CN114478777B - Single-domain antibody for GPA33 and derived protein and application thereof - Google Patents

Single-domain antibody for GPA33 and derived protein and application thereof Download PDF

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CN114478777B
CN114478777B CN202210207351.4A CN202210207351A CN114478777B CN 114478777 B CN114478777 B CN 114478777B CN 202210207351 A CN202210207351 A CN 202210207351A CN 114478777 B CN114478777 B CN 114478777B
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CN114478777A (en
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苏志鹏
孟巾果
王乐飞
张云
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Nanjing Rongjiekang Biotechnology Co ltd
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Nanjing Rongjiekang Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K2317/565—Complementarity determining region [CDR]
    • 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®

Abstract

The invention belongs to the field of immunology, and relates to a single-domain antibody aiming at GPA33, a derivative protein thereof 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:41-SEQ ID NO:47, a heavy chain CDR2 shown in any one of SEQ ID NO:48-SEQ ID NO:55 and a heavy chain CDR3 shown in any one of SEQ ID NO:56-SEQ ID NO: 68. 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 GPA33, the initial affinity of the antibody is obvious, and the antibody can block the specific cell to release the cytokine, and has good binding activity and good drug property through prokaryotic expression.

Description

Single-domain antibody for GPA33 and derived protein and application thereof
Technical Field
The invention relates to the technical field of biotechnology or immunology, and relates to a single domain antibody aiming at GPA33, a derivative protein thereof and application thereof.
Background
Colorectal cancer is one of the most common malignant tumors in western countries and is also the leading cause of cancer death. Statistics data in 2018 show that the number of concurrent cancers in the world is 1810 ten thousand, the number of deaths is 960 ten thousand, wherein the cure rate of intestinal cancer is 6.1%, and the death rate is 9.2%. Because of the high resistance of micro-disseminated colorectal cancer to traditional therapies in recent years, the development of novel therapeutic methods and medicaments is urgent. Generating less immunogenicity, constructing humanized or chimeric antibodies to reduce the immune response of the patient; allowing for antibody reuse, more specific antigen recognition is a goal we pursue.
GPA33 (Cell surface A33 anti, swiss Prot database accession number: Q99795) is a type I single transmembrane protein with a molecular weight of 35.6kDa, and consists of 319 amino acids, and the glycosylation modification position is between 1 and 235 amino acids and is located outside the Cell membrane. GPA33 is a cell surface differentiation antigen belonging to the immunoglobulin superfamily. The GPA33 allele is located on chromosome 1q24 and comprises 7 exons, and the total length of genomic DNA is 37787bp.
GPA33 presents tissue-specific expression. The specific expression of the polypeptide is carried out on gastrointestinal epithelial tissues, is mainly distributed on mucosal epithelial cells, and has lower content in other tissues. Although the function of GPA33 is not fully understood at present, oncology studies have found that GPA33 is highly expressed in more than 95% of colorectal cancers. Since the expression of GPA33 has very high intestinal tissue specificity, the GPA33 not only provides an index for intestinal tumor detection, but also plays an important role in researching the occurrence and progress of intestinal tumor.
French researchers as early as 2009 have found that PPARgamma ligands up-regulate GPA33 mRNA and protein levels in a time and concentration dependent manner. Researchers found that the GPA33 gene is a target gene for PPARgamma in HT29-Cl.16E cells using DNA chip technology; administration of ppary agonist GW7845 to different human colon cancer cells (including HT29-cl.16e, caco2 and SW1116 etc.) showed a positive correlation between the two. The main mechanism involved is that activation of pparγ induces the expression of KLF 4; KLF4 binds to the promoter of GPA33 thereby increasing the expression of GPA 33.
GPA33 also has a certain correlation with osteoarthritis. The current clinical diagnosis of osteoarthritis is based mainly on imaging examinations. However, osteoarthritis has no or few symptoms at an early stage, and only when secondary inflammation, pain, and joint movement are affected, will it seek medical attention. At this time, joint damage occurs for a long time. Developing a method for early diagnosis of osteoarthritis is a highly desirable problem. In 2018, research reports that GPA33 gene expression in normal synovial tissue and osteoarthritis synovial tissue has significant difference, so that GPA33 can be used for developing products for diagnosing osteoarthritis. Experimental staff find that compared with normal synovial tissue, GPA33 gene expression content in the synovial tissue of osteoarthritis is about 10 times higher by using a fluorescent quantitative PCR technology. In addition, the same experimental results were obtained using immunodetection, in situ hybridization, chip or high throughput sequencing. Compared with the traditional means, the gene diagnosis is more timely and sensitive, and the death rate of osteoarthritis is reduced.
Immune disorders refer to the damage of the autoimmune response to the tissues and organs of the individual and the appearance of symptoms. 2021 has been reported to determine the expression pattern of GPA33 in human leukocyte subpopulations using mass spectrometry and flow cytometry. The results show that GPA33 is expressed in B cells, dendritic cells, natural killer cells and innate lymphocytes, with significant cd4+ T cell expression. Primary T cells and cxcr5+ regulatory T cells express high levels of GPA33, and primary cd4+ T cells express moderate levels of GPA33. The expression pattern of GPA33 highlights to some extent the functional heterogeneity of the population of CD4+ central memory T cells. GPA33+CD4+ central memory T cells are completely undifferentiated, true central memory T cells lack immediate effector function, while GPA33+ central memory T cells exhibit rapid effector function. GPA33 expression in traditional CD4+ T cells has been shown to play a role in the localization of the undifferentiated state, self-protection. Meanwhile, research results also show that GPA33 can identify human tTreg cells and provide a safer and more effective adoptive cell therapy strategy for separating the cells. Treg cells can be produced during thymus development (tTreg cells) or can be derived from mature conventional cd4+ T cells (pTreg cells). Mouse studies have shown that tTreg cells are strongly conserved, whereas pTreg cells can be reduced to conventional cd4+ T cells. The results of proteomics and transcriptomics studies showed that GPA33 was expressed on a subset of human Treg cells. GPA33 was acquired later during development of tTreg cells and was not expressed in TGF- β induced Treg cells. GPA33 is able to recognize Treg cells in human blood that lack the ability to produce effector cytokines (IL-2, IFN-gamma, IL-17). GPA33 high expressing Treg cells generally preferentially express the transcription factor Helios of the marked tTreg cells, so that the Treg cells can be amplified in vitro forcefully and stably even in the absence of rapamycin. The amplified GPA33 high-expression Treg cells have inhibitive performance and can not produce pro-inflammatory cytokines.
