Disclosure of Invention
The object of the present invention is to provide a single domain antibody capable of specifically binding to IL-6 and uses thereof.
In a first aspect, the invention provides an anti-IL-6 single domain antibody, which is composed of a heavy chain comprising a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, wherein the amino acid sequence of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 is 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, and CDR3 shown in SEQ ID NO. 19.
The above CDR combinations (1) - (4) correspond in sequence to single domain antibodies 3F7 (SEQ ID NO: 4), 2C6 (SEQ ID NO: 1), 2D7 (SEQ ID NO: 2), 2E10 (SEQ ID NO: 3).
All of the above sequences may be replaced with sequences having "at least 80% homology" 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".
In one embodiment, wherein any one to five of the amino acid residues in any one or more of the CDRs of heavy chain CDR1, CDR2 and CDR3 may be substituted with their conserved amino acids, respectively. Specifically, 1 to 5 amino acid residues in the heavy chain CDR1 can be replaced by the conserved amino acid, 1 to 5 amino acid residues in the heavy chain CDR2 can be replaced by the conserved amino acid, and 1 to 5 amino acid residues in the heavy chain CDR3 can be replaced by the conserved amino acid.
As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies with identical sequences have 100% homology.
In some embodiments, sequences that replace only one or a few amino acids, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to the preceding sequences, may also achieve the object. Such variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and the addition of one or more (typically less than 20, preferably less than 10, more preferably less than 5) amino acids at the C-terminus and/or N-terminus. In fact, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution will preferably be conservative amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which 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, e.g., conservative amino acid substitutions are those in which one or a few amino acids in groups (a) - (d) are substituted for another or a few amino acids in the same group (a) a polar negatively charged residue and its uncharged amide Asp, asn, glu, gln, (b) a polar positively charged residue His, arg, lys, (c) an aromatic residue Phe, trp, tyr, and (d) an aliphatic nonpolar or weakly polar residue Ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are those wherein Asp is substituted by Glu, asn is substituted by Gln or His, glu is substituted by Asp, gln is substituted by Asn, his is substituted by Asn or Gln, arg is substituted by Lys, lys is substituted by Arg, gln, phe is substituted by Met, leu, tyr, trp is substituted by Tyr, tyr is substituted by Phe, trp, ala is substituted by Gly or Ser, ser is substituted by Thr, thr is substituted by Ser, gly is substituted by Ala or Pro, met is substituted by Leu, tyr or Ile, leu is substituted by Ile or Val, val is substituted by Ile or Leu, cys is substituted by Ser. In addition, it is known to those skilled in the art that the framework region sequences FR1-4 are not unalterable and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.
The meaning of "anti-IL-6 single domain antibody" in the present invention includes not only the whole single domain antibody but also fragments, derivatives and analogues of the anti-IL-6 single domain antibody. As used herein, the terms "fragment," "derivative," and "analog" are synonymous and refer to a polypeptide that retains substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an Fc tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.
In a preferred embodiment, the heavy chain further comprises a framework region FR comprising the amino acid sequences of FR1, FR2, FR3 and FR4, the amino acid sequences of framework region FR being:
An FR1 or a variant of FR1 as shown in SEQ ID No. 9, said variant of FR1 comprising up to 5 amino acid substitutions in said FR 1;
10. The FR2 or a variant of FR2 comprising up to 5 amino acid substitutions in said FR 2;
11, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3;
FR4 shown in SEQ ID NO. 12 or a variant of FR4, said variant of FR4 comprising at most 5 amino acid substitutions in said FR 4.
In a second aspect, the invention provides an amino acid sequence of an anti-IL-6 single domain antibody, which is shown in any one of SEQ ID NOS.1-4, or which has at least 80% sequence homology with the amino acid sequence of SEQ ID NOS.1-4, and which is capable of specifically binding to an IL-6 protein.
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 to an amino acid sequence selected from SEQ ID NOS: 1-4 or SEQ ID NOS: 1-4 and is capable of specifically binding to an IL-6 protein.
A third aspect of the invention provides an Fc fusion antibody or a humanized antibody of any of the foregoing anti-IL-6 single domain antibodies.
