CN114621349A - Targeting PD-L1/HSA/CCL5 trispecific nanobody, and derivative and application thereof - Google Patents

Targeting PD-L1/HSA/CCL5 trispecific nanobody, and derivative and application thereof Download PDF

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CN114621349A
CN114621349A CN202210121855.4A CN202210121855A CN114621349A CN 114621349 A CN114621349 A CN 114621349A CN 202210121855 A CN202210121855 A CN 202210121855A CN 114621349 A CN114621349 A CN 114621349A
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CN114621349B (en
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任贺
黄鹤
康广博
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Affiliated Hospital of University of Qingdao
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    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention discloses a targeted PD-L1/HSA/CCL5 trispecific nano antibody, a derivative and an application thereof. The amino acid sequence of the targeted PD-L1/HSA/CCL5 trispecific nano antibody provided by the invention is selected from any one of the amino acid sequences shown in SEQ ID NO.1-SEQ ID NO. 8. The invention also provides a derivative obtained by conjugating the nano antibody with a functional molecule. The nano antibody provided by the invention has extremely strong binding activity and affinity for PD-L1 and CCL 5. The invention further provides application of the targeted PD-L1/HSA/CCL5 trispecific nano antibody and the derivative thereof in preparing medicines or reagents for treating or diagnosing C-FOXP 3-elevated related tumors.

Description

Targeting PD-L1/HSA/CCL5 trispecific nanobody, and derivative and application thereof
Technical Field
The invention relates to a nano antibody for treating or diagnosing pancreatic cancer tumors and a derivative thereof, in particular to a targeted PD-L1/HSA/CCL5 trispecific nano antibody and a derivative thereof, and further designs application of the targeted PD-L1/HSA/CCL5 trispecific nano antibody and the derivative thereof in preparation of a reagent or a medicine for diagnosing or treating tumors, belonging to the field of antibodies for treating or diagnosing tumors.
Background
Pancreatic cancer is a malignant tumor of the digestive tract with high malignancy and difficult diagnosis and treatment, and more than 46 million people die of pancreatic cancer in 2020 world. According to the 2020 world Wide Web for cancer information, pancreatic cancer is the fourth leading cause of amphoteric cancer-related death, with a 5-year survival rate of just 10%. A study aimed at 28 european countries predicted that by 2025, pancreatic cancer will be the third leading cause of cancer death. Pancreatic Ductal Adenocarcinoma (PDAC) is the most common type of pancreatic cancer, accounting for over 90% of pancreatic cancers. However, early PDAC has no obvious symptoms and is difficult to diagnose. Therefore, most PDAC patients are not discovered until late, resulting in the loss of opportunity for early surgical treatment. 80-90% of PDAC patients have unresectable tumors at the time of diagnosis. However, for those patients who receive radical resection and adjuvant chemotherapy, 5-year survival remains low.
Among the malignant tumors that are common worldwide, the incidence of pancreatic cancer is 14 th and the cause of death is 7 th, and in recent years, the incidence of pancreatic cancer is on an increasing trend year by year. The global cancer database statistics show that there are 45 ten thousand cases of confirmed pancreatic cancer and 43 ten thousand cases of pancreatic cancer death globally in 2018. The incidence of pancreatic cancer in different countries is greatly different, and the incidence in developed countries is higher compared with developing countries. The incidence of Chinese pancreatic cancer is at the 10 th position in all malignant tumors and at the 5 th position in the death cause. Pancreatic cancer patients have a 5-year survival rate of about 6% and are one of the worst-prognosis malignancies. The vast majority of pancreatic cancer patients are diagnosed late, with only 20% of pancreatic cancer patients diagnosed with conditions for surgical resection and a 5-year survival rate of 27% for patients with successful surgical resection. Given the rising incidence and low survival rate of pancreatic cancer, it is considered important to identify screening high-risk patients, early diagnosis and improved treatment methods.
Chemotherapy, radiation therapy, or a combination of both are first-line treatment regimens for advanced pancreatic cancer. However, the therapeutic effect of these combination regimens is less than satisfactory. Immune Checkpoint Inhibitors (ICIs) open up new approaches for the treatment of various tumors, and immune checkpoint molecules represented by programmed cell death protein 1(PD-1) are important molecular mechanisms for the negative regulation of the body's immune system. Tumor cells can suppress immune surveillance by T cells and the like by expressing ligand molecules of immune checkpoints at high levels. Aiming at the mechanism, the monoclonal antibody with high affinity is utilized to block the interaction between molecules of an immune checkpoint, so that the activity of killer cells such as T cells and the like is restored to resist the tumor, and the monoclonal antibody becomes an emerging research hotspot for tumor biotherapy. The immune checkpoint molecules are numerous, and the most mature of the current application researches are cytotoxic T lymphocyte-associated protein 4(CTLA-4) and programmed cell death protein 1(PD-1), wherein the application of the latter in clinical application is particularly extensive (the application research of immune checkpoint molecular monoclonal antibody represented by Lijing, PD-1 in tumor treatment progresses, 2018)
However, the effect of the anti-PD-1/PD-L1 or anti-CTLA-4 monoclonal antibody on PDAC is not ideal at present. PD-L1 is expressed in PDACs, whose overexpression is associated with poor prognosis. Preclinical studies show that blocking PD-L1 can inhibit pancreatic cancer development in animal models, suggesting that the PD-1/PD-L1 pathway may be a potential target for treating PDAC. However, clinical trials have shown that targeting the PD-1/PD-L1 pathway with a single drug has little effect on patients with PDACs, suggesting that other targets for treatment of PDACs must be identified in addition to expression of PD-L1.
Recent studies by the present inventors have shown that Cancer forkhead box protein 3(Cancer-FOXP3 or C-FOXP3) recruits Treg cells into PDACs by upregulating CCL5, thereby promoting immune evasion by PDACs. In this study, the inventors demonstrated that PD-L1 was overexpressed in PDAC samples from two independent patients with radical resection. In addition, the finding that C-FOXP3 is co-localized with the expression of PD-L1 in tumor cells at the mRNA and protein levels and correlates with the expression of PD-L1 in tumor cells was confirmed by a cancer genomic profiling database (TCGA). Chromatin immunoprecipitation (ChIP) showed direct binding of C-FOXP3 to the PD-L1 promoter region of pancreatic cancer cells. In addition, overexpression of C-FOXP3 activates the luciferase reporter gene under the control of the PD-L1 promoter. However, mutation of the binding motif-a completely reversed the luciferase activity. In addition, the C-FOXP 3-induced up-regulation of PD-L1 expression can effectively inhibit the activity of CD8+ T cells. Based on the recent discovery that the CCL-5 antibody has a better response to a PDAC model with a high C-FOXP3 level, the inventor further proves that the PD-L1 antibody enhances the anti-tumor effect of CCL-5 blocking in a xenogeneic and orthotopic mouse model with a high C-FOXP3 level (Zhang Ying. chemokine CCL5 and the research progress of a receptor thereof and autoimmune diseases. 2016; Litening. the research progress of the intestinal tract mucosa chemokine. 2020.
