CN114621349B - Targeting PD-L1/HSA/CCL5 trispecific nano-antibody, derivative thereof and application thereof - Google Patents

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

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CN114621349B
CN114621349B CN202210121855.4A CN202210121855A CN114621349B CN 114621349 B CN114621349 B CN 114621349B CN 202210121855 A CN202210121855 A CN 202210121855A CN 114621349 B CN114621349 B CN 114621349B
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CN114621349A (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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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—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
    • A61K47/50—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
    • A61K47/51—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
    • A61K47/68—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
    • A61K47/6835—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Abstract

The invention discloses a targeting PD-L1/HSA/CCL5 trispecific nano antibody and a derivative and application thereof. The amino acid sequence of the targeting PD-L1/HSA/CCL5 trispecific nanometer antibody provided by the invention is selected from any one of 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 to PD-L1 and CCL 5. The invention further provides application of the PD-L1/HSA/CCL5 targeted trispecific nano-antibody and derivatives thereof in preparing medicaments or reagents for treating or diagnosing tumors related to C-FOXP3 elevation.

Description

Targeting PD-L1/HSA/CCL5 trispecific nano-antibody, derivative thereof and application thereof
Technical Field
The invention relates to a nano antibody for treating or diagnosing pancreatic cancer tumor and a derivative thereof, in particular to a targeting PD-L1/HSA/CCL5 trispecific nano antibody and a derivative thereof, and further designs an application of the targeting PD-L1/HSA/CCL5 trispecific nano antibody and a derivative thereof in preparing a reagent or a medicament for diagnosing or treating tumor, belonging to the field of antibodies for treating or diagnosing tumor.
Background
Pancreatic cancer is a malignant tumor of the digestive tract which has high malignancy degree and is difficult to diagnose and treat, and more than 46 tens of thousands of people die from pancreatic cancer worldwide in 2020. According to the global cancer information grid 2020, pancreatic cancer is the fourth leading cause of amphoteric cancer-related death, with 5-year survival rates just reaching 10%. One study in 28 european countries predicts that pancreatic cancer will become the third leading cause of cancer death by 2025. Pancreatic Ductal Adenocarcinoma (PDAC) is the most common type of pancreatic cancer, accounting for over 90% of pancreatic cancers. However, early PDAC had no obvious symptoms and was difficult to diagnose. Thus, most PDAC patients are not found until late, resulting in a loss of opportunity for early surgical treatment. 80-90% of PDAC patients have unresectable tumors at diagnosis. However, survival rates for 5 years remain low for patients who receive radical excision and adjuvant chemotherapy.
Among the malignant tumors common worldwide, pancreatic cancer incidence is 14 th, death causes are 7 th, and in recent years, pancreatic cancer incidence is on an increasing trend year by year. Statistics of the global cancer database show that 45 ten thousand pancreatic cancer diagnosis cases and 43 ten thousand pancreatic cancer death cases are available worldwide in 2018. The pancreatic cancer incidence rate in different countries is greatly different, and compared with the incidence rate in developing countries, the incidence rate in developed countries is higher. The incidence of chinese pancreatic cancer is at the 10 th position in all malignant tumors and at the 5 th position in the cause of death. Pancreatic cancer patients have a survival rate of about 6% for 5 years, and are one of the worst malignant tumors. Most pancreatic cancer patients are diagnosed later, and only 20% of pancreatic cancer patients have surgical resection conditions at the time of diagnosis, with a 5-year survival rate of 27% for patients who can be successfully surgically resected. Given the continuously rising incidence and low survival rate of pancreatic cancer, it is believed to be important to identify patients with screen Cha Gaowei, 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 poor. Immune Checkpoint Inhibitors (ICI) open 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 negative regulation of the immune system of the body. Tumor cells can suppress the immune surveillance effect of T cells and the like by expressing ligand molecules of immune checkpoints at high levels. Aiming at the mechanism, the interaction between molecules of immune checkpoints is blocked by utilizing a monoclonal antibody with high affinity, so that the activity of killer cells such as T cells is recovered to resist tumors, and the monoclonal antibody becomes an emerging research hotspot for tumor biotherapy. The most mature application research of the immune checkpoint molecules is 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 wide (Li. The application research of immune checkpoint molecular monoclonal antibodies represented by PD-1 in tumor treatment is advanced 2018)
However, the effect of anti-PD-1/PD-L1 or anti-CTLA-4 monoclonal antibodies on PDAC is not ideal at present. PD-L1 is expressed in PDAC, and its overexpression is associated with poor prognosis. Preclinical studies indicate that blocking PD-L1 can inhibit pancreatic cancer progression in animal models, suggesting that the PD-1/PD-L1 pathway may be a potential target for treatment of PDAC. However, clinical trials have shown that targeting the PD-1/PD-L1 pathway with a single drug has little effect on PDAC patients, suggesting that in addition to expression of PD-L1, other targets for treatment of PDAC must be determined.
