CN115181181B - anti-CD 25 single domain antibody and application thereof - Google Patents

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

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CN115181181B
CN115181181B CN202210660126.6A CN202210660126A CN115181181B CN 115181181 B CN115181181 B CN 115181181B CN 202210660126 A CN202210660126 A CN 202210660126A CN 115181181 B CN115181181 B CN 115181181B
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CN115181181A (en
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苏志鹏
张云
王乐飞
孟巾果
谢维
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Nanjing Rongjiekang Biotechnology Co ltd
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    • 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
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
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    • 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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    • C07K2317/50—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®
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Abstract

The invention belongs to the field of immunology, and relates to an 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 any one of SEQ ID NO:48-SEQ ID NO:57, a heavy chain CDR2 shown in any one of SEQ ID NO:58-SEQ ID NO:67 and a heavy chain CDR3 shown in any one of SEQ ID NO:68-SEQ ID NO: 78. Compared with the prior art, the invention has the beneficial effects that: the invention uses biological gene engineering technology to screen out the single domain antibody specific to CD25, the antibody has better affinity, and can block the specific cell from releasing cell factor, and has good binding activity through prokaryotic expression and eukaryotic expression, and has certain drug property.

Description

anti-CD 25 single domain antibody and application thereof
Technical Field
The present invention relates to the field of biotechnology or immunology, and in particular to a single domain antibody capable of specifically binding to CD25 (hereinafter, abbreviated as "CD25 single domain antibody"), a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use 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 in autoimmune diseases, 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.
Currently, there is still a lack of strong affinity, pharmaceutically valuable anti-CD 25 single domain antibody products in the prior art.
Disclosure of Invention
The invention aims to provide a single domain antibody capable of specifically binding to CD25 and application thereof.
In a first aspect, the invention provides an anti-CD 25 single domain antibody comprising a heavy chain CDR1 as shown in any one of SEQ ID NO:48-SEQ ID NO:57, a heavy chain CDR2 as shown in any one of SEQ ID NO:58-SEQ ID NO:67, and a heavy chain CDR3 as shown in any one of SEQ ID NO:68-SEQ ID NO: 78. anti-CD 25 single domain antibodies are single domain antibodies directed against CD 25.
Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1) to (11):
(1) CDR1 as shown in SEQ ID NO. 48, CDR2 as shown in SEQ ID NO. 65, CDR3 as shown in SEQ ID NO. 72;
(2) CDR1 shown in SEQ ID NO. 49, CDR2 shown in SEQ ID NO. 67, CDR3 shown in SEQ ID NO. 70;
(3) CDR1 as shown in SEQ ID NO. 50, CDR2 as shown in SEQ ID NO. 63, CDR3 as shown in SEQ ID NO. 68;
(4) CDR1 as shown in SEQ ID NO. 51, CDR2 as shown in SEQ ID NO. 66, CDR3 as shown in SEQ ID NO. 69;
(5) CDR1 as shown in SEQ ID NO. 52, CDR2 as shown in SEQ ID NO. 58, CDR3 as shown in SEQ ID NO. 78;
(6) CDR1 shown in SEQ ID NO. 53, CDR2 shown in SEQ ID NO. 62, CDR3 shown in SEQ ID NO. 76;
(7) CDR1 shown in SEQ ID NO. 54, CDR2 shown in SEQ ID NO. 59, CDR3 shown in SEQ ID NO. 75;
(8) CDR1 as shown in SEQ ID NO. 55, CDR2 as shown in SEQ ID NO. 64, CDR3 as shown in SEQ ID NO. 77;
(9) CDR1 shown in SEQ ID NO. 56, CDR2 shown in SEQ ID NO. 60, CDR3 shown in SEQ ID NO. 71;
(10) CDR1 shown in SEQ ID NO. 56, CDR2 shown in SEQ ID NO. 61, CDR3 shown in SEQ ID NO. 73;
(11) CDR1 shown in SEQ ID NO. 57, CDR2 shown in SEQ ID NO. 60, and CDR3 shown in SEQ ID NO. 74.
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.
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, those skilled in the art will recognize that the framework region sequences FR1-4 that are unique to an antibody are not immutable, and that the sequences of FR1-4 can take the form of conservative sequence variants of the sequences disclosed herein.