In conclusion, C-FOXP 3directly activates PD-L1 as a core transcription factor that mediates the immune escape of PDAC. The combined blocking of PD-L1 and CCL-5 can provide an effective treatment method for PDAC patients with C-FOXP3 increase, and the development of a bispecific antibody targeting CCL5 and PDL1 has important significance in the treatment of pancreatic cancer.
The existing traditional full-length antibody biological preparations have the problems of poor tissue penetration, strong immunogenicity and the like, so the development and application of antibody medicines with miniaturization, humanization and rational design optimization are the development directions of therapeutic antibodies and diagnostic antibodies in the future. The VHH nano antibody (Nanobody) is an outstanding representative of miniaturized antibodies and has the advantages of high affinity, strong specificity, small molecular weight, low immunogenicity and high stability. Bispecific antibodies (BsAb) are another trend in antibody development, which are capable of recognizing and binding two different antigens or epitopes simultaneously, and exhibit the following advantages in terms of therapy: double-target signal blocking, unique or overlapping functions are exerted, and drug resistance and immune escape are effectively prevented; has stronger specificity and targeting property and reduces off-target toxicity; effectively reduces the treatment cost. And the nano antibody is easy to design into a functional form which is multivalent and aims at two or more antigens through a genetic engineering means. Meanwhile, the nano antibody is easy to perform in-vitro affinity maturation in a computer simulation mode so as to obtain an antibody sequence with high affinity. The bispecific single-chain nano antibody constructed by connecting two monovalent small molecule antibodies can improve the efficacy, remarkably increase the binding capacity to a target so as to increase the treatment capacity and prolong the serum half life, and the bispecific nano antibody has become a hot spot of engineering antibody research in recent years. The bispecific nanobodies approved and under development at present mainly focus on the fields of anti-infection, tumor and immune disease treatment and diagnosis, wherein the research in the field of tumor treatment and diagnosis is the most extensive. Therefore, the nano antibody as a miniaturized antibody has the advantages of small molecular weight, high stability, low immunogenicity, easy connection and the like, and has unique advantages when constructing the PDL1/CCL5 bispecific antibody.
CN105814082A discloses a bispecific nanobody comprising a first functional single variable domain and a second immunoglobulin-anchoring single variable domain (ISV) bispecific polypeptide, wherein the first ISV binds a first target on the surface of a cancer cell with low affinity and, when bound, inhibits the function of the first target, and the second ISV binds a second target on the surface of the cell with high affinity, and wherein the first target is different from the second target. However, in this invention, the bispecific nanobody has the potential to selectively target LSCs, normal HSCs and HPGs are not affected by CXCR4 nanobodies. In this protocol, bispecific CXCR4-CD4 polypeptides bind both CXCR4 and CD4, resulting in a strongly increased neutralizing potency against HIV using CXCR 4.
The bispecific nanobody targeting CCL5 and PDL1 lacks evidence for the treatment of pancreatic cancer. During pancreatic carcinogenesis, regulatory T cell (Tregs) key transcriptional regulatory programs are ectopically expressed and function in the pancreatic epithelium. The main expression is that part of pancreatic epithelial cells express a specific transcription molecule of Tregs FOXP3, and pancreatic ductal epithelial cells of FOXP3+ can promote CCR5+ Tregs to infiltrate in pancreatic lesion tissues through a direct transcription regulation chemokine CCL-5, and jointly inhibit the activity of CD8+ killer T cells (Cancer-FOXP3direct activated CCL5 to recombinant FOXP3Treg cells in pancreatic duct adenocarinoma [ J ]. Oncogene, 2017). In addition, FOXP3 (cNacet-FOXP 3, C-FOXP3) which is a pancreatic epithelial tumor is highly positively correlated with PD-L1 expression, and further analysis shows that C-FOXP3 promotes PD-L1 transcription and inhibits CD8+ T cell activity (PD-L1 is a direct target of cancer-FOXP3 in biological products adenosine (PDAC), and combined immunity with antibiotics as an antibody PD-L1 and CCL5 is effective in the area of PDAC [ J ]. Signal transduction and targeted therapy, 2020). Based on the findings, in preclinical tests, the PD-L1 and the CCL-5 combined blocking antibody are adopted for simulation treatment on the C-FOXP3 high-expression pancreatic cancer, so that an obvious curative effect is obtained, a new accurate treatment strategy is provided for reversing the pancreatic cancer immunotherapy drug resistance, and the current clinical situation that the single anti-immune checkpoint treatment response is poor can be solved.
An Immune Checkpoint Inhibitor (ICI) opens up a new way for treating various tumors, PD-L1 is expressed in Pancreatic Ductal Adenocarcinoma (PDAC), and the over-expression of the PD-L1 is related to poor prognosis, but the effect of the anti-PD-1/PD-L1 monoclonal antibody on treating the PDAC is not ideal at present. A prerequisite for effective immune checkpoint inhibitor therapy is high levels of activated Tumor Infiltrating Lymphocytes (TILs) in tumor tissue. However, most PDACs are characterized by lower levels of activated TIL around tumor tissue due to connective tissue stroma and various immunosuppressive cells, such as regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells. Studies have shown that Cancer forkhead box protein 3(Cancer-FOXP3 or C-FOXP3) recruits Treg cells into PDACs by upregulating CCL5, thereby promoting immune evasion by PDACs. The C-FOXP 3directly activates PD-L1, is a core transcription factor for mediating the immune escape of PDAC, and the combined blocking of PD-L1 and CCL-5 can provide an effective treatment method for PDAC patients with C-FOXP3 increase. Therefore, the development of the bispecific nanobody targeting CCL5 and PDL1 has important significance in the treatment of pancreatic cancer.
Disclosure of Invention
One of the purposes of the invention is to provide a targeted PD-L1/HSA/CCL5 trispecific nanobody and a derivative thereof;
the invention also aims to provide a recombinant expression vector containing the targeted PD-L1/HSA/CCL5 trispecific nano-antibody;
it is a further object of the present invention to provide a host cell containing said recombinant expression vector.
The fourth purpose of the invention is to apply the target PD-L1/HSA/CCL5 trispecific nano-antibody and the derivative thereof to the preparation of drugs for treating tumors or the preparation of reagents for diagnosing tumors.
In order to achieve the above object, the technical solution adopted by the present invention comprises:
the first aspect of the invention provides a targeted PD-L1/HSA/CCL5 trispecific nanobody, wherein the amino acid sequence of the targeted PD-L1/HSA/CCL5 trispecific nanobody is selected from any one of the amino acid sequences described in (1) to (3):
(1) any one of SEQ ID No.1 to SEQ ID No. 8;
(2) a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one amino acid sequence of SEQ ID NO.1-SEQ ID NO.8, wherein the protein mutant has the same function as the protein before mutation;
(3) an amino acid sequence having at least 95% identity with the amino acid sequence of any one of SEQ ID No.1-SEQ ID No. 8.
The second aspect of the invention provides a coding gene of the targeted PD-L1/HSA/CCL5 trispecific nanobody.
The third aspect of the present invention provides a recombinant expression vector containing the encoding gene; the expression vector can be a recombinant prokaryotic expression vector, a recombinant eukaryotic expression vector or other recombinant expression vectors.