Recent studies by the present inventors have shown that Cancer fork box protein 3 (Cancer-FOXP 3 or C-FOXP 3) recruits Treg cells into PDACs by upregulating CCL5, thereby promoting immune evasion of PDACs. In this study, the inventors demonstrated that PD-L1 is overexpressed in PDAC samples from two independent radical resected patients. Furthermore, the finding that C-FOXP3 is co-localized with the expression of PD-L1 in tumor cells at the mRNA and protein levels and correlated with the expression of PD-L1 in tumor cells was confirmed by the cancer genomic profile database (TCGA). Chromatin immunoprecipitation (ChIP) showed that C-FOXP3 binds directly to the pancreatic cancer cell PD-L1 promoter region. 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 luciferase activity. In addition, up-regulation of C-FOXP 3-induced PD-L1 expression is effective in inhibiting CD8+ T cell activity. Based on the recent findings by the present inventors that CCL-5 antibodies respond well to PDAC models with high C-FOXP3 levels, the present inventors further demonstrated that PD-L1 antibodies enhanced the anti-tumor effect of CCL-5 blockade in xenogeneic and in situ mouse models with high C-FOXP3 levels (Zhang Ying. Chemokine CCL5 and its receptor with development of autoimmune disease. 2016; li Tianming. Development of intestinal mucosal chemokine study. 2020.
Taken together, direct activation of PD-L1 by C-FOXP3 is a core transcription factor that mediates PDAC immune escape. The combined blocking of PD-L1 and CCL-5 may provide an effective treatment for PDAC patients with elevated C-FOXP3, and the development of bispecific antibodies targeting CCL5 and PDL1 would be of great significance in the treatment of pancreatic cancer.
The traditional full-length antibody biological preparation has the problems of poor tissue penetrability, strong immunogenicity and the like, so the development and application of the antibody medicine with miniaturized, humanized and rationally designed optimization are the development directions of therapeutic antibodies and diagnostic antibodies in the future. VHH nanobodies (nanobodies) have the advantages of high affinity, strong specificity, small molecular weight, low immunogenicity and high stability as outstanding representatives of miniaturized antibodies. Bispecific antibodies (bsabs) are another trend in antibody development that are capable of recognizing and binding two different antigens or epitopes simultaneously, presenting the following advantages in terms of treatment: the double-target signal blocking plays a unique or overlapped function, and effectively prevents drug resistance and immune escape; the method has stronger specificity, targeting property and reduced off-target toxicity; effectively reduces the treatment cost. Nanobodies are easily engineered into multivalent and functional forms for two or more antigens by genetic engineering means. Meanwhile, the nanobody is easy to carry out in vitro affinity maturation in a computer simulation mode so as to obtain an antibody sequence with high affinity. The efficiency of the bispecific single chain nano antibody can be improved by connecting two monovalent small molecular antibodies together, the binding capacity to a target is obviously increased, the therapeutic capacity is further increased, the serum half life of the bispecific nano antibody is prolonged, and the bispecific nano antibody has become a hot spot for engineering antibody research in recent years. Currently, bispecific nanobodies are mainly concentrated in the fields of anti-infection, tumor and immune disease treatment and diagnosis, wherein the research in the fields of tumor treatment and diagnosis is the most extensive. Therefore, the nano antibody has the advantages of small molecular weight, high stability, low immunogenicity, easy connection and the like as a miniaturized antibody, and has unique advantages in constructing PDL1/CCL5 bispecific antibody.
CN105814082a discloses a bispecific nanobody comprising a bispecific polypeptide of a first functional single variable domain and a second immunoglobulin anchoring single variable domain (ISV), wherein the first ISV binds with low affinity to a first target on the surface of a cancer cell and inhibits the function of the first target when bound, and the second ISV binds with high affinity to a second target on the surface of the cell, and wherein the first target is different from the second target. However, in this invention, the bispecific nanobody has the potential to selectively target LSC, normal HSCs and HPGs are not affected by CXCR4 nanobody. In this solution, the bispecific CXCR4-CD4 polypeptide binds both CXCR4 and CD4, resulting in a strongly increased neutralizing potency for HIV using CXCR 4.
The bispecific nanobodies targeting CCL5 and PDL1 are lack of basis in the treatment of pancreatic cancer. During pancreatic carcinogenesis, regulatory T-cell (regulatory T cells, tregs) key transcriptional regulatory programs are expressed and function ectopically in the pancreatic epithelium. Pancreatic ductal epithelial cells, which are mainly characterized by partial pancreatic epithelial cells expressing the Tregs-specific transcriptional molecule FOXP3, foxp3+ promote infiltration of ccr5+ Tregs in pancreatic lesions by direct transcriptional regulatory chemokine CCL-5, together inhibiting the activity of cd8+ killer T cells (Cancer-FOXP 3directly activated CCL5 to recruit FOXP3Treg cells in pancreatic ductal adenocarcinoma [ J ], oncogene, 2017). In addition, pancreatic epithelial tumors FOXP3 (cnacer-FOXP 3, C-FOXP 3) are highly positively correlated with PD-L1 expression, and further analysis found that C-FOXP3 promotes PD-L1 transcription and inhibits CD8+ T cell activity (PD-L1 is a direct target of cancer-FOXP3 in Pancreatic Ductal Adenocarcinoma (PDAC), and combined immunotherapy with antibodies against PD-L1 and CCL5 is effective in the treatment of PDAC [ J ]. Signal transduction and targeted therapy, 2020). Based on the findings, in the preclinical test, PD-L1 and CCL-5 combined blocking antibodies are adopted for simulating treatment aiming at C-FOXP3 high-expression pancreatic cancer, obvious curative effects are obtained, a new accurate treatment strategy is provided for reversing pancreatic cancer immunotherapy drug resistance, or the clinical situation that the single anti-immune checkpoint treatment has poor response effect can be solved.
