WO2024051106A1 - Preparation and use of anti-il4i1 nano antibody - Google Patents

Preparation and use of anti-il4i1 nano antibody Download PDF

Info

Publication number
WO2024051106A1
WO2024051106A1 PCT/CN2023/078088 CN2023078088W WO2024051106A1 WO 2024051106 A1 WO2024051106 A1 WO 2024051106A1 CN 2023078088 W CN2023078088 W CN 2023078088W WO 2024051106 A1 WO2024051106 A1 WO 2024051106A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
determining region
complementary determining
il4i1
cdr2
Prior art date
Application number
PCT/CN2023/078088
Other languages
French (fr)
Chinese (zh)
Inventor
杜继文
薛静
杨燕
袁亚丰
许胤辉
沙思源
王玉芳
查长春
Original Assignee
上海百英生物科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海百英生物科技股份有限公司 filed Critical 上海百英生物科技股份有限公司
Publication of WO2024051106A1 publication Critical patent/WO2024051106A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of molecular biology, and specifically relates to the preparation and application of an anti-IL4i1 nanobody.
  • Interleukin-4-inducible gene 1 is an L-phenylalanine oxidase expressed by antigen-presenting cells. In vitro, IL4I1 inhibits T cell proliferation and cytokine production, promoting The differentiation of CD4+ T cells into regulatory T cells relies in part on their ability to produce H2O2 and consume phenylalanine from the T cell microenvironment. IL4I1 is strongly detected in the tumor bed of most human tumor types, and in some cases, such as certain B-cell lymphomas, it is expressed by the tumor cells themselves.
  • IL4I1 Locally produced IL4I1 promotes tumor growth in mouse models by inhibiting the proliferation and function of anti-tumor CD8+ T cells, changing the balance of immune cells in the tumor microenvironment toward the immunosuppressive population. Furthermore, the location and density of IL4I1 expression in cells correlated with worsening clinical parameters in patients and tended to lead to shorter survival in melanoma patients. The researchers also found that the presence of IL4I1 protein or mRNA was associated with the quality of prognosis in kidney cancer, glioma, colon cancer and breast cancer. These data all suggest that therapeutic strategies aimed at inhibiting IL4I1 expression or reducing its activity in cancer patients may improve immune control in cancer patients.
  • IL4I1 Interleukin-4-Induced-1
  • DKFZ German Cancer Research Center
  • BIOH Berlin Institute of Health
  • Nanobodies are the smallest functional antigen-binding fragments derived from HCAbs in adult camels. They have high stability and high affinity for binding to antigens. They can interact with protein clefts and enzyme active sites, making them act like inhibitors. Therefore, nanobodies can provide new ideas for designing small molecule enzyme inhibitors from peptide mimetic drugs. Since they only have heavy chains, Nanobodies are easier to produce than monoclonal antibodies. Nanobodies' unique properties, such as stability in extreme temperature and pH environments, enable low-cost manufacturing in large quantities.
  • nanobodies have great value in the treatment and diagnosis of diseases, and also have great development prospects in the antibody-targeted diagnosis and treatment of tumors.
  • nanobodies are equivalent to their corresponding scFv in terms of affinity, but have poor performance in solubility, stability, resistance to aggregation, refoldability, expression yield, DNA manipulation, and library construction. and ease of 3-D structure determination beyond scFv.
  • Phage display technology is a method of displaying polypeptides or proteins on the surface of phages, thereby displaying polypeptides or proteins with desired properties.
  • a technique for in vitro screening of peptides or proteins By fusing the gene encoding the target protein into the gene for the phage capsid protein, the target protein can be displayed on the surface of the phage, thus linking genotype and phenotype. Subsequently, several rounds of affinity screening (such as biopanning) are performed in vitro to obtain some phage clones. These phage clones are then amplified and more rounds of screening are performed to further obtain the target protein with strong specificity.
  • RNA sequencing Single-cell RNA sequencing
  • scRNA-seq single-cell RNA sequencing
  • Automated high-throughput single-cell RNA sequencing technology can provide an unbiased and efficient method for high-throughput detection of single-cell transcriptomes to obtain single-cell transcriptome profiles of the microenvironment.
  • droplet-based high-throughput single-cell RNA sequencing technology can increase the number of single-detection cells to tens of thousands, truly realizing high-throughput detection. Therefore, the realization of high-throughput single-cell RNA sequencing technology relies on the application of microfluidic automation technology and high-throughput sequencing technology. The former realizes high-throughput single cell isolation and the latter realizes automated DNA sequencing.
  • Phage display technology, biopanning, high-throughput sequencing, etc. are all commonly used technologies for nanobody production.
  • phage display technology requires phage packaging and panning. This technology is all performed in a prokaryotic expression system, and panning requires After at least 3 rounds, it takes a long time, and compared with the eukaryotic expression system, the prokaryotic expression of the antibody still has certain deficiencies in protein modification and folding, which affects the affinity of the antibody to a certain extent.
  • High-throughput sequencing technology data is large in scale and complex in type, including transcriptome, genome, proteome, etc. The reproducibility is not strong, and data storage and visualization are also issues that need to be optimized. Sample contamination is also a major problem that affects the accuracy of sequencing results.
  • the purpose of the present invention is to provide a method that can give full play to the superior properties of Nanobodies, which not only has excellent specific antigen binding ability, but also overcomes the shortcomings of traditional Nanobody screening methods that are time-consuming and cannot be processed and modified by prokaryotic expression.
  • the screening method is combined with a high-throughput expression platform to screen anti-IL4i1 antibodies and further provide their application in the preparation of tumor treatment drugs and diagnostic preparations.
  • an anti-IL4i1 nanobody the nanobody includes a framework region and a complementarity-determining region; the complementarity-determination region includes CDR1, CDR2, and CDR3, and the amino acid sequence is as follows: the CDR1 sequence of the complementarity-determination region is SEQ ID NO.1, so The complementarity-determining region CDR2 sequence is SEQ ID NO.2, and the complementarity-determining region CDR3 sequence is SEQ ID NO.3; or the complementarity-determining region CDR1 is SEQ ID NO.4, and the complementarity-determining region CDR2 is SEQ ID NO.
  • the complementary determining region CDR3 is SEQ ID NO.6; or the complementary determining region CDR1 is SEQ ID NO.7, the complementary determining region CDR2 is SEQ ID NO.8, the complementary determining region CDR3 is SEQ ID NO.9; or the complementarity determining region CDR1 is SEQ ID NO.7, the complementarity determining region CDR2 is SEQ ID NO.10, the complementation determining region CDR3 is SEQ ID NO.11; or the complementarity determining region CDR3 is SEQ ID NO.11;
  • the complementarity determining region CDR1 is SEQ ID NO.
  • the complementarity determining region CDR2 is SEQ ID NO.13, the complementary determining region CDR3 is SEQ ID NO.14; or the complementary determining region CDR1 is SEQ ID NO.15, the complementary determining region CDR2 is SEQ ID NO.16, the complementary determining region Region CDR3 is SEQ ID NO.17; or the complementarity determining region CDR1 is SEQ ID NO.18, the complementarity determining region CDR2 is SEQ ID NO.19, and the complementarity determining region CDR3 is SEQ ID NO.20; or The complementarity determining region CDR1 is SEQ ID NO.21, the complementarity determining region CDR2 is SEQ ID NO.22, the complementarity determining region CDR3 is SEQ ID NO.23; or the complementarity determining region CDR1 is SEQ ID NO. .24, the complementarity determining region CDR2 is SEQ ID NO.25, and the complementarity determining region CDR3 is SEQ ID NO.26;
  • the anti-IL4i1 Nanobody has an amino acid sequence selected from any one of the following: the amino acid sequence corresponding to ANb32-M384-4M-673 is shown in SEQ ID NO. 27; the amino acid sequence corresponding to ANb32-M384-4M-865 The sequence is shown in SEQ ID NO.28; the amino acid sequence corresponding to ANb32-M384-4M-584 is shown in SEQ ID NO.29; the amino acid sequence corresponding to ANb32-M384-4M-769 is shown in SEQ ID NO.30; ANb32- The amino acid sequence corresponding to M384-4M-761 is shown in SEQ ID NO.31; the amino acid sequence corresponding to ANb32-M384-4M-480 is shown in SEQ ID NO.32; the amino acid sequence corresponding to ANb32-M384-4M-577 is shown as SEQ ID NO.33 is shown; the amino acid sequence corresponding to ANb32-M384-4M-665 is shown in SEQ ID