In a fourth aspect, the invention provides a nucleotide molecule encoding the aforementioned anti-IL-6 single domain antibody, which has a nucleotide sequence as shown in any one of SEQ ID NOS 5-8, or has at least 80% sequence homology with any one of SEQ ID NOS 5-8, respectively.
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 to a nucleotide sequence selected from SEQ ID NO 5-8, and encodes an anti-IL-6 single domain antibody capable of specifically binding to an IL-6 protein.
In a fifth aspect, the invention provides an expression vector comprising a nucleotide molecule encoding an anti-IL-6 single domain antibody or Fc fusion antibody or humanized antibody, the anti-IL-6 single domain antibody having a nucleotide sequence as shown in SEQ ID NOS 5-8 or having at least 80% sequence homology with any one of SEQ ID NOS 5-8, respectively.
In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (the nucleotide molecules encoding the anti-IL-6 single domain antibody or its Fc fusion antibody or humanized antibody are integrated into RJK-V4-hFC1 by genetic engineering means), and other universal expression vectors may be selected as desired.
In a sixth aspect, the invention provides a host cell capable of expressing the aforementioned anti-IL-6 single domain antibody, fc fusion antibody or humanized antibody, or comprising the aforementioned expression vector. Preferably the host cell is a bacterial cell, a fungal cell or a mammalian cell.
In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell, including bacteria, fungi.
In another preferred embodiment, the host cell is selected from the group consisting of E.coli, yeast cells, mammalian cells, phage, or combinations thereof.
In another preferred embodiment, the prokaryotic cell is selected from the group consisting of E.coli, bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or a combination thereof.
In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma, or a combination thereof.
In another preferred embodiment, the eukaryotic cell is selected from the group consisting of insect cells such as armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.
In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.
In another preferred embodiment, the host cell is a suspension 293F cell.
In a seventh aspect, the invention provides a recombinant protein, comprising the aforementioned anti IL-6 single domain antibody. The recombinant protein can be a single domain antibody shown in SEQ ID NO. 1-4, a single domain antibody with at least 80% homology with SEQ ID NO. 1-4, a multi-epitope antibody, a multi-specific antibody and a multivalent antibody, wherein for example, the multi-epitope antibody can be composed of more than one sequence in SEQ ID NO. 1-4, the multivalent antibody can be composed of one sequence in SEQ ID NO. 1-4 repeatedly arranged for a plurality of times, the multi-specific antibody comprises but is not limited to a bispecific antibody and a trispecific antibody, and the recombinant protein can be fragments, derivatives and analogues of the antibodies.
In an eighth aspect, the invention provides a pharmaceutical composition comprising the aforementioned anti-IL-6 single domain antibody and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined by the isoelectric point of the antibody (the pH of the aqueous carrier medium is required to deviate from and from about 2 from the isoelectric point of the antibody).
The pharmaceutical compositions of the invention can be used directly to bind IL-6 protein molecules.
The pharmaceutical composition is used for treating diseases. In a preferred embodiment, the disease Castleman's disease, multicenter Castleman's disease, SARS-CoV-2 acute respiratory disease, smoldering multiple myeloma or multiple myeloma, leukemia.
The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the foregoing single domain antibodies, together with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions.
In a ninth aspect, the invention provides the use of an anti-IL-6 single domain antibody as described above or a pharmaceutical composition as described above in the manufacture of a medicament for the treatment of a disease.
In a preferred embodiment, the disease is Castleman's disease (Cassman's disease), multicenter Castleman's disease, SARS-CoV-2 acute respiratory disease, smoldering multiple myeloma or multiple myeloma, leukemia.
The invention also provides a kit for detecting IL-6 level, which contains the single domain antibody of the IL-6. In a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffers, etc.
In a preferred embodiment, the kit comprises antibodies recognizing IL-6 protein, a lysis medium for lysing the sample, universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.
In a preferred embodiment, the kit further comprises a second antibody and an enzyme or fluorescent or radiolabel for detection, and a buffer.
In a preferred embodiment, the second antibody of the kit may be an antibody (as an anti-antibody) to the aforementioned anti-IL-6 single domain antibody, may be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.
The invention also provides a method of producing a single domain antibody against IL-6 comprising the steps of:
(a) Culturing the host cell of the sixth aspect of the invention under conditions suitable for the production of a single domain antibody, thereby obtaining a culture comprising said anti-IL-6 single domain antibody;
(b) Isolating or recovering said anti-IL-6 single domain antibody from said culture;
(c) Optionally, purifying and/or modifying the anti-IL-6 single domain antibody obtained in step (b).
Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) The single domain antibodies of the invention are specific for IL-6 proteins with the correct spatial structure.
(2) The single domain antibody obtained by the invention has flexible expression system selection, can be expressed in a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells, has low expression cost in the prokaryotic expression system, and can reduce the post production cost.
(3) The single-domain antibody obtained by the invention has simple reconstruction of the multi-combination form of the antibody, can obtain multivalent and multi-specific antibodies through simple serial connection in a genetic engineering mode, has low immune heterogeneity and can not generate stronger immune response under the condition of not undergoing humanized reconstruction.
(4) The single domain antibody obtained by the invention has wider affinity range, and the affinity range can be from nM level to pM level before affinity maturation, so that multiple choices are provided for antibodies with different later uses.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
As used herein, a "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. Thus, a single domain antibody comprises a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.
The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as framework regions.
As used herein, the term "Fc fusion antibody" refers to a protein produced by fusing the Fc segment of an antibody of interest to a functional protein molecule having biological activity using genetic engineering techniques.
The term "humanized antibody" refers to an antibody obtained by fusing a heavy chain variable region of a target antibody (e.g., an animal antibody) with a constant region of a human antibody, or an antibody obtained by grafting complementarity determining regions (CDR 1 to CDR 3 sequences) of a target antibody into a variable region of a human antibody, or an antibody obtained by subjecting a target antibody to amino acid mutation according to the characteristics of human antibody framework regions (FR 1 to FR 4). Humanized antibodies can be synthesized or site-directed mutagenesis.
In the present invention, a single domain antibody against IL-6 can be obtained from a sequence having high sequence homology with CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" with the sequences in SEQ ID NOS.1-4, or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" may be used for the purposes of the invention.
In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in SEQ ID NOs 1-4, e.g., comprising 1,2, 3, 4, 5, 6,7, 8, 9 or 10 conservative amino acid substitutions, may also achieve the object. In fact, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution will preferably be conservative amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which 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, e.g., conservative amino acid substitutions are those in which one or a few amino acids in groups (a) - (d) are substituted for another or a few amino acids in the same group (a) a polar negatively charged residue and its uncharged amide Asp, asn, glu, gln, (b) a polar positively charged residue His, arg, lys, (c) an aromatic residue Phe, trp, tyr, and (d) an aliphatic nonpolar or weakly polar residue Ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are those wherein Asp is substituted by Glu, asn is substituted by Gln or His, glu is substituted by Asp, gln is substituted by Asn, his is substituted by Asn or Gln, arg is substituted by Lys, lys is substituted by Arg, gln, phe is substituted by Met, leu, tyr, trp is substituted by Tyr, tyr is substituted by Phe, trp, ala is substituted by Gly or Ser, ser is substituted by Thr, thr is substituted by Ser, gly is substituted by Ala or Pro, met is substituted by Leu, tyr or Ile, leu is substituted by Ile or Val, val is substituted by Ile or Leu, cys is substituted by Ser. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
The preparation method comprises the steps of preparing target protein and a truncated form of the target protein through a genetic engineering technology, immunizing an inner Mongolian alashan alpaca with the obtained antigen protein, obtaining peripheral blood lymphocytes or spleen cells of the alpaca after multiple immunization, recombining a camel source antibody variable region coding sequence into a phage display carrier through a genetic engineering mode, screening out a specific antibody aiming at the antigen protein through a phage display technology, and further detecting the binding capacity of the specific antibody and the antigen.
The above technical solutions will now be described in detail by way of specific embodiments:
example 1 preparation of human IL-6 protein:
The human recombinant IL-6 protein used in the patent is obtained by self-expression and purification of a company, and the design scheme of an expression vector of the human recombinant IL-6 protein is as follows:
(1) The coding sequence for IL-6, which is identified as NP-000591.1, was retrieved in NCBI and encoded to yield the amino acid sequence accession number P05231.