The meaning of "anti-CD 25 single domain antibody" in the present invention includes not only the whole single domain antibody but also fragments, derivatives and analogues of the anti-CD 25 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 antibody sequence 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 sequences of the framework regions FR are respectively:
23-27, said FR1 variant comprising up to 5 amino acid substitutions in said FR 1;
28-33, said FR2 variant comprising up to 5 amino acid substitutions in said FR 2;
34-44, or a variant of FR3 as set forth in any one of SEQ ID nos. 34-44, said variant of FR3 comprising up to 5 amino acid substitutions in said FR 3;
45-47, said FR4 variant comprising up to 5 amino acid substitutions in said FR 4.
In a second aspect of the invention, there is provided an amino acid sequence of a single domain antibody against CD25, said single domain antibody having the amino acid sequence shown in SEQ ID NO.1-11, respectively, or said single domain antibody having at least 80% sequence homology with the amino acid sequence of SEQ ID NO.1-11 and being capable of specifically binding to a CD25 protein.
In one embodiment, the anti-CD 25 single domain antibody hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-11 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 an Fc fusion or humanized antibody comprising a single domain antibody of any of the foregoing anti-CD 25.
In a fourth aspect, the present invention provides a nucleotide molecule encoding the aforementioned anti-CD 25 single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, having the nucleotide sequence set forth in SEQ ID NO:12-22, or with SEQ ID NO:12-22 has at least 95% sequence homology.
In one embodiment, the nucleic acid molecule encoding the anti-CD 25 single domain antibody hybridizes to a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 12-22 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and encodes an anti-CD 25 single domain antibody capable of specifically binding to CD25 protein.
In a fifth aspect, the present invention provides an expression vector comprising a nucleotide molecule encoding an anti-CD 25 single domain antibody or Fc fusion antibody or humanized antibody, having the nucleotide sequence set forth in SEQ ID NO: 12-22.
In a preferred embodiment, the expression vector used is RJK-V4-hFC1 (the nucleotide molecules encoding the anti-CD 25 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 foregoing anti-CD 25 single domain antibody, fc fusion antibody or humanized antibody, or comprising the foregoing 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: 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 another preferred embodiment, the invention provides a bispecific antibody comprising a first antigen binding moiety that is a single domain antibody as described above against CD25 and a second antigen binding moiety that is another antibody.
In another preferred embodiment, the invention provides a recombinant protein comprising the foregoing anti-CD 25 single domain antibody. The recombinant protein can be a single-domain antibody shown in SEQ ID NO.1-11, a single-domain antibody with at least 80% homology with SEQ ID NO.1-11, a multi-epitope antibody, a multi-specific antibody and a multivalent antibody; for example, the multi-epitope antibody may consist of more than one of the sequences set forth in SEQ ID NOS.1-11; the multivalent antibody can be formed by repeatedly arranging one sequence in SEQ ID NO.1-11 for a plurality of times; such multispecific antibodies include, but are not limited to, bispecific antibodies, and trispecific antibodies; furthermore, the recombinant proteins may be fragments, derivatives and analogues of the aforementioned antibodies.
A seventh aspect of the invention provides a pharmaceutical composition or medicament comprising the aforementioned anti-CD 25 single domain antibody, and/or a pharmaceutically acceptable carrier;
a pharmaceutical composition comprising the foregoing anti-CD 25 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 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 an agent for preventing and/or treating acute immune rejection caused by organ transplantation, comprising the aforementioned anti-CD 25 single domain antibody as an active ingredient.
In another preferred embodiment, the present invention provides a medicament for treating tumors comprising the aforementioned anti-CD 25 single domain antibody as an active ingredient.
In another preferred embodiment, the present invention provides an agent for treating autoimmune diseases, comprising the aforementioned anti-CD 25 single domain antibody as an active ingredient.