In a fourth aspect of the invention, there is provided a recombinant host cell containing said recombinant expression vector. Wherein, the host cell is prokaryotic expression cell, eukaryotic expression cell, fungal cell or yeast cell.
The fifth aspect of the invention provides a derivative of a targeting PD-L1/HSA/CCL5 trispecific nanobody, which comprises a derivative obtained by conjugating the targeting PD-L1/HSA/CCL5 trispecific nanobody with a functional molecule, wherein the functional molecule includes, but is not limited to, one or more of a small molecule drug, a cytotoxic agent, a bioactive protein, a radioisotope or a fluorescent dye.
For example, the derivative obtained by chemically labeling or biological labeling the target PD-L1/HSA/CCL5 trispecific nanobody, or the derivative obtained by coupling the target PD-L1/HSA/CCL5 trispecific nanobody with a solid medium or a semisolid medium.
The obtained derivative can be applied to the preparation of a reagent for tumor molecular imaging diagnosis or a medicine for treating tumors; or the targeted PD-L1/HSA/CCL5 trispecific nano-antibody and the derivative are applied to the preparation of the relevant tumor for diagnosing or treating the C-FOXP3 increase.
The term "fluorescent dye" as used herein refers to a compound that emits visible or infrared light upon excitation by electromagnetic radiation of a relatively short and appropriate wavelength. The fluorescent dye is selected from the group consisting of xanthines, acridines, oxazines, cyanines, styryl dyes, evans blue, coumarins, porphyrins, metal ligand-complexes, fluorescent proteins, nanocrystals, perylenes, boron dipyrromethenes, and phthalocyanines, as well as conjugates and combinations of these classes of dyes.
As used herein, a "radioisotope" is an element that emits alpha, beta, and/or gamma radiation. For example, anti-FAP single domain antibodies, Fc fusion proteins or immunoconjugates are used64Cu,67Ga,68Ga,89Zr,18F,86Y,90Y,111In,99mTc,125I,124I, labeling radioactive isotopes to obtain a molecular image diagnostic medicine for PET (positron emission tomography) or SPECT (single photon emission computed tomography); or anti-single domain antibodies, Fc fusion proteins or immunoconjugates90Y，177Lu,125I,131I,211At,111In,152Sm,166Ho，186Re,188Re,67Cu,212Pb,225Ac,213Bi,212Bi,223Ra,227Th and other radioactive isotopes are used for labeling the medicines for treating FAP related diseases.
In addition, the radioactive isotope can be directly labeled with a target PD-L1/HSA/CCL5 trispecific nanobody or a derivative thereof; the targeted PD-L1/HSA/CCL5 trispecific nanobodies or derivatives thereof may also be indirectly labeled by chelating agents including, but not limited to, 1,4,7, 10-tetraazacyclododecane-N, N' -tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA), N-N "-bis [ 2-hydroxy-5- (carboxyethyl) benzyl ] diethylamine-N, N" -diacetic acid (HBED-CC), 2- (4, 7-bis (carboxymethyl) -1,4, 7-triazono-1-yl) glutaric acid (NOA), 2- (4,7, 10-tris (carboxymethyl) -1,4, 7-10-tetraazacyclododecan-1-yl) glutaric acid (DOTAGA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N ', N "-pentaacetic acid (DTPA), bis- (carboxymethylimidazole) glycine, 6-hydrazinopyridine-3-carboxylic acid (HYNIC), N' - {5- [ acetyl (hydroxy) amino ] pentyl } -N- [5- ({4- [ (5-aminopentyl) (hydroxy) amino ] -4-oxobutanoyl } amino) pentyl ] N-hydroxysuccinamide (DFO), and the like.
Therefore, the sixth aspect of the invention provides the application of the targeted PD-L1/HSA/CCL5 trispecific nanobody, the targeted coding gene of the PD-L1/HSA/CCL5 trispecific nanobody and the targeted derivative of the PD-L1/HSA/CCL5 trispecific nanobody in the preparation of a reagent or a medicament for diagnosing or treating the related tumor with abnormal expression of PD-1 or/and CCR 5.
The seventh aspect of the invention provides a pharmaceutical composition for treating tumors, which consists of a targeted PD-L1/HSA/CCL5 trispecific nanobody or a derivative thereof and a pharmaceutically acceptable carrier and/or excipient; the pharmaceutical composition can also contain effective parts extracted from Chinese medicinal plants for treating tumors, chemotherapeutic drugs or small molecule inhibitors, etc.
The eighth aspect of the invention provides a detection kit for diagnosing or treating tumors, which comprises any one or more of the targeted PD-L1/HSA/CCL5 trispecific nanobodies or derivatives thereof.
The tumor in the invention is a C-FOXP 3-elevated related tumor, preferably pancreatic cancer, more preferably pancreatic ductal adenocarcinoma.
Definitions of terms to which the invention relates
The term "Nanobody" as used herein refers to a fragment, also known as a Nanobody, that contains a single variable domain in an antibody. Like an intact antibody, it binds selectively to a particular antigen. The single domain antibody appears much smaller, approximately only 12-15 kDa, compared to the 150-160 kDa mass of the intact antibody. The first single domain antibody was artificially engineered from a camelid heavy chain antibody, referred to as a "VHH segment".
The term "identity" of sequences as used herein is used interchangeably with "identity" and refers to the degree of similarity between sequences as determined by sequence alignment software, such as BLAST. Methods and software for sequence alignment are well known to those skilled in the art. An engineered nucleotide sequence may be obtained by substitution, deletion and/or addition of one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more) amino acids or bases to a known sequence. For example, by conventional means (e.g., conservative substitutions, etc.), the sequences of SEQ ID NOs: 1-198, and can have greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and substantially the same properties, all within the scope of the present invention. Preferably, the present invention obtains sequence identity by conservative substitutions, but is not limited to conservative substitutions.
The term "amino acid sequence" refers to the sequence of amino acids linked together to form a peptide chain (or polypeptide), and the amino acid sequence can only be read in one orientation. There are more than 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances such as saccharides, lipids, etc. from the amino acid chain, nor is the present invention limited to the amino acids commonly used in 20.
The term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., A, T, G, C in DNA or A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA and mRNA. It is understood that the antibody genes claimed in the present invention also encompass RNA (rRNA, tRNA, mRNA) and their complementary sequences in addition to DNA sequences.
The substitutions described in the present invention may be conservative substitutions, i.e. the substitution of a specific amino acid residue for a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups (e.g., substitutions between Ile, Val, Leu, or Ala), substitutions between polar residues (e.g., substitutions between Lys and Arg, Glu and Asp, Gln and Asn), and the like. Mutants resulting from deletion, substitution, insertion and/or addition of amino acids can be prepared by subjecting DNA encoding a wild-type protein to, for example, site-directed mutagenesis as a well-known technique (see, for example, Nucleic Acid Research, Vol.10, No.20, p.6487-6500, 1982, which is incorporated herein by reference in its entirety).