Currently, there are few targeted clinical drugs against GPA 33. A bispecific antibody (DART) that can recruit T cells was developed by Servers and macrogeneics. DARs are linked by the VH chain of one antibody and the VL chain of another antibody. The VH of the second antibody was linked to the VL of the first antibody to form a bispecific-like antibody that was stabilized to form DART via additional disulfide bonds. MGD007 is a DART protein designed to direct T cells to target GPA33 expressing colon cancer, 7 months 2014, into phase I clinical trial phase (code NCT 02248805). MGD007 is structurally fused to an Fc region and thus has a longer serum half-life. There are related xenograft studies showing that tumor growth is inhibited as low as 4 μg/kg. Cd8+ and cd4+ T cells mediate tumor cell lysis for GPA33 expression, increased activity, increased granzyme and perforin. Notably, the population of suppressor T cells can also be used to mediate lysis of tumor cells expressing GPA 33. Along with CTL activity, activation and expansion of T cells are both represented in a GPA33 dependent manner. In cynomolgus monkeys, the dose of 100 μg/kg for 4 weeks was well tolerated, consistent with the pharmacokinetics of the Fc-containing molecules.
Still other laboratory-independently developed antibodies and related techniques are also in constant validation. In the literature published by the Federal administration institute of Switzerland in 2020, it was mentioned that a GPA33 antibody, A2, generated by phage display technology recognizes the "V" domain of GPA33 and uniformly stains poorly, well-differentiated and highly differentiated colon adenocarcinoma samples. The A2 antibody is a recombinant murine IgG2a subtype, preferentially localized to GPA33 transfected CT26 mouse colon adenocarcinoma cells in immunized mice, evenly distributed within tumor masses, while other antibodies appear as plaque-like in tumor lesions. A2 effectively induces killing of GPA 33-expressing cells in vitro by antibody-dependent cytotoxicity and inhibits growth of GPA 33-positive mouse CT26 and C51 lung metastases in vivo. On the other hand, antibody-based imaging agents are also under constant development as an auxiliary diagnostic tool for solid tumors. University of Sichuan researchers have constructed GPA33scFv-Fc antibodies against GPA33 by fusing GPA33-scFv with the Fc fragment of human IgG1 antibodies. GPA33-scFv-Fc specifically binds GPA33 positive colorectal cancer cells and tumor tissue. After intravenous injection of the near infrared fluorescent probe CF750 labeled GPA33-scFv-Fc into subcutaneous GPA33 positive LS174T tumor graft mice, high contrast images of the tumor graft were recorded dynamically over 24 hours using an optical imaging system. Also, researchers isolated exosomes from GPA33 positive LIM1215 cells and loaded with doxorubicin; meanwhile, GPA33 antibodies (GPA 33 Ab-US) are coated on surface carboxyl superparamagnetic iron oxide nanoparticles (US), and the GPA33 antibodies on the surfaces of the nanoparticles are expected to be combined with GPA33 positive exosomes and form a complex to target GPA33 positive colon cancer cells. The results indicate that GPA33Ab-US-Exo/Dox has good affinity and antiproliferative effect in LIM1215 cells. In addition, in vivo studies show that GPA33Ab-US-Exo/Dox has good tumor targeting capability, can inhibit tumor growth, prolong the survival time of mice and reduce cardiotoxicity.
In summary, there remains a need in the art for antibodies capable of binding with high affinity to GPA33, in particular antibodies specific for GPA33& CD 3.
Nanobodies are a new proposition in the antibody community, and because of the small molecular weight, bivalent, trivalent or bispecific antibodies can be obtained by simple molecular cloning techniques. Because of the small molecular characteristics of the nanobody, the nanobody can achieve high yield in a prokaryotic expression system (escherichia coli) or a eukaryotic expression system (CHO cells, 293 cells and the like). The rapid development of nanobodies has become a potential-unlimited force in antibody drug development, representing an important development direction of antibody drugs from now on.
The nanobody has high affinity and good penetrating power with the target binding site, so that the nanobody can be more easily combined with the receptor in a targeting way and can permeate into tissues with few blood vessels. Meanwhile, in view of the low immunogenicity of the nanobody, the nanobody is repeatedly administered to mice, and no humoral or cellular immunity is caused by detection. In addition, nanobodies can be used to build a variety of molecular structures, allowing for some molecular adjunctive therapies.
Disclosure of Invention
The invention aims to provide a single domain antibody aiming at GPA33 and a derivative protein thereof and application, wherein the single domain antibody and the derivative protein thereof have strong binding capacity with GPA33 protein and can mediate cell internalization or ADCC (advanced cellular cytotoxicity).
The single domain antibody is used as a monoclonal antibody, has higher affinity generally and better stability than the traditional monoclonal antibody, and has the advantages of the traditional monoclonal antibody. Therefore, the invention utilizes the advantages to obtain the single domain antibody aiming at GPA33, has strong binding capacity with GPA33 protein after prokaryotic expression, and discovers that the single domain antibody can mediate cell internalization or ADCC action after eukaryotic expression and has certain drug forming property.
In a first aspect of the invention there is provided a single domain antibody to GPA33, said single domain antibody comprising a heavy chain CDR1 as shown in any one of SEQ ID NO:41-SEQ ID NO:47, a heavy chain CDR2 as shown in any one of SEQ ID NO:48-SEQ ID NO:55 and a heavy chain CDR3 as shown in any one of SEQ ID NO:56-SEQ ID NO: 68.