(2) The nucleotide sequence of the 30 th to 212 th amino acid of the IL-6 protein (the 30 th to 212 th amino acid is the extracellular domain of the IL-6 protein) is cloned into a vector pcDNA3.4 by utilizing a gene synthesis mode. And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out mass extraction on the recombinant plasmid after confirming no errors, removing endotoxin, and carrying out target protein expression and purification on transfected suspension 293F cells, wherein the purity reaches more than 90%, and meets the animal immunization requirement.
Example 2 construction of a single domain antibody library against IL-6 protein:
1mg of the recombinant human IL-6 protein obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, and an inner Mongolian Alexal camel was immunized once a week for a total of 7 consecutive immunizations, and the remaining six immunizations were animal immunized with 1mg of IL-6 protein mixed with Freund's incomplete adjuvant in equal volumes except for the first immunization, in order to intensively stimulate the camel to produce antibodies against IL-6 protein.
After the animal immunization is finished, 150mL of camel peripheral blood lymphocytes are extracted, and RNA of the cells is extracted. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified by a nested PCR reaction using the cDNA as a template.
Then, the vector pMECS and the VHH fragment are digested with restriction enzymes, respectively, and then the digested fragments and the vector are linked. The ligated fragments were electrotransformed into competent cells TG1, phage display libraries of IL-6 protein were constructed and the library capacity was determined, the library capacity was about 1X 109, and the correct insertion rate of the library at the fragment of interest was detected by colony PCR identification.
The results showed that 28 clones amplified bands of predicted size and 2 clones amplified incorrectly after PCR amplification of 30 randomly selected colonies from the library, so that the correct insertion rate was 28.about.30.times.100.about.93%.
Example 3 screening of Single-domain antibodies against IL-6 protein:
200. Mu.L of the recombinant TG1 cells of example 2 were cultured in 2 XTY medium, during which 40. Mu.L of helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phage, the phage was precipitated the next day with PEG/NaCl, and the amplified phage was collected by centrifugation.
500. Mu.g of IL-6 protein diluted in 100mM NaHCO3 at pH8.3 was coupled to the ELISA plate, left overnight at 4℃while negative control wells (medium control) were established, 200. Mu.L of 3% skim milk was added the next day, blocked at room temperature for 2h, 100. Mu.L of amplified phage library (approximately 2X 1011 phage particles) was added after the end of the blocking, and allowed to act at room temperature for 1h, and after 1h of action, washed 15 times with PBS+0.05% Tween-20 to wash out unbound phage.
Phage specifically binding to IL-6 protein was dissociated with trypsin at a final concentration of 25mg/mL, and E.coli TG1 cells in the logarithmic growth phase were infected, cultured at 37℃for 1h, phage were generated and collected for the next round of screening, and the same screening process was repeated for 1 round, and enrichment was gradually obtained.
When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 1.
In fig. 1, P/n=number of monoclonal bacteria grown after phage infection TG1 bacteria by biopanning positive Kong Xi/number of monoclonal bacteria grown after phage infection TG1 bacteria by negative Kong Xi, which gradually increases after enrichment occurs, and I/e=total number of phage added to positive wells per round of biopanning/total number of phage removed from positive Kong Xi per round of biopanning, which gradually approaches 1 after enrichment occurs.
Example 4 screening of specific positive clones for IL-6 by phage enzyme-linked immunosorbent assay (ELISA):
Screening was performed according to the screening method described in example 3 above for 2 rounds of screening against a single domain antibody against IL-6 protein, the phage enrichment factor against IL-6 protein was 10 or more, 384 single colonies were selected from positive clones obtained by screening after the end of screening, inoculated into 96-well plates containing 2 XSTY medium of 100. Mu.g/mL ampicillin, respectively, and a blank was set, and after 37℃to the logarithmic phase, IPTG was added at a final concentration of 1mM, and cultured overnight at 28 ℃.
Crude antibodies were obtained by osmotic swelling, IL-6 recombinant proteins were released into 100mM NaHCO3, pH8.3, respectively, and 100. Mu.g of the proteins were coated in ELISA plates (ELISA plates) at 4℃overnight. Transferring 100 μl of the obtained crude antibody extract to ELISA plate added with antigen, incubating at room temperature for 1 hr, washing unbound antibody with PBST, adding MouseAnti-HAtagAntibody (HRP) (mouse anti-HA horseradish peroxidase labeled antibody diluted at 1:2000), incubating at room temperature for 1 hr, washing unbound antibody with PBST, adding horse radish peroxidase chromogenic solution, reacting at 37deg.C for 15min, adding stop solution, and reading absorption value at 450nm wavelength on enzyme-labeled instrument.
