CN115181182B - Humanized anti-CD 25 single domain antibody and application thereof - Google Patents

Humanized anti-CD 25 single domain antibody and application thereof Download PDF

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CN115181182B
CN115181182B CN202210660177.9A CN202210660177A CN115181182B CN 115181182 B CN115181182 B CN 115181182B CN 202210660177 A CN202210660177 A CN 202210660177A CN 115181182 B CN115181182 B CN 115181182B
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ser
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CN115181182A (en
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苏志鹏
王乐飞
孟巾果
张云
谢维
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Nanjing Rongjiekang Biotechnology Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention belongs to the field of immunology, and relates to a humanized anti-CD 25 single domain antibody and application thereof. The single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1 shown in SEQ ID NO. 17, a heavy chain CDR2 shown in SEQ ID NO. 18 and a heavy chain CDR3 shown in SEQ ID NO. 19, and the heavy chain further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4, and the framework region FR sequence is subjected to humanization. Compared with the prior art, the invention has the beneficial effects that: the anti-CD 25 single domain antibody provided by the invention is subjected to humanized transformation, so that the affinity and tumor cell proliferation blocking effect of the anti-CD 25 single domain antibody are maintained, and the immune side reaction is effectively reduced.

Description

Humanized anti-CD 25 single domain antibody and application thereof
Technical Field
The invention relates to the technical field of biotechnology or immunology, in particular to a humanized single domain antibody capable of specifically binding with CD25, a pharmaceutical composition containing the humanized single domain antibody as an active ingredient and a pharmaceutical treatment application thereof.
Background
CD25 is the alpha subunit of the human interleukin 2 (IL 2) receptor, IL-2 is an important cytokine in the immune response, and its receptor consists of three subunits, alpha, beta and gamma; the single alpha subunit is a medium affinity receptor of IL-2, and when the alpha subunit is combined with beta and gamma heterodimers, the complete high affinity receptor is formed, the signal of the IL-2 is received, and the T lymphocyte is activated to start mitosis, so that CD25 is mainly involved in the activation of the T cell and the activation of an immune system, and various immune responses and immune transplantation rejection reactions are initiated. In addition, excessive activation of T cells can cause uncontrolled proliferation of T cells and the initiation of T cell lymphomas.
Resting T cells, B cells and monocytes are capable of expressing small amounts of CD25 molecules. However, the receptor is capable of rapid transcription and expression upon activation (elery et al (2002) Cytokine Growth Factor rev.13 (1): 27-40; morris et al (2000) Ann. Rheum. Dis.59 (suppl. 1): IL 09-14). Cells expressing the high affinity IL-2R are capable of over-expression of CD25 (CD 25-subunit), which results in binding characteristics of high and low affinity IL-2 (Waldmann et al (1993) Blood 82 (6): 1701-12; de Jong et al (1996) J. Immunol.156 (4): 1339-48): CD25 is a variant of certain autoimmune diseases such as rheumatoid arthritis, scleroderma and uveitis, and dermatological diseases such as psoriasis and atopic dermatitis, and various lymphomas such as T cell leukemia and Hodgkin's disease expressed by high levels of T cells (Waldmann (1993) Immunol. Today 14 (6): 264-70; kuttler et al (1999) J. Mol. 77 (1): 226-9): CD25 is a variant of host cell type, and CD25 is associated with the expression of fluid (2002-84) and other such as human T cell leukemia and Hodgkin disease (1993).
Thus, CD25 is an important goal of antibody-mediated therapy, e.g., for reducing inflammation from immune disorders, treating swelling pain, and preventing graft rejection. However, while the results obtained, and the clinical experience obtained so far, clearly define that CD25 is a useful target for immunotherapy, they also show that the murine and chimeric antibodies currently available do not constitute ideal therapeutic agents. Thus, there is a need for additional therapeutic antibodies against CD25 that are effective in preventing and/or treating a variety of diseases associated with CD25 expressing cells.
Currently, CD25 inhibitors are mainly used in the clinic for autoimmune diseases and immune rejection in organ transplantation, and are expected to be further expanded to the field of tumor treatment in the future. Daclizumab (daclizumab) is a humanized, selectively highly affinity monoclonal antibody drug that binds T cell surface interleukin 2 (IL-2) receptor subunit CD25 developed by the cooperation of the hundreds of jakobs (Biogen) and AbbVie. The Li Zhushan antibodies are marketed twice in succession, first in 2009, the indication is immunosuppression of organ transplantation, and later in 2009, the antibodies are marketed due to commercialization problems. Later on, the hundred-bond reduced ADCC by defucosylation of the drug, and darifenacin was "revived" in 2016 for the treatment of Multiple Sclerosis (MS). Multiple sclerosis is an autoimmune disease, and clinical manifestations include vision disorders, paralysis of limbs, paresthesia, etc. In multiple sclerosis patients, CD25 is expressed at high levels and T cells are activated abnormally. Darlizumab is effective in modulating immune disorders by modulating the IL-2 pathway, reducing abnormally activated T cells. However, 3 months 2018, baijian and ibovi announced global withdrawal of dabigatran. The reason for the market return is mainly the existence of serious adverse events of darifenacin, including inflammatory brain dysfunction and death of patients caused by hepatotoxicity. Basiliximab (sully) is a human/mouse chimeric monoclonal antibody which can directionally antagonize the receptor alpha chain (CD 25) of IL-2, block the combination of T cells and IL-2 and inhibit the proliferation of Teff cells. In 2002, basiliximab was marketed in domestic batches, and the indication was to prevent early acute organ rejection after kidney transplantation. In 2019, basiliximab was included in medical insurance.