In another preferred embodiment, the invention provides a targeting agent that specifically targets the CD25 antigen comprising: the aforementioned anti-CD 25 single domain antibodies, and functional drugs linked, conjugated or adsorbed thereto. Functional drug refers to an anti-cancer drug (e.g., chemotherapeutic), radiation therapy (by applying radiation externally to the body or by administering a radiation conjugated compound), an anti-tumor antigen or marker antibody (antigen or marker is an antibody such as CD4, CD38, CA125, PSMA, c-MET, VEGF, CD137, VEGFR2, CD20, HER2, HER3, SLAMF7, CD326, CAIX, CD40, CD47, or EGF receptor), a checkpoint inhibitor or an immunomodulatory antibody (e.g., an antibody that targets PD-1, PD-L1, TIM3, CD38, GITR, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG3, ICOS, TIM3, GAL9, CD28, AP2M1, SHP-2, OX-40, VISTA, TIGIT, BTLA, HVEM, CD160, etc.), a vaccine (specifically, a cancer vaccine such as GVAX), an adjuvant, a cancer cell, or one or more compounds that target cancer cells or stimulate an immune response to cancer cells, or any other combination thereof.
In a tenth aspect, the invention provides a kit for detecting CD25 levels, comprising the aforementioned anti-CD 25 single domain antibody. 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 CD25 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 single domain antibody against CD25, may be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.
In an eleventh aspect of the invention, there is provided a method of producing a single domain antibody against CD25 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-CD 25 single domain antibody; and
(b) Isolating or recovering said anti-CD 25 single domain antibody from said culture; and
(c) Optionally purifying and/or modifying the single domain antibody of CD25 obtained in step (b).
In a twelfth aspect, the present invention provides the use of the aforementioned anti-CD 25 single domain antibody or the aforementioned pharmaceutical composition or agent for the preparation of a medicament for the treatment of autoimmune diseases or tumors, or for the prevention and/or treatment of immune rejection of organ transplants.
The invention also provides the use of the aforementioned anti-CD 25 single domain antibody or the aforementioned pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of immune rejection of organ transplantation. Immune rejection of organ transplants includes, but is not limited to, acute immune rejection following kidney, heart, lung, liver, kidney, pancreas, small intestine, skin, cornea, and vascular transplants.
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 a preferred embodiment, the disease is an autoimmune disease (e.g., multiple sclerosis, rheumatoid arthritis, scleroderma, and uveitis, and dermatological disorders such as psoriasis and atopic dermatitis, psoriasis).
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 CD25 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 invention provides 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.
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 of the antibody antigen binding response curve assay (a portion of clones) in example 12;
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 (another clone);
FIG. 5 shows ADCC (Tab, hIgG) induced by specific single domain antibodies and tool antibodies in example 13;
FIG. 6 is a graph showing ADCC (a portion of clones) induced by specific single domain antibodies and tool antibodies in example 13;
FIG. 7 shows ADCC (another partial clone) induced by specific single domain antibodies and tool antibodies in example 13;
FIG. 8 shows ADCC (still another part of cloning) induced by specific single domain antibodies and tool antibodies in example 13.
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 novel 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 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.
In some embodiments, the polypeptide that hybridizes to SEQ ID NO:1-11, 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 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 and application of the specific antibody in treatment of autoimmune diseases.
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 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 amino acid of 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 proteins
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 the VHH fragment were digested separately using restriction enzymes, and the digested fragments and vector were ligated. 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 mug of CD25 protein is coupled on an ELISA plate and placed at 4 DEG COvernight, 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 with PBSTAdding 100 μl of Mouse Anti-HA tag Antibody (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.
The gene sequences of the individual clones were analyzed according to the sequence alignment software vector nti, the strains with identical CDR1, CDR2 and CDR3 sequences were regarded as the same clone, and the strains with different sequences were regarded as different clones, finally obtaining a single domain antibody specific for the CD25 protein (single domain antibody of SEQ ID No.1-11, also including 1H7 with sequences not shown).
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 sequences SEQ ID NO.1-11 of the single domain antibodies correspond to the single domain antibodies 1A5,1B5,1E1,1H1,2H3,3E11,3F5,3G3,4A4,4D9,4H7 one by one in sequence. The nucleotide sequences SEQ ID NO.12-22 of the single domain antibodies also correspond one-to-one with the single domain antibodies 1A5,1B5,1E1,1H1,2H3,3E11,3F5,3G3,4A4,4D9,4H7 in sequence.
CDR and FR sequences of the 11 single domain antibodies are shown in tables 1-7. The amino acid sequences of the 11 single-domain antibodies are shown as SEQ ID NO.1-11, and the DNA sequences for encoding the 11 single-domain antibodies are shown as SEQ ID NO. 12-22.