Specifically, the heavy chain includes a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3; the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 are one of the following (1) - (20):
(1) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 50, CDR3 shown in SEQ ID NO. 62;
(2) CDR1 as shown in SEQ ID NO. 46, CDR2 as shown in SEQ ID NO. 50, CDR3 as shown in SEQ ID NO. 63;
(3) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 50, CDR3 shown in SEQ ID NO. 63;
(4) CDR1 as shown in SEQ ID NO. 45, CDR2 as shown in SEQ ID NO. 50, CDR3 as shown in SEQ ID NO. 63;
(5) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 52, CDR3 shown in SEQ ID NO. 63;
(6) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, CDR3 shown in SEQ ID NO. 63;
(7) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, CDR3 shown in SEQ ID NO. 61;
(8) CDR1 as shown in SEQ ID NO. 42, CDR2 as shown in SEQ ID NO. 53, CDR3 as shown in SEQ ID NO. 66;
(9) CDR1 as shown in SEQ ID NO. 42, CDR2 as shown in SEQ ID NO. 53, CDR3 as shown in SEQ ID NO. 67;
(10) CDR1 as shown in SEQ ID NO. 42, CDR2 as shown in SEQ ID NO. 53, CDR3 as shown in SEQ ID NO. 65;
(11) CDR1 shown in SEQ ID NO. 44, CDR2 shown in SEQ ID NO. 53, CDR3 shown in SEQ ID NO. 64;
(12) CDR1 as shown in SEQ ID NO. 43, CDR2 as shown in SEQ ID NO. 54, CDR3 as shown in SEQ ID NO. 59;
(13) CDR1 shown in SEQ ID NO. 41, CDR2 shown in SEQ ID NO. 48, CDR3 shown in SEQ ID NO. 68;
(14) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, CDR3 shown in SEQ ID NO. 60;
(15) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, CDR3 shown in SEQ ID NO. 57;
(16) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 51, CDR3 shown in SEQ ID NO. 56;
(17) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 49, CDR3 shown in SEQ ID NO. 56;
(18) CDR1 as shown in SEQ ID NO. 47, CDR2 as shown in SEQ ID NO. 49, CDR3 as shown in SEQ ID NO. 58;
(19) CDR1 shown in SEQ ID NO. 47, CDR2 shown in SEQ ID NO. 55, CDR3 shown in SEQ ID NO. 58;
(20) CDR1 as shown in SEQ ID NO. 45, CDR2 as shown in SEQ ID NO. 49, and CDR3 as shown in SEQ ID NO. 58.
The CDR combinations of (1) - (20) above correspond in sequence to SEQ ID NO.1-20, respectively.
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 a preferred embodiment, the sequence of the single domain antibody 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:
69-72, or a variant of FR1 as set forth in SEQ ID No. 69-72, said variant of FR1 comprising up to 3 amino acid substitutions in said FR 1;
73-77, said FR2 variant comprising up to 3 amino acid substitutions in said FR 2;
78-89, said FR3 variant comprising up to 3 amino acid substitutions in said FR 3;
90-91, said FR4 variant comprising up to 3 amino acid substitutions in said FR 4.
In one embodiment, the single domain antibody to GPA33 hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-20 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and is capable of specifically binding to a GPA33 protein.
In another preferred embodiment, the single domain antibody directed against GPA33 hybridizes to a polypeptide selected from the group consisting of SEQ ID NOs: 1-20, and is capable of specifically binding to a GPA33 protein.
In a second aspect of the invention there is provided a single domain antibody against GPA33, said single domain antibody having the amino acid sequence shown in SEQ ID NO.1-20, respectively, or said single domain antibody having at least 95% sequence homology with the amino acid sequences of SEQ ID NO. 1-20.
In one embodiment, the nucleic acid molecule encoding the single domain antibody to GPA33 hybridizes with a nucleic acid molecule selected from the group consisting of SEQ ID NOs: 21-40 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology and encodes a binding domain directed against a GPA33 single domain antibody capable of specifically binding to a GPA33 protein.
Preferably, the single domain antibody has a coding sequence as shown in SEQ ID NO.21-40, respectively, or has at least 95% sequence homology with SEQ ID NO. 21-40.
A third aspect of the invention is to provide the aforementioned Fc fusion or humanized antibody against a single domain antibody to GPA 33.
In a fourth aspect, the present invention provides a nucleotide molecule encoding the aforementioned single domain antibody against GPA33, having the nucleotide sequence set forth in SEQ ID NO:21-40 or has at least 95% sequence homology with SEQ ID No. 21-40.
In a fifth aspect, the invention provides an expression vector comprising a nucleotide molecule encoding the aforementioned single domain antibody or the aforementioned Fc fusion antibody or the aforementioned nucleotide molecule.
In a sixth aspect, the invention provides a host cell or non-human organism which can express the single domain antibody against GPA33 as described above, or which comprises the expression vector as described above.
The present invention also provides a method of producing a single domain antibody or Fc fusion antibody thereof to GPA33 comprising the steps of: (a) Culturing the aforementioned host cell under conditions suitable for the production of a single domain antibody or Fc fusion antibody thereof, thereby obtaining a culture comprising said single domain antibody or Fc fusion antibody directed against GPA 33; (b) Isolating or recovering the single domain antibody against GPA33 or Fc fusion antibody thereof from the culture; and (c) optionally purifying and/or modifying the single domain antibody against GPA33 or Fc fusion antibody obtained in step (b).
A seventh aspect of the present invention provides a pharmaceutical composition comprising: (i) An Fc fusion antibody as described previously for a single domain antibody against GPA33, or a single domain antibody as described previously for GPA 33; and (ii) one or more pharmaceutically acceptable excipients.
The invention also provides application of the single domain antibody for GPA33 in preparing a medicine or an anti-tumor medicine or an anti-arthritis medicine for inhibiting GPA33 gene expression. The medicament for inhibiting GPA33 gene expression can be applied to any diseases with high GPA33 gene expression. Preferably, the neoplasm includes, but is not limited to, colorectal cancer. The arthritis may be osteoarthritis.
The invention also provides the use of a single domain antibody directed against GPA33 in the manufacture of a medicament for mediating cellular internalization or ADCC.
The invention also provides the use of the aforementioned single domain antibody against GPA33, or the aforementioned Fc fusion antibody against a single domain antibody of GPA33, for the preparation of a reagent, assay plate or kit (e.g., an ELISA kit); wherein the reagent, assay plate or kit is for: detecting the presence and/or amount of GPA33 protein in the sample.