In 2019, humanized anti-CD 25 monoclonal antibody injection of Sansheng Guojian was marketed as Jianziping, and the indication was to prevent acute rejection caused by kidney transplantation. Benproperine acts specifically on the alpha chain of the activated T cell IL2 receptor, antagonizing T cell proliferation mediated by IL-2 binding to the receptor by binding to CD 25.
The humanized single-domain antibody has the FR region with partial amino acid sequence replaced with human sequence, and has affinity and specificity maintained, lowered heterology and other advantages.
Currently, there is still a lack of strong affinity, pharmaceutically valuable humanized anti-CD 25 single domain antibody products in the prior art.
Disclosure of Invention
The object of the present invention is to provide a humanized single domain antibody capable of specifically binding to CD25 and uses thereof.
In a first aspect, the present invention provides a humanized anti-CD 25 single domain antibody, said single domain antibody comprising a heavy chain CDR1 as set forth in SEQ ID NO. 17, a heavy chain CDR2 as set forth in SEQ ID NO. 18 and a heavy chain CDR3 as set forth in SEQ ID NO. 19, said heavy chain further comprising a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequence of the framework region FR is one of the following (1) to (16):
(1) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 26; FR4 shown in SEQ ID NO. 31;
(2) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 25; FR4 shown in SEQ ID NO. 31;
(3) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 24; FR4 shown in SEQ ID NO. 31;
(4) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 23; FR4 shown in SEQ ID NO. 31;
(5) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 30; FR4 shown in SEQ ID NO. 31;
(6) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 29; FR4 shown in SEQ ID NO. 31;
(7) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 28; FR4 shown in SEQ ID NO. 31;
(8) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 27; FR4 shown in SEQ ID NO. 31;
(9) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 26; FR4 shown in SEQ ID NO. 31;
(10) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 25; FR4 shown in SEQ ID NO. 31;
(11) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 24; FR4 shown in SEQ ID NO. 31;
(12) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 23; FR4 shown in SEQ ID NO. 31;
(13) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 30; FR4 shown in SEQ ID NO. 31;
(14) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 29; FR4 shown in SEQ ID NO. 31;
(15) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 28; FR4 shown in SEQ ID NO. 31;
(16) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 27; FR4 shown in SEQ ID NO. 31.
All of the above sequences may be replaced by sequences having "at least 80% homology" to 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".
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. In particular, in the heavy chain CDR1, 1 to 5 amino acid residues may be replaced by their conserved amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues may be replaced by their conserved amino acids; in the heavy chain CDR3, 1 to 5 amino acid residues may be replaced by their conserved amino acids.
In one embodiment, wherein the amino acid sequence of any one of the framework regions of FR1, FR2, FR3 and FR4, one to five of any amino acid residues may be substituted with their conserved amino acids, respectively.
The inventor of the invention obtains the humanized CD25 single domain antibody which keeps the affinity and ADCC effect and effectively reduces the immune side reaction through scientific and reasonable humanized reconstruction at a proper position on the basis of the camel-source anti-CD 25 single domain antibody of SEQ ID No. 32.
The complementarity determining regions and the backbones are connected in the following order to form the primary sequence structure of the single domain antibody: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
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. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. In fact, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution would preferably be conservative amino acid substitutions, 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 conserved amino acid, which may be generally described as an amino acid substitution of an amino acid residue with another amino acid residue having a similar chemical structure, has little or no effect on the function, activity, or other biological property 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 the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced 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 "humanized anti-CD 25 single domain antibody" in the present invention includes not only whole single domain antibodies but also fragments, derivatives and analogues of humanized anti-CD 25 single domain antibodies. 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 second aspect of the invention there is provided a humanized anti-CD 25 single domain antibody having the amino acid sequences as set out in SEQ ID nos. 1-16, respectively, or having at least 80% sequence homology with the amino acid sequences of SEQ ID nos. 1-16 and being capable of specifically binding to CD25 protein.
In one embodiment, the humanized anti-CD 25 single domain antibody hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-16 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding to CD25 protein.
In a third aspect, the invention provides a nucleotide molecule encoding the aforementioned humanized anti-CD 25 single domain antibody.
In a fourth aspect the invention provides a bispecific antibody comprising a first antigen binding moiety which is a humanized anti-CD 25 single domain antibody as described above and a second antigen binding moiety. The second antigen binding portion is another antibody, which may be an antibody (which may be a monoclonal antibody, a polyclonal antibody, a single domain antibody, or any other form of antibody) to PD-1, CD 4.
In a fifth aspect the invention provides a recombinant protein comprising a humanized anti-CD 25 single domain antibody as described above.