TABLE 1 CDR1 sequences of 11 single domain antibodies
TABLE 2 CDR2 sequences of 11 single domain antibodies
TABLE 3 CDR3 sequences of 11 single domain antibodies
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TABLE 4 FR1 sequences of 11 single domain antibodies
TABLE 5 FR2 sequences of 11 single domain antibodies
TABLE 6 FR3 sequences of 11 single domain antibodies
TABLE 7 FR4 sequences of 11 single domain antibodies
Example 5: purification and expression of specific single domain antibody of CD25 protein in host bacterium escherichia coli
Plasmids of the different clones obtained by sequencing (pMECS-VHH) in example 4 were electrotransformed into E.coli HB2151 and plated onto LB+amp+glucose-containing culture plates, which were incubated overnight at 37 ℃; individual colonies were selected and inoculated in 5mL of LB medium containing ampicillin, 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 1M IPTG, shake culturing at 28deg.C overnight; centrifuging, collecting escherichia coli, and obtaining an antibody crude extract by using a permeation swelling method;
purifying the antibody by nickel column affinity chromatography, wherein the purified part of single domain antibody comprises VHH 1-11.
The single domain antibodies of VHH1-11 correspond in sequence to the amino acid sequences SEQ ID NO.1-11, i.e.VHH 1-11 correspond respectively: 1A5,1B5,1E1,1H1,2H3,3E11,3F5,3G3,4A4,4D9,4H7.
Example 6: construction of Fc fusion antibody eukaryotic expression vector of anti-CD 25 single domain antibody
(1) Subcloning the target sequence obtained in example 4 into a eukaryotic expression vector: the antibodies selected in example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;
(2) The nucleotide sequence (SEQ ID NO: 11-22) is synthesized 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, the SEQ ID NO:11-22 is inserted into a multiple cloning site MCS of the vector), and 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: single domain antibodies against CD25 protein are expressed in suspension ExpiCHO-S cells
(1) 3 days before transfection at 2.5X10 5 ExpiCHO-S cell passage and expansion culture/mL 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 anti-CD 25 single domain antibody prepared in example 6) complex for 1-5 minutes at room temperature, then adding gently into the prepared cell suspension, and gently agitating shake flask during the 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-293CD05 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-293CD05 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 an Fc fusion antibody eukaryotic expression vector of the 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 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) 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 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 293Expression 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 specific single domain antibodies (eukaryotic expression) to CD25 protein
(1) 50. Mu.L of 1. Mu.g/mLCD 25 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 and Tab controls are also respectively arranged; the VHH-hFc was obtained by purifying the Fc fusion antibody of the specific single domain antibody of the CD25 protein prepared in example 8 in example 9.
(4) Washing the plate; add 50. Mu.L of single domain antibody diluted in step (3), double wells and incubate at 37℃for 1h.
(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 and 4, where part of the antibody clone had two EC50 data, since two replicates were performed.
It can be seen that the single domain antibodies 1A5,1B5,1E1,1H1,2H3,3E11,3F5,3G3,4A4,4D9,4H7 are excellent in both binding potency and specificity for CD25 protein.
Example 13: ADCC action induced by specific single domain antibodies and tool antibodies
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, tab1, tab2, tab3 and a single-domain antibody sample, 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 an Fc fusion antibody of a specific single domain antibody of CD25 protein prepared in example 8, which was purified 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. 5, 6, 7, 8.