Immune Checkpoint Inhibitors (ICI) open new approaches for the treatment of various tumors, PD-L1 is expressed in Pancreatic Ductal Adenocarcinoma (PDAC), and its overexpression is associated with poor prognosis, but the current effect of anti-PD-1/PD-L1 mab on treatment of PDAC is not ideal. A prerequisite for effective immune checkpoint inhibitor treatment is high levels of activated Tumor Infiltrating Lymphocytes (TILs) in the tumor tissue. However, most PDACs are characterized by lower levels of TIL activated around tumor tissue due to connective tissue interstitium and various immunosuppressive cells, such as regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells. Studies have shown that Cancer fork box protein 3 (Cancer-FOXP 3 or C-FOXP 3) recruits Treg cells into PDACs by upregulating CCL5, thereby promoting immune evasion of PDACs. The direct activation of PD-L1 by C-FOXP3 is a core transcription factor mediating PDAC immune escape, and the combined blocking of PD-L1 and CCL-5 may provide an effective treatment for PDAC patients with C-FOXP3 elevation. Therefore, developing bispecific nanobodies targeting CCL5 and PDL1 would have significant implications in the treatment of pancreatic cancer.
Disclosure of Invention
One of the purposes of the present invention is to provide a targeting PD-L1/HSA/CCL5 trispecific nanobody and derivatives thereof;
the second object of the invention is to provide a recombinant expression vector containing the targeting PD-L1/HSA/CCL5 trispecific nanobody;
the third object of the present invention is to provide a host cell containing the recombinant expression vector.
The invention also provides a method for preparing the targeting PD-L1/HSA/CCL5 trispecific nano antibody and a derivative thereof, which are applied to preparing a medicament for treating tumors or a reagent for diagnosing tumors.
In order to achieve the above purpose, the technical scheme adopted by the invention comprises the following steps:
in a first aspect, the present invention provides a PD-L1/HSA/CCL 5-targeted trispecific nanobody, the amino acid sequence of which is selected from any one of the amino acid sequences set forth in (1) - (3):
(1) Any one of SEQ ID No.1-SEQ ID No. 8;
(2) A protein mutant obtained by deleting, substituting, inserting and/or adding one or more amino acids in any one of the amino acid sequences of SEQ ID No.1-SEQ ID No.8, wherein the protein mutant has the same function as a protein before mutation;
(3) An amino acid sequence having at least 95% identity to the amino acid sequence of any one of SEQ ID No.1 to SEQ ID No. 8.
In a second aspect, the invention provides a coding gene of the PD-L1/HSA/CCL5 targeted trispecific nanobody.
In a third aspect, the present invention provides a recombinant expression vector comprising the coding gene; the expression vector may be a recombinant prokaryotic expression vector, a recombinant eukaryotic expression vector or other recombinant expression vectors.
In a fourth aspect, the present invention provides a recombinant host cell comprising said recombinant expression vector. Wherein the host cell is a prokaryotic expression cell, a eukaryotic expression cell, a fungal cell or a yeast cell.
In a fifth aspect, the invention provides a derivative of a PD-L1/HSA/CCL5 trispecific nanobody, comprising a derivative obtained by conjugating the PD-L1/HSA/CCL5 trispecific nanobody targeted to a functional molecule, wherein the functional molecule comprises, 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 biologically labeling the targeting PD-L1/HSA/CCL5 trispecific nanobody or the derivative obtained by coupling the targeting PD-L1/HSA/CCL5 trispecific nanobody with a solid medium or a semisolid medium.
The obtained derivative can be applied to preparation of tumor molecular imaging diagnosis reagents or tumor treatment medicines; or the targeting PD-L1/HSA/CCL5 trispecific nano-antibody and the derivative are applied to preparing and diagnosing or treating the relevant tumors with the C-FOXP3 elevation.
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 suitable wavelength. The fluorescent dye is selected from xanthine, acridine, oxazine, cyanine, styryl dye, evans blue, coumarin, porphyrin, metal ligand-complex, fluorescent protein, nanocrystal, perylene, borodipyrromethene, and phthalocyanine, and conjugates and combinations of these types 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 used 64 Cu, 67 Ga, 68 Ga, 89 Zr, 18 F, 86 Y, 90 Y, 111 In, 99m Tc, 125 I, 124 Labeling radioisotope such as I to obtain a molecular imaging diagnostic drug for PET (positron emission tomography) or SPECT; or using anti-single domain antibodies, fc fusion proteins and immunoconjugates 90 Y， 177 Lu, 125 I, 131 I, 211 At, 111 In, 152 Sm, 166 Ho， 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bi, 223 Ra, 227 The radioactive isotopes such as Th are labeled and used for the therapeutic drugs for FAP-related diseases.
In addition, the radioisotope can directly label the PD-L1/HSA/CCL5 targeted trispecific nanobody or a derivative thereof; the PD-L1/HSA/CCL5 trispecific nanobodies or derivatives thereof may also be indirectly labeled with chelators including, but not limited to, 1,4,7, 10-tetraazacyclododecane-N, N ', N ' -tetraacetic acid (DOTA), ethylenediamine tetraacetic 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-trisazo-1-yl) glutaric acid (NODAGA), 2- (4, 7, 10-tris (carboxymethyl) -1,4, 7-10-tetraazacyclododecane-1-yl) glutaric acid (DOTAGA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N, N, N ', N "-pentaacetic acid (DTPA), bis- (carboxymethyl) -1,4, 7-tetraazo-1-tetraacetic acid (DOPA) and (DTPA) 3-phenylhydrazine, any one or more of N' - {5- [ acetyl (hydroxy) amino ] pentyl } -N- [5- ({ 4- [ (5-aminopentyl) (hydroxy) amino ] -4-oxobutanoyl } amino) pentyl ] N-hydroxysuccinamide (DFO) and the like.