The single domain antibody is a VHH, which comprises only the antibody heavy chain and does not comprise the antibody light chain.
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 GPA33, the initial affinity of the antibody is obvious, and the effect of blocking the specific cell to release the cytokine is excellent, the invention has good binding activity through prokaryotic expression, has better drug forming property, and has the following advantages:
(1) 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.
(2) 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.
(3) 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
FIG. 1 is a SDS-PAGE analysis of human recombinant GPA33 protein;
FIG. 2VHH sequence insertion analysis;
FIG. 3 library enrichment case of targeted GPA33 panning;
FIG. 4 SDS-PAGE of GPA33 target portion prokaryotic expression antibodies;
FIG. 5 SDS-PAGE of eukaryotic expression antibodies of GPA33 target portions;
FIG. 6GPA33 target antibody antigen binding activity;
FIG. 7GPA33 target tool antibody antigen binding activity;
FIG. 8GPA33 target antibody species cross-reactive antigen binding activity;
figure 9 ADCC activity of GPA33 target nanobody.
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.
Single domain antibodies (sdabs, also called nanobodies or VHHs by the developer Ablynx) are 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.
In the present invention, a nanobody against GPA33 can be obtained from a sequence having high sequence homology with CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences in (1) - (20) may achieve the object of the invention (i.e., derived proteins).
In some embodiments, the polypeptide that hybridizes to SEQ ID NO:1-20, 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, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, 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:
example 1: preparation of human GPA33 recombinant extracellular domain 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 the expression vector of the human recombinant GPA33 protein is as follows:
(1) The coding sequence for GPA33, which is identified as NM-005814.2, was retrieved from NCBI and encoded to produce the amino acid sequence accession No. NP-005805.1,Uniprot ID as Q99795.
(2) The amino acid sequences corresponding to NP 005805.1 were analyzed for the transmembrane region and extracellular end of the protein via TMHMM and SMART websites, respectively.
(3) The analysis result shows that the extracellular end of GPA33 protein is 1-235 amino acids, wherein 1-21 is the signal peptide of the protein.
(4) The nucleotide sequence encoding amino acids 22-235 of GPA33 protein was cloned into the vector pcDNA3.4 by means of gene synthesis.
(5) And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out batch extraction on the recombinant plasmid after confirming no errors, removing endotoxin, carrying out expression and purification of target protein by transfecting suspension 293F, wherein the SDS-PAGE analysis result of the purified GPA33 recombinant protein is shown in figure 1, and the purity of the purified protein is as high as 90%, so that the requirement of animal immunity is met.
Example 2: construction of a single domain antibody library against GPA33 protein:
1mg of the recombinant GPA33 protein obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, and an inner Mongolian Alexal camel was immunized once a week for 7 consecutive immunizations, and the remaining six immunizations were animal immunizations with 1mg of GPA33 protein mixed with an equal volume of Freund's incomplete adjuvant except for the first immunization, in order to intensively stimulate the camel to produce antibodies against GPA33 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 phage display library of GPA33 protein and measurement of library capacity, library size of about 1X 10 9 At the same time, the correct insertion rate of the library into the target fragment was detected by colony PCR identification, and the results are shown in FIG. 2.
The results showed that 28 clones amplified a band of 600bp (predicted size) and 2 clones amplified an incorrect band after PCR amplification of 30 randomly selected colonies from the library, so the correct insertion rate was 28.times.30%. Apprxeq.93.3%.
Example 3: single domain antibody screening against GPA33 protein:
200. Mu.L of the recombinant TG1 cells of example 2 were cultured in 2 XTY medium, during which 40. Mu.L of helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phage, the phage was precipitated the next day with PEG/NaCl, and the amplified phage was collected by centrifugation.
NaHCO diluted at 100mM pH8.3 3 500 mug of GPA33 protein is coupled on an ELISA plate, and the plate is placed at 4 ℃ overnight, and a negative control hole (culture medium control) is also established; add 2 the next day00 μl of 3% skim milk was 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 combined with GPA33 protein is dissociated by trypsin with a final concentration of 25mg/mL, and E.coli TG1 cells in logarithmic growth phase are infected, cultured for 1h at 37 ℃, phage are generated and collected for the next round of screening, the same screening process is repeated for 1 round, enrichment is gradually obtained, and when the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 3.
In fig. 3, P/N = number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal by biopanning/number of monoclonal bacteria grown after infection of TG1 bacteria by phage with positive Kong Xi removal, this parameter gradually increases after enrichment has occurred; 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 against GPA33 by phage enzyme-linked immunosorbent assay (ELISA):
screening was performed according to the screening method described in example 3 above for 3 rounds of screening against the single domain antibody against GPA33 protein, the phage enrichment factor against GPA33 protein was 10 or more, 384 single colonies were selected from positive clones obtained by screening after the end of screening, inoculated into 96-well plates containing 2 XSTY medium of 100. Mu.g/mL ampicillin, respectively, and a blank was set, and after 37℃to the logarithmic phase, IPTG was added at a final concentration of 1mM and cultured overnight at 28 ℃.
Obtaining a crude extract antibody by using a permeation swelling method; GPA33 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; unbound Antibody was washed off with PBST, 100. Mu.l of Mouse Anti-HA tag Anti-body (HRP) (Mouse Anti-HA horseradish peroxidase) diluted 1:2000 was addedLabeled antibody, thermo Fisher), incubated 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.
Gene sequences of the respective clones were analyzed by sequence alignment software VectorNTI, strains with the same CDR1, CDR2 and CDR3 sequences were regarded as the same clone, and strains with different sequences were regarded as different clones, and finally single domain antibodies specific for GPA33 proteins (SEQ ID NOS.1-20 and other single domain antibodies of 1G9, 1H5, 1H7, 2A10, 2A3, 3C6, 3F7, 3H10, 4B3, 4B5, 2D5, 2G5, 3B6, etc., the sequences not shown) were obtained.
The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally the single-domain antibody protein is obtained.
CDR and FR sequences of the 20 single domain antibodies are shown in tables 1-6.