The lower EC50 concentration indicates a stronger antibody-mediated ADCC function, and the above results indicate that the antibodies prepared according to the present invention 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
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<211> 122
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
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 Ser Arg Arg Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asn Thr
35 40 45
Ala Gly Gly His Thr Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala Ala Glu Thr Gly
85 90 95
Ser Tyr Cys Tyr Ala His Val Pro Glu Ser Ala Asp Phe Gly Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 5
<211> 125
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
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 His Thr Ser Ser Ser Asn Arg Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Pro Glu Gly Val Ala Val Ile Ser Thr
35 40 45
Gly Gly Gly Arg Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Gly Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Ala Ser
85 90 95
Ser Ile Ala Ile Ser Arg Phe Trp Thr Trp Pro Pro Lys Glu Phe Phe
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 121
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Ala Ser Gly Tyr Thr Tyr Ser Arg Arg Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Ser
35 40 45
Gly Asn Gly Val Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn 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 Gln Gly Asp
85 90 95
Tyr Cys Tyr Ala His Ala Pro Glu Pro Ala Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln 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 Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Arg Asn Cys Val Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Ser
35 40 45
Gly Gly Gly Tyr Ser Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Phe Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Glu Pro Ala
85 90 95
Ser Cys Phe Pro His Val Pro Thr Glu Val Asp Phe Gly Tyr Trp Gly
100 105 110
Arg Gly Thr Gln 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 Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Gly Ser Arg Tyr Thr Ser Ile Thr Gly 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 His Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ala Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Val Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Gly Met Tyr Tyr Cys Ala Ala Glu Ala Ser
85 90 95
Pro Cys Phe Pro His Asn Pro Gln Val Ala Asp Phe Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 9
<211> 117
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu Thr Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser Gly Phe Thr Phe Asp Asp Ser Asp Met Gly Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser Ile Met Arg Ser
35 40 45
Asp Arg Ser Thr Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Val Tyr His Cys Ala Ala Ala Gly Ala Ile
85 90 95
Ser Ile Ala Ala Ala Cys Pro Thr His Trp Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 10
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
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 Pro Tyr Arg Arg Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Ser
35 40 45
Ala Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Asp Asp Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
65 70 75 80
Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Leu Asn Ala Pro Thr
85 90 95
Trp Glu Cys Arg Ala Gly Gly Ala Pro Ala Phe Ala Tyr Ser Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 11
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Glu Ser Thr Tyr Arg Arg Thr Tyr Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Asp Gly Val Ala Val Ile Tyr Thr
35 40 45
Gly Asn Gly Val Thr Trp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr
50 55 60
Ile Ser Gln Asp Asn 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 Ser Met
85 90 95
Arg Gly Arg Asn Val Leu His Ser Gly Phe Tyr His Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 12
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
gagtctggag gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
ggatacacct atagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggtcacacat actatgccga ctccgttgtg 180
aagggccgat tcaccatctc ccaagacaac gccaagaaca cggtgtatct gcaaatgaac 240
agcctgaaac ctgaggacac tgccatgtac tactgtgcgg cagatagtgg ttactgctac 300
gcggaggaac cggccccaga ccgatttggt tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 13
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gagtctggag gaggctcggt gcaggctgga gggtctctaa gactctcctg tgcagcctct 60
ggatacacct acagtagcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggatacacct actatgccga ctccgtggag 180
ggccgattca ccatctccca agacaaagcc aagaacacgg tgtatctgca aatgaacagc 240
ctgaaacctg aggacactgc catgtactac tgtgcggcag atacgggtcg ctgcttcccg 300
cacgtccctt atgaagttga ctttggttac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 14
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gagtctggag gaggctcggt ggaggctgga gggtctctga gactctcctg tgcacgccct 60
ggatacgact acgataccga ctacatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggagttg cgactattta tactcaaaaa ggtttcacat ggtatgccga ttctgtgaag 180
ggccgattca ccatctccca agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgaaacctg aggacactgc catgtactac tgtgcggcag ccgattacgg ggctgcgacg 300
tccaaattga acccatcaaa gtataacgcg tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 15
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gagtctgggg gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