In the present specification, "one or more amino acids" refers to amino acids that can be deleted, substituted, inserted, and/or added by a site-directed mutagenesis method, and is not limited, but is preferably 20 or less, 15 or less, 10 or less, or 7 or less, and more preferably 5 or less. In the case of site-directed mutagenesis, for example, in addition to the desired variation, i.e., a specific mismatch, synthetic oligonucleotide primers complementary to the single-stranded phage DNA to be mutated can be used in the following manner. That is, a strand complementary to the phage is synthesized using the synthetic oligonucleotide as a primer, and the resulting double-stranded DNA is used to transform a host cell. The culture of the transformed bacteria was plated on agar and plaques were formed from phage-containing single cells. Then, plaques hybridized with the probe were collected and cultured to recover DNA. Further, there are methods of deleting, substituting, inserting and/or adding one or more amino acids from an amino acid sequence of a biologically active peptide such as an enzyme while maintaining its activity, and in addition to the above-mentioned site-directed mutagenesis, there are also methods of treating a gene with a mutagenesis source, and methods of selectively cleaving a gene, then deleting, substituting, inserting or adding a selected nucleotide, and then ligating it.
The term "recombinant Expression vectors" refers to vectors in which Expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic backbone of a cloning vector to enable Expression of a desired gene. The expression vector comprises four parts: target gene, promoter, terminator and marker gene. The present invention includes, but is not limited to, prokaryotic, eukaryotic, or other cellular expression vectors.
The terms "mutation" and "mutant" have their usual meanings herein, and refer to a genetic, naturally occurring or introduced change in a nucleic acid or polypeptide sequence, which has the same meaning as is commonly known to those of skill in the art.
The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome.
Drawings
FIG. 1 is a structural schematic diagram of a tri-specific nanobody targeting PDL1/CCL5 constructed based on an anti-HSA nanobody.
FIG. 2 is a photograph of a PD-L1-GST SDS-PAGE gel electrophoresis; PD-L1 has a size of 26Kd, and due to glycosylation, the protein migrates at a speed of 30-35Kd under reducing conditions, and the GST tag has a size of 26 Kd; "M" is protein marker; "Whole" is whole bacteria lysate, "Top" is lysate supernatant, "sink" is lysate precipitate, "punch" is breakthrough peak, and number 1-10 is elution peak.
FIG. 3 is a CCL5-GST SDS-PAGE gel electrophoresis; the size of the CCL5 protein is 8Kd, the size of the GST tag is 26Kd, and the target band is about 34 Kd; the same band exists around 26Kd, and the result is that part of the protein GST label is detached; "M" is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-8 are elution peaks.
FIG. 4 is a photograph of a HSA-GST SDS-PAGE gel electrophoresis; the size of the HSA protein is 69Kd, the size of the GST tag is 26Kd, and the target band is about 95 Kd; "M" is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-10 are elution peaks.
FIG. 5 is a BC8P2 SDS-PAGE gel; "M" is protein marker; "Whole" is whole bacteria lysate, "Top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and No. 1-9 is elution peak.
FIG. 6 is a SDS-PAGE gel of BC8P 3; "M" is protein marker; "Whole" is whole bacteria lysate, "Top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and No. 1-9 is elution peak.
FIG. 7 is a SDS-PAGE gel of BC11P 2; "M" is protein marker; "Whole" is whole bacteria lysate, "Top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and No. 1-9 is elution peak.
FIG. 8 is a SDS-PAGE gel of BC11P 3; "M" is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-10 are elution peaks.
FIG. 9 is a SDS-PAGE gel of BP2C 8; "M" is protein marker; "Whole" is whole bacteria lysate, "Top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and No. 1-9 is elution peak.
FIG. 10 is a SDS-PAGE gel of BP2C 11; "M" is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-10 are elution peaks.
FIG. 11 is a SDS-PAGE gel of BP3C 8; m is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-10 are elution peaks.
FIG. 12 is a SDS-PAGE gel of BP3C 11; "M" is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-10 are elution peaks.
FIG. 13 shows the results of ELISA to verify the binding activity of the antibody to PD-L1 at concentration gradients.
Fig. 14 is a graph of the results of ELISA to verify the binding activity of antibodies to CCL5 at concentration gradients.
FIG. 15 shows the results of ELISA to verify the binding activity of antibody to HSA under concentration gradient.
FIG. 16 is an SPR sensorgram of the interaction between BC8P2 and PD-L1.
FIG. 17 is an SPR sensorgram of the interaction between BC8P3 and PD-L1.
FIG. 18 is an SPR sensorgram of the interaction between BC11P2 and PD-L1.
FIG. 19 is an SPR sensorgram of the interaction between BC11P3 and PD-L1.
FIG. 20 is an SPR sensorgram of the interaction between BP2C8 and PD-L1.
FIG. 21 is an SPR sensorgram of the interaction between BP2C11 and PD-L1.
FIG. 22 is an SPR sensorgram of the interaction between BP3C8 and PD-L1.
FIG. 23 is an SPR sensorgram of the interaction between BP3C11 and PD-L1.
FIG. 24 is an SPR sensorgram of the interaction between BC8P2 and CCL 5.
FIG. 25 is an SPR sensorgram of the interaction between BC8P3 and CCL 5.
FIG. 26 is an SPR sensorgram of the interaction between BC11P2 and CCL 5.
FIG. 27 is an SPR sensorgram of the interaction between BC11P3 and CCL 5.
FIG. 28 is an SPR sensorgram of the interaction between BP2C8 and CCL 5.
FIG. 29 is an SPR sensorgram of the interaction between BP2C11 and CCL 5.
FIG. 30 is an SPR sensorgram of the interaction between BP3C8 and CCL 5.
FIG. 31 is an SPR sensorgram of the interaction between BP3C11 and CCL 5.
FIG. 32 is a SDS-PAGE gel of nickel ion after purification; m is protein marker; "Whole" is whole bacteria lysate, "top" is lysate supernatant, "sediment" is lysate sediment, "breakthrough" is breakthrough peak, and numbers 1-10 are elution peaks.
FIG. 33 is a SDS-PAGE gel electrophoresis of molecular sieve purification; m is protein marker, and No. 1-10 are elution peaks.
Detailed Description
The present invention is further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become more apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Biological materials and reagents
1. Cell lines and production strains
The PDL1, HSA and CCL5 antigens of the invention are prepared by using HEK-293F cell line. Human embryonic kidney cells HEK-293F (RRID: CVCL _6642) were obtained from the American cell culture center. All human cell lines were validated by STR analysis over the last three years. Mycoplasma contamination was excluded in these cell lines. Placing HEK-293F cells in 5% CO2 constant temperature shaker at 37 deg.C and 120rpm for shake culture (during passage, cell count and cell survival rate should be observed, and density of 3-6 × 10 should be selected as much as possible6cells/mL of high viability cells were subcultured. Cell density and viability were determined prior to transient transfection. To ensure transfection efficiency, it is advisable to use a growth in the exponential phase (density about 2-4X 10)6One/ml), survival greater than 98%. Cell density>2.0×106Adding fresh KOP293 culture solution to dilute the cells to 2 × 10/mL6One per mL. The flask was placed in 5% CO2After shaking culture at constant temperature of 37 ℃ and 120rpm for 10min in a constant temperature shaking table, transfection was started. Taking 200mL of transfected cell suspension as an example, preparing a 15mL sterile centrifuge tube, adding 10mL of transfection medium and 200 mu g of sterile plasmid DNA into one of the 50mL sterile centrifuge tubes, and gently blowing, beating and uniformly mixing; adding 10mL of transfection medium and 1mL of transfection reagent into the other centrifugal tube, and gently blowing, beating and uniformly mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and gently blowing, beating and uniformly mixing; standing for 10 minutes at room temperature to prepare a plasmid-vector compound; taking out cells from constant temperature shaking table, and addingThe good plasmid-vector complex is put back into a CO2 constant temperature shaking table for shake culture. 1mL 293 cell protein expression enhancer and 4mL nutritional additives can be added 24 hours after transfection, 50mL fresh medium can be added four days after transfection, and fermentation broth can be collected seven days after transfection.
