CN112574309B - anti-PD-L1 nano antibody and application thereof - Google Patents

anti-PD-L1 nano antibody and application thereof Download PDF

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CN112574309B
CN112574309B CN202011309419.7A CN202011309419A CN112574309B CN 112574309 B CN112574309 B CN 112574309B CN 202011309419 A CN202011309419 A CN 202011309419A CN 112574309 B CN112574309 B CN 112574309B
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屈向东
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Qiyu Biotechnology Shanghai Co ltd
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Abstract

The invention provides an anti-PD-L1 nanobody and application thereof, wherein the anti-PD-L1 nanobody at least comprises a VHH fragment, and the VHH fragment comprises three amino acid fragments of CDR1, CDR2 and CDR 3. The anti-PD-L1 nano antibody and the Fc fusion protein thereof have strong specificity, high affinity, weak immunogenicity to human and obvious anti-tumor effect.

Description

anti-PD-L1 nano antibody and application thereof
Cross Reference to Related Applications
The present application claims priority to a trifunctional fusion protein containing a TGF- β inhibitor and its use, filed on 5 th month 2019, 12, under the patent application number cn201911235055.X, the entire contents of which are incorporated herein by reference.
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to an anti-PD-L1 nano antibody and Fc fusion protein thereof, and application of the anti-PD-L1 nano antibody and Fc fusion protein thereof.
Background
In classical immune surveillance theory, the immune system can recognize and eliminate tumor antigens. Immune clearance can be stabilized if the immune system is able to completely eliminate tumor cells. If tumor cells escape clearance of the immune system by mutation, the immune system will rebalance. During this process, the immunogenicity of the tumor cells gradually decreases. The proliferative capacity of tumor cells becomes weaker under the stress of the immune system, making detection of tumor cells more difficult.
Activation of oncogenes causes tumor cells to alter themselves and the tumor microenvironment, such that the balance between the immune system and the tumor cells is broken. When the immune system and tumor cells enter the escape phase, the malignancy of the tumor cells increases, while the tumor cells lose MHC molecules so that they are prevented from being recognized and eliminated by the immune cells. Tumor microenvironments may also inhibit the immune system by releasing immunosuppressive factors, such as IL-10, TGF-beta, and the like. Immunosuppressive proteins (e.g., programmed death ligand-1, PD-L1) are also highly expressed on the tumor cell surface, and when effector T cells bind to tumor cells, PD-L1 interacts with PD-1 and induces apoptosis of T cells, which is one of the main causes of tumor tolerance to the immune system, and tumor rapidly grows and metastasizes. If the host's immune system is artificially activated and redirected to tumor cells, tumor tissue can theoretically be cleared, and the theory of immunotherapy has been widely demonstrated in clinical therapy.
Immunotherapy can be divided into two categories: specific and non-specific treatments. Specific therapies within the class include the following therapeutic strategies: tumor vaccines activate antigens of immune cells to a patient by injecting tumors. The tumor vaccine comprises: inactivated tumor cell vaccines, tumor antigen vaccines, tumor DNA vaccines, dendritic Cell (DC) vaccines and bacterial vaccines. Specific ACT immunotherapy mainly includes three therapeutic approaches:
a) Tumor Infiltrating Lymphocytes (TIL): lymphocytes are isolated from tumor tissue and cultured in vitro. TIL can secrete IL-2 with specific anti-tumor capabilities.
b) T Cell Receptor (TCR) treatment: t cells recognize tumor antigens by their single chain antibody fragments (scFv), and single chain antibody fragment TCRs are cloned into normal T cells by viral vectors. Thus, normal T cells become specific tumor killing T cells.
c) CAR-T: t cells are genetically modified to obtain T cells with tumor specific receptors. Unlike conventional T cell recognition mechanisms, CAR-T cells recognize tumor antigens without restriction by MHC molecules. Thus, CAR-T cells can overcome the immune escape mechanism of tumors by increasing co-stimulatory signaling molecules, enhancing the killing capacity of T cells against tumor cells.
In non-specific ACT immunotherapy, there are two main methods of treatment: lymphokine activated killer (lymphokine activated killer, LAK) cell therapy and cytokine induced killer (cytokine induced killer, CIK) cell therapy, respectively.
a) LAK cell therapy: LAK cells stimulate immune cells, including NK cells and T cells, etc., in peripheral blood lymphocytes with IL-2 on the one hand, and on the other hand, by overexpressing FAS ligands, enhance the ability to recognize target cells and kill tumor cells by releasing perforin and granzyme cells.
b) CIK cell therapy: CIK cells are derived from Peripheral Blood Lymphocytes (PBLs) of a patient or healthy person and are expanded under in vitro conditions under stimulation with anti-CD 3 antibodies, IFN-gamma and IL-2. CIK cells exert antitumor effects mainly through FasL and perforin.
Immune checkpoints are protective molecules in the human immune system that prevent inflammatory injury in the normal body due to T cell over-activation. The tumor cells can utilize the characteristic, over-express immune checkpoint molecules, inhibit the immune response of the organism, and evade the monitoring and killing of the immune system of the human body, thereby promoting the growth of the tumor cells. Immune checkpoint inhibitor treatment can achieve an anti-tumor effect by inhibiting immune checkpoint activity in the tumor microenvironment, reactivating the immune response of T cells to the tumor. Complete activation of T cells is regulated by a "dual signal" system: the first signal comes from the specific binding of its own TCR (T cell receptor) to the MHC of the antigen, i.e. the T cell recognizes the antigen; the second signal is derived from a costimulatory molecule, which is involved in the interaction of the costimulatory molecule expressed by an antigen-presenting cell (APC) with a corresponding receptor or ligand (e.g. CD 28) on the surface of a T cell. For example, CD28-B7 is a positive co-stimulatory signal, while negative co-stimulatory molecules are predominantly CTLA4-B7 pathway and PD-1/PD-L1 pathway. After tumor cell invasion, this inhibition pathway is favored by tumor cells to inhibit T cell activation, thereby evading the clearing effect of the immune system.