Thus, the sixth aspect of the invention provides application of the PD-L1/HSA/CCL5 trispecific nanobody targeted, the PD-L1/HSA/CCL5 trispecific nanobody targeted encoding gene and the PD-L1/HSA/CCL5 trispecific nanobody targeted derivatives in preparation of reagents or medicines for diagnosing or treating tumors related to 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 targeting 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 tumor, chemotherapeutics or small molecule inhibitors, etc.
In an eighth aspect, the invention provides a kit for diagnosing or treating a tumor, which comprises any one or more of the PD-L1/HSA/CCL 5-targeted trispecific nanobodies or derivatives thereof.
The tumor described in the present invention is a C-FOXP 3-elevated tumor, preferably pancreatic cancer, more preferably pancreatic ductal adenocarcinoma.
Definition of terms in connection with the present invention
The term "Nanobody" as used herein refers to a fragment comprising a single variable domain in an antibody, also known as a Nanobody (Nanobody). As with intact antibodies, it can bind selectively to specific antigens. Single domain antibodies appear much smaller than the 150-160kDa mass of intact antibodies, approximately only 12-15kDa. The first single domain antibody was engineered from a heavy chain antibody of a camel and is referred to as the "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 the invention SEQ ID NO:1-198, and having substantially the same properties as those having greater than 80%, greater than 85%, greater than 90%, greater than 95%, or greater than 99% sequence identity thereto, and which are within the scope of the present invention. Preferably, the present invention achieves sequence identity through conservative substitutions, but is not limited to conservative substitutions.
The term "amino acid sequence" refers to the order in which amino acids are linked to one another to form a peptide chain (or polypeptide), and the amino acid sequence can be read in only one direction. There are 100 different types of amino acids, 20 of which are commonly used, and the present invention does not exclude other substances on the amino acid chain, such as sugar, lipid, etc., and the present invention is not limited to the commonly used amino acids in 20.
The term "nucleotide sequence" refers to the arrangement of bases in DNA or RNA, i.e., A, T, G, C in DNA, or A, U, G, C in mRNA, including rRNA, tRNA, mRNA. It should be understood that the antibody genes claimed in the present invention encompass RNA (rRNA, tRNA, mRNA) and their complements in addition to DNA sequences.
The substitutions described in the present invention may be conservative substitutions, i.e. the substitution of a particular amino acid residue for one having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include those between aliphatic group-containing amino acid residues (e.g., ile, val, leu or inter-substitution between Ala), those between polar residues (e.g., between Lys and Arg, glu and Asp, gln and Asn), and the like. Mutants resulting from the deletion, substitution, insertion and/or addition of amino acids can be made by subjecting the DNA encoding the wild-type protein to site-directed mutagenesis as known in the art (see, for example, nucleic Acid Research, vol.10, no.20, p.6487-6500, 1982, 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, more preferably 5 or less. In the case of the site-directed mutagenesis method, for example, in addition to the desired mutation, i.e., the specific inconsistency, the synthesis of oligonucleotide primers complementary to the single-stranded phage DNA to be mutated can be performed as follows. That is, a strand complementary to phage is synthesized using the above synthetic oligonucleotide as a primer, and the obtained double-stranded DNA is used to transform a host cell. Cultures of transformed bacteria were plated on agar to form plaques from phage-containing single cells. Then, plaques hybridized with the probe were collected, cultured, and DNA was recovered. Furthermore, methods of deleting, substituting, inserting and/or adding one or more amino acids to the amino acid sequence of a biologically active peptide such as an enzyme while maintaining the activity thereof include methods of treating a gene with a mutagenesis source in addition to the above-mentioned site-directed mutagenesis, and methods of selectively cleaving a gene, deleting, substituting, inserting or adding a selected nucleotide, and then ligating.
The term "recombinant expression vector (Expression vectors)" refers to a vector in which expression elements (e.g., promoter, RBS, terminator, etc.) are added to the basic skeleton of a cloning vector so that a desired gene can be expressed. Expression vector four parts: a target gene, a promoter, a terminator and a marker gene. The invention includes, but is not limited to, prokaryotic expression vectors, eukaryotic expression vectors, or other cellular expression vectors.
The terms "mutation" and "mutant" have their usual meaning herein, referring to genetic, naturally occurring or introduced changes in a nucleic acid or polypeptide sequence, which are in the same sense as commonly known to those skilled 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 to insert to produce a recombinant host cell, such as 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 schematic diagram of a structure of a PDL1/CCL5 targeting nanobody constructed based on an anti-HSA nanobody.
FIG. 2 is a PD-L1-GST SDS-PAGE coacervation electrophoresis; PD-L1 is 26Kd in size, and due to glycosylation, the protein migrates at a speed of 30-35Kd under reducing conditions, and GST tag is 26Kd in size; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 3 is a gel electrophoresis chart of CCL5-GST SDS-PAGE; CCL5 protein is 8Kd in size, GST tag is 26Kd in size, and the target strip is located at about 34 Kd; a band is also arranged around 26Kd, which is the result of partial protein GST label falling off; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-8.