TABLE 3 CDR3 sequences of 20 single domain antibodies
TABLE 4 FR1 sequences of 20 single domain antibodies
TABLE 5 FR2 sequences of 20 single domain antibodies
TABLE 6 FR3 sequences of 20 single domain antibodies
The amino acid sequences SEQ ID NO.1-20 of the single domain antibodies are in one-to-one correspondence with the single domain antibodies 1A2, 2H6, 4F11, 1E10, 3C4, 1A9, 1D7, 1B12, 4E2, 4C4, 2B2, 2E2, 4F7, 3G9, 2C2, 1A6, 2A4, 1A3, 2A12, 4B9 in sequence.
The FR4 sequence of the 4F7 single domain antibody is SEQ ID NO:90, the FR4 sequences of the remaining 19 single domain antibodies are all SEQ ID NO:91.
example 5: purification and expression of specific single domain antibody of GPA33 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;
the antibodies were purified by nickel column affinity chromatography and the purified fraction of single domain antibodies, shown in FIG. 4, included VHHs 1-18.
The single domain antibodies of VHH1-18 correspond to the amino acid sequences SEQ ID NO.1-18, i.e.VHH 1-18 correspond to: 1A2, 2H6, 4F11, 1E10, 3C4, 1A9, 1D7, 1B12, 4E2, 4C4, 2B2, 2E2, 4F7, 3G9, 2C2, 1A6, 2A4, 1A3.
Example 6: construction of Fc fusion antibody eukaryotic expression vector of specific single domain antibody of GPA33 protein
(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) Synthesizing the nucleotide sequence (SEQ ID NO: 21-40) subjected to codon optimization into a vector RJK-V4-hFC designed and modified by the company in a sequence synthesis mode to obtain a recombinant eukaryotic expression vector, wherein the modification 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: fc fusion antibody of specific single domain antibody of GPA33 protein 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 upside downHomogeneous ExpiFectamine 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; the plasmid DNA is an Fc fusion antibody eukaryotic expression vector of a specific single domain antibody of GPA33 protein, and is prepared in the example 6;
(7) Incubating the ExpiFectamine CHO/plasmid DNA complex for 1-5 minutes at room temperature, then gently adding to the prepared cell suspension, gently agitating the 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 Fc fusion antibodies of specific single domain antibodies of GPA33 proteins 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; the plasmid DNA was diluted with 4mL of Opt-MEM medium, mixed back and forth, and filtered with a 0.22 μm filter. 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) Protein expression supernatants were collected about 7 days after transfection (cell viability below 70%).
Example 9: purification of human Fc recombinant 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) After the above-mentioned neutralized protein solution was dialyzed, SDS-PAGE analysis was performed to confirm that the protein purity was 95% or higher and the concentration was 0.5mg/mL or higher, and then the protein solution was stored at a low temperature for further use, and the SDS-PAGE results of a part of the antibodies are shown in FIG. 5. In FIG. 5, 1-7 are single domain antibodies 1A2, 1A3, 1A6, 1E10, 2A4, 2A12, 2B2, respectively.
Example 10: construction of nanobody eukaryotic expression vector RJK-V4-hFc
The targeting vector RJK-V4-hFC used in the invention is modified by the company after fusing the Fc region in the heavy chain coding sequence (NCBI Access No.: AB 776838.1) of human IgG on the basis of the invitrogen commercial vector pCDNA3.4 (vector data link: https:// packages. Thermoformer. 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 IgG 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 a6 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: binding dose-response curve assay for specific single domain antibodies (prokaryotic expression) of GPA33 proteins
(1) 50. Mu.L of 1. Mu.g/mL GPA33 was coated overnight at 4 ℃.
(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 1h.
(3) VHH was diluted to 2. Mu.g/mL, then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. The VHH is a specific single domain antibody of the prokaryotic expressed GPA33 protein obtained in example 5;
(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; mu.L of murine anti-HA tag-HRP 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 as shown in fig. 6, it was seen that all of 20 single domain antibodies against GPA33 protein were excellent in GPA33 protein binding potency and specificity.
In FIG. 6, for example, 4B9-1,2B5-2 represents the first expression of the 4B9 single domain antibody and the second expression of the 2B5 single domain antibody, respectively. The "-1", "-2" symbols are merely a distinction between the number of expressions (the sequence of 4B9-1,2B5-2 is the same as 4B9 and 2B5, respectively), and the other "-1", "-2" symbols in this figure are all for this purpose.
Example 12: expression and purification of Tool antibodies (Tabs) targeting human GPA33
The Tab (MGD 007) sequence was from the international immunogenetics database 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) was added and incubated at 8% CO2 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 this method was not less than 2mg/ml in purity of more than 94% and had an EC50 of about 0.16nM in combination with GPA33 (ACRO, cat#FO1-H52H 1), and the results are shown in FIG. 7.
Example 13: determination of the Cross-species binding Effect Curve of Fc fusion antibodies of specific Single-domain antibodies of GPA33 proteins
(1) 50. Mu.L, 1. Mu.g/mL human, cynomolgus monkey, mouse GPA33 were individually coated overnight at 4 ℃.
(2) Washing the plate; 200. Mu.L of 5% milk was added and blocked at 37℃for 1h.
(3) VHH-hFc was diluted to 2. Mu.g/mL, followed by a 5-fold gradient of diluted antibody for a total of 8 concentration gradients. The VHH-hFc here was obtained by purifying the Fc fusion antibody of the specific single domain antibody of GPA33 protein of example 8 by the purification of 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 goat anti-human IgG-HRP 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) Plotting the curve and calculating the EC50 as shown in fig. 8; wherein hIgG designates a isotype control, immunoglobulin molecules that do not bind to any target are commercially available; tab was prepared from example 12; it can be seen that the 20 single domain antibodies directed against GPA33 of the invention are excellent in cross-binding potency to the species of GPA33 protein.