ggatacacca gcaggcgcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattaa tactgctggt ggtcacacat actatgccga ctccgtggag 180
ggccgattca ccatctccca agacaacgcc aggaacacgg tgtatctgca aatgaacagc 240
ctgaaacctg aggacactgg catgtactac tgtgcggcag agactggtag ttactgctac 300
gcgcatgtcc ccgagtcagc tgactttggt tactgggggc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 16
<211> 375
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gagtctggag gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
ggccacacct ctagtagcaa ccgtatgggc tggttccgcc aggctccagg gaaggagccc 120
gagggggtcg cagttattag tactggtggc ggtcgcacat tgtatgccga ctccgtgaag 180
ggccgattca ccatctccca agacaacgcc aagaacacgc tgggtctgca aatgaacagc 240
ctgaaacctg aggacactgc catgtactac tgtgctgcgg ccgcgtcatc tatagcgatc 300
tccagatttt ggacctggcc cccgaaagaa ttttttgatt actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 17
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gagtctgggg gaggctcggt gcaggctgga gggtctctga gactctcctg tgtagcctct 60
ggatacacct acagtaggcg ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattta tagtggtaat ggtgtcacat actatcccga ctccgtgaag 180
ggccgattca ccatctccca agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgaaacctg aggacactgc catgtactac tgtgcggctc agggtgatta ctgctacgcc 300
cacgccccag agccggctga ctttgggtac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 18
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
gagtctggag gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
ggatacacct acagtcgcaa ctgtgtgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tagcggtggt ggttactcat actatgccga ctccgtgaag 180
ggccgattca ccatctccca agacaacgcc aagaacacgg tgtttctcca aatgaacagc 240
ctgaaacctg aggacactgc cgtgtactac tgtgcggcag agccggctag ctgcttcccg 300
cacgttccta ctgaagttga ctttggttac tggggccggg ggacccaggt caccgtctcc 360
tca 363
<210> 19
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
gagtctggag gaggctcggt gcaggctgga gggtctctga gactctcctg tgtaggctct 60
agatacacct ccattaccgg ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtcg cagctattga tactggtggt ggtcacacat actatgccga ctccgtgaag 180
ggccgattca ccatcgccca agacaacgcc aagaatacgg tgtatctgca agtgaacagc 240
ctgaaacctg aggacactgg catgtactac tgtgcggcag aggcctcccc ctgtttccct 300
cacaacccac aagttgctga ctttggttac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 20
<211> 351
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gagtctgggg gaggctcggt gcaggctgga gagactctga gactctcctg tacagcctcc 60
ggattcactt ttgatgattc tgacatgggc tggtaccgcc aggctccagg gaatgagtgc 120
gagttggtct caattatgcg tagtgatcgt agcacatact atgcagcctc cgtgaagggc 180
cgattcacca tctcccaaga caacgccaag aacacggtgt atctgcaaat gaacagcctg 240
aaacctgagg acacagccgt gtatcactgt gcggcagctg gggctatttc tatagcggct 300
gcgtgcccca cccattggtg gggccagggg acccaggtca ccgtctcctc a 351
<210> 21
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gagtctgggg gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
ggatacccct acagacgcaa ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120
gagggggtag ccgctattga tagtgcgggc accacaacct acgcagactc cgtgaagggc 180
cgattcacca tctccaaaga cgacgccaag aacactctgt atctgcaaat gaacagcctg 240
aaacctgagg acactgccat gtactactgt gcgctaaatg ccccgacgtg ggaatgccgg 300
gccggggggg cacccgcctt tgcttactcg ggccagggga cccaggtcac cgtctcctca 360
<210> 22
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gagtctgggg gaggctcggt gcaggctgga gggtctctga gactctcctg tgcagcctct 60
gaatccacct accgtagaac ctacatggcc tggttccgcc aggctccagg gaaggagcgc 120
gacggggtcg cggttattta tactggtaat ggggtcacat ggtatgccga ctccgtggag 180
ggccgattca ccatctccca agacaacgcc aagaacacgg tgtatctgca aatgaacagc 240
ctgaaacctg aggacactgc catgtactac tgtgcggcag actcaatgcg aggtcgtaat 300
gtcctacaca gcggattcta tcactactgg ggccagggga cccaggtcac cgtctcctca 360
<210> 23
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Glu Ser Gly Gly Gly Ser Val Glu Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Arg Pro
20
<210> 24
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu Thr Leu Arg Leu Ser
1 5 10 15
Cys Thr Ala Ser
20
<210> 25
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 26
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Ala Ser
20
<210> 27
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Val Gly Ser
20
<210> 28
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Gly Val Ala
1 5 10 15
Val
<210> 29
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 29
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Pro Glu Gly Val Ala
1 5 10 15
Val
<210> 30
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 30
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 31
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 31
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Thr
<210> 32
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 32
Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu Val Ser
1 5 10 15
Ile
<210> 33
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 34
<211> 39
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 34
Tyr Tyr Ala Asp Ser Val Val Lys Gly Arg Phe Thr Ile Ser Gln Asp
1 5 10 15
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu
20 25 30
Asp Thr Ala Met Tyr Tyr Cys
35
<210> 35
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 35
Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Gln Asp Lys
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 36
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Trp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 37