FIG. 4 is a diagram of HSA-GST SDS-PAGE coacervation electrophoresis; HSA protein has a size of 69Kd, GST tag has a size of 26Kd, and the target band is located at about 95 Kd; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 5 is a diagram of BC8P2 SDS-PAGE gel electrophoresis; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-9.
FIG. 6 is a photograph of a BC8P3 SDS-PAGE gel; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-9.
FIG. 7 is a diagram of BC11P2 SDS-PAGE gel electrophoresis; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-9.
FIG. 8 is a photograph of a BC11P3 SDS-PAGE gel; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 9 is a BP2C8 SDS-PAGE gel; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-9.
FIG. 10 is a BP2C11 SDS-PAGE gel; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 11 is a BP3C8 SDS-PAGE gel; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 12 is a BP3C11 SDS-PAGE gel; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 13 is a graph showing the results of ELISA for verifying the binding activity of an antibody to PD-L1 under a concentration gradient.
FIG. 14 is a graph showing the results of ELISA for verifying the binding activity of antibodies to CCL5 under concentration gradient.
FIG. 15 shows the results of ELISA for verifying the binding activity of antibodies to HSA under concentration gradients.
FIG. 16 is a SPR sensorgram of interaction between BC8P2 and PD-L1.
FIG. 17 is a SPR sensorgram of interaction between BC8P3 and PD-L1.
FIG. 18 is a SPR sensorgram of interaction between BC11P2 and PD-L1.
FIG. 19 is a SPR sensorgram of interaction between BC11P3 and PD-L1.
FIG. 20 is a SPR sensorgram of the interaction between BP2C8 and PD-L1.
FIG. 21 is a SPR sensorgram of the interaction between BP2C11 and PD-L1.
FIG. 22 is a SPR sensorgram of the interaction between BP3C8 and PD-L1.
FIG. 23 is a SPR sensorgram of the interaction between BP3C11 and PD-L1.
FIG. 24 is a SPR sensorgram of interactions between BC8P2 and CCL 5.
FIG. 25 is a SPR sensorgram of interactions between BC8P3 and CCL 5.
FIG. 26 is a SPR sensorgram of interactions between BC11P2 and CCL 5.
FIG. 27 is a SPR sensorgram of interactions between BC11P3 and CCL 5.
FIG. 28 is a SPR sensorgram of the interaction between BP2C8 and CCL 5.
FIG. 29 is a SPR sensorgram of the interaction between BP2C11 and CCL 5.
FIG. 30 is a SPR sensorgram of the interaction between BP3C8 and CCL 5.
FIG. 31 is a SPR sensorgram of the interaction between BP3C11 and CCL 5.
FIG. 32 is a SDS-PAGE gel after nickel ion purification; "M" is a protein marker; the whole is the whole bacteria lysate, the upper part is the lysate supernatant, the sediment is the lysate sediment, the penetrating part is the penetrating peak, and the eluting peaks are numbered 1-10.
FIG. 33 is a SDS-PAGE gel after purification of the molecular sieves; "M" is protein marker, and numbers 1-10 are elution peaks.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Biological material and reagent
1. Cell lines and production strains
In the invention, HEK-293F cell lines are used for preparing PDL1, HSA and CCL5 antigens. Human embryonic kidney cells HEK-293F (RRID: CVCL_6642) were taken from the U.S. cell culture center. Over the last three years, all human cell lines passed the verification of STR analysis. Mycoplasma contamination was excluded from these cell lines. HEK-293F cells are placed in a 5% CO2 constant temperature shaking table for constant temperature shaking culture at 37 ℃ and 120rpm (during passage, cell counting and cell viability observation are needed, and the density is selected as much as 3-6×10) 6 High-activity cells of cells/mL were subcultured. Cell density and viability were determined prior to transient transfection of cells. To ensure the transfection effect, it is recommended to use a growth in exponential phase (density of about 2-4X 10 6 Cell transfection at a rate of greater than 98% per milliliter). If the cell density is>2.0×10 6 Fresh KOP293 medium was added to dilute the cell density to 2X 10 per mL 6 And each mL. Shake flask placed in 5% CO 2 In a constant temperature shaking table, the transfection is started after 10min of constant temperature shaking culture at 37 ℃ and 120 rpm. 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 sterile centrifuge tubes, and gently blowing and uniformly mixing; taking the other branch of the separation tube, adding 10mL of transfection medium and 1mL of transfection reagent, and gently blowing and uniformly mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and lightly blowing and uniformly mixing; standing for 10 minutes at room temperature to prepare a plasmid-carrier compound; and (5) taking out the cells from the constant temperature shaking table, adding the prepared plasmid-carrier complex, and putting the plasmid-carrier complex back into the CO2 constant temperature shaking table for shake culture. 24 hours after transfection, 1mL 293 cell protein expression enhancer and 4mL nutritional additives may be added, and after four days of transfection, 50mL fresh medium may be added, and after seven days of transfection, the fermentation broth may be collected.
The three-specificity nano-antibodies of the targeting PDL1/CCL5 constructed based on the anti-HSA nano-antibody all adopt TransB (DE 3) expression bacteria.