In a sixth aspect the invention provides an expression vector comprising a nucleic acid molecule encoding the aforementioned humanized anti-CD 25 single domain antibody or the aforementioned recombinant protein.
In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (the nucleotide molecules encoding the humanized anti-CD 25 single domain antibody are integrated into RJK-V4-hFC1 by genetic engineering means), and other universal expression vectors may be selected as desired.
In a seventh aspect, the invention provides a host cell capable of expressing a humanized anti-CD 25 single domain antibody as described above or a recombinant protein as described above, or comprising an expression vector as described above. 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: coli, yeast cells, mammalian cells, phage, or combinations thereof.
In another preferred embodiment, the prokaryotic cell is selected from the group consisting of: coli, bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or combinations 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 myxoplasma gondii, 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 an eighth aspect, the invention provides a kit for detecting the level of CD25 protein, said kit comprising the humanized anti-CD 25 single domain antibody described above.
In a ninth aspect, the invention provides a pharmaceutical composition comprising a humanized anti-CD 25 single domain antibody as described above and/or 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 formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intravenous and transdermal (directly applied or plastered on affected part).
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 vectors include (but are not limited to): saline, buffer, glucose, 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 another preferred embodiment, the present invention provides a medicament for treating tumors comprising the aforementioned humanized anti-CD 25 single domain antibody as an active ingredient.
In another preferred embodiment, the present invention provides a medicament for treating autoimmune diseases comprising the aforementioned humanized anti-CD 25 single domain antibody as an active ingredient.
In a tenth aspect of the invention, there is provided a method of producing a humanized anti-CD 25 single domain antibody 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 humanized anti-CD 25 single domain antibody; and
(b) Isolating or recovering said humanized anti-CD 25 single domain antibody from said culture; and
(c) Optionally purifying and/or modifying the humanized single domain antibody of CD25 obtained in step (b).
A twelfth aspect of the invention provides the use of a single domain antibody or pharmaceutical composition against the aforementioned humanized anti-CD 25 for the manufacture of a medicament for the treatment or prophylaxis of a disease.
In a preferred embodiment, the disease is a tumor, or the disease is a disorder associated with aberrant expression of CD 25. Abnormal expression of CD25 generally refers to expression of CD25 at higher than normal levels.
Such as B-cell malignancy, lymphoma (hodgkin's lymphoma (Hodgkins Lymphoma), non-hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, myeloma), myeloproliferative disorders, solid tumors (e.g., breast cancer, squamous cell carcinoma, colon cancer, head and neck cancer, lung cancer, genitourinary cancer, rectal cancer, stomach cancer, sarcoma, melanoma, esophageal cancer, liver cancer, testicular cancer, cervical cancer, mast cell tumor, hemangioma, eye cancer, laryngeal cancer, oral cancer, mesothelioma, skin cancer, rectal cancer, laryngeal cancer, bladder cancer, breast cancer, uterine cancer, prostate cancer, lung cancer, pancreatic cancer, kidney cancer, stomach cancer, gastric cancer, non-small cell lung cancer, kidney cancer, brain cancer, and ovarian cancer).
In another preferred embodiment, the aforementioned diseases are selected from the group consisting of rheumatoid arthritis, scleroderma and uveitis, dermatological diseases, such as psoriasis and atopic dermatitis, and various lymphomas, multiple sclerosis.
In another preferred embodiment, the disease-preventing agent is mainly used for preventing the occurrence of acute rejection after kidney transplantation. In addition, acute immune rejection after heart, lung, liver, kidney, pancreas, small intestine, skin, cornea and blood vessel transplantation can be prevented.
Advantageous effects
Compared with the prior art, the invention has the beneficial effects that: the anti-CD 25 single domain antibody provided by the invention is subjected to humanized transformation, so that the affinity and tumor cell proliferation blocking effect of the anti-CD 25 single domain antibody are maintained, and the immune side reaction is effectively reduced.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a library enrichment profile of the targeted CD25 antibody screen of example 3;
FIG. 2 is a graph showing the quantitative response curves of the binding of the antibody antigen in example 12 (Tab, hIgG, a portion of clones);
FIG. 3 is a graph of the antibody antigen binding response curve assay of example 12 (another clone);
FIG. 4 is a graph of the antibody antigen binding response curve assay of example 12 (yet another part of cloning);
FIG. 5 is a graph of the antibody antigen binding response curve assay of example 12 (another clone);
FIG. 6 shows the ADCC effect induced by the humanized single domain antibody and the tool antibody (Tab, hIgG, a portion of clones) of example 13;
FIG. 7 is the humanized single domain antibody and tool antibody induced ADCC (another partial clone) of example 13;
FIG. 8 is the humanized single domain antibody and tool antibody induced ADCC (yet another partial clone) of example 13;
FIG. 9 is the ADCC effect induced by the humanized single domain antibody and the tool antibody of example 13 (another partial clone);
FIG. 10 is a schematic diagram of amino acid sequence of humanized antibody after resolution according to CDR and FR;
FIG. 11 is a schematic representation of the complete amino acid sequences of 16 humanized antibodies.