The tri-specific nanobody targeting PDL1/CCL5 constructed based on the anti-HSA nanobody adopts Trans B (DE3) expression bacteria.
DH5 alpha competent cells for plasmid cloning were purchased from Beijing Quanjin, a century Co., Ltd, and TransB competent cells for Nanobody expression were purchased from Beijing Quanjin. The constructed expression plasmid and pSOX plasmid are co-transfected into Trans B (DE3) competent cells, and the engineering bacteria are inoculated into a 5mL LB liquid medium test tube, wherein the inoculation amount is 1 percent, and the dosage of the ampicillin and the chloramphenicol is one thousandth, and the cells are cultured for about 8 hours at the temperature of 37 ℃ and the speed of 220rpm in a constant temperature shaking table. 2mL of the activated bacterial suspension was inoculated into 200mL of an Erlenmeyer flask containing LB medium in an amount of 1%, 200. mu.L of mother solutions of ampicillin and chloramphenicol were added, respectively, and cultured in a constant temperature shaker at 37 ℃ and 220 rpm. When the thallus grows to OD600 of about 0.4, adding 4mL of arabinose into LB culture medium, culturing at 30 ℃ and 220rpm for 45min, adding 80 mu L of isopropyl-beta-D-thiogalactoside (IPTG) solution, and culturing at 20 ℃ and 160rpm for 16 h. The culture was centrifuged at 8000rpm for 15min at 4 ℃ to collect the cells. The PBS was resuspended in the cells, centrifuged again twice, and the cells weighed and stored in a-20 ℃ freezer. After dissolving each 1g of the cells in about 20mL of PBS, the cells were disrupted by ultrasonic irradiation at 180W for 25min at a frequency of 3s to 5 s. Followed by centrifugation at 10,000rpm for 20min at 4 ℃. And reserving a small amount of whole bacteria liquid before centrifugation, and precipitate and supernatant after centrifugation. The supernatant was filtered through a 0.45 μm filter and purified by the AKTA prime protein purification system.
2. Reagent
1) LB culture medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl are added into about 900mL of deionized water, the pH value is adjusted to 7.4 by dissolution, the volume is fixed to 1L, and the mixture is subpackaged and sterilized in a high-pressure steam sterilization pot at 121 ℃ for 20 min. And (3) subpackaging the solid culture medium in a liquid culture medium, adding 15g of agar powder into each liter of culture medium, and sterilizing by high-pressure steam.
2) PBS solution: 0.14mol NaCl (8.18g), 3mmol KCl (0.22g), 0.01mol Na2HPO4(1.42g), 2mmol KH2PO4(0.27g) were dissolved in about 900mL deionized water, adjusted to pH7.4 and then made to volume 1L, and sterilized in an autoclave at 121 ℃ for 20 min.
3)0.5mol/L IPTG inducer: 0.12g IPTG was dissolved in 1mL deionized water; filtering with 0.22 μm filter membrane, and storing in a refrigerator at-20 deg.C.
4)100mg/mL Amp solution: 100mg of ampicillin is dissolved in 1mL of deionized water; filtering with 0.22 μm filter membrane, and storing in a refrigerator at-20 deg.C.
5)250mg/mL chloramphenicol solution: 250mg of chloramphenicol was dissolved in 1mL of deionized water; filtering with 0.22 μm filter membrane, and storing in a refrigerator at-20 deg.C.
6) Equilibration buffer (50mmol/L imidazole buffer): 0.5mol of NaCl (29.22g), 0.01mol of Na2HPO4(1.4196g), 0.01mol of NaH2PO4(1.1998g) and 50mmol of imidazole (3.404g) were dissolved in 900mL of deionized water, adjusted to pH7.4, and then the solution was diluted to 1L and sterilized at 121 ℃ for 20 min.
7) Elution buffer (500mmol/L imidazole buffer): the imidazole concentration was increased to 500mmol (34.04g) of equilibration buffer and sterilized at 121 ℃ for 20 min.
8)250mg/mL arabinose solution: dissolving 12.5g of arabinose in deionized water, fixing the volume to 50ml, filtering with a 0.22 mu m filter membrane, and storing in a refrigerator at the temperature of minus 20 ℃.
Example 1 design of a PD-L1/HSA/CCL 5-targeted trispecific nanobody, construction of a plasmid, and expression and purification of a protein
1. Test method
The amino acid sequences of Human Serum Album (HSA), Programmed cell death 1ligand 1(PD-L1), regulated uplink activation normal T cell expressed and secreted factor (CCL5) are subjected to eukaryotic (Human) expression optimization and gene synthesis, and the synthesized gene fragment is subcloned into eukaryotic expression vector pcDNA3.1-N-GST-TEV. Recombinant proteins were produced by the HEK-293F cellular protein expression system and purified using a GSTApfF column and AKTA primer plus (GE Healthcare) affinity chromatography. The basic principle of purification is that the target protein is adsorbed on a chromatographic column through the coordination interaction between a GST label on the surface of the protein and reduced glutathione covalently bound on the chromatographic column, the protein is separated from a protein mixed solution, and then the target protein is eluted by high-concentration reduced glutathione.
BC8P2(SEQ ID No.1), BC8P3(SEQ ID No.2), BC11P2(SEQ ID No.3), BC11P3(SEQ ID No.4), BP2C8(SEQ ID No.5), BP2C11(SEQ ID No.6), BP3C8(SEQ ID No.7) and BP3C11(SEQ ID No.8) are subjected to total 8 trispecific nano-antibodies, the amino acid sequence is subjected to prokaryotic (Escherichia coli) expression optimization, the nano-antibody sequence is synthesized and cloned into a prokaryotic expression vector pET-32a (+) by bioengineering companies, and a His tag is connected to the C end of the antibody. The constructed expression plasmid and pSOX plasmid were co-transfected into Trans B (DE3) competent cells for protein expression. The fermented cells were sonicated to extract the supernatant, which was purified by AKTA prime protein purification System and 5mL His Trap from GETMAnd (4) carrying out affinity chromatography purification on the HP prepacked column to obtain the corresponding nano antibody. The purification process is as follows:
1) the ethanol in the instrument is washed clean by deionized water filtered by a 0.45 mu m filter membrane through the water washing program of the instrument.
2) A nickel ion affinity chromatography column with a column volume of 5mL was mounted on the instrument and the column was rinsed clean of ethanol with deionized water until each indicator line remained stationary at the baseline position, requiring about 50mL of deionized water.