Example 14: single domain antibodies specific for GPA33 and tool antibody induced ADCC (antibody dependent cell mediated cytotoxicity):
collecting Colo205 cells of 3-4 passages after resuscitating, and spreading into 96-well plates according to 20000 holes;
preparing Tab, hIgG and VHH-hFc samples into a solution with the highest concentration of 10 mug/mL, and carrying out 10-time gradient dilution to obtain 7 concentrations; wherein hIgG designates a isotype control, immunoglobulin molecules that do not bind to any target are commercially available; tab was prepared from example 12; the VHH-hFc was obtained by purifying Fc fusion antibody of specific single domain antibody of GPA33 protein in example 8 by example 9;
adding the antibody solution diluted in a gradient manner into a cell culture hole according to the equal volume of the cell suspension;
For sample wells and E/T wells (antibody concentration 0), jurkat-NFAT-luc-FcgammaRIIIa cells were collected and added to cell culture wells at 20000 cells per well;
after 6h incubation, cell killing was detected with One-Glo kit, and luminescence read;
calculation of the fold induction (Fold of Induction) = (sample-BG)/(E/T-BG)
Based on target cell killing and concentration, four-parameter fitting was performed to calculate EC50 concentrations for each antibody-mediated ADCC, and the results are shown in fig. 9. It can be seen that the 20 single domain antibodies of the invention are effective in mediating ADCC.
The mixed solution obtained in the above step is read to obtain a fluorescence value by a flow cytometer.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Sequence listing
<110> Nanjing Rongjiekang biotechnology Co., ltd
<120> Single-domain antibody against GPA33, protein derived therefrom and use thereof
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Cys Ala Ala Ser Gly Tyr Thr Tyr Thr Pro Asn Cys Met Gly Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Leu Val Thr
35 40 45
Gly Thr Asn Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Leu Asp Glu Asp Lys Asn Thr Met Tyr Leu Gln Met Ser Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Thr Ala Ala
85 90 95
Cys Thr Val Leu Arg Gly Arg Ser Phe Pro Glu Glu Phe Asn Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 12
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Pro Gly Tyr Thr Tyr Ser Val Asn Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Met Pro Ile
35 40 45
Arg Thr Asp Arg Thr Tyr Tyr Ala Asp Phe Val Lys Gly Arg Phe Thr
50 55 60
Ile Ala Gln Asp Asn Ala Lys Asn Thr Ile Tyr Leu Glu Met Asn Gly
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Met Asp
85 90 95
Gly Cys Thr Arg Trp Leu Pro Arg Ser Glu Tyr Arg Tyr Trp Gly Gln
100 105 110
Gly Thr Gln 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 Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Val Ser Gly His Thr Phe Cys Ser Tyr Asp Met Ser Trp Tyr
20 25 30
Arg Gln Ala Pro Gly Lys Glu Pro Glu Phe Val Ser Asp Ile Asp Ser
35 40 45
His Ala Ile Thr Ser Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Thr
50 55 60
Ser Gln Asp Asn Val Ala Asn Thr Tyr Thr Val Tyr Leu Gln Met Asn
65 70 75 80
Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Lys Thr Gln Arg
85 90 95
Gly Arg Arg Cys Gly Glu Ala Ser Trp Ser Pro Leu Asn Tyr Arg Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 14
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Tyr Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Ile Tyr Thr
35 40 45
Gly Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Asn Asn
85 90 95
Glu Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 15
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Tyr Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Ile Tyr Thr
35 40 45
Gly Ser Gly Asn Thr His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys Ala Ala Ala Lys Ala
85 90 95
Asp Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Phe Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 16
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Tyr Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Ile Tyr Thr
35 40 45
Gly Ser Ser Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Lys Ala
85 90 95
Asp Tyr Cys Ala Gly Leu Asn Ser Tyr Gly Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 17
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Tyr Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Ile Tyr Thr
35 40 45
Gly Ser Gly Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Lys Ala
85 90 95
Asp Tyr Cys Ala Gly Leu Asn Ser Tyr Gly Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 18
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Tyr Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Ile Tyr Thr
35 40 45
Gly Ser Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Lys Ala
85 90 95
Asp Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 19
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Tyr Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Thr Tyr Thr
35 40 45
Gly Ser Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Lys Ala
85 90 95
Asp Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 20
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 20
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser Arg Ser Thr Tyr Gly Ser Val Cys Met Ala Trp Phe
20 25 30
Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala Phe Ile Tyr Thr
35 40 45
Gly Ser Gly Asn Thr Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr
50 55 60
Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser
65 70 75 80
Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ala Lys Ala
85 90 95
Asp Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 21
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaggaccg cctacgccga cagcgtgcag 180
ggcaggttca ccatcagcag ggacaacgcc aacgccaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcgtgaaca gctacggcta caaccactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 22
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacatct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaggaccg cctacgccga cagcgtgcag 180
ggcaggttca ccatcagcag ggacaacgcc aacgccaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcgtgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 23
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaggaccg cctacgccga cagcgtgcag 180
ggcaggttca ccatcagcag ggacaacgcc aacgccaccc tgtacctgca gatgagcagc 240
ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcgtgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 24
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggagcacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaggaccg cctacgccga cagcgtgcag 180
ggcaggttca ccatcagcag ggacaacgcc aacgccaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcgtgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 25
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc agcaggaccg cctacgccga cagcgtgcag 180
ggcaggttca ccatcagcag ggacaacgcc aacgccaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcgtgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 26
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacacca ggtacgccga cagcgtgaag 180
ggcaggttca ccatcagcag ggactacgcc aacaacaccc tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgg catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcgtgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 27
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacacca ggtacgccga cagcgtgaag 180
ggcaggttca ccatcagcag ggactacgcc aacaacaccc tgtacctgca gatgaacacc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaacagcga gtactgcagc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 28