When the OD value of the sample well is 5 times or more greater than that of the control well, positive clone wells are judged, and the positive clone wells are transferred to LB medium containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced.
Gene sequences of the respective clones were analyzed according to sequence alignment software VectorNTI, strains with the same CDR1, CDR2 and CDR3 sequences were regarded as the same clone, and strains with different sequences were regarded as different clones, and finally single domain antibodies specific for IL-6 proteins (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, 2C 7) were obtained.
The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally the single-domain antibody protein is obtained.
The amino acid sequence of the single domain antibody 2C6,2D7,2E10,3F7 is shown as SEQ ID NO. 1-4, and the nucleotide sequence is shown as SEQ ID NO. 5-8.
CDR sequences of the 4 single domain antibodies are shown in tables 1-3. The FR1 sequences of the 4 single-domain antibodies are shown as SEQ ID NO. 9, the FR2 sequences of the 4 single-domain antibodies are shown as SEQ ID NO. 10, the FR3 sequences of the 4 single-domain antibodies are shown as SEQ ID NO. 11, and the FR4 sequences of the 4 single-domain antibodies are shown as SEQ ID NO. 12.
TABLE 14 CDR1 sequences of single domain antibodies
TABLE 24 CDR2 sequences of single domain antibodies
TABLE 34 CDR3 sequences of single domain antibodies
EXAMPLE 5 purification and expression of specific Single-domain antibodies against IL-6 protein in E.coli
Plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in example 4 were electrotransformed into E.coli HB2151 and plated on LB+amp+glucose-containing culture plates, cultured overnight at 37℃and single colonies were selected and inoculated in 5mL ampicillin-containing LB medium and shake cultured overnight at 37 ℃.
Inoculating 1mL of overnight culture strain into 330mLTB culture solution, shake culturing at 37deg.C until OD600nm reaches 0.6-0.9, adding 1MIPTG, shake culturing at 28deg.C overnight, centrifuging, collecting Escherichia coli, and obtaining crude extractive solution of antibody by osmotic swelling method.
The antibodies were purified by nickel column affinity chromatography.
EXAMPLE 6 construction of Fc fusion antibody eukaryotic expression vector of anti-IL-6 Single-domain antibody
(1) Subcloning the target sequence obtained in example 4 into eukaryotic expression vector, and subjecting the antibody screened in example 4 to Sanger sequencing to obtain nucleotide sequence;
(2) The above nucleotide sequences (SEQ ID NOS: 5-8 and nucleotide sequences of other single domain antibody clones not showing sequences) were synthesized into the vector RJK-V4-hFC1 designed and modified by the present company by means of sequence synthesis to obtain a recombinant eukaryotic expression vector, the modification method of which is described in example 10;
(3) Converting the recombinant eukaryotic expression vector constructed in the step (2) into DH5 alpha escherichia coli, culturing to extract plasmids, and removing endotoxin;
(4) Sequencing and identifying the extracted plasmid;
(5) The recombinant vector after confirmation was prepared for subsequent eukaryotic cell transfection and expression, and after expression of the Fc protein of VHH by the method of example 7 or 8, the above antibody was purified by the method of example 9.
Example 7 expression of anti-IL-6 protein Single Domain antibodies in suspension ExpiCHO-S cells
(1) Passaging and expanding ExpiCHO-STM cells at 2.5X105/mL cells 3 days prior to transfection, transferring the calculated desired cell volume to a 500mL shake flask containing fresh pre-warmed 120mL (final volume) ExpiCHOTM expression medium, bringing the cell concentration to about 4X 106-6×106 viable cells/mL;
(2) The day before transfection, expiCHO-STM cells were diluted to 3.5X106 viable cells/mL and allowed to incubate overnight;
(3) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 106-10×106 viable cells/mL prior to transfection;
(4) Cells were diluted to 6×106 viable cells/mL with fresh ExpiCHOTM expression medium pre-warmed to 37 ℃. The calculated desired cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) ExpiCHOTM expression medium;
(5) Mixing ExpiFectamineTM CHO reagent gently upside down, diluting ExpiFectamineTM CHO reagent with 3.7mLOptiPROTM medium, and stirring or mixing;
(6) Diluting plasmid DNA with refrigerated 4mLOptiPROTM culture medium, and mixing;
(7) Incubating ExpiFectamineCHO/plasmid DNA (plasmid DNA is Fc fusion antibody eukaryotic expression vector of anti-IL-6 single domain antibody prepared in example 6) complex for 1-5 min at room temperature, then adding gently into the prepared cell suspension, and gently agitating shake flask during addition;
(8) Shake culturing cells in 37 ℃ and 8% co2 in humidified air;
(9) 600 μ lExpiFectamineTM CHOEnhancer and 24mLExpiCHO feed were added on day 1 (18-22 hours post transfection);
(10) Supernatants were collected about 8 days after transfection (cell viability below 70%).