PD-1 (CD 279) was reported earlier in 1992, the human PD-1 encoding gene PDCD1 was located at 2q37.3, full length 2097bp, and consisted of 6 exons, the translation product was a PD-1 precursor protein consisting of 288 amino acids, and the mature protein was obtained after cleavage of the signal peptide consisting of the first 20 amino acids. PD-1 comprises an IgV domain of an extracellular immunoglobulin variable region, a hydrophobic transmembrane domain and an intracellular domain, and the N-terminal ITIM motif of the intracellular tail domain comprises 2 phosphorylation sites and the C-terminal ITSM motif. PD-1 is a membrane protein belonging to the CD28 immunoglobulin superfamily, mainly expressed on the surface of activated T cells, and also expressed in low abundance on thymus CD4-CD8-T cells, activated NK cells and monocytes. PD-1 has 2 ligands, PD-L1 (CD 274, B7-H1) and PD-L2 (CD 273, B7-DC) of the B7 family of proteins, respectively, with 40% identity in the PD-L1 and PD-L2 amino acid sequences. The difference between the two is mainly that the expression modes are different, the constitutive low expression of PD-L1 is carried out on APC, non-hematopoietic cells (such as vascular endothelial cells and islet cells) and immune-free parts (such as placenta, testis and eyes), and inflammatory cytokines such as type I and type II interferon, TNF-alpha, VEGF and the like can induce the expression of PD-L1. PD-L2 is expressed only in activated macrophages and dendritic cells. After the PD-1 and the PD-L1 are combined with activated T cells, the ITSM motif of the PD-1 is subjected to tyrosine phosphorylation, so that dephosphorylation of downstream protein kinase Syk and PI3K is further caused, activation of downstream AKT, ERK and other channels is inhibited, and finally transcription and translation of genes and cytokines required by T cell activation are inhibited, so that the effect of negatively regulating the activity of the T cells is exerted.
In tumor cells, tumor cells and tumor microenvironments negatively regulate T cell activity by up-regulating PD-L1 expression and binding to PD-1 on the surface of tumor-specific cd8+ T cells, inhibiting immune response. Tumor cells can up-regulate PD-L1 expression by 4 pathways: 1. amplification of the gene encoding PD-L1 (9p24.1); EGFR, MAPK, PI3K-Akt signaling pathway activation, HIF-1 transcription factors and the like can up-regulate PD-L1 expression from the transcription level; induction of epstein barr virus (epstein barr virus positive gastric cancer and nasopharyngeal carcinoma are shown by high expression of PD-L1); and 4. Epigenetic regulation. In the tumor microenvironment, the stimulation of inflammatory factors such as interferon-gamma and the like can induce the expression of PD-L1 and PD-L2. Inflammatory factors can induce other cells in the tumor microenvironment, including macrophages, dendritic cells and stromal cells to express PD-L1 and PD-L2, while tumor-infiltrating T cells capable of recognizing tumor antigens can secrete interferon-gamma, thereby inducing up-regulation of PD-L1 expression, a process known as "adaptive immune resistance" by which tumor cells can achieve self-protection. There is increasing evidence that tumors utilize PD-1 dependent immunosuppression to evade immunity. High expression of PD-L1 and PD-L2 has been found in a variety of solid tumors and hematological malignancies. In addition, there is a strong correlation between the expression of PD-Ls and the poor prognosis of tumor cells, demonstrating that it includes esophageal, gastric, renal, ovarian, bladder, pancreatic, and melanoma, among others.
PD-1 therapeutic monoclonal antibodies currently on the market are approved by the FDA, including Nivolumab (Opdivo, month 9 of 2014), pembrolizumab (Kertruda, month 12 of 2014) and Cemiplimab (Libtayo, month 9 of 2018), and PD-L1 therapeutic monoclonal antibodies on the market are approved by the FDA, including Atezolizumab (Tecentriq, month 9 of 2014), avelumab (Bavendio, month 5 of 2016) and Duravulumab (Impinzi, month 5 of 2017), and approved indications are shown in the following table.
Figure BDA0002789311500000041
In addition, PD-1 mAbs such as Pidilizumab, AMP-224, AMP-514 and PDR001, and PD-L1 mAbs such as BMS-936559 and CK-301 are under development and clinical trials.
However, in the existing monoclonal antibodies, the affinity does not reach the ideal state, and the immunogenicity is strong due to the large volume.
Disclosure of Invention
The invention aims at providing a novel anti-PD-L1 nanobody and Fc fusion protein thereof and application of the anti-PD-L1 nanobody and Fc fusion protein thereof according to the existing requirements in the field.
In one aspect, the invention provides an anti-PD-L1 nanobody and derivatives thereof, wherein the anti-PD-L1 nanobody comprises at least one VHH fragment, wherein the VHH fragment comprises three amino acid fragments CDR1, CDR2 and CDR3, and wherein CDR1, CDR2 and CDR3 each have the following amino acid sequences:
Further, the anti-PD-L1 nanobody is characterized in that the amino acid sequence of the anti-PD-L1 nanobody is shown in SEQ ID NO:1 to SEQ ID NO: shown at 20.
Further, the anti-PD-L1 nanobody according to the invention is characterized in that, in the amino acid sequence of the anti-PD-L1 nanobody, at least 80% (e.g., 90%, 95%, 98%, 99% or 99.9%, etc.) of the amino acid sequence other than CDR1, CDR2 and CDR3 is identical to the amino acid sequence of SEQ ID NO:1 to SEQ ID NO:20, and the amino acid sequence shown in the formula 20 is identical.
Further, the anti-PD-L1 nanobody according to the invention, characterized in that the derivative is derived from fusion with a polypeptide, including serum albumin and fragments, or serum albumin binding proteins; fusion with FCRn binding protein; and chemical coupling with polymers such as polyethylene glycol.