3) The column was washed with equilibration buffer at a flow rate of 5mL/min until the lines leveled, requiring about 70mL of equilibration buffer.
4) The sample was loaded at a flow rate of 1mL/min and the breakthrough peak was taken, after which the sample was washed with equilibration buffer at a flow rate of 1mL/min for two minutes to allow the entire sample to flow through the column, and then the column was equilibrated at a flow rate of 4 mL/min.
5) Eluting the protein by using an elution buffer solution at the flow rate of 4mL/min, and collecting an elution peak, wherein the protein of the elution peak is the nano antibody.
6) After all bound proteins were eluted, the column was rinsed with deionized water until the salt ion concentration was zero.
7) Washing with 50mL of 20% ethanol, soaking the column in ethanol, taking off the column, storing at 4 deg.C, and closing the apparatus.
The nano antibody injection after nickel ion affinity purification is further purified by an AKTA Pure 25 protein chromatography purification system and a (2) Superdex 7510/300 GL high-performance column of GE company. The purification process is as follows:
1) the inlets of the A1 and B1 pumps were placed in deionized water, and the A pump and the B pump were rinsed with deionized water by a pump rinsing procedure. The B1 pump inlet was then placed in PBS and the solution in the B pump was replaced by PBS by a pump wash procedure.
2) A15 ml centrifuge tube was placed on the collector for subsequent collection of the sample (also through the waste port, there was about 0.8ml lag from the UV detection signal to the waste port, but the collector would alarm without the centrifuge tube), a loading loop (500ul) of appropriate volume was installed and the loading loop was flushed with deionized water and PBS sequentially with the syringe under load.
3) And setting alarm pressure, wherein the system pressure is 20Mpa, and the column pressure is 1.8 Mpa. A low flow rate (0.1ml/min) was set and the column was connected to the system to ensure that no air bubbles entered the column during the process.
4) Two column volumes (50ml) were washed with deionized water and two column volumes (50ml) were washed flat with PBS, the flow rate was adjusted to 0.4ml/min, taking care that the column pressure did not exceed 1.8MPa throughout the process.
5) In the load state, a sample is injected into a sample loading ring by a syringe (the volume of the sample is larger than the volume of the sample loading ring), the mode is adjusted to an inject mode to start sample loading, the mode is adjusted to a load mode after sample loading is finished, and peak collection conditions are set for sample collection (or collection from a waste liquid port by observing an ultraviolet peak, one tube is collected every 500ul)
6) After the experiment was completed, the pump A and the pump B were first washed with deionized water, then PBS in the column was washed with deionized water, then the pump A and the pump B were washed with 20% ethanol, and then the column was filled with 20% ethanol.
7) And (5) taking down the column, finishing the program operation, and storing the column well and the experimental data well. Shut down procedure, computer and instrument.
2. Test results
FIGS. 2-4 are SDS-PAGE gel electrophoreses of PD-L1-GST, CCL5-GST, and HSA-GST, respectively; FIGS. 5 to 12 are SDS-PAGE gel electrophoresis images of BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11, respectively.
Test example 1 binding Activity of Targeted PD-L1/HSA/CCL5 trispecific Nanobody to antigen test 1 test method
Binding activity of the three-specific nano-antibody BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and HSA is verified by an indirect ELISA experiment by using GST-tagged HSA (SEQ ID No.9), PDL1(SEQ ID No.10) and CCL5(SEQ ID No.11) as antigens and BC8P2, BC8P3, BC11P2, BC11P 368, BP3C11 as primary antibodies and using HRP-tagged anti-His murine monoclonal antibody as a secondary antibody.
1) Coating: diluting the antigen to a final concentration of 50. mu.g/mL, adding 100. mu.L of the antigen to each well of a 96-well plate, and placing the 96-well plate in a refrigerator at 4 ℃ for overnight coating; the negative control was BSA.
2) Washing: the next day, discarding the coating solution; adding 200 μ L PBST solution into each well, slightly shaking for 3min, and discarding the solution; three washes in total.
3) And (3) sealing: add 200. mu.L of blocking solution to each well and incubate in an oven at 37 ℃ for 2.5 hours.
4) Washing: discarding the confining liquid; PBST solution was added for washing, as in step 2).
5) Adding a primary antibody: setting the concentration gradient of the nano antibody to be seven times of dilution concentrations of 100nM, 500nM, 100nM, 10nM, 1nM, 100pM and 10pM, sequentially adding 100 microliter of nano antibody solution into each hole, and setting three parallel holes; incubate in an oven at 37 ℃ for 2.5 hours.
6) Washing: discarding the liquid in the pores; adding PBST solution, slightly shaking for 5min, and discarding the solution; a total of five washes were performed.
7) Adding a secondary antibody: diluting the secondary antibody according to the proportion of 1:1000, and adding the diluted secondary antibody into a 96-well plate, wherein each well is 100 mu L; incubate in an oven at 37 ℃ for 1 hour.
8) Color development: discarding the liquid in the pores; adding PBST solution for washing, and the step is the same as 6); after the liquid in the holes is completely thrown by force, 100 mu L of TMB color development liquid is added into each hole; incubate the oven with light at 37 ℃ for 20 minutes.
9) Termination reaction and measurement: adding 100 mu L of stop solution into each hole to stop the reaction; the OD450 value of each well was determined.
2. Test results
Fig. 13 shows results of binding activity of BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11 trispecific nanobodies to PD-L1 under ELISA-verified concentration gradient, fig. 14 shows results of binding activity of BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11 trispecific nanobodies to CCL5 under ELISA-verified concentration gradient, and fig. 15 shows results of binding activity of BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11 trispecific nanobodies to HSA under ELISA-verified concentration gradient. According to the test results, BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11 trispecific nanobodies have extremely strong binding activity on CCL5, HAS and PD-L1.
Test example 2 affinity determination test for PD-L1/HSA/CCL 5-targeting trispecific Nanobody
1. Test method
SPR experiments are commonly used to accurately determine the affinity constant KD of antigen-antibody reactions. The assay used a Biacore T200 instrument (GE Healthcare) for SPR affinity determination with a chip model of CM-5 at 25 ℃. Purified GST-tagged PD-L1, CCL5 or HSA protein (2 units in 10mM HEPES, pH7.4,150mM NaCl,3mM EDTA, 0.005% P20) was covalently bound to a CM5 sensor chip at a fixed density of approximately 350 Resonance Units (RUs). An irrelevant biotinylated protein was immobilized on a reference surface matched to the RU values to control non-specific binding. The affinity chromatography and purified nanobody were diluted by BIAcore running buffer (10mM HEPES, pH7.4,150mM NaCl,3mM EDTA, 0.05% (v/v) P20) to set nanobody concentration gradient at 5-fold dilution concentration of 100. mu.g/mL-6.25. mu.g/mL, and one concentration setting was selected to repeat the experiment; the setup program was loaded while measurements were taken at a flow rate of 40 ℃. The running buffer without nanobodies was then passed through the chip, allowing spontaneous dissociation at the same flow rate. After each run, the sensor chip was regenerated by injection of 10mM glycine, pH 2.0. After the experiment is finished, the computer is off, the obtained data are imported into instrument matching software for analysis, a 1:1 Langmuir binding model is adopted, and after the data are fitted, an affinity constant K is calculatedDThe value is obtained.