DH5 alpha competent cells used for plasmid cloning were purchased from Beijing as century Inc., and TransB competent cells used for nanobody expression were purchased from Beijing full gold Co. The constructed expression plasmid and pSOX plasmid are co-transfected into Trans B (DE 3) competent cells, engineering bacteria are inoculated into a 5mL LB liquid culture medium test tube, the inoculation amount is 1 percent, the consumption of ampicillin and chloramphenicol is one thousandth, and the engineering bacteria are cultured for about 8 hours at the speed of 220rpm in a constant temperature shaking table at the temperature of 37 ℃. 2mL of the activated bacteria were inoculated into 200mL conical flasks of LB medium at an inoculum size of 1%, 200. Mu.L of ampicillin and chloramphenicol mother liquor were added, respectively, and cultured in a constant temperature shaker at 37℃and 220 rpm. When the cells grew until the OD600 was about 0.4, 4mL of arabinose was added to the LB medium, and after 45min of culture at 30℃and 220rpm, 80. Mu.L of isopropyl-. Beta. -D-thiogalactoside (IPTG) solution was added, and the mixture was cultured at 20℃and 160rpm for 16 hours. The culture broth was centrifuged at 8000rpm at 4℃for 15min, and the cells were collected. The cells were resuspended in PBS and washed twice again by centrifugation, and the cells were weighed and stored in a-20deg.C refrigerator. After about 20mL of PBS was used for dissolving every 1g of the cells, the cells were broken by 180W ultrasound for 25min at a frequency of 3s to 5s. Followed by centrifugation at 10,000rpm at 4℃for 20min. And (5) a small amount of whole bacterial liquid before centrifugation, sediment and supernatant after centrifugation are reserved. The supernatant was filtered through a 0.45 μm filter and purified by the AKTA prime protein purification system.
2. Reagent(s)
1) LB medium: 10g of tryptone, 5g of yeast extract and 10g of NaCl are added into about 900mL of deionized water, the pH is adjusted to 7.4 by dissolution, the volume is fixed to 1L, and the mixture is packaged and sterilized for 20min at 121 ℃ in a high-pressure steam sterilizing pot. The solid culture medium is prepared by adding 15g of agar powder into each liter of culture medium after the liquid culture medium is subpackaged, and sterilizing by high-pressure steam.
2) PBS solution: 0.14mol of NaCl (8.18 g), 3mmol of KCl (0.22 g), 0.01mol of Na2HPO4 (1.42 g), 2mmol of KH2PO4 (0.27 g) were dissolved in about 900mL of deionized water, the pH was adjusted to 7.4, the volume was adjusted to 1L, and the autoclave was sterilized at 121℃for 20 minutes.
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 refrigerator at-20deg.C.
4) 100mg/mL Amp solution: 100mg ampicillin was dissolved in 1mL deionized water; filtering with 0.22 μm filter membrane, and storing in refrigerator at-20deg.C.
5) Chloramphenicol solution at 250 mg/mL: 250mg chloramphenicol was dissolved in 1mL deionized water; filtering with 0.22 μm filter membrane, and storing in refrigerator at-20deg.C.
6) Equilibration buffer (50 mmol/L imidazole buffer): 0.5mol NaCl (29.22 g), 0.01mol Na2HPO4 (1.4196 g), 0.01mol NaH2PO4 (1.1998 g), 50mmol imidazole (3.404 g) were dissolved in 900mL deionized water, adjusted to pH=7.4, and then fixed to 1L, and sterilized at 121℃for 20min.
7) Elution buffer (500 mmol/L imidazole buffer): the imidazole concentration was increased to 500mmol (34.04 g) in equilibration buffer and sterilized at 121℃for 20min.
8) 250mg/mL arabinose solution: 12.5g of arabinose is dissolved in deionized water, the volume is fixed to 50ml, and after filtration through a 0.22 mu m filter membrane, the mixture is preserved in a refrigerator at the temperature of minus 20 ℃.
Example 1 design of targeting PD-L1/HSA/CCL5 trispecific nanobodies, construction of plasmids, expression and purification of proteins
1. Test method
The amino acid sequences of human Human Serum Albumin (HSA), programmed cell death 1ligand 1 (PD-L1), regulated upon activation normal T cell expressed and secreted factor (CCL 5) were subjected to eukaryotic (human) expression optimization and gene synthesis, and the synthesized gene fragment was subcloned into eukaryotic expression vector pcDNA3.1-N-GST-TEV. Recombinant proteins were produced by HEK-293F cell protein expression systems and purified using GSTrapFF chromatography columns and AKTA primer plus (GE Healthcare) affinity chromatography. The basic principle of purification is that GST label on the surface of protein and reduced glutathione covalently bound on chromatographic column form coordination interaction, so that the target protein is adsorbed on the chromatographic column, the protein is separated from protein mixed liquor, 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), BP3C11 (SEQ ID No. 8) were combined for 8 trispecific nanobodies, the amino acid sequences were optimized for prokaryotic (E.coli) expression, the nanobody sequences were synthesized by the Bioengineering company and cloned into the prokaryotic expression vector pET-32a (+) with the His tag attached at the C-terminus of the antibody. Expression plasmid to be constructed and pSOX plasmidProtein expression was performed in cotransfected Trans B (DE 3) competent cells. The supernatant was extracted by sonication of the fermented cells, and was purified by AKTA prime protein purification System and 5mL His Trap from GE company TM And (3) performing affinity chromatography purification on the HP pre-packed column to obtain the corresponding nano antibody. The purification procedure was as follows:
1) The instrument was washed clean of ethanol by deionized water filtered through a 0.45 μm filter.