  • the nucleotide sequences encoding the anti-IL4i1 Nanobody are as follows: the nucleotide sequence corresponding to ANb32-M384-4M-673 is shown in SEQ ID NO. 36; the nucleotide sequence corresponding to ANb32-M384-4M-865 The nucleotide sequence is shown in SEQ ID NO.37; the nucleotide sequence corresponding to ANb32-M384-4M-584 is shown in SEQ ID NO.38; ANb32- The nucleotide sequence corresponding to M384-4M-769 is shown in SEQ ID NO.39; the nucleotide sequence corresponding to ANb32-M384-4M-761 is shown in SEQ ID NO.40; the corresponding nucleotide sequence ANb32-M384-4M-480 The nucleotide sequence of is shown in SEQ ID NO.41; the nucleotide sequence corresponding to ANb32-M384-4M-5
  • the present invention also provides a molecular expression vector, which contains one of the nucleotide sequences of SEQ ID NO. 36 to SEQ ID NO. 44.
  • the invention also provides a host cell containing the expression vector, and the cell is a eukaryotic cell, preferably a mammalian cell.
  • the invention also provides a method for preparing the nanobody.
  • the preparation process is as follows:
  • step S2 Use the antigen obtained in step S1 to immunize the alpaca, and use droplet microfluidic technology to screen to obtain B cells that can secrete the target antibody;
  • step S3 Use the B cells obtained in step S2 as raw materials, extract RNA, and reverse-transcribe it into cDNA.
  • step S4 Use the IL4i1-VHH monoclonal expression library constructed in step S3 as a template to extract the plasmid, further transfect the cells, and screen positive clones to obtain the result.
  • the eukaryotic expression vector in step S3 is a PcDNA3.4 vector.
  • the invention also provides an application of the nanobody in preparing drugs for preventing and treating tumors.
  • the invention also provides an application of the nanobody in preparing a tumor detection reagent.
  • the anti-IL4i1 nanobody provided by the present invention has the following advantages:
  • amino acid sequence of the anti-IL4i1 nanoparticles provided by the present invention is arranged in the form of framework sequence + CDR1 + framework sequence + CDR2 + framework sequence + CDR3.
  • the present invention combines the use of cell microfluidic technology to obtain antibody sequences more intuitively, obtain a large number of IL4i1 nanobody genes in a short period of time, realize large-scale production of nanobodies, and is conducive to further development of IL4i1 research utilization;
  • the present invention uses a high-throughput mammalian cell expression system to express IL4i1 nanobodies, which effectively reduces the development and production costs of IL4i1 antibodies, shortens the antibody expression time, increases throughput, and improves efficiency.
  • Figure 1 shows the ELISA test results
  • Figure 2 is a partial diagram of the results of cloning the target fragment
  • Figure 3 shows the results of monoclonal ELISA
  • Figure 4 shows the positive clone screening results.
  • the present invention first constructs IL4i1 antigen and immunizes alpacas four times to obtain alpaca PBMC cells; secondly, screen and separate PBMC cells through microfluidic technology; capture single cells for RNA, and obtain antibodies through nested PCR. Gene fragments are constructed to construct eukaryotic expression vectors; then high-throughput expression of antibodies is induced through a mammalian cell high-throughput expression system; and finally through ELISA and enzyme activity inhibition detection (using Red Catalase Assay Kit (Red Catalase Assay Kit), and sequencing results were analyzed to obtain antibodies with high sensitivity and enzyme activity inhibition.
  • anti-IL4i1 nanobodies There are 9 kinds of anti-IL4i1 nanobodies, namely ANb32-M384-4M-673, ANb32-M384-4M-865, ANb32-M384-4M-584, ANb32-M384-4M-769, ANb32-M384-4M- 761, ANb32-M384-4M-480, ANb32-M384-4M-577, ANb32-M384-4M-665, ANb32-M384-4M-672.
  • ANb32-M384-4M-673 namely ANb32-M384-4M-673, ANb32-M384-4M-865, ANb32-M384-4M-584, ANb32-M384-4M-769, ANb32-M384-4M- 761, ANb32-M384-4M-480, ANb32-M384-4M-577, AN
  • the preparation process of the anti-IL4i1 nanobody is as follows:
  • step S2 Use the mixture of the modified antigen and Freund's adjuvant obtained in step S1 to immunize the alpaca four times, and screen the B cells that can secrete VHH and have binding activity through droplet microfluidic technology;
  • Alpacas were primed with an emulsified mixture of 200 ⁇ g, human IL4i1/His protein and 200 ⁇ L Freund's complete adjuvant, and on days 21, 42, and 63, human IL4i1/His protein (i.e., the modified protein obtained in step S1 Antigen) and 200 ⁇ L of Freund's incomplete adjuvant were boosted 3 times.
  • human IL4i1/His protein i.e., the modified protein obtained in step S1 Antigen
  • 200 ⁇ L of Freund's incomplete adjuvant were boosted 3 times.
  • blood was collected to detect the Anti-IL4i1/His serum titer.
  • 50 mL of blood was collected for microfluidic platform testing. Single cell sorting.
  • the Anti-IL4i1/His serum titer was detected by ELISA.
  • the IL4i1/His protein at a concentration of 2 ⁇ g/mL was used to coat the enzyme plate.
  • 100 ⁇ L of 2-fold gradient diluted serum was added to each well (the control was pre-immune alpaca serum), 37 Incubate at °C for 1.5h, Wash twice, add 1:10000 diluted horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody to each well, incubate at 37°C for 1 hour, wash 5 times, add 100 ⁇ L TMB substrate, 37 Incubate at °C for 10 minutes, stop the reaction with 50 ⁇ L of 0.1M H2SO4, and measure OD 450nm.
  • the results are shown in Figure 1.
  • the serum titer detected by ELISA was a dilution value of 2 times that of the blank control at OD450.
  • the antiserum titer after 4 immunizations was 102,400. It can be seen that this antigen can induce alpacas to produce high-titer antiserum specifically against IL4i1 protein.
  • Sorted cells One week after the fourth immunization, 50 mL of blood was collected and used on the microfluidic platform to sort functional antibody-secreting cells.
  • Droplet microfluidic technology can package cells and antigens in picolitre-level units. In dispersed oil droplets, the generation speed of thousands of monodispersed droplets per second can be achieved, and each microdroplet obtained from single cells is independent, achieving environmental independence between cells and preventing cross-contamination; in Fluorescently labeled alpaca secondary antibodies are used in the oil droplets to recognize the IL4i1 nanobody. At the same time, if the VHH antibodies secreted by the cells can recognize the fluorescently labeled antigen, a FRET signal can be formed and sorted out. Screening can secrete VHH with binding activity of B cells.
  • step S3 Use the B cells obtained in step S2 as raw materials, extract RNA, and reverse-transcribe it into cDNA.
  • the B cells screened in step S2 as a template, use the Trizol method to extract RNA, and use oligo(dT) to reverse it into cDNA.
  • the amplification system of the target fragment is as follows: 2, the amplification procedure is shown in Table 3 below.
  • the target fragment cloning results are shown in Figure 2 (partial results); the alpaca VHH gene was cloned into the eukaryotic expression vector pCDNA3.4 and transformed into competent cells Top 10 to obtain a VHH monoclonal expression library.
  • sequence information of the upstream primer DFL-(03-16) is as follows:
  • DFL-09 AGATCTACACATGGCCCAGCCGCAS
  • DFL-11 ATCTACACATGGCCCAGGTGCA
  • DFL-12 GAGATCTACACATGGCCGAGGTGCAGY
  • DFL-14 GATCTACACATGGCCGCGGTA
  • DFL-15 GATCTACACATGGCCGAGTTGCAAC
  • DFL-16 CGAGATCTACACATGGCCAGYTKGGTG
  • sequence information of downstream primer DFL-06 is: CATACGAGAT ACTAGTTGAGGAGACR
  • Y represents C or T base
  • K represents G or T base
  • R represents A or G base
  • S represents C or G base.
  • step S4 Use the IL4i1-VHH monoclonal expression library constructed in step S3 as a template to extract the plasmid, further transfect the cells, and screen positive clones to obtain the result.
  • Transfect the plasmid into mammalian cells HEK-293 add the cell suspension to the corresponding 96-well cell culture plate, approximately 5 ⁇ 104 cells per well, and culture in a 37°C, 5% CO2 cell culture incubator, continuously Culture for 72h.
  • the cell supernatant after transfection was tested for binding to IL4i1 protein. That is binding ELISA.
  • the specific detection process is as follows: the antigen IL4i1 is coated with 2 ⁇ g/mL at 4°C overnight, 100 ⁇ L of cell supernatant per well is incubated at 25°C for 1 hour, the secondary antibody is added and incubated at 25°C for 1 hour, and the color development is stopped and the reading is completed.
  • the results of the monoclonal ELISA showed that 377 positive clones were obtained from a total of 961 monoclones in ten 96-well cell plates (as shown in Figure 3, part of the results are shown), and these sequences were sequenced and compared to eliminate repeated sequences.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Urology & Nephrology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)

Abstract

A preparation and use of an anti-IL4i1 nano antibody. The anti-IL4i1 nano antibody has three unique complementarity-determining regions CDR1, CDR2, and CDR3, and nine sequences of the anti-IL4i1 nano antibody are selected in total. Further provided are a coding sequence for coding the nano antibody or a VHH chain thereof, a corresponding expression vector, a host cell capable of expressing the nano antibody, and a production method for the anti-IL4i1 nano antibody.