2. Test results
The test results are shown in table 1 and fig. 16 to 31.
TABLE 1 affinity constants of trispecific nanobodies for PD-L1 and CCL5
KD/nM BC8P2 BC8P3 BC11P2 BC11P3 BP2C8 BP2C11 BP3C8 BP3C11
PD-L1 3.1 6.9 3.2 6.9 31 9.6 16 2.3
CCL5 14 6.8 6.5 8.6 9.7 3.9 8.8 5.3
As can be seen from the affinity measurement results in table 1 and fig. 16 to 31, the trispecific nanobodies BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11 all have very strong affinity for PD-L1 and CCL 5.
Test example 3 Scale preparation of antibody biologicals (Pilot plant test)
After the thalli are harvested in pilot scale, the purity of the thalli is over 80 percent after Ni ion affinity chromatography purification, the purity of the thalli is over 98 percent after SEC purification, and a figure 32 is an SDS-PAGE gel electrophoresis picture after nickel ion purification. FIG. 33 is a SDS-PAGE gel electrophoresis of molecular sieve purification;
the yields after antibody purification are shown in table 2.
TABLE 2 yield of antibody biologicals on pilot scale
Figure BDA0003498827170000161
Sequence listing
<110> affiliated Hospital of Qingdao university
<120> targeted PD-L1/HSA/CCL5 tri-specific nano antibody and derivative and application thereof
<130> 0026
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 394
<212> PRT
<213> Artifical sequence
<400> 1
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Thr Met
20 25 30
Asp Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Thr Ile Ser Arg Ser Gly Val Gly Thr Phe Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Ser Asp Thr Ala Leu Tyr Tyr
85 90 95
Cys Ala Ala Arg Pro Asp Tyr Thr Leu Gly Thr Ser Ser Tyr Asp Tyr
100 105 110
Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
130 135 140
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu
145 150 155 160
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp
165 170 175
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser
180 185 190
Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe
195 200 205
Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn
210 215 220
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly
225 230 235 240
Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
260 265 270
Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu
275 280 285
Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Gly Ser Asp Met
290 295 300
Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val Ser Thr
305 310 315 320
Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
325 330 335
Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
340 345 350
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu
355 360 365
His Cys Thr Gly Ser Trp Ala Leu Ile Met Gly Gln Gly Thr Gln Val
370 375 380
Thr Val Ser Ser His His His His His His
385 390
<210> 2
<211> 403
<212> PRT
<213> Artifical sequence
<400> 2
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Thr Met
20 25 30
Asp Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Thr Ile Ser Arg Ser Gly Val Gly Thr Phe Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Ser Asp Thr Ala Leu Tyr Tyr
85 90 95
Cys Ala Ala Arg Pro Asp Tyr Thr Leu Gly Thr Ser Ser Tyr Asp Tyr
100 105 110
Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
130 135 140
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu
145 150 155 160
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp
165 170 175
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser
180 185 190
Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe
195 200 205
Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn
210 215 220
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly
225 230 235 240
Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
260 265 270
Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu
275 280 285
Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly Ser Asp Met
290 295 300
Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val Ser Thr
305 310 315 320
Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
325 330 335
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu
340 345 350
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg
355 360 365
Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys Lys Ala Asn
370 375 380
Ser Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His
385 390 395 400
His His His
<210> 3
<211> 394
<212> PRT
<213> Artifical sequence
<400> 3
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asp Asp
20 25 30
Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Ser Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Asn Leu Asn Pro Glu Asp Thr Ala Ala Tyr Tyr
85 90 95
Cys Ala Ala Ala Pro Pro Asp Cys Thr Tyr Tyr Pro Ala Thr Pro Ile
100 105 110
Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
130 135 140
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu
145 150 155 160
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp
165 170 175
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser
180 185 190
Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe
195 200 205
Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn
210 215 220
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly
225 230 235 240
Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
260 265 270
Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu
275 280 285
Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Gly Ser Asp Met
290 295 300
Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val Ser Thr
305 310 315 320
Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
325 330 335
Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
340 345 350
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu
355 360 365
His Cys Thr Gly Ser Trp Ala Leu Ile Met Gly Gln Gly Thr Gln Val
370 375 380
Thr Val Ser Ser His His His His His His
385 390
<210> 4
<211> 403
<212> PRT
<213> Artifical sequence
<400> 4
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asp Asp
20 25 30
Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Ser Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Asn Leu Asn Pro Glu Asp Thr Ala Ala Tyr Tyr
85 90 95
Cys Ala Ala Ala Pro Pro Asp Cys Thr Tyr Tyr Pro Ala Thr Pro Ile
100 105 110
Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu
130 135 140
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu
145 150 155 160
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp
165 170 175
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ser Ile Ser
180 185 190
Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe
195 200 205
Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn
210 215 220
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly
225 230 235 240
Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly
245 250 255
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
260 265 270
Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu
275 280 285
Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly Ser Asp Met
290 295 300
Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu Val Ser Thr
305 310 315 320
Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg
325 330 335
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu
340 345 350
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Arg
355 360 365
Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys Lys Ala Asn
370 375 380
Ser Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His
385 390 395 400
His His His
<210> 5
<211> 394
<212> PRT
<213> Artifical sequence
<400> 5
Met Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Gly
20 25 30
Ser Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu
35 40 45
Val Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu His Cys Thr Gly Ser Trp Ala Leu Ile Met Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
130 135 140
Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
145 150 155 160
Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly
165 170 175
Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr
180 185 190
Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
195 200 205
Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
210 215 220
Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser
225 230 235 240
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
260 265 270
Gly Gly Leu Val Gln Ala Gly Asp Ser Leu Arg Leu Ser Cys Ala Thr
275 280 285
Ser Gly Arg Thr Phe Thr Met Asp Gly Met Gly Trp Phe Arg Gln Ala
290 295 300
Pro Gly Lys Glu Arg Glu Phe Val Ala Thr Ile Ser Arg Ser Gly Val
305 310 315 320
Gly Thr Phe Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
325 330 335
Asp Asn Ala Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro
340 345 350
Ser Asp Thr Ala Leu Tyr Tyr Cys Ala Ala Arg Pro Asp Tyr Thr Leu
355 360 365
Gly Thr Ser Ser Tyr Asp Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val
370 375 380
Thr Val Ser Ser His His His His His His
385 390
<210> 6
<211> 394
<212> PRT
<213> Artifical sequence
<400> 6
Met Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Thr Leu Ser Cys Val Ala Ser Gly Phe Thr Phe Asp Gly
20 25 30
Ser Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu
35 40 45
Val Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Leu His Cys Thr Gly Ser Trp Ala Leu Ile Met Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
130 135 140
Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
145 150 155 160
Phe Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly
165 170 175
Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr
180 185 190
Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
195 200 205
Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
210 215 220
Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser
225 230 235 240
Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
245 250 255
Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
260 265 270
Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala
275 280 285
Ser Gly Phe Asn Phe Asp Asp Tyr Ala Ile Gly Trp Phe Arg Gln Ala