2) A nickel ion affinity chromatography column with a column volume of 5mL was mounted to the instrument and deionized water was used to rinse clean the column of ethanol 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 each line was leveled, requiring about 70mL equilibration buffer.
4) Loading at a flow rate of 1mL/min, taking a penetrating peak, flushing with an equilibration buffer at a flow rate of 1mL/min for two minutes after loading, allowing the sample to flow through the chromatographic column completely, and then equilibrating the column at a flow rate of 4 mL/min.
5) Washing the protein with eluting buffer solution at a flow rate of 4mL/min, and taking out the eluting peak, wherein the protein of the eluting peak is the nano antibody.
6) After all bound proteins are eluted, the solution is rinsed with deionized water until the salt ion concentration is zeroed.
7) Washing with 50mL of 20% ethanol, soaking the column in ethanol, removing the column, preserving at 4deg.C, and closing the instrument.
The nanobody injection after nickel ion affinity purification is further purified by an AKTA Pure 25 protein chromatography purification system and a (2) Superdex 75/300 GL high-efficiency column of GE company. The purification procedure was as follows:
1) The pump inlets A1 and B1 were placed in deionized water and the pumps A and B were rinsed with deionized water by a pump rinse procedure. The B1 pump inlet was then placed in PBS and the solution in the B pump was replaced with PBS by a pump wash procedure.
2) A15 ml centrifuge tube was placed on the collector for subsequent collection of samples (also through the waste port, there was about 0.8ml lag from UV detection signal to waste port, but the collector would not have a centrifuge tube following alarm), a loading ring (500 ul) of appropriate volume was installed, and the loading ring was rinsed successively with deionized water and PBS with the syringe in the loaded state.
3) An alarm pressure was set, the system pressure was 20MPa, and the column pressure pre column pressure was 1.8MPa. A low flow rate (0.1 ml/min) was set to connect the column to the system, ensuring that no bubbles entered the column during this process.
4) The two column volumes (50 ml) were rinsed with deionized water and the two column volumes (50 ml) were rinsed with PBS to flush the lines, the flow rate was adjusted to 0.4ml/min, taking care that Quan Chengzhu pressure did not exceed 1.8MPa.
5) The sample is driven into a loading ring (the volume of the sample is larger than the volume of the loading ring) by a syringe under the load state, the sample is loaded after the sample loading is started after the sample loading is finished and the load mode is set, and the sample collection is carried out by setting the peak collection condition (the sample can be collected from a waste liquid port by observing ultraviolet peaks, and a tube is collected every 500 ul)
6) After the experiment was completed, the pumps a and B were washed with deionized water, the PBS in the column was washed with deionized water, the pumps a and B were washed with 20% ethanol, and the column was filled with 20% ethanol.
7) And taking down the column, ending the program operation, and storing the column and experimental data. Shut down the program, computer and instrument.
2. Test results
FIGS. 2 to 4 are SDS-PAGE gel electrophoresis of PD-L1-GST, CCL5-GST and HSA-GST, respectively; FIGS. 5 to 12 are SDS-PAGE gel electrophoresis of BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11, respectively.
Test example 1 binding Activity of Targeted PD-L1/HSA/CCL5 trispecific nanobody against antigen test 1 test method
Binding activity of three specific nano antibodies of BC8P2, BC8P3, BC11P2, BC 2P 3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8, BP3C11 with GST-tagged HSA (SEQ ID No. 9), PDL1 (SEQ ID No. 10) and CCL5 (SEQ ID No. 11) on PDL1, CCL5 and HSA was verified by indirect ELISA experiments using anti-His murine monoclonal antibodies with HRP tags as primary antibodies.
1) Coating: diluting the antigen to a final concentration of 50 μg/mL, adding 100 μl to each well of the 96-well plate, and coating overnight at 4deg.C; the negative control was BSA.
2) Washing: the next day, the coating liquid is discarded; 200. Mu.L of PBST solution was added to each well, gently shaken for 3min, and the solution was discarded; the washing was performed three times.
3) Closing: 200. Mu.L of blocking solution was added to each well and incubated in an oven at 37℃for 2.5 hours.
4) Washing: discarding the sealing liquid; wash with PBST solution, step 2).
5) Adding an antibody: setting the concentration gradient of the nano antibody to be 100nM, 500nM, 100nM, 10nM, 1nM, 100pM and 10pM, sequentially adding 100 mu L of nano antibody solution into each well, and setting three parallel holes; the mixture was placed in an oven at 37℃for 2.5 hours.
6) Washing: discarding the liquid in the hole; adding PBST solution, gently shaking for 5min, and discarding the solution; the washing was performed five times in total.
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 100 mu L of secondary antibody is added into each well; the mixture was placed in an oven at 37℃for 1 hour.
8) Color development: discarding the liquid in the hole; adding PBST solution for washing, wherein the step is the same as the step 6); after the liquid in the holes is thrown out forcefully, 100 mu L of TMB color development liquid is added into each hole; the oven was incubated at 37℃for 20 minutes in the dark.
9) Termination reaction and measurement: 100. Mu.L of stop solution was added to each well to terminate the reaction; OD450 values were determined per well.