Description

一种抗IL4i1纳米抗体的制备及其应用Preparation and application of an anti-IL4i1 nanobody 技术领域Technical field
本发明属于分子生物学技术领域，具体涉及一种抗IL4i1纳米抗体的制备及其应用。The invention belongs to the technical field of molecular biology, and specifically relates to the preparation and application of an anti-IL4i1 nanobody.
背景技术Background technique
白细胞介素-4诱导基因1(IL4I1)是一种由抗原提呈细胞表达的L-苯丙氨酸氧化酶。在体外，IL4I1抑制T细胞增殖和细胞因子的产生，促进CD4+T细胞分化为调节性T细胞，有一部分原因依靠其产生H2O2和从T细胞微环境中消耗苯丙氨酸的能力。IL4I1在大多数人类肿瘤类型的肿瘤床中都能强烈地检测到，在某些情况下，如某些B细胞淋巴瘤，它是由肿瘤细胞本身表达的。局部产生IL4I1通过抑制抗肿瘤CD8+T细胞的增殖和功能，改变肿瘤微环境中免疫细胞向免疫抑制人群的平衡，促进小鼠模型中肿瘤的生长。此外，IL4I1在细胞中表达的位置和密度与患者变差的临床参数相关，并倾向于导致黑色素瘤患者更短的生存期。研究者还发现，IL4I1蛋白或mRNA的存在与肾癌、神经胶质瘤、结肠癌和乳腺癌预后质量有关。这些数据都表明，旨在抑制肿瘤患者IL4I1表达或降低其活性的治疗策略可能会提高肿瘤患者的免疫控制。而且这种策略似乎不伤害宿主，因为有研究表明KO小鼠中IL4I1的缺失似乎不会导致特定的病理或过早死亡。因此，靶向IL4I1可能会是一种新的肿瘤免疫治疗策略。在Cell杂志发表的一项研究中，来自德国癌症研究中心(DKFZ)和柏林健康研究所(BIH)的科学家们发现，肿瘤中大量产生的一种代谢酶IL4I1(Interleukin-4-Induced-1)能促进肿瘤细胞的扩散并抑制免疫系统。他们的研究表明IL4I1是一种代谢免疫检查点，IL4i1抑制剂在将来有可能会为癌症治疗带来新的机遇。Interleukin-4-inducible gene 1 (IL4I1) is an L-phenylalanine oxidase expressed by antigen-presenting cells. In vitro, IL4I1 inhibits T cell proliferation and cytokine production, promoting The differentiation of CD4+ T cells into regulatory T cells relies in part on their ability to produce H2O2 and consume phenylalanine from the T cell microenvironment. IL4I1 is strongly detected in the tumor bed of most human tumor types, and in some cases, such as certain B-cell lymphomas, it is expressed by the tumor cells themselves. Locally produced IL4I1 promotes tumor growth in mouse models by inhibiting the proliferation and function of anti-tumor CD8+ T cells, changing the balance of immune cells in the tumor microenvironment toward the immunosuppressive population. Furthermore, the location and density of IL4I1 expression in cells correlated with worsening clinical parameters in patients and tended to lead to shorter survival in melanoma patients. The researchers also found that the presence of IL4I1 protein or mRNA was associated with the quality of prognosis in kidney cancer, glioma, colon cancer and breast cancer. These data all suggest that therapeutic strategies aimed at inhibiting IL4I1 expression or reducing its activity in cancer patients may improve immune control in cancer patients. This strategy does not appear to harm the host, as it has been shown that deletion of IL4I1 in KO mice does not appear to cause specific pathology or premature death. Therefore, targeting IL4I1 may be a new tumor immunotherapy strategy. In a study published in the journal Cell, scientists from the German Cancer Research Center (DKFZ) and the Berlin Institute of Health (BIH) discovered that IL4I1 (Interleukin-4-Induced-1), a metabolic enzyme produced in large quantities in tumors, Can promote the spread of tumor cells and suppress the immune system. Their research shows that IL4I1 is a metabolic immune checkpoint, and IL4i1 inhibitors may bring new opportunities for cancer treatment in the future.
纳米抗体有来源于成年骆驼体内HCAbs的最小的功能性抗原结合片段，具有高度稳定性和与抗原结合的高亲合力，能与蛋白裂隙和酶活性位点相互作用，使之作用类似抑制剂。因此，纳米抗体可以为从肽模拟药物设计小分子酶抑制物提供新的思路。由于仅有重链，纳米抗体的制造较单克隆抗体容易。纳米抗体的独特性质，如处于极端温度和pH环境中的稳定性，可以低成本制造大产量。Nanobodies are the smallest functional antigen-binding fragments derived from HCAbs in adult camels. They have high stability and high affinity for binding to antigens. They can interact with protein clefts and enzyme active sites, making them act like inhibitors. Therefore, nanobodies can provide new ideas for designing small molecule enzyme inhibitors from peptide mimetic drugs. Since they only have heavy chains, Nanobodies are easier to produce than monoclonal antibodies. Nanobodies' unique properties, such as stability in extreme temperature and pH environments, enable low-cost manufacturing in large quantities.
因此，纳米抗体在疾病的治疗和诊断中具有很大的价值，在肿瘤的抗体靶向诊断和治疗中也具有很大的发展前景。相对于常规的四链抗体的scFv而言，纳米抗体在亲和力方面与其对应的scFv相当，但在可溶性、稳定性、对聚集的抗性、可重折叠性、表达产率以及DNA操作、文库构建和3-D结构测定的容易性方面超越scFv。Therefore, nanobodies have great value in the treatment and diagnosis of diseases, and also have great development prospects in the antibody-targeted diagnosis and treatment of tumors. Compared with conventional four-chain antibody scFv, nanobodies are equivalent to their corresponding scFv in terms of affinity, but have poor performance in solubility, stability, resistance to aggregation, refoldability, expression yield, DNA manipulation, and library construction. and ease of 3-D structure determination beyond scFv.
噬菌体展示技术是一种将多肽或蛋白质展示在噬菌体的表面，从而将所需性质的多 肽或蛋白质进行体外筛选的技术。将编码目的蛋白的基因融合到噬菌体衣壳蛋白的基因之中，就能使目的蛋白展示在噬菌体的表面，从而将基因型和表型联系起来。随后，在体外进行几轮亲和筛选(如生物淘选)得到一些噬菌体克隆，再将这些噬菌体克隆扩增，进行更多轮的筛选，便可进一步得到特异性较强的目的蛋白。Phage display technology is a method of displaying polypeptides or proteins on the surface of phages, thereby displaying polypeptides or proteins with desired properties. A technique for in vitro screening of peptides or proteins. By fusing the gene encoding the target protein into the gene for the phage capsid protein, the target protein can be displayed on the surface of the phage, thus linking genotype and phenotype. Subsequently, several rounds of affinity screening (such as biopanning) are performed in vitro to obtain some phage clones. These phage clones are then amplified and more rounds of screening are performed to further obtain the target protein with strong specificity.
目前最广泛使用的单细胞分析技术是高通量单细胞RNA测序(Single-cell RNA sequencing,scRNA-seq)。自动化的高通量单细胞RNA测序技术可以提供一种无偏见和高效的方法对单细胞转录组进行高通量检测，以获得微环境的单细胞转录组图谱。基于液滴的高通量单细胞RNA测序技术出现，可以将单次检测细胞数量提高到上万个，真正实现了高通量检测。因此，高通量的单细胞RNA测序技术的实现依赖于微流控自动化技术和高通量测序技术的应用，前者实现高通量的单细胞分离而后者实现自动化的DNA测序。Currently, the most widely used single cell analysis technology is high-throughput single-cell RNA sequencing (Single-cell RNA sequencing, scRNA-seq). Automated high-throughput single-cell RNA sequencing technology can provide an unbiased and efficient method for high-throughput detection of single-cell transcriptomes to obtain single-cell transcriptome profiles of the microenvironment. The emergence of droplet-based high-throughput single-cell RNA sequencing technology can increase the number of single-detection cells to tens of thousands, truly realizing high-throughput detection. Therefore, the realization of high-throughput single-cell RNA sequencing technology relies on the application of microfluidic automation technology and high-throughput sequencing technology. The former realizes high-throughput single cell isolation and the latter realizes automated DNA sequencing.
噬菌体展示技术、生物淘洗、高通量测序等都是进行纳米抗体生产的常用技术，其中噬菌体展示技术由于需要噬菌体的包装及淘选，此技术均是在原核表达系统中进行，淘选需要经过至少3轮，所以耗时较长，且原核表达的抗体相对于真核表达系统对蛋白的修饰与折叠仍有一定的不足，一定程度上影响了抗体的亲和力。高通量测序技术数据规模庞大，类型复杂，包括转录组，基因组，蛋白质组等，可重复性不强，数据储存与可视化也是待优化的问题。样品污染也是影响测序结果准确度的一大问题。Phage display technology, biopanning, high-throughput sequencing, etc. are all commonly used technologies for nanobody production. Among them, phage display technology requires phage packaging and panning. This technology is all performed in a prokaryotic expression system, and panning requires After at least 3 rounds, it takes a long time, and compared with the eukaryotic expression system, the prokaryotic expression of the antibody still has certain deficiencies in protein modification and folding, which affects the affinity of the antibody to a certain extent. High-throughput sequencing technology data is large in scale and complex in type, including transcriptome, genome, proteome, etc. The reproducibility is not strong, and data storage and visualization are also issues that need to be optimized. Sample contamination is also a major problem that affects the accuracy of sequencing results.