290 295 300
Pro Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Ser Ser Ser Asp Gly
305 310 315 320
Ser Thr Tyr Ser Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ser
325 330 335
Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Asn Leu Asn Pro
340 345 350
Glu Asp Thr Ala Ala Tyr Tyr Cys Ala Ala Ala Pro Pro Asp Cys Thr
355 360 365
Tyr Tyr Pro Ala Thr Pro Ile Tyr Tyr Trp Gly Gln Gly Thr Gln Val
370 375 380
Thr Val Ser Ser His His His His His His
385 390
<210> 7
<211> 403
<212> PRT
<213> Artifical sequence
<400> 7
Met Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly
20 25 30
Ser Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu
35 40 45
Val Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys
100 105 110
Lys Ala Asn Ser Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser
145 150 155 160
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly
165 170 175
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
180 185 190
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys
195 200 205
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu
210 215 220
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr
225 230 235 240
Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val
245 250 255
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
260 265 270
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
275 280 285
Asp Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Thr Phe Thr Met
290 295 300
Asp Gly Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
305 310 315 320
Val Ala Thr Ile Ser Arg Ser Gly Val Gly Thr Phe Tyr Ala Asp Ser
325 330 335
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Val
340 345 350
Tyr Leu Gln Met Asn Ser Leu Lys Pro Ser Asp Thr Ala Leu Tyr Tyr
355 360 365
Cys Ala Ala Arg Pro Asp Tyr Thr Leu Gly Thr Ser Ser Tyr Asp Tyr
370 375 380
Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His
385 390 395 400
His His His
<210> 8
<211> 403
<212> PRT
<213> Artifical sequence
<400> 8
Met Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Asp Gly
20 25 30
Ser Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Thr Glu Cys Glu Leu
35 40 45
Val Ser Thr Ile Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Leu Pro His Ile Asp Val Val Ala Thr Ala Lys Gly Cys
100 105 110
Lys Ala Asn Ser Tyr Leu Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
130 135 140
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asn Ser
145 150 155 160
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe Gly
165 170 175
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser
180 185 190
Ser Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp Ser Val Lys
195 200 205
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr Leu
210 215 220
Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr
225 230 235 240
Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu Val Thr Val
245 250 255
Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
260 265 270
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
275 280 285
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Phe Asp Asp
290 295 300
Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
305 310 315 320
Val Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Ser Ala Asp Ser
325 330 335
Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val
340 345 350
Tyr Leu Gln Met Asn Asn Leu Asn Pro Glu Asp Thr Ala Ala Tyr Tyr
355 360 365
Cys Ala Ala Ala Pro Pro Asp Cys Thr Tyr Tyr Pro Ala Thr Pro Ile
370 375 380
Tyr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser His His His
385 390 395 400
His His His
<210> 9
<211> 591
<212> PRT
<213> Homo sapiens
<400> 9
Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His Arg
1 5 10 15
Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala
20 25 30
Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu
35 40 45
Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser
50 55 60
Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu
65 70 75 80
Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys
85 90 95
Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys
100 105 110
Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val
115 120 125
Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr
130 135 140
Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu
145 150 155 160
Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln
165 170 175
Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg
180 185 190
Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser
195 200 205
Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg
210 215 220
Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu
225 230 235 240
Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu
245 250 255
Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu
260 265 270
Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro
275 280 285
Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met
290 295 300
Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp
305 310 315 320
Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe
325 330 335
Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu
340 345 350
Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala
355 360 365
Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
370 375 380
Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu
385 390 395 400
Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg
405 410 415
Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val
420 425 430
Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu
435 440 445
Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn
450 455 460
Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr
465 470 475 480
Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala
485 490 495
Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr
500 505 510
Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln
515 520 525
Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys
530 535 540
Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe
545 550 555 560
Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu
565 570 575
Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
580 585 590
<210> 10
<211> 220
<212> PRT
<213> Homo sapiens
<400> 10
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser
1 5 10 15
Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu
20 25 30
Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln
35 40 45
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg
50 55 60
Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala
65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys
85 90 95
Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val
100 105 110
Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro
115 120 125
Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys
130 135 140
Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys
145 150 155 160
Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr
165 170 175
Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
180 185 190
Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile
195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg
210 215 220
<210> 11
<211> 68
<212> PRT
<213> Homo sapiens
<400> 11
Ser Pro Tyr Ser Ser Asp Thr Thr Pro Cys Cys Phe Ala Tyr Ile Ala
1 5 10 15
Arg Pro Leu Pro Arg Ala His Ile Lys Glu Tyr Phe Tyr Thr Ser Gly
20 25 30
Lys Cys Ser Asn Pro Ala Val Val Phe Val Thr Arg Lys Asn Arg Gln
35 40 45
Val Cys Ala Asn Pro Glu Lys Lys Trp Val Arg Glu Tyr Ile Asn Ser
50 55 60
Leu Glu Met Ser
65

Claims (10)

1. The target PD-L1/HSA/CCL5 trispecific nanobody is characterized in that the amino acid sequence thereof is selected from any one of the amino acid sequences in (1) to (3):
(1) any one of the amino acid sequences shown in SEQ ID NO.1-SEQ ID NO. 8;
or (2) a protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.1-SEQ ID NO.8, wherein the protein mutant has the same function with the protein before mutation;
or (3) an amino acid sequence having at least 90% identity with any one of the amino acid sequences shown in SEQ ID NO.1-SEQ ID NO. 8.
2. The coding gene of the targeted PD-L1/HSA/CCL5 tri-specific nanobody of claim 1.
3. A recombinant expression vector comprising the coding gene of claim 2.
4. A host cell comprising the recombinant expression vector of claim 3.
5. The derivative obtained by conjugating the targeted PD-L1/HSA/CCL5 tri-specific nanobody of claim 1 with a functional molecule.
6. The derivative of claim 5, wherein the functional molecule includes, but is not limited to, one or more of a small molecule drug, a cytotoxic drug, a bioactive protein, a radioisotope, or a fluorescent dye.
7. The use of the targeted PD-L1/HSA/CCL5 trispecific nanobody of claim 1 in the preparation of a medicament or reagent for the treatment or diagnosis of C-FOXP3 elevated tumor-related diseases; preferably, the C-FOXP 3-elevated related tumors comprise pancreatic cancer.
8. The use of a derivative according to claim 5 or 6 for the preparation of a medicament or reagent for the treatment or diagnosis of a tumor associated with elevated C-FOXP 3; preferably, the tumor associated with C-FOXP3 elevation is pancreatic cancer.
9. A pharmaceutical composition for treating tumor, comprising the targeted PD-L1/HSA/CCL5 trispecific nanobody antibody of claim 1, or the derivative of claim 5 or 6, and a pharmaceutically acceptable carrier and/or excipient; preferably, the tumor is a tumor associated with elevated C-FOXP3, and more preferably, the tumor is a pancreatic cancer.
10. A diagnostic kit for detecting tumors, comprising the targeted PD-L1/HSA/CCL5 trispecific nanobody antibody of claim 1, or the derivative of claim 5 or 6; preferably, the tumor is a C-FOXP 3-elevated related tumor, more preferably, the tumor is a pancreatic cancer.
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CN117343194B (en) 2024-03-05
CN114621349B (en) 2023-12-01

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