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 "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.
The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (e.g., an animal antibody) to the constant region of a human antibody, or by grafting the complementarity determining regions (CDR 1 to CDR3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting a target antibody to amino acid mutation according to the characteristics of the framework regions (FR 1 to FR 4) of a human antibody. Humanized antibodies can be synthesized or site-directed mutagenesis.
The antibodies shown in SEQ ID NOS.1-16 herein are antibodies obtained by mutating amino acids of a target antibody according to the characteristics of human antibody framework regions (FR 1-4).
In some embodiments, sequences having "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences in SEQ ID No.1-16 may achieve the object of the invention.
In some embodiments, the polypeptide that hybridizes to SEQ ID NO:1-16, e.g., comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, may also achieve the object of the invention. 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 the following groups (a) - (d) are substituted for another or a few amino acids in the same group: (a) a polar negatively charged residue and an uncharged amide thereof: asp, asn, glu, gln; (b) a polar positively charged residue: his, arg, lys; (c) aromatic residues: phe, trp, tyr; (d) aliphatic nonpolar or low polar residues: ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are as follows: asp is substituted with Glu; asn is substituted with Gln or His; glu is substituted with Asp; gln is substituted with Asn; his is substituted with Asn or Gln; arg is replaced by Lys; lys is substituted by Arg, gln; phe is replaced by Met, leu, tyr; trp is substituted with Tyr; tyr is substituted with Phe, trp; substitution of Ala with Gly or Ser; ser is substituted by Thr; thr is replaced by Ser; substitution of Gly with Ala or Pro; met is substituted with Leu, tyr or Ile; leu is substituted with Ile or Val; lie is substituted with Leu or Val; val is substituted with Ile or Leu; cys is replaced 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.
Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.
The humanized single domain antibodies of the invention are specific for CD25 protein with the correct spatial structure. The humanized 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 later production cost. The humanized single-domain antibody obtained by the invention has simple multi-combination form transformation, multivalent and multi-specificity antibodies can be obtained by simple serial connection in a genetic engineering mode, and has low immune heterogeneity, and stronger immune response can not be generated under the condition of not undergoing the humanized transformation. The invention provides humanized single domain antibodies with a broader range of affinities, ranging from nM to pM, which provide multiple options for later use of the antibodies without affinity maturation.
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 the phage display technology, and further detecting the binding capacity of the specific antibody and the antigen protein and application of the specific antibody in treatment including autoimmune diseases, tumors and transplant rejection.
The above technical solutions will now be described in detail by way of specific embodiments:
the features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1 preparation of single domain antibodies directed against human recombinant CD25 protein.
The human recombinant extracellular domain 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 CD25 protein is as follows:
(1) The coding sequence for CD25, which is identified as NM-000417.2, was retrieved from NCBI and encoded to produce the amino acid sequence accession number NP-000408.1,Uniprot ID as P01589.
(2) The nucleotide sequence encoding the CD25 protein was cloned into the vector pcDNA3.4 by means of gene synthesis. 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 expression and purification of target protein by transfecting 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 of CD25 protein
1mg of the recombinant human CD25 protein purified 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 CD25 protein mixed with Freund's incomplete adjuvant in equal volumes, except for the first immunization, in order to concentrate the stimulation of the camel to produce antibodies against the CD25 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 pMECS vector and VHH fragment are respectively digested with restriction enzymes, and then the digested fragments and VHH fragment are digestedAnd (5) linking the carriers. Electrotransformation of the ligated fragments into competent cells TG1, construction of a phage display library of CD25 protein and measurement of the library capacity, which was approximately 1X 10 9 At the same time, the correct insertion rate of the library at the fragment of interest was detected by colony PCR identification.
The results showed that after PCR amplification of 15 randomly selected colonies from the library, 14 clones could be amplified as predicted size bands and 1 clone was incorrectly amplified, so the correct insertion rate was (14/15). Times.100%. Apprxeq.93.3%.
Example 3: screening of Single-Domain antibodies against CD25 proteins
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.
NaHCO diluted at 100mM pH8.3 3 500 μg of CD25 protein coupled to an ELISA plate, and left overnight at 4deg.C while negative control wells (medium control) were established; the next day 200 μl of 3% skim milk was added and blocked at room temperature for 2h; after blocking was completed, 100. Mu.l of amplified phage library (approximately 2X 10 11 Individual phage particles), 1h at room temperature; after 1 hour of action, the unbound phage were washed off by washing 15 times with PBS+0.05% Tween-20.
The phage specifically binding to CD25 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 1 hour, 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 infection of TG1 bacteria by phage with positive Kong Xi removal from biopanning/number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal, which parameter increases gradually after enrichment occurs; I/E = total phage added to positive wells per round of biopanning/total phage removed from positive Kong Xi per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred.
Example 4: screening of specific positive clones for CD25 by phage enzyme-linked immunosorbent assay (ELISA):
screening was performed 3 rounds against single domain antibodies against CD25 protein according to the screening method described in example 3 above, phage enrichment factor against CD25 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 control was set, and after culturing at 37℃to logarithmic phase, IPTG was added at a final concentration of 1mM, and culturing was carried out overnight at 28 ℃.