发明内容Contents of the invention
本发明的目的就是提供一种能够充分发挥纳米抗体的优越性能，既具有优异的特异性抗原结合能力，又能克服传统纳米抗体筛选方法耗时长，原核表达不能加工修饰的缺点，通过微流控筛选的方法结合高通量表达的平台，筛选到抗IL4i1抗体，并进一步提供其在肿瘤治疗药物和诊断制剂制备中的应用。The purpose of the present invention is to provide a method that can give full play to the superior properties of Nanobodies, which not only has excellent specific antigen binding ability, but also overcomes the shortcomings of traditional Nanobody screening methods that are time-consuming and cannot be processed and modified by prokaryotic expression. Through microfluidic control The screening method is combined with a high-throughput expression platform to screen anti-IL4i1 antibodies and further provide their application in the preparation of tumor treatment drugs and diagnostic preparations.
针对上述目的，本发明采用的技术方案为：To achieve the above objectives, the technical solutions adopted by the present invention are:
一种抗IL4i1纳米抗体，所述纳米抗体包括框架区和互补决定区；所述互补决定区包括CDR1、CDR2、CDR3，氨基酸序列如下：所述互补决定区CDR1序列为SEQ ID NO.1，所述互补决定区CDR2序列为SEQ ID NO.2，所述互补决定区CDR3序列为SEQ ID NO.3；或者所述互补决定区CDR1为SEQ ID NO.4，所述互补决定区CDR2为SEQ ID NO.5，所述互补决定区CDR3为SEQ ID NO.6；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ ID NO.8，所述互补决定区CDR3为SEQ ID NO.9；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ ID NO.10，所述互补决定区CDR3为SEQ ID NO.11；或者所述互补决定区CDR1为SEQ ID NO.12，所述互补决定区CDR2为 SEQ ID NO.13，所述互补决定区CDR3为SEQ ID NO.14；或者所述互补决定区CDR1为SEQ ID NO.15，所述互补决定区CDR2为SEQ ID NO.16，所述互补决定区CDR3为SEQ ID NO.17；或者所述互补决定区CDR1为SEQ ID NO.18，所述互补决定区CDR2为SEQ ID NO.19，所述互补决定区CDR3为SEQ ID NO.20；或者所述互补决定区CDR1为SEQ ID NO.21，所述互补决定区CDR2为SEQ ID NO.22，所述互补决定区CDR3为SEQ ID NO.23；或者所述互补决定区CDR1为SEQ ID NO.24，所述互补决定区CDR2为SEQ ID NO.25，所述互补决定区CDR3为SEQ ID NO.26；具体序列信息如下表1所示。An anti-IL4i1 nanobody, the nanobody includes a framework region and a complementarity-determining region; the complementarity-determination region includes CDR1, CDR2, and CDR3, and the amino acid sequence is as follows: the CDR1 sequence of the complementarity-determination region is SEQ ID NO.1, so The complementarity-determining region CDR2 sequence is SEQ ID NO.2, and the complementarity-determining region CDR3 sequence is SEQ ID NO.3; or the complementarity-determining region CDR1 is SEQ ID NO.4, and the complementarity-determining region CDR2 is SEQ ID NO. NO.5, the complementary determining region CDR3 is SEQ ID NO.6; or the complementary determining region CDR1 is SEQ ID NO.7, the complementary determining region CDR2 is SEQ ID NO.8, the complementary determining region CDR3 is SEQ ID NO.9; or the complementarity determining region CDR1 is SEQ ID NO.7, the complementarity determining region CDR2 is SEQ ID NO.10, the complementation determining region CDR3 is SEQ ID NO.11; or the complementarity determining region CDR3 is SEQ ID NO.11; The complementarity determining region CDR1 is SEQ ID NO. 12, and the complementarity determining region CDR2 is SEQ ID NO.13, the complementary determining region CDR3 is SEQ ID NO.14; or the complementary determining region CDR1 is SEQ ID NO.15, the complementary determining region CDR2 is SEQ ID NO.16, the complementary determining region Region CDR3 is SEQ ID NO.17; or the complementarity determining region CDR1 is SEQ ID NO.18, the complementarity determining region CDR2 is SEQ ID NO.19, and the complementarity determining region CDR3 is SEQ ID NO.20; or The complementarity determining region CDR1 is SEQ ID NO.21, the complementarity determining region CDR2 is SEQ ID NO.22, the complementarity determining region CDR3 is SEQ ID NO.23; or the complementarity determining region CDR1 is SEQ ID NO. .24, the complementarity determining region CDR2 is SEQ ID NO.25, and the complementarity determining region CDR3 is SEQ ID NO.26; the specific sequence information is shown in Table 1 below.
表1本发明IL4i1纳米抗体互补决定区具体序列
Table 1 The specific sequence of the complementarity determining region of IL4i1 Nanobody of the present invention
优选地，所述抗IL4i1纳米抗体具有选自以下任一种的氨基酸序列：ANb32-M384-4M-673对应的氨基酸序列如SEQ ID NO.27所示；ANb32-M384-4M-865对应的氨基酸序列如SEQ ID NO.28；ANb32-M384-4M-584对应的氨基酸序列如SEQ ID NO.29所示；ANb32-M384-4M-769对应的氨基酸序列如SEQ ID NO.30所示；ANb32-M384-4M-761对应的氨基酸序列如SEQ ID NO.31所示；ANb32-M384-4M-480对应的氨基酸序列如SEQ ID NO.32所示；ANb32-M384-4M-577对应的氨基酸序列如SEQ ID NO.33所示；ANb32-M384-4M-665对应的氨基酸序列如SEQ ID NO.34所示；ANb32-M384-4M-672对应的氨基酸序列如SEQ ID NO.35所示。Preferably, the anti-IL4i1 Nanobody has an amino acid sequence selected from any one of the following: the amino acid sequence corresponding to ANb32-M384-4M-673 is shown in SEQ ID NO. 27; the amino acid sequence corresponding to ANb32-M384-4M-865 The sequence is shown in SEQ ID NO.28; the amino acid sequence corresponding to ANb32-M384-4M-584 is shown in SEQ ID NO.29; the amino acid sequence corresponding to ANb32-M384-4M-769 is shown in SEQ ID NO.30; ANb32- The amino acid sequence corresponding to M384-4M-761 is shown in SEQ ID NO.31; the amino acid sequence corresponding to ANb32-M384-4M-480 is shown in SEQ ID NO.32; the amino acid sequence corresponding to ANb32-M384-4M-577 is shown as SEQ ID NO.33 is shown; the amino acid sequence corresponding to ANb32-M384-4M-665 is shown in SEQ ID NO.34; the amino acid sequence corresponding to ANb32-M384-4M-672 is shown in SEQ ID NO.35.
优选地，编码所述抗IL4i1纳米抗体的核苷酸序列分别如下：ANb32-M384-4M-673对应的核苷酸序列如SEQ ID NO.36所示；ANb32-M384-4M-865对应的核苷酸序列如SEQ ID NO.37所示；ANb32-M384-4M-584对应的核苷酸序列如SEQ ID NO.38所示；ANb32- M384-4M-769对应的核苷酸序列如SEQ ID NO.39所示；ANb32-M384-4M-761对应的核苷酸序列如SEQ ID NO.40所示；ANb32-M384-4M-480对应的核苷酸序列如SEQ ID NO.41所示；ANb32-M384-4M-577对应的核苷酸序列如SEQ ID NO.42所示；ANb32-M384-4M-665对应的核苷酸序列如SEQ ID NO.43所示；ANb32-M384-4M-672对应的核苷酸序列如SEQ ID NO.44所示。Preferably, the nucleotide sequences encoding the anti-IL4i1 Nanobody are as follows: the nucleotide sequence corresponding to ANb32-M384-4M-673 is shown in SEQ ID NO. 36; the nucleotide sequence corresponding to ANb32-M384-4M-865 The nucleotide sequence is shown in SEQ ID NO.37; the nucleotide sequence corresponding to ANb32-M384-4M-584 is shown in SEQ ID NO.38; ANb32- The nucleotide sequence corresponding to M384-4M-769 is shown in SEQ ID NO.39; the nucleotide sequence corresponding to ANb32-M384-4M-761 is shown in SEQ ID NO.40; the corresponding nucleotide sequence ANb32-M384-4M-480 The nucleotide sequence of is shown in SEQ ID NO.41; the nucleotide sequence corresponding to ANb32-M384-4M-577 is shown in SEQ ID NO.42; the nucleotide sequence corresponding to ANb32-M384-4M-665 is shown as SEQ ID NO.43 is shown; the nucleotide sequence corresponding to ANb32-M384-4M-672 is shown in SEQ ID NO.44.
本发明还提供了一种分子表达载体，所述载体包含所述SEQ ID NO.36～SEQ ID NO.44的核苷酸序列中的一种。The present invention also provides a molecular expression vector, which contains one of the nucleotide sequences of SEQ ID NO. 36 to SEQ ID NO. 44.
本发明还提供了一种含有所述表达载体的宿主细胞，所述细胞为真核细胞，优选为哺乳动物细胞。The invention also provides a host cell containing the expression vector, and the cell is a eukaryotic cell, preferably a mammalian cell.