In another aspect, the present invention also provides an Fc fusion protein of an anti-PD-L1 nanobody, wherein the Fc fusion protein of an anti-PD-L1 nanobody comprises an anti-PD-L1 nanobody as described above and an Fc segment selected from the group consisting of human IgG1, igG2, igG3, igG4, and mutants thereof, for example, as shown in SEQ ID No. 61 or SEQ ID No. 62.
In another aspect, the present invention also provides a humanized anti-PD-L1 nanobody and a derivative thereof, which is characterized in that the humanized anti-PD-L1 nanobody is obtained by modifying the anti-PD-L1 nanobody as described above, and has the amino acid sequence of SEQ ID NO:43 to SEQ ID NO: 57.
In another aspect, the present invention also provides an Fc fusion protein of a humanized anti-PD-L1 nanobody, characterized in that the Fc fusion protein of a humanized anti-PD-L1 nanobody comprises an anti-PD-L1 nanobody as described above and an Fc segment selected from the group consisting of human IgG1, igG2, igG3, igG4 and mutants thereof, for example as shown in SEQ ID No. 61 or SEQ ID No. 62.
Further, the humanized anti-PD-L1 nanobody according to the invention, characterized in that the derivative is derived from fusion with a polypeptide, including serum albumin and fragments, or serum albumin binding proteins; fusion with FCRn binding protein; and chemical coupling with polymers such as polyethylene glycol.
In another aspect, the invention also provides an anti-PD-L1 nanobody or Fc fusion protein thereof as described above, and the use of a humanized anti-PD-L1 nanobody or Fc fusion protein thereof in the preparation of a kit for blocking the binding of PD-L1 and PD-1.
On the other hand, the invention also provides the anti-PD-L1 nanobody or Fc fusion protein thereof and the application of the humanized anti-PD-L1 nanobody or Fc fusion protein thereof in preparing medicaments for inhibiting tumor growth.
In another aspect, the invention also provides an anti-PD-L1 nanobody or Fc fusion protein thereof as described above, and the use of a humanized anti-PD-L1 nanobody or Fc fusion protein thereof in the preparation of a medicament for treating cancer, infection or an immunomodulatory disease.
Further, the use according to the invention is characterized in that the cancer or tumor is colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, renal cancer, cervical cancer, bone marrow cancer, lymphoma, leukemia, thyroid cancer, endometrial hyperplasia, uterine cancer, bladder cancer, neuroendocrine tumor, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, fibrosarcoma of the carina skin, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.
The beneficial effects of the invention are that
The anti-PD-L1 nano antibody and the Fc fusion protein thereof have strong specificity, high affinity, weak immunogenicity to human and obvious anti-tumor effect.
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The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 shows the results of ELISA detection of binding of PD-L1 nanobody Fc fusion protein QP326-QP333 to human PD-L1 protein;
FIG. 2 shows the results of ELISA detection of binding of PD-L1 nanobody Fc fusion proteins QP334-QP341 to human PD-L1 protein;
FIG. 3 shows the results of ELISA detection of binding of PD-L1 nanobody Fc fusion protein QP342-QP345 to human PD-L1 protein;
FIG. 4 shows the results of FACS detection of binding of PD-L1 nanobody Fc fusion protein to human non-small lung cancer cell HCC827 that naturally expresses human PD-L1;
FIG. 5 shows the results of ELISA detection of PD-L1 nanobody Fc fusion protein QP326-QP335 blocking human PD-L1 and PD-1 protein binding;
FIG. 6 shows the results of ELISA detection of PD-L1 nanobody Fc fusion protein QP336-QP345 blocking human PD-L1 and PD-1 protein binding;
FIG. 7 shows the results of ELISA detection of PD-L1 nanobody Fc fusion proteins QP326-QP329, and QP331-QP333 blocking human PD-L1 and CD80 protein binding;
FIG. 8 shows the results of ELISA detection of PD-L1 nanobody Fc fusion proteins QP334, QP341, and QP343-QP345 blocking human PD-L1 and CD80 protein binding;
FIG. 9 shows the results of ELISA detection of PD-L1 nanobody Fc fusion protein QP326-QP329, QP331-QP333 binding to cynomolgus monkey PD-L1 protein;
FIG. 10 shows the results of ELISA detection of binding of PD-L1 nanobody Fc fusion proteins QP334, QP341, and QP343-QP345 to cynomolgus PD-L1 protein;
FIG. 11 shows the results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP341, and QP508-QP512 binding to human PD-L1 protein;
FIG. 12 shows the results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP344, and QP513-QP517 binding to human PD-L1 protein;
FIG. 13 shows the results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP332, and QP518-QP522 binding to human PD-L1 protein;
FIG. 14 shows the results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP341, and QP508-QP512 blocking human PD-L1 and PD-1 protein binding;
FIG. 15 shows the results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP344, and QP513-QP517 blocking human PD-L1 and PD-1 protein binding;
FIG. 16 shows the results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP332, and QP518-QP522 blocking human PD-L1 and PD-1 protein binding;
FIG. 17 shows the results of detection of binding of the Fc fusion protein of the humanized PD-L1 nanobody to the cynomolgus PD-L1 protein by ELISA;
FIG. 18 shows the results of detection of binding of the Fc fusion proteins QP509 and QP3447 of the humanized PD-L1 nanobody to human PD-L1 protein by ELISA;
FIG. 19 shows the results of detection of Fc fusion proteins of humanized PD-L1 nanobodies by ELISA to block binding of human PD-L1 protein to PD-1 protein;
FIG. 20 shows the results of detection by FACS of binding of PD-L1 nanobody FC fusion protein QP3447 to human non-small lung cancer cell HCC827 naturally expressing human PD-L1;
fig. 21 to 24 show the results of the humanized nanobody Fc fusion proteins QP509 and QP3447 promoting T cell proliferation in a mixed lymphocyte reaction;
fig. 25 and 26 show the results of evaluating the inhibitory effect of anti-PD-L1 nanobodies on tumor growth in a transgenic murine model.