Obtaining a crude extract antibody by using a permeation swelling method; the CD25 recombinant protein was released to 100mM NaHCO pH8.3, respectively 3 100. Mu.g of protein was coated in an ELISA plate (ELISA plate) at 4℃overnight. Transferring 100 mu L of the obtained crude antibody extract to an ELISA plate added with antigen, and incubating for 1h at room temperature; washing unbound Antibody with PBST, adding 100 μl of Mouse Anti-HA tag Anti-body (HRP) (Mouse Anti-HA horseradish peroxidase labeled Antibody, thermo Fisher) diluted 1:2000, and incubating for 1h at room temperature; washing off unbound antibody with PBST, adding horseradish peroxidase chromogenic solution, reacting at 37deg.C for 15min, adding stop solution, and reading absorption value at 450nm wavelength on an enzyme-labeled instrument.
When the OD value of the sample hole is more than 5 times that of the control hole, judging that the sample hole is a positive cloning hole; the positive clone well was transferred to LB medium containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced.
And analyzing the gene sequence of each clone strain according to sequence alignment software VectorNTI, and regarding strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain and strains with different sequences as different clone strains to finally obtain the single domain antibody 1B5 specific to the CD25 protein.
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 1B5 is obtained.
The amino acid sequence of the 1B5 single domain antibody is shown as SEQ ID NO. 32.
Example 5: humanization of single domain antibodies against CD25
The humanization method is completed by adopting a method of carrying out high-throughput screening on the mutation library of the antibody framework region constructed based on the analysis result of big data. The method comprises the following detailed steps:
(1) Sequence analysis of human/camel antibody data: performing amino acid preference analysis on 13873 Nb (Human) sequences downloaded in batches from NCBI websites, and simultaneously performing amino acid preference analysis on 2000 nanometer antibody sequences of the company to obtain amino terminal proportion data of each site of a framework region;
(2) Comprehensive weighting analysis of human camel sources: the source/camel source antibody sequences are uniformly numbered according to the IMGT numbering rule and are in one-to-one correspondence, the analysis results of the amino acid proportion in the two species are combined, weighting analysis is carried out according to the weight of 10% of the humanized 90% camel source, the proportion of the amino acid of each site after weighting is counted, and the sequence is ordered from high to low; according to the final weighting result, only preserving the amino acid types with the proportion of more than 10% at a single site of the framework region, and calculating the final weight of the amino acid with the proportion of more than 10% according to the standard that the proportion is integrated to be 1 after preservation, so as to be used as the design basis of a subsequent amino acid custom library;
(3) Scheme design of amino acid custom library: the method comprises the steps of providing an independent site to be mutated, providing n as the number of amino acids of which the number is more than 10%, providing V as the ratio of the highest value to the lowest value of the proportion of the amino acids of which the number is more than 10%, and judging the properties of the site to be mutated: if V is more than or equal to 3 and n is less than or equal to 2, the locus is considered to be a 'high concentration locus', otherwise, the locus is considered to be a 'medium-low concentration locus'. According to the method, the customized amino acid library is divided into two libraries with high concentration, medium concentration and low concentration, and the construction of the amino acid customized library is respectively carried out, wherein the final weight in the step (2) is the reference basis of the types and the proportions of the site amino acids in the library.
(4) High throughput screening of amino acid custom libraries:
and (3) constructing a humanized antibody library for the antibody strain, aiming at the constructed library, respectively panning with corresponding antigens, and finally obtaining an antibody sequence with higher affinity and higher humanization degree.
The following 16 humanized antibodies were obtained: the amino acid sequence of the framework region FR is one of the following (1) to (16):
(1) 1B5-F1: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 26; FR4 shown in SEQ ID NO. 31;
(2) 1B5-F2: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 25; FR4 shown in SEQ ID NO. 31;
(3) 1B5-F3: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 24; FR4 shown in SEQ ID NO. 31;
(4) 1B5-F4: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 23; FR4 shown in SEQ ID NO. 31;
(5) 1B5-F5: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 30; FR4 shown in SEQ ID NO. 31;
(6) 1B5-F6: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 29; FR4 shown in SEQ ID NO. 31;
(7) 1B5-F7: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 28; FR4 shown in SEQ ID NO. 31;
(8) 1B5-F8: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 27; FR4 shown in SEQ ID NO. 31;
(9) 1B5-F9: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 26; FR4 shown in SEQ ID NO. 31;
(10) 1B5-F10: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 25; FR4 shown in SEQ ID NO. 31;
(11) 1B5-F11: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 24; FR4 shown in SEQ ID NO. 31;
(12) 1B5-F12: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 23; FR4 shown in SEQ ID NO. 31;
(13) 1B5-F13: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 30; FR4 shown in SEQ ID NO. 31;
(14) 1B5-F14: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 29; FR4 shown in SEQ ID NO. 31;
(15) 1B5-F15: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 28; FR4 shown in SEQ ID NO. 31;
(16) 1B5-F16: FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 27; FR4 shown in SEQ ID NO. 31.