本发明还提供了一种所述纳米抗体的制备方法，制备过程如下：The invention also provides a method for preparing the nanobody. The preparation process is as follows:
S1、依据IL4i1的蛋白序列和基因序列信息，分析并设计免疫抗原，在其C端连接His-tag，获得经修饰的抗原；S1. Based on the protein sequence and gene sequence information of IL4i1, analyze and design the immune antigen, connect the His-tag to its C-terminal, and obtain the modified antigen;
S2、用步骤S1获得的抗原对羊驼进行免疫，通过液滴微流控技术筛选得到可以分泌目标抗体的B细胞；S2. Use the antigen obtained in step S1 to immunize the alpaca, and use droplet microfluidic technology to screen to obtain B cells that can secrete the target antibody;
S3、以步骤S2获得的B细胞为原材料，提取RNA，并反转录成cDNA，经PCR获得目的基因片段，然后将目的基因片段克隆至真核表达载体中，并转化入感受态细胞，构建IL4i1-VHH单克隆表达库；S3. Use the B cells obtained in step S2 as raw materials, extract RNA, and reverse-transcribe it into cDNA. Obtain the target gene fragment through PCR, then clone the target gene fragment into a eukaryotic expression vector, and transform it into competent cells to construct IL4i1-VHH monoclonal expression library;
S4、以步骤S3所构建的IL4i1-VHH单克隆表达库为模板，提取质粒，并进一步经细胞转染，筛选阳性克隆，即得。S4. Use the IL4i1-VHH monoclonal expression library constructed in step S3 as a template to extract the plasmid, further transfect the cells, and screen positive clones to obtain the result.
优选地，步骤S3所述真核表达载体为PcDNA3.4载体。Preferably, the eukaryotic expression vector in step S3 is a PcDNA3.4 vector.
本发明还提供了一种所述纳米抗体在制备预防和治疗肿瘤药物中的应用。The invention also provides an application of the nanobody in preparing drugs for preventing and treating tumors.
本发明还提供了一种所述的纳米抗体在制备肿瘤检测试剂中的应用。The invention also provides an application of the nanobody in preparing a tumor detection reagent.
与现有技术相比，本发明提供的抗IL4i1纳米抗体具有如下优势：Compared with the existing technology, the anti-IL4i1 nanobody provided by the present invention has the following advantages:
本发明提供的抗IL4i1纳米，其氨基酸序列的排列方式为框架序列+CDR1+框架序列+CDR2+框架序列+CDR3的方式排列。The amino acid sequence of the anti-IL4i1 nanoparticles provided by the present invention is arranged in the form of framework sequence + CDR1 + framework sequence + CDR2 + framework sequence + CDR3.
(1)本发明结合使用细胞微流控技术，能够比较直观的获得抗体序列，在较短时间内获得了大量的IL4i1纳米抗体基因，实现了纳米抗体的大规模生产，有利于IL4i1的进一步深入研究利用；(1) The present invention combines the use of cell microfluidic technology to obtain antibody sequences more intuitively, obtain a large number of IL4i1 nanobody genes in a short period of time, realize large-scale production of nanobodies, and is conducive to further development of IL4i1 research utilization;
(2)本发明利用高通量哺乳动物细胞表达系统对IL4i1纳米抗体表达，有效地降低了IL4i1抗体的开发和生产成本，缩减了抗体表达时间，同时增加了通量，提高了效率。 (2) The present invention uses a high-throughput mammalian cell expression system to express IL4i1 nanobodies, which effectively reduces the development and production costs of IL4i1 antibodies, shortens the antibody expression time, increases throughput, and improves efficiency.
附图说明Description of the drawings
图1为ELISA检测结果图；Figure 1 shows the ELISA test results;
图2为目的片段克隆部分结果图；Figure 2 is a partial diagram of the results of cloning the target fragment;
图3为单克隆ELISA检测结果图；Figure 3 shows the results of monoclonal ELISA;
图4为阳性克隆筛选结果图。Figure 4 shows the positive clone screening results.
具体实施方式Detailed ways
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术方案均在本发明的保护范围之内。本实施例中未注明具体技术或条件者，按照本领域常规技术方法和仪器说明书内容进行操作；所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The present invention will be further explained below in conjunction with specific examples. However, it should be noted that the following examples are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same or similar to the present invention are included in the present invention. within the scope of protection. If no specific techniques or conditions are indicated in this example, the operation shall be carried out in accordance with conventional technical methods in the field and the contents of the instrument instructions; if the manufacturers of the reagents or instruments used are not indicated, they are conventional products that can be purchased commercially.
本发明首先构建IL4i1抗原对羊驼进行四次免疫后，获得羊驼PBMC细胞；其次通过微流控技术对PBMC细胞进行筛选分离；对单个细胞进行RNA的捕获，通过巢氏PCR的方法获得抗体基因片段，构建真核表达载体；然后通过哺乳动物细胞高通量表达系统诱导抗体高通量表达；最后通过ELISA与酶活抑制检测(利用Red Catalase Assay Kit试剂盒进行)，及测序结果分析获得了具有高的灵敏度和酶活抑制的抗体。The present invention first constructs IL4i1 antigen and immunizes alpacas four times to obtain alpaca PBMC cells; secondly, screen and separate PBMC cells through microfluidic technology; capture single cells for RNA, and obtain antibodies through nested PCR. Gene fragments are constructed to construct eukaryotic expression vectors; then high-throughput expression of antibodies is induced through a mammalian cell high-throughput expression system; and finally through ELISA and enzyme activity inhibition detection (using Red Catalase Assay Kit (Red Catalase Assay Kit), and sequencing results were analyzed to obtain antibodies with high sensitivity and enzyme activity inhibition.
实施例一种抗IL4i1纳米抗体Example of an anti-IL4i1 Nanobody
所述抗IL4i1纳米抗体共9种，分别为ANb32-M384-4M-673、ANb32-M384-4M-865、ANb32-M384-4M-584、ANb32-M384-4M-769、ANb32-M384-4M-761、ANb32-M384-4M-480、ANb32-M384-4M-577、ANb32-M384-4M-665、ANb32-M384-4M-672。There are 9 kinds of anti-IL4i1 nanobodies, namely ANb32-M384-4M-673, ANb32-M384-4M-865, ANb32-M384-4M-584, ANb32-M384-4M-769, ANb32-M384-4M- 761, ANb32-M384-4M-480, ANb32-M384-4M-577, ANb32-M384-4M-665, ANb32-M384-4M-672.
所述抗IL4i1纳米抗体的制备过程如下：The preparation process of the anti-IL4i1 nanobody is as follows:
S1、依据IL4i1的蛋白序列和基因序列信息，分析并设计可有效诱导羊驼产生针对human IL4i1的特异性抗体的抗原，在其C端连接His-tag，获得经修饰的抗原；S1. Based on the protein sequence and gene sequence information of IL4i1, analyze and design an antigen that can effectively induce alpacas to produce specific antibodies against human IL4i1, and connect His-tag to its C-terminus to obtain a modified antigen;
S2、用步骤S1获得的经修饰的抗原与弗氏佐剂的混合液对羊驼进行四次免疫，通过液滴微流控技术筛选得到可以分泌VHH有结合活性的B细胞；S2. Use the mixture of the modified antigen and Freund's adjuvant obtained in step S1 to immunize the alpaca four times, and screen the B cells that can secrete VHH and have binding activity through droplet microfluidic technology;
用200μg、human IL4i1/His蛋白与200μL弗氏完全佐剂的乳化混合物对羊驼进行初免，在第21天、42天、63天用human IL4i1/His蛋白(即步骤S1获得的经修饰的抗原)与200μL弗氏不完全佐剂加强免疫3次，每次免疫1周后，采血检测Anti-IL4i1/His血清滴度；第4次免疫1周后，采血50mL用于微流控平台进行单细胞分选。Alpacas were primed with an emulsified mixture of 200 μg, human IL4i1/His protein and 200 μL Freund's complete adjuvant, and on days 21, 42, and 63, human IL4i1/His protein (i.e., the modified protein obtained in step S1 Antigen) and 200 μL of Freund's incomplete adjuvant were boosted 3 times. One week after each immunization, blood was collected to detect the Anti-IL4i1/His serum titer. One week after the fourth immunization, 50 mL of blood was collected for microfluidic platform testing. Single cell sorting.