Detailed Description
The following describes specific embodiments of the present invention in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the invention, are not intended to limit the invention.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The experimental method in which specific conditions are not specified in the present embodiment is generally performed under conventional conditions or under conditions recommended by the manufacturer of the raw materials or commodities. Such as molecular cloning, laboratory manuals, cold spring harbor laboratories, contemporary molecular biology methods, cell biology, and the like. The reagents of specific origin are not noted and are commercially available conventional reagents.
Example 1: alpaca immunity and construction of phage immune library
Alpaca immunization: one healthy alpaca (alcaga pacos, lama pacos) was immunized with PD-L1 extracellular domain recombinant protein (sense Qiaogn, 10084-H08H), 0.11mg protein per immunization, total 5 times, first Freund's complete adjuvant CFA was used, and 2 nd to 5 th times were mixed with antigen by Freund's incomplete adjuvant IFA emulsification and then injected subcutaneously at multiple points.
Building a warehouse: and (3) taking 50ml of peripheral blood to separate Peripheral Blood Mononuclear Cells (PBMC), extracting total RNA of the PBMC, performing reverse transcription to form cDNA by RT-PCR, designing a primer for PCR amplification of VHH genes, and constructing an immune library. Peripheral blood lymphocytes were isolated, 50mL of peripheral blood was collected, PBMCs were isolated according to the instructions for lymphocyte isolation, and PBMC total RNA was extracted with TRIzol reagent. Reverse transcription, see
Figure BDA0002789311500000091
III First-Strand Synthesis System for RT-PCR instructions, 8. Mu.g RNA was transcribed. The first round of nested PCR, the VHH fragment of about 750bp is recovered by tapping, and the second round of nested PCR is performed. The phage library vector is constructed by using pComb3XSS, the pComb3XSS phagemid vector is cut by using SfiI single enzyme, and the pComb3XSS is cut into two large fragments of 1672bp (SS stuffer) and 3301bp (vector target fragment), and the vector HAs His tag and HA tag, so that the purification and detection are convenient. Ligation of vector with fragment of interest the vector was ligated toThe target fragments are respectively subjected to enzyme digestion by sfiI at 50 ℃ overnight, and then the target fragments are recovered by tapping. The molar ratio of the connection is Vector: vhh=1:3. The electrotransformation is carried out for 10 times, 1mL of 2YT culture medium is added for resuscitation immediately after the electrotransformation, 100ml of resuscitated product is added, the total is recovered at 37 ℃,180rpm is recovered for 45min, 100 mu L of the measured reservoir capacity is taken, the rest is centrifuged, 5mL of 2YT is added for resuspension, and the mixture is coated on a 200mm flat plate for 8 blocks. Next day 10 -5 There were 73 clones in total, so the pool size was 7.3X10 8 (73*100*10 5 )。
Example 2: nano antibody immune library screening identification
Round 2 screening was performed with PDL1 extracellular domain recombinant protein.
First round of elutriation screening: PDL1-hFc (protein number QP 004) and hFc protein were coated in the immune tubes, 50nM,1ml, overnight at 4 ℃. Closing: the immune tubes were blocked with 2% Mill/PBS at 37℃for 1 hour. And (3) reducing: the blocking solution was decanted and 900. Mu.l of 2% Mill/PBS was added to the coated hFc tube, and 100ul phage immunopool was added and the tube was spun at room temperature for 1 hour. Combining: phage immunopools were transferred to PDL1-hFc coated immune tubes and spun for 1 hour at room temperature. Washing: the 1 XPBST wash the immune tubes 5 times and the 1 XPBS wash the immune tubes 5 times. Eluting: 800 μl of 100mM TEA, 10min at room temperature. And (3) neutralization: the eluate was transferred to a 1.5ml EP tube and 400. Mu.l of 1M pH 7.4Tris was added. Infection: the phage eluted after neutralization was added to 10ml TG1 with od600=0.5. 4min at 37 ℃. Measuring titer, and coating the rest bacterial liquid on a large plate. Incubated overnight at 37 ℃. First round of elutriation output: 3.7e+06.
Packaging phage library: 2 XTY medium was scraped and inoculated into 50ml of 2 XTY medium+Amp+1% glucose to an OD of about 0.1,37 ℃at 200rpm, and grown for about 1 hour for 20 minutes to an OD of about 0.4 to 0.6. 500 mu l M13KO7 was added and infected at 37℃for 40min, the supernatant was removed by centrifugation and 100ml of 2 XTY medium+Amp+Kana were resuspended in pellet at 30℃for 200rpm overnight. Collecting phage libraries: the cultured broth was centrifuged overnight, and the supernatant was added to 1/4 volume of 5 XPEG/NaCl. Centrifuging at 4deg.C for 20min, discarding supernatant, centrifuging again, absorbing excess supernatant, adding 1ml PBS per 50ml supernatant to resuspend phagosome precipitate, centrifuging at 13000g for 10min, and transferring supernatant to new EP tube. This is the second round of phage library input phage for the next panning.
A second wheel (2 nd) of the elutriation screen: PDL1-hFc (protein No. QP 004) 10nM and hFc protein 50nM,2 ng/. Mu.l, 1ml, overnight at 4℃were coated in the immune tube, respectively. Closing: the immune tubes were blocked with 2% Mill/PBS at 37℃for 1 hour. And (3) reducing: the blocking solution was decanted off and 2ml of 2% Mill/PBS was added to the coated hFc immune tube, and 400. Mu.l of the input phage was added and the tube was spun at room temperature for 1 hour. Combining: the supernatant was transferred to PDL1-hFc coated immune tubes and spun at room temperature for 1 hour. Cleaning: the 1 XPBSs wash the immune tubes 10 times and then the 1XPBS wash the immune tubes 10 times. Eluting: 800 μl of 100mM TEA, 10min at room temperature. And (3) neutralization: the eluate was transferred to a 1.5ml EP tube and 400. Mu.l of 1M pH 7.4Tris was added. Infection: the phage eluted after neutralization was added to 10ml TG1 with od600=0.5. 4min at 37 ℃. Measuring titer, and coating the rest bacterial liquid on a large plate. Incubated overnight at 37 ℃. Second round of panning results: 8.5E+08.