The humanized antibody of 1B 5-F1-1B 5-F16 has a heavy chain CDR1 shown in SEQ ID NO. 17, a heavy chain CDR2 shown in SEQ ID NO. 18 and a heavy chain CDR3 shown in SEQ ID NO. 19. All humanized antibodies have the same CDR1, CDR2, CDR3 as the CDR1, CDR2, CDR3 of the non-humanized 1B5 single domain antibody. Humanization is a sequence directed to the FR framework region. The structure of the humanized antibody remains: the amino acid sequences of the humanized antibodies of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4,1B5-F1 to 1B5-F16 after resolution according to CDR and FR are shown in FIG. 10.
Wherein SEQ ID NO. 20: ESGGGLVKPGGSLRLSCAAS; SEQ ID NO. 31: WGQGTLVTVSS.
The humanized antibodies 1B5-F1 to 1B5-F16 have the FR2 sequences shown in Table 1, the FR3 sequences shown in Table 2, the CDR1 sequences shown in Table 3, the CDR2 sequences shown in Table 4, the CDR3 sequences shown in Table 5 and the complete amino acid sequences shown in FIG. 11.
The nucleotide sequences of the humanized antibodies 1B 5-F1-1B 5-F16 are SEQ ID NO. 33-SEQ ID NO. 48 in sequence.
TABLE 1 FR2 sequences of 16 humanized antibodies
TABLE 2 FR3 sequences of 16 humanized antibodies
TABLE 3 CDR1 sequences of 16 humanized antibodies
TABLE 4 CDR2 sequences of 16 humanized antibodies
TABLE 5 CDR3 sequences of 16 humanized antibodies
Example 6: construction of Fc fusion antibody eukaryotic expression vector of humanized anti-CD 25 single domain antibody
(1) Designing a corresponding nucleotide sequence according to the target sequence obtained in example 5;
(2) Synthesizing the nucleotide sequence (SEQ ID NO: 33-48) into a vector RJK-V4-hFC1 designed and modified by the company in a sequence synthesis mode to obtain a recombinant eukaryotic expression vector (specifically, inserting one of SEQ ID NO:33-48 into a multiple cloning site MCS of the vector), wherein the construction method of the vector is as 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: humanized anti-CD 25 protein single domain antibodies expressed in suspension ExpiCHO-S cells
(1) 3 days before transfection at 2.5X10 5 Cell transfer/mLGeneration and expansion culture of ExpiCHO-S TM The cells, calculated desired cell volume, were transferred to an ExpiCHO containing fresh pre-warmed 120mL (final volume) TM 500mL shake flask of expression medium; to achieve a cell concentration of about 4X 10 6 -6×10 6 Living cells/mL;
(2) One day prior to transfection, expiCHO-S was used TM Cell dilution concentration to 3.5X10 6 Living cells/mL, allowing the cells to incubate overnight;
(3) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 before transfection 6 -10×10 6 Living cells/mL;
(4) Fresh ExpiCHO preheated to 37 ℃ TM Dilution of cells to 6X 10 in expression Medium 6 Each living cell/mL. The calculated desired cell volume was transferred to 100mL (final volume) of expcho filled with fresh pre-warmed TM 500mL shake flask of expression medium;
(5) Gently mixing the mixture with the mixture of the Expifectamine in a reverse manner TM CHO reagent with 3.7mL OptiPRO TM Dilution of Expifectamine in Medium TM CHO reagent, whipping or mixing;
(6) With refrigerated 4mL OptiPRO TM Diluting plasmid DNA with culture medium, and mixing;
(7) Incubating ExpiFectamine CHO/plasmid DNA (plasmid DNA is Fc fusion antibody eukaryotic expression vector of humanized anti-CD 25 single domain antibody prepared in example 6) complex for 1-5 min at room temperature, then adding gently to the prepared cell suspension, and gently agitating shake flask during addition;
(8) The cells were incubated at 37℃with 8% CO 2 Shake culturing in humidified air;
(9) Mu.l of Expifectamine was added on day 1 (18-22 hours post transfection) TM CHO enhancement and 24mL of expi CHO feed.
(10) Supernatants were collected about 8 days after transfection (cell viability below 70%).
Example 8: expression of anti-CD 25 protein single domain antibodies in suspension 293F cells
Recombinant single domain antibody expression experimental procedure (500 mL shake flask for example):
(1) 3 days before transfection at 2.5X10 5 The cells were passaged/mL and expanded 293F cells, and the calculated desired cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 120mL (final volume) OPM-293 CD05 Medium. To achieve a cell concentration of about 2X 10 6 -3×10 6 Living cells/mL.
(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 before transfection 6 -3×10 6 Living cells/mL.
(3) Dilution of cells to 1X 10 with pre-warmed OPM-293 CD05 Medium 6 Each living cell/mL. The calculated cell volume required was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.
(4) Diluting PEI (1 mg/mL) reagent with 4mL of Opti-MEM culture medium, and stirring or blowing to mix uniformly; plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the humanized anti-CD 25 single domain antibody prepared in example 6) was diluted with 4mL of Opt-MEM medium, mixed by vortexing, 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) The cells were incubated at 37℃with 5% CO 2 Shake culturing at 120 rpm.