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 37
Tyr Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Arg Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Met Tyr Tyr Cys
35
<210> 38
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 38
Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Leu Gly Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 39
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Tyr Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 40
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 40
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Phe Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 41
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ala Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Val Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Met Tyr Tyr Cys
35
<210> 42
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Tyr Tyr Ala Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr His Cys
35
<210> 43
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asp
1 5 10 15
Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 44
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Trp Tyr Ala Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 45
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 46
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 47
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 48
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 48
Glu Ser Thr Tyr Arg Arg Thr Tyr
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
Gly Phe Thr Phe Asp Asp Ser Asp
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Gly His Thr Ser Ser Ser Asn Arg
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Gly Tyr Asp Tyr Asp Thr Asp Tyr
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Gly Tyr Pro Tyr Arg Arg Asn Cys
1 5
<210> 53
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 53
Gly Tyr Thr Ser Arg Arg Asn Cys
1 5
<210> 54
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 54
Gly Tyr Thr Tyr Ser Arg Asn Cys
1 5
<210> 55
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 55
Gly Tyr Thr Tyr Ser Arg Arg Cys
1 5
<210> 56
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 56
Gly Tyr Thr Tyr Ser Ser Asn Cys
1 5
<210> 57
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 57
Arg Tyr Thr Ser Ile Thr Gly Cys
1 5
<210> 58
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 58
Ile Asp Ser Ala Gly Thr Thr
1 5
<210> 59
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 59
Ile Asp Ser Gly Gly Gly Tyr Ser
1 5
<210> 60
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 60
Ile Asp Thr Gly Gly Gly His Thr
1 5
<210> 61
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 61
Ile Asp Thr Gly Gly Gly Tyr Thr
1 5
<210> 62
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 62
Ile Asn Thr Ala Gly Gly His Thr
1 5
<210> 63
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 63
Ile Ser Thr Gly Gly Gly Arg Thr
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 64
Ile Tyr Ser Gly Asn Gly Val Thr
1 5
<210> 65
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 65
Ile Tyr Thr Gly Asn Gly Val Thr
1 5
<210> 66
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 66
Ile Tyr Thr Gln Lys Gly Phe Thr
1 5
<210> 67
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 67
Met Arg Ser Asp Arg Ser Thr
1 5
<210> 68
<211> 23
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 68
Ala Ala Ala Ala Ser Ser Ile Ala Ile Ser Arg Phe Trp Thr Trp Pro
1 5 10 15
Pro Lys Glu Phe Phe Asp Tyr
20
<210> 69
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 69
Ala Ala Ala Asp Tyr Gly Ala Ala Thr Ser Lys Leu Asn Pro Ser Lys
1 5 10 15
Tyr Asn Ala
<210> 70
<211> 16
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 70
Ala Ala Ala Gly Ala Ile Ser Ile Ala Ala Ala Cys Pro Thr His Trp
1 5 10 15
<210> 71
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 71
Ala Ala Asp Ser Gly Tyr Cys Tyr Ala Glu Glu Pro Ala Pro Asp Arg
1 5 10 15
Phe Gly Tyr
<210> 72
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 72
Ala Ala Asp Ser Met Arg Gly Arg Asn Val Leu His Ser Gly Phe Tyr
1 5 10 15
His Tyr
<210> 73
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 73
Ala Ala Asp Thr Gly Arg Cys Phe Pro His Val Pro Tyr Glu Val Asp
1 5 10 15
Phe Gly Tyr
<210> 74
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 74
Ala Ala Glu Ala Ser Pro Cys Phe Pro His Asn Pro Gln Val Ala Asp
1 5 10 15
Phe Gly Tyr
<210> 75
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 75
Ala Ala Glu Pro Ala Ser Cys Phe Pro His Val Pro Thr Glu Val Asp
1 5 10 15
Phe Gly Tyr
<210> 76
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 76
Ala Ala Glu Thr Gly Ser Tyr Cys Tyr Ala His Val Pro Glu Ser Ala
1 5 10 15
Asp Phe Gly Tyr
20
<210> 77
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 77
Ala Ala Gln Gly Asp Tyr Cys Tyr Ala His Ala Pro Glu Pro Ala Asp
1 5 10 15
Phe Gly Tyr
<210> 78
<211> 19
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 78
Ala Leu Asn Ala Pro Thr Trp Glu Cys Arg Ala Gly Gly Ala Pro Ala
1 5 10 15
Phe Ala Tyr

Claims (9)

1. An anti-CD 25 single domain antibody, characterized in that: the single domain antibody is composed of a heavy chain, the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, and the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are (1) or (2) as follows:
(1) CDR1 as shown in SEQ ID NO. 48, CDR2 as shown in SEQ ID NO. 65, CDR3 as shown in SEQ ID NO. 72;
(2) CDR1 shown in SEQ ID NO. 56, CDR2 shown in SEQ ID NO. 61, and CDR3 shown in SEQ ID NO. 73.