Anti-IL4i1/His血清效价通过ELISA检测，用浓度为2μg/mL的IL4i1/His蛋白包被酶标板，每孔加入2倍梯度稀释的血清100μL(对照为免疫前羊驼血清)，37℃孵育1.5h， 洗涤2次，每孔加入1：10000稀释的辣根过氧化物酶标记的Goat anti-Alpaca IgG(H+L)二抗，37℃孵育1h，洗涤5次后，加100μL TMB底物，37℃孵育10min，50μL 0.1M的H2SO4中止反应，测定OD 450nm。结果如图1所示，ELISA检测血清效价为在OD450是空白对照的2倍的稀释数值，4免后的抗血清效价为102400。由此可见，该抗原可诱导羊驼产生特异性针对IL4i1蛋白的高滴度抗血清。The Anti-IL4i1/His serum titer was detected by ELISA. The IL4i1/His protein at a concentration of 2 μg/mL was used to coat the enzyme plate. 100 μL of 2-fold gradient diluted serum was added to each well (the control was pre-immune alpaca serum), 37 Incubate at ℃ for 1.5h, Wash twice, add 1:10000 diluted horseradish peroxidase-labeled Goat anti-Alpaca IgG (H+L) secondary antibody to each well, incubate at 37°C for 1 hour, wash 5 times, add 100 μL TMB substrate, 37 Incubate at ℃ for 10 minutes, stop the reaction with 50 μL of 0.1M H2SO4, and measure OD 450nm. The results are shown in Figure 1. The serum titer detected by ELISA was a dilution value of 2 times that of the blank control at OD450. The antiserum titer after 4 immunizations was 102,400. It can be seen that this antigen can induce alpacas to produce high-titer antiserum specifically against IL4i1 protein.
分选细胞：第4次免疫1周后，采血50mL用于微流控平台进行有功能的抗体分泌细胞分选，液滴微流控技术可以将细胞与抗原包裹在一个个皮升级别的单分散油滴中，可以达到每秒数千个单分散液滴的生成速度，获取单细胞每个微液滴都是独立的，实现了细胞与细胞之间的环境独立，互不交叉污染；在油滴内通过荧光标记的羊驼二抗去识别IL4i1纳米抗体，同时如果细胞分泌的VHH抗体能够识别荧光标记的抗原，就能形成FRET信号而被分选出来，筛选得到可以分泌VHH有结合活性的B细胞。Sorted cells: One week after the fourth immunization, 50 mL of blood was collected and used on the microfluidic platform to sort functional antibody-secreting cells. Droplet microfluidic technology can package cells and antigens in picolitre-level units. In dispersed oil droplets, the generation speed of thousands of monodispersed droplets per second can be achieved, and each microdroplet obtained from single cells is independent, achieving environmental independence between cells and preventing cross-contamination; in Fluorescently labeled alpaca secondary antibodies are used in the oil droplets to recognize the IL4i1 nanobody. At the same time, if the VHH antibodies secreted by the cells can recognize the fluorescently labeled antigen, a FRET signal can be formed and sorted out. Screening can secrete VHH with binding activity of B cells.
S3、以步骤S2获得的B细胞为原材料，提取RNA，并反转录成cDNA，经PCR获得目的基因片段，然后将目的基因片段克隆至真核表达载体中，并转化入感受态细胞，构建IL4i1-VHH单克隆表达库；S3. Use the B cells obtained in step S2 as raw materials, extract RNA, and reverse-transcribe it into cDNA. Obtain the target gene fragment through PCR, then clone the target gene fragment into a eukaryotic expression vector, and transform it into competent cells to construct IL4i1-VHH monoclonal expression library;
以步骤S2筛选得到的B细胞为模板，利用Trizol法提取RNA，并利用oligo(dT)反转为cDNA，通过引物扩增，以及分子克隆等技术获得目的片段，目的片段的扩增体系如下表2所示，扩增程序如下表3所示。目的片段克隆结果如图2(部分结果)；将羊驼的VHH基因克隆至真核表达载体pCDNA3.4中，转化至感受态细胞Top 10中，得到VHH单克隆表达库。为了进一步鉴定IL4i1-VHH单克隆表达库是否构建成功，在涂的单克隆菌板中，挑选10个克隆进行测序，测序结果显示，阳性克隆率和序列多样性为100％；比对结果显示，差异序列大多在CDR结合区。经检测，该构建得到一个IL4i1-VHH单克隆表达库。Use the B cells screened in step S2 as a template, use the Trizol method to extract RNA, and use oligo(dT) to reverse it into cDNA. Use primer amplification, molecular cloning and other techniques to obtain the target fragment. The amplification system of the target fragment is as follows: 2, the amplification procedure is shown in Table 3 below. The target fragment cloning results are shown in Figure 2 (partial results); the alpaca VHH gene was cloned into the eukaryotic expression vector pCDNA3.4 and transformed into competent cells Top 10 to obtain a VHH monoclonal expression library. In order to further identify whether the IL4i1-VHH single-clonal expression library was successfully constructed, 10 clones were selected for sequencing from the single-clonal bacteria plate. The sequencing results showed that the positive clone rate and sequence diversity were 100%; the comparison results showed that, Most of the differential sequences are in the CDR binding region. After testing, this construction resulted in an IL4i1-VHH monoclonal expression library.
上游引物DFL-(03-16)的序列信息分别如下：The sequence information of the upstream primer DFL-(03-16) is as follows:
DFL-03：AGATCTACACATGGCCCAGGTGCDFL-03: AGATCTACACATGGCCCAGGTGC
DFL-04：GAGATCTACACATGGCCCAGKTGCAGCDFL-04: GAGATCTACACATGGCCCAGKTGCAGC
DFL-05：TACACATGGCCGAGGTGCAGDFL-05: TACACATGGCCGAGGTGCAG
DFL-07：ATCTACACATGGCCCAGTCGCADFL-07: ATCTACACATGGCCCAGTCGCA
DFL-08：CTACACATGGCCCAGCCGCASDFL-08: CTACACATGGCCCAGCCGCAS
DFL-09：AGATCTACACATGGCCCAGCCGCASDFL-09: AGATCTACACATGGCCCAGCCGCAS
DFL-10：ATCTACACATGGCCCAGTTGDFL-10: ATCTACACATGGCCCAGTTG
DFL-11：ATCTACACATGGCCCAGGTGCA DFL-11: ATCTACACATGGCCCAGGTGCA
DFL-12：GAGATCTACACATGGCCGAGGTGCAGYDFL-12: GAGATCTACACATGGCCGAGGTGCAGY
DFL-13：CGAGATCTACACATGGCCGAGGTRSDFL-13:CGAGATCTACACATGGCCGAGGTRS
DFL-14：GATCTACACATGGCCGCGGTADFL-14: GATCTACACATGGCCGCGGTA
DFL-15：GATCTACACATGGCCGAGTTGCAACDFL-15: GATCTACACATGGCCGAGTTGCAAC
DFL-16：CGAGATCTACACATGGCCAGYTKGGTGDFL-16:CGAGATCTACACATGGCCAGYTKGGTG
下游引物DFL-06的序列信息为：CATACGAGAT ACTAGTTGAGGAGACRThe sequence information of downstream primer DFL-06 is: CATACGAGAT ACTAGTTGAGGAGACR
其中，Y代表C或T碱基，K代表G或T碱基，R代表A或G碱基，S代表C或G碱基。Among them, Y represents C or T base, K represents G or T base, R represents A or G base, and S represents C or G base.
表2目的片段扩增体系
Table 2 Target fragment amplification system
表3扩增程序
Table 3 Amplification procedure
S4、以步骤S3所构建的IL4i1-VHH单克隆表达库为模板，提取质粒，并进一步经细胞转染，筛选阳性克隆，即得。S4. Use the IL4i1-VHH monoclonal expression library constructed in step S3 as a template to extract the plasmid, further transfect the cells, and screen positive clones to obtain the result.
3.4细胞转染与筛选：3.4 Cell transfection and screening:
将质粒转染至哺乳动物细胞HEK-293中，将细胞悬液分别加入对应的96孔细胞培养板中，每孔约5×104个细胞，37℃、5％CO2细胞培养箱内培养，连续培养72h。Transfect the plasmid into mammalian cells HEK-293, add the cell suspension to the corresponding 96-well cell culture plate, approximately 5×104 cells per well, and culture in a 37°C, 5% CO2 cell culture incubator, continuously Culture for 72h.
转染后的细胞上清与IL4i1蛋白进行结合检测。即binding ELISA，具体检测过程为：抗原IL4i1包被2μg/mL 4℃过夜，细胞上清每孔100μL，25℃孵育1h，加入二抗25℃孵育1h，显色终止读数即可。单克隆ELISA结果显示，十板96孔细胞板共961个单克隆得到377个阳性克隆(如图3所示，部分结果展示)，并将这些序列进行测序比对剔除重复序列。为了进一步验证文库中结合IL4i1-VHH蛋白的阳性抗体，将细胞上清进行酶活抑制实验，结果显示，377个ELISA阳性克隆中有9个抑制活性较好的阳性抗体(如图4所 示，部分结果展示)。The cell supernatant after transfection was tested for binding to IL4i1 protein. That is binding ELISA. The specific detection process is as follows: the antigen IL4i1 is coated with 2 μg/mL at 4°C overnight, 100 μL of cell supernatant per well is incubated at 25°C for 1 hour, the secondary antibody is added and incubated at 25°C for 1 hour, and the color development is stopped and the reading is completed. The results of the monoclonal ELISA showed that 377 positive clones were obtained from a total of 961 monoclones in ten 96-well cell plates (as shown in Figure 3, part of the results are shown), and these sequences were sequenced and compared to eliminate repeated sequences. In order to further verify the positive antibodies that bind IL4i1-VHH protein in the library, the cell supernatants were subjected to enzyme activity inhibition experiments. The results showed that 9 of the 377 ELISA positive clones had positive antibodies with better inhibitory activity (as shown in Figure 4 display, partial results are shown).
最后，需要说明的是，上述实施例仅例示性说明本发明的原理、性能及功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。 Finally, it should be noted that the above embodiments are only illustrative of the principles, performance, and efficacy of the present invention, and are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (9)