(7) 5mL OPM-CHO PFF05 feed was added 24h, 72h post transfection.
(8) Supernatants were collected about 7 days after transfection (cell viability below 70%).
Example 9: purification of humanized anti-CD 25 protein single domain antibodies
(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 using a Protein purifier to perform affinity chromatography, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human Fc and Protein A;
(3) Passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is combined with the packing;
(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;
(5) The target protein combined on the column is subjected to a system by using a low pH buffer solution;
(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 vector RJK-V4-hFC1
The mentioned nanobody universal targeting vector RJK-V4-hFC1 is modified by the company after fusion of the Fc region in the heavy chain coding sequence of human IgG1 on the basis of the invitrogen commercial vector pCDNA3.4 (vector data link: https:// packages. Thermofiser. Com/TFS-packages/LSG/manual/pcdna3_4_topo_ta_cloning_kit_man. Pdf), i.e. the vector comprises the Hinge region (Hinge) CH2 and CH3 regions of the 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 Cloning Site) 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.
Example 11: expression and purification of Tool antibodies (Tabs) targeting human CD25
Herein, tab1 is camidanlumab tesirine, tab2 is daclizumab (dalizumab), tab3 is Basiliximab (Basiliximab), and the sequence is from IMGT.
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 (Macherey Nagel, cat# 740412.50). According to the addition of 100. Mu.g of plasmid per 100mL of cells (40. Mu.g of heavy chain+60. Mu.g of light chain), PEI was transiently expressed in 293F cells (medium: freeStyle 293 Expression medium,Thermo,Cat#12338026+F-68, thermo, cat # 24040032); after 6-24 h of transfection 5% by volume of 10% Peptone (Sigma, cat#P0521-100G), 8% CO were added 2 Culturing at 130rpm for about 7-8 days; when the cell viability was reduced to 50%, the expression supernatant was collected and purified using a gravity column of ProteinA (GE, cat#17-5438-02); after PBS dialysis, concentration was determined using Nanodrop, SEC to identify purity, and indirect ELISA to verify binding capacity; tab obtained by the method has the concentration of not less than 2mg/ml and the purity of more than 95 percent.
Example 12: binding dose-response curve assay for humanized specific single domain antibodies (eukaryotic expression) to CD25 protein
(1) 50. Mu.L of 1. Mu.g/mL CD25 protein (i.e., human IL-2 Ra) was coated overnight at 4 ℃.
(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 2h.
(3) VHH-hFc was diluted to 2ug/mL and then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. In addition, hIgG (actually hIgG 1) and Tab controls were also set up; the VHH-hFc was obtained by purifying the Fc fusion antibody of the humanized single domain antibody of the CD25 protein obtained in example 8 in example 9.
(4) Washing the plate; 50. Mu.L of the humanized single domain antibody diluted in step (3) was added, and the wells were double-plated and incubated at 37℃for 1 hour.
(5) Washing the plate; 50. Mu.L of HRP-coat anti-hIgG secondary antibody was added and incubated at 37℃for 30min.
(6) Washing the plate (washing several times); 50. Mu.L of TMB which had previously recovered the room temperature was added thereto, and the reaction was continued at the normal temperature in the dark for 15 minutes.
(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.
(8) The EC50 was calculated by plotting the curves and the results are shown in figures 2, 3, 4 and 5.
It can be seen that 16 humanized single domain antibodies are excellent in binding potency and specificity for CD25 protein.
Example 13: ADCC action induced by humanized single domain antibody and tool antibody
The method comprises the following steps:
(1) Collecting SU-DHL-1 cells of 3-4 passages after resuscitating, and spreading into 96-well plates according to 10000 holes;
(2) Preparing a solution with the highest concentration of 10 mug/mL from hIgG (actually hIgG 1), tab1, tab2, tab3 and humanized single-domain antibody samples, and carrying out 10-time gradient dilution to finally obtain 7 concentrations; hIgG designates a isotype control, immunoglobulin molecules that do not bind to any target, are commercially available, as follows. The single domain antibody herein was obtained by purifying the Fc fusion antibody of the humanized single domain antibody of CD25 protein obtained in example 8 in example 9. Tab1, tab2 and Tab3 were prepared in the same manner as in example 11.
(3) Adding the antibody solution diluted in a gradient manner into a cell culture hole according to the equal volume of the cell suspension;
(4) For sample wells and E/T wells (antibody concentration 0), PBMC cells were collected and added to the cell culture wells at 250000 cells per well, twice the volume of the target cell suspension; for MAX wells, two times the volume of target cell suspension per well was added; for MIN wells, twice the volume of the target cell suspension was added to each well;
(5) After 6h incubation, detecting cell killing by using an LDH kit, and reading absorbance;
(6) The EC50 concentration for each antibody-mediated ADCC was calculated from the target cell killing rate and concentration by four-parameter fitting according to the formula = (sample-E/T)/(MAX-MIN), and the results are shown in fig. 6, 7, 8, 9.