Immune library screening and identification: through 2 rounds of panning, monoclonal clones were picked for phage ELISA, and positive clones binding PDL1 were screened. The positive clones were sequenced to obtain the VHH sequence of the antibody PD-L1.
VHH antibody secretory expression: in 96-well plates, 150 μl of 2 XYT+ampicillin+1% glucose medium per well was inoculated with the second round of screened monoclonal and incubated for 3 hours at 37 ℃. Mu.l were transferred to a new 96-well plate, 25. Mu.l 50% glycerol was added and frozen. To the master plate, 2 XYT+ampicillin+3 mM IPTG, 50. Mu.l/well was added. Culturing at 30℃and 220r overnight.
Binding ELISA: PDL1-hFc (protein No. QP 004) was coated at 2. Mu.g/ml, 50. Mu.l/well, overnight at 4 ℃. ELISA plates were blocked with 3% BSA at 37℃for 1 hour. The culture broth was centrifuged overnight and the supernatant was transferred to a new 96-well plate. In PDL1-hFc plate, the supernatant was incubated at 25. Mu.l/well+5% BSA at 25. Mu.l/well, RT,1h. PBST 3 times, PBS 6 times. Incubating a secondary antibody: HRP-anti-HA (1:5000), 50 ul/well, RT,1h. PBST 6 times, PBS 3 times. TMB color development, H 2 SO 4 And (5) terminating. Partial clones were selected for sequencing validation.
Blocking ELISA: the PD1-hFc protein (protein No. QP 1138) was coated at 5. Mu.g/ml, 50. Mu.l/well, overnight at 4 ℃.3% BSAELISA plates were blocked at 37℃for 1 hour. The culture broth was centrifuged overnight and the supernatant was transferred to a new 96-well plate. Supernatant and Biotin-PDL1-FC (protein No. Biotin-QP 004.3) protein 0.3. Mu.g/ml were mixed according to 1: mix at 1 ratio, 50 μl/well, RT,1h. PBST 3 times, PBS 6 times. Incubating a secondary antibody: HRP-streptavidin (1:5000), 50. Mu.l/well, RT,1h. PBST 6 times, PBS 3 times. TMB color development, H 2 SO 4 And (5) terminating.
The positive clone ELISA results are shown in table 1 below:
TABLE 1
Figure BDA0002789311500000111
Selecting a clone which is positively combined with PDL1-hFc (protein number QP 004) and blocks PD1/PD-L1 from being combined, namely a positive clone which is specifically combined with PD-L1 protein, sequencing to obtain a VHH sequence of an antibody PD-L1 antibody, and regarding the sequences with the same CDR1, 2 and 3 as the same clone to obtain 20 unique nanometer antibody sequences, wherein the amino acid sequences are shown as SEQ ID NO: 1-20.
Example 3: construction of FC fusion protein by nano antibody, cloning, expression and purification of protein
Cloning design construction: 20 clones were transformed into PD-L1-FC fusion proteins with the C-terminus of human IgG1 FC. The reconstructed plasmid is expressed in HEK293 cells, and purified by protein A affinity chromatography to obtain 20 candidate PD-L1 VHH-FC fusion proteins QP326-QP345 with the sequence of SEQ ID NO:1-20 is connected with a human IgG1 Fc segment with the sequence SEQ ID NO: 61. Numbering of the Fc fusion proteins of the corresponding nanobodies is by suffix Fc to the corresponding nanobodies.
In addition, reference is made to QP322 in the examples, and reference may be made to patent CN201910567277.5, whose anti-PD-L1 VHH sequences are:
EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS
in addition, in the examples, atezolizumab (Atezolizumab) was used as a positive control, protein number was QP11801181, and the light and heavy chain sequences consisted of SEQ ID NO:58 and SEQ ID NO:59, respectively.
Protein expression: the culture density of 293E cells is maintained between 0.2 and 3×10 6 Culturing in maintenance stage culture medium (GIBCO Freestyle 293 expression culture medium) between/ml, centrifuging the transfected cells, and adjusting cell density to 0.5-0.8X10 6 /ml. On the day of transfection, 293E cell density was 1-1.5X10 6 /ml. Plasmid and transfection reagent PEI were prepared, the amount of plasmid to be transfected was 100. Mu.g/100 ml cells, and the mass ratio of PEI to plasmid was used was 2:1. Mixing the plasmid and PEI, standing for 15min, and not more than 20min. The plasmid and PEI mixture was slowly added to 293E cells and 8% CO was added 2 Culturing in a shaking table at 120rpm and 37 ℃ for the fifth day of transfection, and centrifuging at 4700rpm for 20min by a horizontal centrifuge to collect cell supernatant.
Protein a affinity chromatography purification: passing the solution through a column with a balance liquid, wherein the actual volume is 20ml, and the pH and the conductivity of the solution flowing out of the final instrument are consistent with those of the balance liquid, and the flow rate is 1ml/min; loading the supernatant of the culture solution after centrifugation on a column, wherein the sample is 40ml, and the flow rate is 0.33ml/min; passing the solution through a column with a balance liquid, wherein the actual volume is 20ml, and the pH and the conductivity of the solution flowing out of the final instrument are consistent with those of the balance liquid, and the flow rate is 0.33ml/min; the elution was passed through the column, the elution peak (PAC-EP) was collected starting when UV280 was rising to 15mAU, and the collection was stopped when UV280 was falling to 15mAU, at a flow rate of 1ml/min. After sample collection was completed, PAC-EP was neutralized with pH adjusting solution.
Example 4: ELISA detection of binding of PD-L1 nanobody Fc fusion protein to human PD-L1 protein
The experimental steps are as follows:
And (3) wrapping the plate: coating positive antibody alemtuzumab (Atezolizumab) 2 μg/ml, PD-L1 VHH-FC 1 μg/ml (13.3 nM), 60 μl/well, overnight at 4deg.C, PBS 3;
closing: 5% mill/PBS, 200. Mu.l/well, 25℃for 1h;
antigen: incubation of PD-L1-his protein QP003, 4-fold dilution from 5. Mu.g/ml, 8 gradients, 60. Mu.l/well, 25 ℃,1h, PBST 5;
and (2) secondary antibody: anti-his HRP,1:8000 dilutions, 60 μl/well, 25 ℃,1h, pbst 5;
color development: TMB 100. Mu.l/well, 5-10min,2M H 2 SO 4 The reaction was stopped and read at 450 nm.