  1. 一种抗IL4i1纳米抗体，其特征在于，所述纳米抗体包括框架区和互补决定区；所述互补决定区包括CDR1、CDR2、CDR3，氨基酸序列如下：所述互补决定区CDR1序列为SEQ ID NO.1，所述互补决定区CDR2序列为SEQ ID NO.2，所述互补决定区CDR3序列为SEQ ID NO.3；或者所述互补决定区CDR1为SEQ ID NO.4，所述互补决定区CDR2为SEQ ID NO.5，所述互补决定区CDR3为SEQ ID NO.6；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ ID NO.8，所述互补决定区CDR3为SEQ ID NO.9；或者所述互补决定区CDR1为SEQ ID NO.7，所述互补决定区CDR2为SEQ ID NO.10，所述互补决定区CDR3为SEQ ID NO.11；或者所述互补决定区CDR1为SEQ ID NO.12，所述互补决定区CDR2为SEQ ID NO.13，所述互补决定区CDR3为SEQ ID NO.14；或者所述互补决定区CDR1为SEQ ID NO.15，所述互补决定区CDR2为SEQ ID NO.16，所述互补决定区CDR3为SEQ ID NO.17；或者所述互补决定区CDR1为SEQ ID NO.18，所述互补决定区CDR2为SEQ ID NO.19，所述互补决定区CDR3为SEQ ID NO.20；或者所述互补决定区CDR1为SEQ ID NO.21，所述互补决定区CDR2为SEQ ID NO.22，所述互补决定区CDR3为SEQ ID NO.23；或者所述互补决定区CDR1为SEQ ID NO.24，所述互补决定区CDR2为SEQ ID NO.25，所述互补决定区CDR3为SEQ ID NO.26。An anti-IL4i1 nanobody, characterized in that the nanobody includes a framework region and a complementarity determining region; the complementarity determining region includes CDR1, CDR2, and CDR3, and the amino acid sequence is as follows: the complementarity determining region CDR1 sequence is SEQ ID NO .1. The CDR2 sequence of the complementary determining region is SEQ ID NO.2, and the CDR3 sequence of the complementary determining region is SEQ ID NO.3; or the CDR1 of the complementary determining region is SEQ ID NO.4, and the complementary determining region CDR1 is SEQ ID NO.4. CDR2 is SEQ ID NO.5, and the complementary determining region CDR3 is SEQ ID NO.6; or the complementary determining region CDR1 is SEQ ID NO.7, and the complementary determining region CDR2 is SEQ ID NO.8, and the complementary determining region CDR2 is SEQ ID NO.8. The complementary determining region CDR3 is SEQ ID NO.9; or the complementary determining region CDR1 is SEQ ID NO.7, the complementary determining region CDR2 is SEQ ID NO.10, and the complementary determining region CDR3 is SEQ ID NO.11 ; Or the complementary determining region CDR1 is SEQ ID NO.12, the complementary determining region CDR2 is SEQ ID NO.13, the complementary determining region CDR3 is SEQ ID NO.14; or the complementary determining region CDR1 is SEQ ID NO.15, the complementary determining region CDR2 is SEQ ID NO.16, the complementary determining region CDR3 is SEQ ID NO.17; or the complementary determining region CDR1 is SEQ ID NO.18, the complementary determining region CDR2 is SEQ ID NO.19, and the complementary determining region CDR3 is SEQ ID NO.20; or the complementary determining region CDR1 is SEQ ID NO.21, and the complementary determining region CDR2 is SEQ ID NO.22, and the complementary determining region CDR2 is SEQ ID NO.22. The complementary determining region CDR3 is SEQ ID NO.23; or the complementary determining region CDR1 is SEQ ID NO.24, the complementary determining region CDR2 is SEQ ID NO.25, and the complementary determining region CDR3 is SEQ ID NO.26 .
  2. 如权利要求1所述的抗IL4i1纳米抗体，其特征在于，所述抗IL4i1纳米抗体具有选自以下任一种的氨基酸序列：SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35。The anti-IL4i1 Nanobody as claimed in claim 1, wherein the anti-IL4i1 Nanobody has an amino acid sequence selected from any one of the following: SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29 , SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35.
  3. 如权利要求2所述的抗IL4i1纳米抗体，其特征在于，编码所述抗IL4i1纳米抗体氨基酸序列的核苷酸序列为下列序列之一：SEQ ID NO.36、SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44。The anti-IL4i1 Nanobody as claimed in claim 2, wherein the nucleotide sequence encoding the amino acid sequence of the anti-IL4i1 Nanobody is one of the following sequences: SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44.
  4. 一种分子表达载体，其特征在于，所述载体包含权利要求3所述SEQ ID NO.36～SEQ ID NO.44的核苷酸序列中的一种。A molecular expression vector, characterized in that the vector contains one of the nucleotide sequences of SEQ ID NO. 36 to SEQ ID NO. 44 described in claim 3.
  5. 一种含有权利要求4所述表达载体的宿主细胞，其特征在于，所述细胞为真核细胞。A host cell containing the expression vector of claim 4, characterized in that the cell is a eukaryotic cell.
  6. 一种如权利要求1-5任一项所述抗IL4i1纳米抗体的制备方法，其特征在于，制备过程如下：A method for preparing anti-IL4i1 Nanobody according to any one of claims 1 to 5, characterized in that the preparation process is as follows:
    S1、依据IL4i1的蛋白序列和基因序列信息，分析并设计免疫抗原，在其C端连接His-tag，获得经修饰的抗原；S1. Based on the protein sequence and gene sequence information of IL4i1, analyze and design the immune antigen, connect the His-tag to its C-terminal, and obtain the modified antigen;
    S2、用步骤S1获得的抗原对羊驼进行免疫，通过液滴微流控技术筛选得到可以分泌目标抗 体的B细胞；S2. Use the antigen obtained in step S1 to immunize alpacas, and screen through droplet microfluidic technology to obtain targets that can secrete the target antibody. B cells of the body;
    S3、以步骤S2获得的B细胞为原材料，提取RNA，并反转录成cDNA，经PCR获得目的基因片段，然后将目的基因片段克隆至真核表达载体中，并转化入感受态细胞，构建IL4i1-VHH单克隆表达库；S3. Use the B cells obtained in step S2 as raw materials, extract RNA, and reverse-transcribe it into cDNA. Obtain the target gene fragment through PCR, then clone the target gene fragment into a eukaryotic expression vector, and transform it into competent cells to construct IL4i1-VHH monoclonal expression library;
    S4、以步骤S3所构建的IL4i1-VHH单克隆表达库为模板，提取质粒，并进一步经细胞转染，筛选阳性克隆，即得。S4. Use the IL4i1-VHH monoclonal expression library constructed in step S3 as a template to extract the plasmid, further transfect the cells, and screen positive clones to obtain the result.
  7. 如权利要求6所述的制备方法，其特征在于，步骤S3所述真核表达载体为PcDNA3.4载体。The preparation method according to claim 6, characterized in that the eukaryotic expression vector in step S3 is a PcDNA3.4 vector.
  8. 一种权利要求1所述抗IL4i1纳米抗体在制备预防和治疗肿瘤药物中的应用。An application of the anti-IL4i1 nanobody according to claim 1 in the preparation of drugs for the prevention and treatment of tumors.
  9. 一种权利要求1所述的抗IL4i1纳米抗体在制备肿瘤检测试剂中的应用。 An application of the anti-IL4i1 nanobody according to claim 1 in the preparation of tumor detection reagents.
PCT/CN2023/078088 2022-09-09 2023-02-24 Preparation and use of anti-il4i1 nano antibody WO2024051106A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211103782.2 2022-09-09
CN202211103782.2A CN115785277B (en) 2022-09-09 2022-09-09 Preparation and application of anti-IL4i1 nano antibody