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
gaaagcggcg gcggcagcgt gcaggcgggc ggcagcctgc gcctgagctg cgcggcgagc 60
ggctatacct atagcccgaa ctgcatggcg tggtttcgcc aggcgccggg caaagaacgc 120
gaaggcgtgg cgaccctggt gaccggcacc aaccgcacct attatgcgga tagcgtgaaa 180
ggccgcttta ccattagcca ggataaagat aaaattgcga tgtatctgca gatgaacagc 240
ctgaaaccgg aagataccgc gatgtattat tgcgcgacca ccgcggcgtg caccgtgctg 300
cgcggccgca cctttccgga agaatttaac tattggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 29
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagccccaa ctgcatggcc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccaccctggt gaccggcacc aacaggacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaaggac aagatcgcca tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccacca ccgccgcctg caccgtgctg 300
aggggcagga ccttccccgt ggagttcaac tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 30
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
ggctacacct acagccccaa ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccaccctggt gaccggcacc aacaggacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcca ggacaaggac aaggacacca tgtacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgcca ccgccgcctg caccgtgctg 300
aggggcagga ccttccccga ggagttcaac tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 31
<211> 366
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
gagagcggcg gcggcagcgt gcaggccggc ggccccctga ggctgagctg cgccgccagc 60
ggctacacct acacccccaa ctgcatgggc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccaccctggt gaccggcacc aacaggacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcct ggacgaggac aagaacacca tgtacctgca gatgagcagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgcca ccgccgcctg caccgtgctg 300
aggggcagga gcttccccga ggagttcaac tactggggcc agggcaccca ggtgaccgtg 360
agcagc 366
<210> 32
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccccc 60
ggctacacct acagcgtgaa ctgcatggcc tggttcaggc aggcccccgg caaggagagg 120
gagggcgtgg ccgccatgcc catcaggacc gacaggacct actacgccga cttcgtgaag 180
ggcaggttca ccatcgccca ggacaacgcc aagaacacca tctacctgga gatgaacggc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccatggacgg ctgcaccagg 300
tggctgccca ggagcgagta caggtactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 33
<211> 363
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gagagcggcg gcggcctggt gcagcccggc ggcagcctga ggctgagctg cgccgtgagc 60
ggccacacct tctgcagcta cgacatgagc tggtacaggc aggcccccgg caaggagccc 120
gagttcgtga gcgacatcga cagccacgcc atcaccagct acagcgacag cgtgaagggc 180
aggttcacca ccagccagga caacgtggcc aacacctaca ccgtgtacct gcagatgaac 240
agcctgaagc ccgaggacac cgccatgtac tactgcaaga cccagagggg caggaggtgc 300
ggcgaggcca gctggagccc cctgaactac aggggccagg gcacccaggt gaccgtgagc 360
agc 363
<210> 34
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaacaacga gtactgcagc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 35
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacaccc actacgccga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacagcgc catgtactac tgcgccgccg ccaaggccga ctactgcagc 300
ggcctgaaca gctacggctt caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 36
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc agcaacacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaaggccga ctactgcgcc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 37
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacacct actacgccga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaaggccga ctactgcgcc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 38
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacacct actacaggga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaaggccga ctactgcagc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 39
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 39
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggtacacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcaccta caccggcagc ggcaacacct actacaggga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaaggccga ctactgcagc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 40
<211> 360
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
gagagcggcg gcggcagcgt gcaggccggc ggcagcctga ggctgagctg cgccgccagc 60
aggagcacct acggcagcgt gtgcatggcc tggttcaggc aggcccccgg caagcagagg 120
gagggcgtgg ccttcatcta caccggcagc ggcaacacct actacaggga cagcgtgaag 180
ggcaggttca ccatcagcag ggacaacgcc aacaacaccc tgcacctgca gatgaacagc 240
ctgaagcccg aggacaccgc catgtactac tgcgccgccg ccaaggccga ctactgcagc 300
ggcctgaaca gctacggcta caactactgg ggccagggca cccaggtgac cgtgagcagc 360
<210> 41
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 41
Gly His Thr Phe Cys Ser Tyr Asp
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Gly Tyr Thr Tyr Ser Pro Asn Cys
1 5
<210> 43
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 43
Gly Tyr Thr Tyr Ser Val Asn Cys
1 5
<210> 44
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 44
Gly Tyr Thr Tyr Thr Pro Asn Cys
1 5
<210> 45
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Arg Ser Thr Tyr Gly Ser Val Cys
1 5
<210> 46
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 46
Arg Tyr Ile Tyr Gly Ser Val Cys
1 5
<210> 47
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 47
Arg Tyr Thr Tyr Gly Ser Val Cys
1 5
<210> 48
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 48
Ile Asp Ser His Ala Ile Thr
1 5
<210> 49
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
Ile Tyr Thr Gly Ser Gly Asn Thr
1 5
<210> 50
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Ile Tyr Thr Gly Ser Gly Arg Thr
1 5
<210> 51
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 51
Ile Tyr Thr Gly Ser Ser Asn Thr
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 52
Ile Tyr Thr Gly Ser Ser Arg Thr
1 5
<210> 53
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 53
Leu Val Thr Gly Thr Asn Arg Thr
1 5
<210> 54
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 54
Met Pro Ile Arg Thr Asp Arg Thr
1 5
<210> 55
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 55
Thr Tyr Thr Gly Ser Gly Asn Thr
1 5
<210> 56
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 56
Ala Ala Ala Lys Ala Asp Tyr Cys Ala Gly Leu Asn Ser Tyr Gly Tyr
1 5 10 15
Asn Tyr
<210> 57
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 57
Ala Ala Ala Lys Ala Asp Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Phe
1 5 10 15
Asn Tyr
<210> 58
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 58
Ala Ala Ala Lys Ala Asp Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr
1 5 10 15
Asn Tyr
<210> 59
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 59
Ala Ala Ala Met Asp Gly Cys Thr Arg Trp Leu Pro Arg Ser Glu Tyr
1 5 10 15
Arg Tyr
<210> 60
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 60
Ala Ala Ala Asn Asn Glu Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr
1 5 10 15
Asn Tyr
<210> 61
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 61
Ala Ala Ala Asn Ser Glu Tyr Cys Ser Gly Leu Asn Ser Tyr Gly Tyr
1 5 10 15
Asn Tyr
<210> 62
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 62
Ala Ala Ala Asn Ser Glu Tyr Cys Ser Gly Val Asn Ser Tyr Gly Tyr
1 5 10 15
Asn His
<210> 63
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 63
Ala Ala Ala Asn Ser Glu Tyr Cys Ser Gly Val Asn Ser Tyr Gly Tyr
1 5 10 15
Asn Tyr
<210> 64
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 64
Ala Ala Thr Ala Ala Cys Thr Val Leu Arg Gly Arg Ser Phe Pro Glu
1 5 10 15
Glu Phe Asn Tyr
20
<210> 65
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 65
Ala Ala Thr Ala Ala Cys Thr Val Leu Arg Gly Arg Thr Phe Pro Glu