The results are shown in FIGS. 1-3, where the PD-L1 nanobody Fc fusion proteins all bind human PD-L1 protein.
Example 5: SPR detection of binding affinity of PD-L1 nanobody Fc fusion protein to human PD-L1 protein
Surface Plasmon Resonance (SPR) detection affinity the affinity of the molecules to be detected with the proteins human PD-L1 and human PD-L1 was determined by Biacore T200 (GE).
Antigen and control antibody information is as follows:
antigen: human PD-L1 recombinant protein (Yiqiao Shenzhou, 10084-H08H) with the number of QPJ;
control antibodies: alemtuzumab (Atezolizumab), numbered QP11801181.
SPR affinity results are shown in table 2 below:
table 2: biacore detects the binding affinity of PD-L1 nanobody Fc fusion protein to human PD-L1 protein.
Numbering device Abs Ka(1/Ms) Kd(1/s) KD(M)
1 Alemtuzumab 2.30E+05 2.06E-04 8.97E-10
2 QP322 9.98E+05 4.55E-03 4.56E-09
3 QP326 8.14E+05 1.05E-03 1.28E-09
4 QP327 8.98E+05 9.47E-04 1.05E-09
5 QP328 7.16E+05 1.03E-03 1.44E-09
6 QP329 7.37E+05 9.52E-04 1.29E-09
7 QP331 1.26E+06 2.58E-03 2.05E-09
8 QP332 1.21E+06 4.27E-04 3.53E-10
9 QP333 9.48E+05 3.80E-04 4.00E-10
10 QP334 6.53E+05 1.14E-03 1.74E-09
11 QP341 3.14E+06 4.39E-04 1.40E-10
12 QP343 2.53E+06 4.14E-04 1.63E-10
13 QP344 1.81E+06 4.26E-04 2.35E-10
14 QP345 1.88E+06 4.02E-04 2.14E-10
Results: the screened PD-L1 nano antibody Fc fusion proteins are combined with human PD-L1 proteins.
Example 6: FACS detection of binding of PD-L1 nanobody Fc fusion protein to human non-small lung cancer cell HCC827 naturally expressing human PD-L1
The human non-small cell lung cancer cell HCC827 naturally expresses PD-L1 at high levels. HCC827 cells (confluence 80%) grown in log phase were prepared, concentration adjusted and plated in costar 96 well plates, 1E5 cells/well, 1 x PBS washed once, added 3% bsa,250 μl/well, incubated for 1h at 37 ℃; adding the antibody to be detected by 4-fold gradient dilution (33.33 nM to 0.008nM, corresponding to a mass concentration of 5 mug/mL to 0.001 mug/mL), 50 mug/hole, and incubating on ice for 1h; plates were washed 2 times with 1 XPBS, PE-anti-hFc (1:200) was added, 50 μl/well and incubated on ice for 1h; plates were washed 3 times with 1 XPBS and read on the machine.
As a result, as shown in FIG. 4, FACS detects that the PD-L1 nanobody Fc fusion proteins all bind to human non-small lung cancer cell HCC827, which naturally expresses human PD-L1.
Example 7: ELISA detection of PD-L1 nanobody Fc fusion protein to block human PD-L1 and PD-1 protein binding
Coating protein QP1138 (PD 1-FC) 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. Respectively preparing 2 mug/ml PDL 1-mouse FC and antibodies with different concentrations, uniformly mixing the two components in equal volumes, and incubating for 1h at room temperature. PBST was washed 3 times and PBS was washed 3 times. Incubating a secondary antibody: HRP-mouse IgG (1:5000) 50 μl/well, PBST washed 6 times, PBS washed 3 times. Color development: TMB 100. Mu.l/well was developed for 10min.2M H 2 SO 4 50 μl/Kong Zhongzhi.
The results are shown in FIGS. 5 and 6, and the PD-L1 nanobody Fc fusion protein can block the binding of human PD-L1 and PD-1 proteins.
Example 8: ELISA detection of PD-L1 nanobody Fc fusion protein to block binding of human PD-L1 and CD80 proteins
Coating protein CD80-hFC 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. 2 mug/ml PDL 1-mouse FC and antibodies with different concentrations are respectively prepared, diluted by 3 times, evenly mixed in equal volume and incubated for 1h at room temperature. PBST was washed 3 times and PBS was washed 3 times. Incubating a secondary antibody: HRP-mouse IgG (1:5000) 50 μl/well, PBST washed 6 times, PBS washed 3 times. Color development: TMB 100. Mu.l/well was developed for 10min.2M H 2 SO 4 50 μl/Kong Zhongzhi.
The results are shown in FIGS. 7 and 8, where the PD-L1 nanobody Fc fusion protein blocks human PD-L1 and CD80 protein binding.
Example 9: ELISA detection of binding of PD-L1 nanobody Fc fusion protein to cynomolgus monkey PD-L1 protein
Rabbit His antibody (gold Style, A00174) was coated at 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. PDL1-his (sinobio 90251-C08H) was incubated at 1. Mu.g/ml, 50. Mu.l/well, 1H at 25℃and washed 3 times with PBS. Incubation antibody QP11801181 initial concentration 20 μg/ml, QP322, QP326 … … initial concentration 10 μg/ml,1:4 dilutions were made for 8 gradients. 50 μl/well, 1h at 25℃and 6 times with PBS. Secondary antibody HRP-anti-human FC was incubated, diluted 1:5000 times, 60. Mu.l/well, 1h at 25℃and PBST washed 6 times. TMB color development, H 2 SO 4 The reaction was terminated. The microplate reader was set up with a 450nm reading.