Example 8 expression of anti-IL-6 protein Single Domain antibodies in suspension 293F cells
Recombinant single domain antibody expression experimental procedure (500 mL shake flask for example):
(1) 293F cells were passaged and expanded at 2.5X105/mL 3 days prior to transfection, and the calculated required cell volume was transferred to 500mL shake flasks with fresh pre-warmed 120mL (final volume) OPM-293CD05 Medium. The cell concentration was brought to about 2X 106-3×106 viable cells/mL.
(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 106-3×106 viable cells/mL prior to transfection.
(3) Cells were diluted to 1X 106 viable cells/mL with pre-warmed OPM-293CD05 Medium. The calculated cell volume required was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.
(4) PEI (1 mg/mL) reagent was diluted with 4mLOpti-MEM medium, swirled or blown to mix well, plasmid DNA (plasmid DNA was the Fc fusion antibody eukaryotic expression vector of the anti-IL-6 single domain antibody prepared in example 6) was diluted with 4mLOpt-MEM medium, swirled to mix well, and filtered with a 0.22um filter head. Incubate at room temperature for 5min.
(5) Diluted PEI reagent was added to the diluted DNA and mixed upside down. PEI/plasmid DNA complexes were incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, during which time the shake flask was gently swirled.
(6) Cells were shake cultured at 37℃with 5% CO2 at 120 rpm.
(7) 5MLOPM-CHOPFF05 feed was added 24h, 72h after transfection.
(8) Supernatants were collected about 7 days after transfection (cell viability below 70%).
EXAMPLE 9 purification of Single-domain antibodies against IL-6 protein
(1) The protein expression supernatant obtained in example 7 or 8 was filtered with a disposable filter head of 0.45 μm to remove insoluble impurities;
(2) Purifying the filtrate by affinity chromatography using a protein purifier, and purifying by using agarose filler coupled with protein A by utilizing the binding capacity of human Fc and the protein A;
(3) Passing the filtrate through a pre-cartridge of ProteinA at a flow rate of 1 mL/min, wherein the target protein in the filtrate is bound to the packing;
(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;
(5) Eluting the target protein bound on the column with a low pH buffer;
(6) Rapidly adding the eluent into Tris-HCl solution with pH of 9.0 for neutralization;
(7) And (3) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein purity is above 95%, and preserving the protein at a low temperature for later use after the concentration is above 0.5 mg/mL.
Example 10 construction of Single-Domain antibody eukaryotic expression vectors
The mentioned nanobody universal targeting vector RJK-V4-hFC1 is modified by the company after fusion of Fc segment in heavy chain coding sequence of human IgG1 based on the commercial vector pCDNA3.4 (vector data link: https:// packages. Thermo-filter. Com/TFS-packages/LSG/manuals/pcdna 3_4_topo_ta_cloning_kit_man. Pdf), i.e. the vector contains Hinge region (Hinge) CH2 and CH3 region of IgG1 heavy chain. The concrete improvement scheme is as follows:
(1) Selecting restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;
(2) Introducing multiple cloning sites (MCS, multiple CloningSite) and a 6 XHis tag at the 5 'end and the 3' end of the coding sequence of the Fc fragment respectively by means of overlapping PCR;
(3) Amplifying the fragments by PCR using a pair of primers with XbaI and AgeI cleavage sites, respectively;
(4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction enzymes XbaI and AgeI, respectively;
(5) And (3) connecting the digested vector and the inserted fragment under the action of T4 ligase, then converting the connection product into escherichia coli, amplifying, and checking by sequencing to obtain the recombinant plasmid.