2. Test results
FIG. 13 shows the results of binding activities of the three-specific nano-antibodies BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8, BP3C11 on PD-L1 in ELISA assay concentration gradients, FIG. 14 shows the results of binding activities of the three-specific nano-antibodies BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8, BP3C11 on CCL5 in ELISA assay concentration gradients, and FIG. 15 shows the results of binding activities of the three-specific nano-antibodies BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8, BP3C11 on HSA in ELISA assay concentration gradients. According to the test results, BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8 and BP3C11 trispecific nanobodies have extremely strong binding activity to CCL5, HAS and PD-L1.
Test example 2 affinity assay for PD-L1/HSA/CCL 5-targeting trispecific nanobodies
1. Test method
SPR experiments are commonly used to accurately determine the affinity constant KD of an antigen-antibody reaction. The SPR affinity was determined in this test using a Biacore T200 instrument (GE Healthcare), chip model CM-5, at 25 ℃. Purified GST-tagged PD-L1, CCL5 or HSA proteins (2 units in 10mM HEPES, pH7.4,150mM NaCl,3mM EDTA,0.005%P20) were covalently bound to CM5 sensor chip at an immobilization density of about 350 Resonance Units (RUs). An unrelated biotinylated protein was immobilized on a reference surface matched to RU values to control non-specific binding. Diluting affinity chromatography and purified nanobody by BIAcore running buffer (10mM HEPES,pH 7.4,150mM NaCl,3mM EDTA,0.05% (v/v) P20), setting the concentration gradient of the nanobody to be 5 times of dilution concentration of 100 mug/mL-6.25 mug/mL, and selecting one concentration setting to repeat experiment; the program was set up for loading while measurements were made at a flow rate of 40 ℃. The running buffer without nanobody was then passed through the chip, allowing spontaneous dissociation at the same flow rate. After each run, 10mM pH 2.0 glycine was injected to regenerate the sensor chip. After the experiment is finished, the machine is started, the obtained data is imported into instrument matched software for analysis, a Langmuir binding model of 1:1 is adopted, and affinity constants K are calculated after data fitting D Values.
2. Test results
The test results are shown in Table 1 and FIGS. 16-31.
TABLE 1 affinity constants of trispecific nanobodies for PD-L1 and CCL5
K D /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 table 1 and the affinity measurements of fig. 16-31, BC8P2, BC8P3, BC11P2, BC11P3, BP2C8, BP2C11, BP3C8, BP3C11 trispecific nanobodies have very strong affinity for both PD-L1 and CCL 5.
Test example 3 Scale preparation of antibody biological preparation (pilot plant)
After the bacterial cells were harvested on a pilot scale, the purity was 80% or higher after purification by Ni ion affinity chromatography and 98% or higher after purification by SEC, and FIG. 32 is an SDS-PAGE gel electrophoresis chart after purification of Ni ions. FIG. 33 is a SDS-PAGE gel after purification of the molecular sieves;
the yields after antibody purification are shown in table 2.
Table 2 production of antibody biologicals at pilot scale
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Sequence listing
<110> Qingdao university affiliated Hospital
<120> targeting PD-L1/HSA/CCL5 trispecific nano-antibody, 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 (12)

1. The targeting PD-L1/HSA/CCL5 trispecific nano-antibody is characterized in that the amino acid sequence is shown as SEQ ID NO. 5.
2. The gene encoding the PD-L1/HSA/CCL5 trispecific 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 PD-L1/HSA/CCL 5-targeted trispecific nanobody of claim 1 with a functional molecule.
6. The derivative of claim 5, wherein the functional molecule comprises one or more of a small molecule drug, a cytotoxic drug, a bioactive protein, a radioisotope, or a fluorescent dye.
7. Use of the PD-L1/HSA/CCL5 trispecific nanobody of claim 1 in the manufacture of a medicament or agent for treating or diagnosing a tumor associated with elevated C-FOXP 3; wherein the tumor associated with elevated C-FOXP3 includes pancreatic cancer.
8. Use of a derivative according to claim 5 or 6 for the manufacture of a medicament or agent for the treatment or diagnosis of a tumor associated with elevated C-FOXP 3; wherein the tumor associated with the increased C-FOXP3 is pancreatic cancer.
9. A pharmaceutical composition for treating a tumor, comprising the PD-L1/HSA/CCL 5-targeted trispecific nanobody of claim 1, or the derivative of claim 5 or 6, and a pharmaceutically acceptable carrier and/or excipient; wherein the tumor is a tumor associated with elevated C-FOXP 3.
10. The pharmaceutical composition of claim 9, wherein the tumor is pancreatic cancer.
11. A diagnostic kit for detecting a tumor comprising the PD-L1/HSA/CCL 5-targeted trispecific nanobody of claim 1, or the derivative of claim 5 or 6; wherein the tumor is a tumor associated with elevated C-FOXP 3.
12. The diagnostic kit of claim 11, wherein the tumor is pancreatic cancer.
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CN109265548A (en) * 2018-09-13 2019-01-25 东南大学 Anti- PD-L1 nano antibody and its coded sequence, preparation method and application
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EP2097451A2 (en) * 2006-12-22 2009-09-09 Ablynx N.V. Anti-chemokine (ccl2, ccl3, ccl5, cxcl11, cxcl12) single-domain antibodies

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CN109265548A (en) * 2018-09-13 2019-01-25 东南大学 Anti- PD-L1 nano antibody and its coded sequence, preparation method and application
CN113461824A (en) * 2020-03-31 2021-10-01 普米斯生物技术(珠海)有限公司 Platform for constructing multispecific antibody

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