The results are shown in fig. 9 and 10, wherein the PD-L1 nanobody Fc fusion proteins bind to cynomolgus monkey PD-L1 protein.
Example 10: humanized design of nano antibody, cloning, expressing and purifying protein
Nanobody humanized design: by comparing the germline gene database of the heavy and light chain variable region of the IMGT human antibody with MOE software, heavy and light chain variable region germline genes with high homology with QP341, QP344 and QP332 are respectively selected as templates, and CDRs of the murine antibody are respectively transplanted into corresponding human templates to form variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Some important amino acid residues are selected for back mutation combination. Wherein the amino acid residues are determined and annotated by the Kabat numbering system. Designing a primer PCR to build each humanized antibody VH gene fragment, and then carrying out homologous recombination with an expression vector pQD with a signal peptide and a constant region gene (FC) fragment to construct an antibody full-length expression vector VH-FC-pQD. The VH/VK gene fragment required by recombination is synthesized by designing a plurality of primers by using online software DNAWorks (v3.2.4) (http:// helix web. Nih. Gov/DNAWorks /). 5'-30bp signal peptide +VH +30bp FC-3'. The gene fragment required for VH/VK recombination was obtained by two-step PCR amplification using the above designed primers according to TaKaRa Primer STAR GXL DNA polymerase protocol. Construction and cleavage of expression vector pQD carrying Signal peptide and constant region Gene (FC) fragment the expression vector pQD carrying Signal peptide and constant region Gene (FC) fragment was designed using the characteristic that the recognition sequence differs from the cleavage site by restriction enzymes such as BsmBI. BsmBI enzyme cutting carrier, cutting glue and recovering for standby. Recombinant construction of expression vector VH-FC-pQD. VH containing recombinant gene fragment and BsmBI restriction enzyme recovery expression vector pQD (with signal peptide and constant region gene (FC) fragment) according to 3:1 proportion is respectively added into DH5H competent cells, ice bath is carried out for 30min at 0 ℃, heat shock is carried out for 90s at 42 ℃, 5 times of LB medium is added, incubation is carried out for 45min at 37 ℃, LB-Amp plates are coated, culture is carried out at 37 ℃ overnight, and monoclonal is picked up and sent to sequencing to obtain each target clone.
The humanized design light and heavy chain variable region sequences and protein expression numbers for each clone are shown in Table 3 below, where all antibodies fuse human IgG1-FC constant regions at their C-termini.
TABLE 3 QP341, QP344, QP332 humanized design
Figure BDA0002789311500000161
Cloning and constructing: the above humanized design clone was converted to a PD-L1-FC fusion protein with the C-terminus of human IgG1 FC. The reconstructed plasmid is expressed in HEK293 cells and purified by protein A affinity chromatography to obtain 15 humanized PD-L1 VHH-FC fusion proteins QP509-QP522 with the sequence of SEQ ID NO:1-20 is connected with human IgG1 FC segment at the rear end, and the sequence is shown in SEQ ID NO: shown at 61.
Protein expression: the culture density of 293E cells is maintained between 0.2 and 3×10 6 Culturing in maintenance stage culture medium (GIBCO Freestyle 293 expression culture medium) between/ml, centrifuging the transfected cells, and adjusting cell density to 0.5-0.8X10 6 /ml. On the day of transfection, 293E cell density was 1-1.5X10 6 /ml. Plasmid and transfection reagent PEI were prepared, the amount of plasmid to be transfected was 100. Mu.g/100 ml cells, and the mass ratio of PEI to plasmid was 2:1. mixing the plasmid and PEI, standing for 15min, and not more than 20min. The plasmid and PEI mixture was slowly added to 293E cells and 8% CO was added 2 Culturing in a shaking table at 120rpm and 37 ℃ for the fifth day of transfection, and centrifuging at 4700rpm for 20min by a horizontal centrifuge to collect cell supernatant.
Protein a affinity chromatography purification: passing the solution through a column with a balance liquid, wherein the actual volume is 20ml, and the pH and the conductivity of the solution flowing out of the final instrument are consistent with those of the balance liquid, and the flow rate is 1ml/min; loading the supernatant of the culture solution after centrifugation on a column, wherein the sample is 40ml, and the flow rate is 0.33ml/min; passing the solution through a column with a balance liquid, wherein the actual volume is 20ml, and the pH and the conductivity of the solution flowing out of the final instrument are consistent with those of the balance liquid, and the flow rate is 0.33ml/min; the elution was passed through the column and the elution peak (PAC-EP) was collected starting when the UV280 was rising to 15mAU and stopped when the UV280 was falling to 15mAU at a flow rate of 1ml/min. After sample collection was completed, PAC-EP was neutralized with pH adjusting solution.
Example 11: ELISA detection of binding of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein
The humanized nanobody QP509 and other molecules are coated with 1 mug/ml, and the positive control alemtuzumab (Atezolizumab) is coated with 2 mug/ml. Overnight at 4℃and 3 washes with PBS. 5% milk blocked, RT 1h. The starting concentration of Biotin-QP004 (Biotin-PD-L1 hFC) was incubated at 0.5. Mu.g/ml, diluted 5-fold, RT 1h, and PBST washed 3 times. HRP-streptavidin was 5000-fold diluted and PBST washed 5 times. TMB was developed for 10min.450nm reading.
The results are shown in FIGS. 11-13, and ELISA detection of humanized PD-L1 nanobody Fc fusion protein binding to human PD-L1 protein, selection of binding and low EC50 value molecules for further validation.
Example 12: ELISA detection of humanized PD-L1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein binding
Coating protein QP1138 (PD 1-FC) 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. 2 mug/ml PDL 1-mouse FC and 15 mug/ml humanized nano-antibodies with different concentrations QP509 and the like are respectively prepared, and positive control alemtuzumab (Atezolizumab) 30 mug/ml, 1:3, diluting, uniformly mixing in equal volume, and incubating for 1h at room temperature. PBST was washed 3 times and PBS was washed 3 times. Incubating a secondary antibody: HRP-mouse IgG (1:5000) 50 μl/well, PBST washed 6 times, PBS washed 3 times. Color development: TMB 100. Mu.l/well was developed for 10min.2M H 2 SO 4 50 μl/Kong Zhongzhi.