The name with hFC1 refers to an Fc fusion antibody obtained by eukaryotic expression after cloning the corresponding single domain antibody sequence to RJK-V4-hFC1 (for example, the Fc fusion antibody obtained by eukaryotic expression after cloning the nucleotide sequence corresponding to 2C6 single domain antibody of 2C6-hFC1 to RJK-V4-hFC 1).
Example 11 expression and purification of tool antibodies (Toolantibody, tab 1) targeting human IL-6
Here, tab1 is Siltuximab (cetuximab).
The searched sequences were commissioned for mammalian cell expression system codon optimization by general biosystems (Anhui) Inc., and cloned into pcDNA3.1 vector. After resistance selection, plasmid positive bacteria were selected for amplification and plasmids were extracted using a plasmid extraction kit (MACHEREYNAGEL, cat# 740412.50). According to the method, 100 mug of plasmid (40 mug heavy chain+60 mug light chain) is added to 100mL of cells, PEI is used for transient expression in 293F cells (culture medium: freeStyle293Expressionmedium, thermo, cat #12338026+F-68, thermo, cat # 24040032), 10% peptone (Sigma, cat # P0521-100G) with 5% volume is added after transfection for 6-24 hours, 8% CO2 rpm is added for culturing for about 7-8 days, expression supernatant is collected when the cell activity is reduced to 50%, ELISA is used for purification by using ProteinA (GE, cat # 17-5438-02) gravity column, after PBS dialysis, the concentration is measured by using Nanodrop, the purity is identified by SEC, and the binding capacity is indirectly verified;
Tab1 obtained by the method has the concentration not less than 2mg/ml and the purity more than 95%.
Example 12 determination of antigen binding response of antibodies
This example was performed using standard enzyme-linked immunosorbent assay (ELISA) protocols.
(1) 50. Mu.L of 1. Mu.g/mL IL-6 protein was coated at 4℃overnight.
(2) Washing the plate, adding 200 μl of 5% milk, and sealing at 37deg.C for 2 hr.
(3) VHH-hFc was diluted to 2ug/mL and then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. The VHH-hFc was purified from the anti-IL-6 protein single domain antibody prepared in example 8 (which was expressed as a fusion with Fc) in example 9. In addition, hIgG and Tab1 controls were also set, respectively, tab1 was prepared from example 11;
(4) Washing the plate, adding 50 mu L of the single-domain antibody obtained by dilution in the step (3), and incubating for 1h at 37 ℃ with two duplicate wells.
(5) Washing the plate, adding 50 mu L HRP-Goat anti hIgG secondary antibody, and incubating at 37 ℃ for 30min.
(6) Washing the plate (washing several times), adding 50 μl TMB for recovering normal temperature in advance, and reacting at normal temperature in dark place for 15min.
(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.
(8) The EC50 was calculated by plotting curves as shown in fig. 2,3, 4, 5, where hIgG designates a type control, immunoglobulin molecules that do not bind to any target, and are commercially available.
The 4 single domain antibodies of the invention are all excellent in IL-6 protein binding potency and specificity.
Example 13 detection of IL-6-induced proliferation of TF1 cells in antibodies (eukaryotic samples).
The following operations are carried out according to methods common to those skilled in the art:
(1) Spreading TF-1 cells which are passaged 3-4 times after resuscitating into 96-well plates according to 10000 holes per well;
(2) Tab1 (prepared in example 11) and Fc-fused single domain antibody (purified in example 9) were prepared as 10. Mu.g/mL solutions, and subjected to 5-fold gradient dilution;
(3) Respectively mixing the Tab1 and the single-domain antibody which are subjected to gradient dilution with IL-6 with the concentration of 1.985ng/ml according to the ratio of 1:1 to prepare a mixed solution;
(4) Adding the mixed solution obtained in the previous step into a cell culture hole according to the equal volume of the cell culture solution;
(5) After incubation for 72h, detecting the cell viability by using a luminescence method cell viability detection kit;
(6) The EC50 concentrations of the different single domain antibodies for neutralizing IL-6 to induce TF-1 cell proliferation are calculated according to the detection results, and the results are shown in FIG. 6, FIG. 7, FIG. 8 and FIG. 9.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.