1 5 10 15
Glu Phe Asn Tyr
20
<210> 66
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 66
Ala Thr Thr Ala Ala Cys Thr Val Leu Arg Gly Arg Thr Phe Pro Glu
1 5 10 15
Glu Phe Asn Tyr
20
<210> 67
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 67
Ala Thr Thr Ala Ala Cys Thr Val Leu Arg Gly Arg Thr Phe Pro Val
1 5 10 15
Glu Phe Asn Tyr
20
<210> 68
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 68
Lys Thr Gln Arg Gly Arg Arg Cys Gly Glu Ala Ser Trp Ser Pro Leu
1 5 10 15
Asn Tyr
<210> 69
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 69
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Val Ser
20
<210> 70
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 70
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Pro Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Ser
20
<210> 71
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 71
Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser
1 5 10 15
Cys Ala Ala Pro
20
<210> 72
<211> 20
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 72
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> 73
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 73
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Ala
<210> 74
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 74
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Thr
<210> 75
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 75
Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val Ala
1 5 10 15
Phe
<210> 76
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 76
Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala
1 5 10 15
Thr
<210> 77
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 77
Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Glu Pro Glu Phe Val Ser
1 5 10 15
Asp
<210> 78
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 78
Ala Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Asn Ala Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Met Tyr Tyr Cys
35
<210> 79
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 79
Ala Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Asn Ala Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Met Tyr Tyr Cys
35
<210> 80
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 80
His Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Ser Ala Met Tyr Tyr Cys
35
<210> 81
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 81
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr
1 5 10 15
Ala Asn Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Gly Met Tyr Tyr Cys
35
<210> 82
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 82
Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr
1 5 10 15
Ala Asn Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 83
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 83
Ser Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Thr Ser Gln Asp Asn
1 5 10 15
Val Ala Asn Thr Tyr Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
20 25 30
Glu Asp Thr Ala Met Tyr Tyr Cys
35 40
<210> 84
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 84
Tyr Tyr Ala Asp Phe Val Lys Gly Arg Phe Thr Ile Ala Gln Asp Asn
1 5 10 15
Ala Lys Asn Thr Ile Tyr Leu Glu Met Asn Gly Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 85
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 85
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Leu Asp Glu
1 5 10 15
Asp Lys Asn Thr Met Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 86
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 86
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys
1 5 10 15
Asp Lys Asp Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 87
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 87
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys
1 5 10 15
Asp Lys Ile Ala Met Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 88
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 88
Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 89
<211> 38
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 89
Tyr Tyr Arg Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Asn Asn Thr Leu His Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Met Tyr Tyr Cys
35
<210> 90
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 90
Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10
<210> 91
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 91
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
1 5 10

Claims (7)

1. A single domain antibody to GPA33, characterized in that: the single domain antibody is composed of a heavy chain, wherein the heavy chain comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3;
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. 42, CDR2 as shown in SEQ ID NO. 53, CDR3 as shown in SEQ ID NO. 67;
(2) CDR1 shown in SEQ ID NO. 42, CDR2 shown in SEQ ID NO. 53, and CDR3 shown in SEQ ID NO. 65.
2. The single domain antibody of GPA33 of claim 1, wherein: the antibody sequence also includes 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 sequence of the framework region FR is: FR1 shown in SEQ ID NO. 72, FR2 shown in SEQ ID NO. 74, FR3 shown in SEQ ID NO. 87 and FR4 shown in SEQ ID NO. 91;
when the amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are (2), the amino acid sequence of the framework region FR is: FR1 shown in SEQ ID NO. 72, FR2 shown in SEQ ID NO. 76, FR3 shown in SEQ ID NO. 86 and FR4 shown in SEQ ID NO. 91.
3. A single domain antibody to GPA33, characterized in that: the amino acid sequence of the single domain antibody is shown as SEQ ID NO.9 or 10.
4. An Fc fusion antibody or a humanized antibody of the single domain antibody of GPA33 of any of claims 1-3.
5. A nucleotide molecule encoding the GPA33 single domain antibody of any of claims 1-3, characterized in that: the nucleotide sequence is shown as SEQ ID NO:29 or 30.
6. An expression vector comprising a nucleotide molecule encoding the single domain antibody of any one of claims 1-3 or the Fc fusion antibody of claim 4 or the nucleotide molecule of claim 5.
7. A host cell capable of expressing the single domain antibody of GPA33 as defined in any one of claims 1 to 3, or comprising the expression vector of claim 6.
CN202210207351.4A 2022-03-03 2022-03-03 Single-domain antibody for GPA33 and derived protein and application thereof Active CN114478777B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110540591A (en) * 2019-08-09 2019-12-06 无锡傲锐东源生物科技有限公司 anti-Glycoprotein A33 (glycoprotin A33) monoclonal antibody and immunodetection application thereof
WO2022108976A2 (en) * 2020-11-18 2022-05-27 Memorial Sloan Kettering Cancer Center Anti-gpa33 multi-specific antibodies and uses thereof
WO2022121928A1 (en) * 2020-12-09 2022-06-16 江苏先声药业有限公司 Anti-egfr nanobody and use thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2896723C (en) * 2012-12-28 2024-02-13 Precision Biologics, Inc. Humanized monoclonal antibodies and methods of use for the diagnosis and treatment of colon and pancreas cancer
US20220054544A1 (en) * 2018-09-21 2022-02-24 Harpoon Therapeutics, Inc. Conditionally active receptors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110540591A (en) * 2019-08-09 2019-12-06 无锡傲锐东源生物科技有限公司 anti-Glycoprotein A33 (glycoprotin A33) monoclonal antibody and immunodetection application thereof
WO2022108976A2 (en) * 2020-11-18 2022-05-27 Memorial Sloan Kettering Cancer Center Anti-gpa33 multi-specific antibodies and uses thereof
WO2022121928A1 (en) * 2020-12-09 2022-06-16 江苏先声药业有限公司 Anti-egfr nanobody and use thereof

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