2. A single domain anti-CD 25 antibody according to claim 1, characterized in that: the single domain antibody also comprises a framework region FR; the framework regions FR include the amino acid sequences of FR1, FR2, FR3 and FR 4;
when the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are (1), the amino acid sequences of the framework region FR are respectively:
25, or a variant of FR1 as set forth in SEQ ID No. 25, said variant of FR1 comprising up to 5 amino acid substitutions in said FR 1;
28, said FR2 variant comprising up to 5 amino acid substitutions in said FR 2;
44, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3;
46. FR4 or a variant of FR4 shown in SEQ ID NO. 46, said variant of FR4 comprising a substitution of at most 5 amino acids in said FR 4;
when the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are (2), the amino acid sequences of the framework region FR are respectively:
25, or a variant of FR1 as set forth in SEQ ID No. 25, said variant of FR1 comprising up to 5 amino acid substitutions in said FR 1;
30, said FR2 variant comprising up to 5 amino acid substitutions in said FR 2;
35, said FR3 variant comprising up to 5 amino acid substitutions in said FR 3;
FR4 shown in SEQ ID NO. 46 or a variant of FR4, said variant of FR4 comprising at most 5 amino acid substitutions in said FR 4.
3. An anti-CD 25 single domain antibody, characterized in that: the amino acid sequence of the single domain antibody is shown as SEQ ID NO.2 or 11 respectively.
4. An Fc fusion antibody consisting of the anti-CD 25 single domain antibody of any one of claims 1 to 3 and an Fc fused thereto.
5. A nucleotide molecule encoding the anti-CD 25 single domain antibody of any one of claims 1 to 3, characterized in that: the nucleotide sequences of the nucleotide sequences are respectively shown in SEQ ID NO:13 or 22.
6. An expression vector, characterized in that: comprising a single domain antibody encoding an anti-CD 25 according to any one of claims 1 to 3 or an Fc fusion antibody according to claim 4 or a nucleotide molecule according to claim 5.
7. A host cell, characterized in that: which can express the single domain antibody against CD25 of any one of claims 1 to 3 or the Fc fusion antibody of claim 4, or which comprises the expression vector of claim 6.
8. A pharmaceutical composition or medicament, characterized in that: the pharmaceutical composition or agent comprising an anti-CD 25 single domain antibody according to any one of claims 1 to 3, and/or a pharmaceutically acceptable carrier.
9. Use of an anti-CD 25 single domain antibody according to any one of claims 1 to 3 or a pharmaceutical composition or agent according to claim 8 for the manufacture of a medicament for the treatment of lymphoma.
CN202210660126.6A 2022-06-13 2022-06-13 anti-CD 25 single domain antibody and application thereof Active CN115181181B (en)

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CN103333248A (en) * 2013-06-07 2013-10-02 东南大学 CD25 nanometer antibody as well as coding sequence and application thereof
CN115181182A (en) * 2022-06-13 2022-10-14 南京融捷康生物科技有限公司 Humanized anti-CD 25 single-domain antibody and application thereof

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EP3216804A3 (en) * 2013-03-15 2017-12-20 AbbVie Biotechnology Ltd. Anti-cd25 antibodies and their uses
US11873341B2 (en) * 2018-03-13 2024-01-16 Tusk Therapeutics Ltd. Anti-CD25 for tumour specific cell depletion

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CN103333248A (en) * 2013-06-07 2013-10-02 东南大学 CD25 nanometer antibody as well as coding sequence and application thereof
CN115181182A (en) * 2022-06-13 2022-10-14 南京融捷康生物科技有限公司 Humanized anti-CD 25 single-domain antibody and application thereof

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