The lower EC50 concentration indicates a stronger antibody-mediated ADCC function, and the above results indicate that the 16 humanized single-domain antibodies prepared by the present invention all have ADCC function.
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.
Sequence listing
<110> Nanjing Rongjiekang biotechnology Co., ltd
<120> a humanized anti-CD 25 single domain antibody and use thereof
<160> 48
<170> SIPOSequenceListing 1.0
<210> 1
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 2
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 3
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 4
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 5
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 6
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 7
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 9
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 10
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 11
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 12
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 13
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 14
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 15
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 16
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 17
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Gly Tyr Thr Tyr Ser Ser Asn Cys
1 5
<210> 18
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Ile Asp Thr Gly Gly Gly Tyr Thr
1 5
<210> 19
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Ala Ala Asp Thr Gly Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp
1 5 10 15
Phe Gly Tyr
<210> 20
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 21
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 22
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Met Gly Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 23
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 24
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 25
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 26
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 27
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 28
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 29
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 30
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 31
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
1 5 10
<210> 32
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Thr
35 40 45
Gly Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Lys Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp Thr Gly
85 90 95
Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 33
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgacctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 34
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgagccg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 35
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 36
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgcgcgg aggacactgc cgtttactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 37
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaactcc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 38
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgcgccg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 39
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 40
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgcgcgg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 41
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 42
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 42
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgcgccg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 43
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 44
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacgcc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgcgccg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 45
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 45
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 46
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgg tgtatctgca aatgaacagc 240
ctgcgcgccg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 47
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgccctg aggacactgc cgtttactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363
<210> 48
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 48
gagtctggag gaggcctagt gaagccagga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tgggtgcgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtgaaa 180
ggccgattca ccatctcccg agacaacagc aagaacacgc tgtatctgca aatgaacagc 240
ctgcgcgccg aggacactgc cgtctactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggaccctggt caccgtctcc 360
tca 363

Claims (9)

1. A humanized anti-CD 25 single domain antibody, characterized in that: the humanized anti-CD 25 single domain antibody consists of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1 shown in SEQ ID NO. 17, a heavy chain CDR2 shown in SEQ ID NO. 18 and a heavy chain CDR3 shown in SEQ ID NO. 19, and the heavy chain further comprises a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4; the amino acid sequence of the framework region FR is one of the following (1) to (16):
(1) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 26; FR4 shown in SEQ ID NO. 31;
(2) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 25; FR4 shown in SEQ ID NO. 31;
(3) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 24; FR4 shown in SEQ ID NO. 31;
(4) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 23; FR4 shown in SEQ ID NO. 31;
(5) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 30; FR4 shown in SEQ ID NO. 31;
(6) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 29; FR4 shown in SEQ ID NO. 31;
(7) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 28; FR4 shown in SEQ ID NO. 31;
(8) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 21 and FR3 shown in SEQ ID NO. 27; FR4 shown in SEQ ID NO. 31;
(9) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 26; FR4 shown in SEQ ID NO. 31;
(10) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 25; FR4 shown in SEQ ID NO. 31;
(11) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 24; FR4 shown in SEQ ID NO. 31;
(12) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 23; FR4 shown in SEQ ID NO. 31;
(13) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 30; FR4 shown in SEQ ID NO. 31;
(14) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 29; FR4 shown in SEQ ID NO. 31;
(15) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 28; FR4 shown in SEQ ID NO. 31;
(16) FR1 shown in SEQ ID NO. 20, FR2 shown in SEQ ID NO. 22 and FR3 shown in SEQ ID NO. 27; FR4 shown in SEQ ID NO. 31.
2. A humanized anti-CD 25 single domain antibody, characterized in that: the amino acid sequences of the humanized anti-CD 25 single domain antibodies are shown in any one of SEQ ID NO.1-16 respectively.
3. A nucleotide molecule encoding the humanized anti-CD 25 single domain antibody of claim 1 or 2.
4. A recombinant protein, characterized in that: consists of the humanized anti-CD 25 single domain antibody of claim 1 or 2 and an Fc fused to said humanized anti-CD 25 single domain antibody.
5. An expression vector, characterized in that: comprising a nucleic acid molecule encoding the humanized anti-CD 25 single domain antibody of claim 1 or 2 or the recombinant protein of claim 4.
6. A host cell, characterized in that: which can express the humanized single-domain antibody against CD25 of claim 1 or 2 or the recombinant protein of claim 4, or which comprises the expression vector of claim 5.
7. A kit for detecting CD25 protein levels, comprising: the kit comprising the humanized anti-CD 25 single domain antibody of claim 1 or 2.
8. A pharmaceutical composition characterized by: the pharmaceutical composition comprises a single domain antibody selected from the humanized anti-CD 25 antibody of claim 1 or 2, and/or a pharmaceutically acceptable carrier.
9. Use of a humanized anti-CD 25 single domain antibody as described in claim 1 or 2 or a pharmaceutical composition as described in claim 8 for the preparation of a medicament for the treatment or prevention of a disease, characterized in that: the disease is lymphoma.
CN202210660177.9A 2022-06-13 2022-06-13 Humanized anti-CD 25 single domain antibody and application thereof Active CN115181182B (en)

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