °C for 30 min.

[0165] (6) Wash the plate (wash several times); add 50 μL of TMB pre-restored to room temperature and react at room temperature in the dark for 15 min.

[0166] (7) Add 50 μL of termination solution (1N HCl) and save the readings on the microplate reader.

[0167] (8) Plot the curve and calculate the EC50, as Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 shown, where hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target, and is obtained by commercial purchase.

[0168] The 4 single-domain antibodies of the present invention all have excellent binding potency and specificity to IL-6 protein.

[0169] Example 13: Detection of the neutralization of IL-6-induced TF1 cell proliferation by the antibody (eukaryotic sample).

[0170] Perform the following operations according to the method commonly used by those skilled in the art:

[0171] (1) Seed TF-1 cells that have been passaged 3 - 4 times after resuscitation into a 96-well plate at 10,000 cells per well;

[0172] (2) Prepare solutions of Tab1 (prepared in Example 11) and the Fc-fused single-domain antibody (the anti-IL-6 protein single-domain antibody prepared in Example 8 and purified by Example 9) at 10 μg / mL respectively, and perform 5-fold serial dilutions;

[0173] (3) Mix the serially diluted Tab1 and single-domain antibody with IL-6 at a concentration of 1.985 ng / ml in a 1:1 ratio to prepare a mixture;

[0174] (4) Add the mixture in the above step to the cell culture wells in an equal volume of cell culture medium;

[0175] (5) After incubating for 72 h, detect the cell viability using a luminescence-based cell viability detection kit;

[0176] (6) Calculate the EC50 concentration of different single-domain antibodies for neutralizing IL-6-induced TF-1 cell proliferation according to the detection results, and the results are as Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 shown.

[0177] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A single-domain antibody against IL-6, characterized in that: The single-domain antibody described above is composed of a heavy chain, and the heavy chain includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are one of the following (1)-(4): (1) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO: 16, CDR3 shown in SEQ ID NO:17; (2) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO: 16, CDR3 shown in SEQ ID NO:19; (3) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO: 16, CDR3 shown in SEQ ID NO:18; (4) CDR1 shown in SEQ ID NO:15, CDR2 shown in SEQ ID NO:16, CDR3 shown in SEQ ID NO:

19.

2. The anti-IL-6 single domain antibody according to claim 1, characterized in that: The heavy chain further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are respectively: FR1 shown in SEQ ID NO:9 or a variant of FR1, and the variant of FR1 contains at most 5 amino acid substitutions in the FR1; FR2 shown in SEQ ID NO:10 or a variant of FR2, and the variant of FR2 contains at most 5 amino acid substitutions in the FR2; FR3 shown in SEQ ID NO:11 or a variant of FR3, and the variant of FR3 contains at most 5 amino acid substitutions in the FR3; FR4 shown in SEQ ID NO:12 or a variant of FR4, and the variant of FR4 contains at most 5 amino acid substitutions in the FR4.

3. A single-domain antibody against IL-6, characterized in that: The amino acid sequences of the single-domain antibody are respectively shown in any one of SEQ ID NO:1-4.

4. The Fc fusion antibody or humanized antibody of the anti-IL-6 single-domain antibody according to any one of claims 1 to 3.

5. A nucleotide molecule encoding the anti-IL-6 single domain antibody according to any one of claims 1 to 3, characterized in that: Its nucleotide sequence is respectively shown in any one of SEQ ID NO:5-8.

6. An expression vector, characterized in that: It contains a nucleotide molecule encoding the anti-IL-6 single-domain antibody according to any one of claims 1 to 3 or the Fc fusion antibody or humanized antibody according to claim 4.

7. A host cell, characterized in that: It can express the anti-IL-6 single-domain antibody according to any one of claims 1 to 3 or the Fc fusion antibody or humanized antibody according to claim 4, or it contains the expression vector according to claim 6.

8. A pharmaceutical composition, characterized in that: The pharmaceutical composition described above contains the anti-IL-6 single-domain antibody selected from any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

9. Use of the anti-IL-6 single domain antibody according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating a disease, characterized in that: The diseases described above are multicentric Castleman disease, SARS-CoV-2 acute respiratory disease, multiple myeloma, or leukemia.

Images