Publications (1)

Publication Number Publication Date
WO2024051106A1 true WO2024051106A1 (en) 2024-03-14

Family

ID=85431899

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/078088 WO2024051106A1 (en) 2022-09-09 2023-02-24 Preparation and use of anti-il4i1 nano antibody

Country Status (2)

Country Link
CN (1) CN115785277B (en)
WO (1) WO2024051106A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066858A1 (en) * 2008-12-10 2010-06-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment and the prognosis of cancer
US20170246259A1 (en) * 2014-09-10 2017-08-31 Georgetown University Compositions and Methods of Using Interleukin-4 Induced Gene 1 (IL-4I1)
US20190135911A1 (en) * 2016-04-26 2019-05-09 Five Prime Therapeutics, Inc. Treating respiratory diseases by targeting interleukin 4 induced 1 (il4i1)
US20200166512A1 (en) * 2018-11-23 2020-05-28 Centre National De La Recherche Scientifique Composition and methods for detecting cancer
CN114126634A (en) * 2019-04-10 2022-03-01 德国癌症研究公共权益基金会 Interleukin-4 inducible gene 1(IL4I1) as biomarker and use thereof
CN114901837A (en) * 2019-12-10 2022-08-12 德国癌症研究公共权益基金会 Interleukin-4 inducible gene 1(IL4I1) and corresponding metabolites as biomarkers for cancer

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140315743A1 (en) * 2011-10-03 2014-10-23 Karen Chapman Methods and Compositions for the Treatment and Diagnosis of Ovarian Cancer
JP2016536002A (en) * 2013-09-06 2016-11-24 セダーズ−シナイ メディカル センター System, device, and method for anti-TL1A therapy
TWI794761B (en) * 2015-08-28 2023-03-01 德商英麥提克生物技術股份有限公司 Novel peptides, combination of peptides and scaffolds for use in immunotherapeutic treatment of various cancers
GB201906202D0 (en) * 2019-05-02 2019-06-19 Autolus Ltd Cell
CA3189257A1 (en) * 2020-08-18 2022-02-24 Universitat Zurich A cd25-biased anti-il-2 antibody
WO2022040506A2 (en) * 2020-08-21 2022-02-24 Yale University Nanobody compositions and methods of use of the same

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010066858A1 (en) * 2008-12-10 2010-06-17 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods for the treatment and the prognosis of cancer
US20170246259A1 (en) * 2014-09-10 2017-08-31 Georgetown University Compositions and Methods of Using Interleukin-4 Induced Gene 1 (IL-4I1)
US20190135911A1 (en) * 2016-04-26 2019-05-09 Five Prime Therapeutics, Inc. Treating respiratory diseases by targeting interleukin 4 induced 1 (il4i1)
US20200166512A1 (en) * 2018-11-23 2020-05-28 Centre National De La Recherche Scientifique Composition and methods for detecting cancer
CN114126634A (en) * 2019-04-10 2022-03-01 德国癌症研究公共权益基金会 Interleukin-4 inducible gene 1(IL4I1) as biomarker and use thereof
CN114901837A (en) * 2019-12-10 2022-08-12 德国癌症研究公共权益基金会 Interleukin-4 inducible gene 1(IL4I1) and corresponding metabolites as biomarkers for cancer

Also Published As

Publication number Publication date
CN115785277B (en) 2023-06-20
CN115785277A (en) 2023-03-14

Similar Documents

Publication Publication Date Title
WO2017206773A1 (en) Method for finding antibody having specific function
CN113234166B (en) AFP-resistant nano antibody 1C5 and application thereof
CN111875702B (en) anti-CREG monoclonal antibody and application
JP2016222725A (en) Methods and systems for generating, validating and using monoclonal antibodies
WO2020125136A1 (en) Recombinant antibody of anti-human n-terminal brain natriuretic peptide precursor
CN113173994B (en) AFP-resistant nano antibody 1A7 and application thereof
CN112979814B (en) anti-HE 4 nano antibody 3C8 and application thereof
JP7439108B2 (en) Recombinant antibody against human cardiac troponin I
KR102686392B1 (en) Anti-species-specific malaria parasite lactate dehydrogenase antibody
WO2024051106A1 (en) Preparation and use of anti-il4i1 nano antibody
CN111471109A (en) Anti-fibroblast growth factor receptor 4 monoclonal antibody and preparation method and application thereof
CN112979815B (en) anti-HE 4 nano antibody 1G8 and application thereof
CN113214399B (en) AFP-resistant nano antibody 2F5 and application thereof
CN113583133B (en) AFP-resistant nano antibody 1B9 and application thereof
CN113045663B (en) anti-HE 4 nano antibody 1A8 and application thereof
CN112679607B (en) Preparation method of troponin I E13 single-chain antibody
CN111349172B (en) Recombinant antibody of anti-human creatine kinase isoenzyme CK-MB
WO2024145974A1 (en) Tigit nanobody, and preparation method therefor and use thereof
CN112574303A (en) Antibody for resisting C-reactive protein
WO2024051096A1 (en) Anti-cd40 nanobody, and preparation method therefor and use thereof
CN116496395B (en) Monoclonal antibody combined with Dsg3 and application thereof
CN117003880B (en) Anti-thiocyanate fluorescein monoclonal antibody and application thereof
CN116987194B (en) Anti-idiotype nano antibody of mimic epitope peptide of human ST2 antigen and application thereof
CN112094350B (en) Monoclonal antibody of mouse anti-cell surface glycoprotein CD326 applicable to tumor cell capture
CN113461822B (en) Scl-70 antibody or binding fragment thereof, screening method thereof, and detection kit comprising same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23861814

Country of ref document: EP

Kind code of ref document: A1