The results are shown in FIGS. 14-16, and ELISA detection of humanized PD-L1 nanobody Fc fusion protein blocked binding of human PD-L1 protein to PD-1 protein, and further confirmed.
Example 13: SPR detection of binding affinity of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein
Surface Plasmon Resonance (SPR) detection affinity the affinity of the molecules to be detected with the proteins human PD-L1 and human PD-L1 was determined by Biacore T200 (GE).
Antigen and control antibody information is as follows:
antigen: human PD-L1 recombinant protein (sinobiologic, 10084-H08H);
control antibodies: alemtuzumab (Atezolizumab).
SPR affinity results are shown in table 4 below:
TABLE 4 Table 4
Abs Ka(1/Ms) Kd(1/s) KD(M)
Alemtuzumab 1.13E+05 3.04E-04 2.70E-09
QP341 2.18E+06 3.16E-04 1.45E-10
QP509 1.69E+06 4.21E-04 2.49E-10
QP512 1.70E+06 2.98E-04 1.76E-10
QP344 1.73E+06 3.25E-04 1.88E-10
QP517 1.11E+06 3.61E-04 3.26E-10
QP332 1.01E+06 3.62E-04 3.59E-10
QP521 7.57E+05 4.08E-04 5.39E-10
QP522 7.07E+05 2.77E-04 3.92E-10
The humanized PD-L1 nanobody Fc fusion protein binds to human PD-L1 protein.
Example 14: ELISA detection of binding of humanized PD-L1 nanobody Fc fusion protein to cynomolgus monkey PD-L1 protein
Rabbit His antibody (gold Style, A00174) was coated at 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. PDL1-his (sinobio 90251-C08H) was incubated at 1. Mu.g/ml, 50. Mu.l/well, 1H at 25℃and washed 3 times with PBS. Incubation positive control alemtuzumab (Atezolizumab) starting concentration 20 μg/ml, QP322, QP326 … … starting concentration 10 μg/ml,1:4 dilutions were made for 8 gradients. 50 μl/well, 1h at 25℃and 6 times with PBS. Secondary antibody HRP-anti-human FC was incubated, diluted 1:5000 times, 60. Mu.l/well, 1h at 25℃and PBST washed 6 times. TMB color development, H 2 SO 4 The reaction was terminated. The microplate reader was set up with a 450nm reading.
The results are shown in FIG. 17, where the humanized PD-L1 nanobody Fc fusion protein binds to cynomolgus PD-L1 protein.
To evaluate PD-L1 in vitro functional activity, two forms of humanized nanobody Fc fusion proteins were designed, specifically, the first form was a QP509 VHH fusion at the N-terminus of the Fc protein, with the protein numbering being QP509 (QP 509 VHH-hFC); the second form is to fuse QP509 VHH at the C-terminal of the human IgG1 FC segment mutation eliminating FC γR function (EU count L234A/L235A/K338A), protein number QP3447 (hFC (L234A/L235A/K338A) -QP509 VHH), amino acid sequence of QP3447 is shown in SEQ ID NO: shown at 60.
Example 15: SPR detection of binding affinity of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein
Surface Plasmon Resonance (SPR) detection affinity the affinity of the molecules to be detected with the proteins human PD-L1 and human PD-L1 was determined by Biacore T200 (GE).
Antigen and control antibody information is as follows:
antigen: human PD-L1 recombinant protein (SinoBio, 10084-H08H)
Control antibodies: alemtuzumab (ROCHE).
The experimental results are shown in table 5.
Table 5: QP3447 and the like bind with human PD-L1.
Proteins ka(1/Ms) kd(1/s) KD(M)
QP3447 8.58E+05 4.11E-04 4.79E-10
QP509 1.36E+06 4.07E-04 2.99E-10
Alemtuzumab 2.07E+05 2.87E-04 1.39E-09
Example 16: ELISA detection of QP3447 binding to human PD-L1 protein
Rabbit His antibody (gold Style, A00174) was coated at 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. Human PDL1-his (sinobio 10084-H08H) was incubated at 1. Mu.g/ml, 50. Mu.l/well, 1H at 25℃and washed 3 times with PBS. Positive controls, atrazumab (Atezolizumab), QP509, QP3447 starting concentrations 67nm, were incubated, diluted 5-fold for 7 gradients, and the last well diluted 100-fold. 50 μl/well, 1h at 25℃and 6 times with PBS. Secondary antibody HRP-anti-human FC was incubated, diluted 1:5000 times, 60. Mu.l/well, 1h at 25℃and PBST washed 6 times. TMB color development, H 2 SO 4 The reaction was terminated. The microplate reader was set up with a 450nm reading.
The results of ELISA detection of binding of the Fc fusion proteins QP509 and QP3447 of the humanized PD-L1 nanobody to human PD-L1 protein are shown in FIG. 18.
Example 17: ELISA detection of humanized PD-L1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein binding
Coating protein QP1138 (PD 1-FC) 2. Mu.g/ml 50. Mu.l/well overnight at 4 ℃. PBS was washed 3 times. Closing: 3% BSA 250. Mu.l/well, incubated for 1h at room temperature. 2. Mu.g/ml PDL 1-mouse FC fusion protein was formulated, 50. Mu.l/well incubated for 1h at room temperature, washed 3 times with PBST and 3 times with PBS. Different concentrations of QP509, QP3447 and positive control alemtuzumab (Atezolizumab) 333nM starting concentrations were incubated, 5-fold diluted 8 concentration gradients, and incubated for 1h at room temperature. PBST was washed 3 times and PBS was washed 3 times. Incubating a secondary antibody: HRP-mouse IgG (1:5000) 50 μl/well, PBST washed 6 times, PBS washed 3 times. Color development: TMB 100. Mu.l/well was developed for 10min.2M H 2 SO 4 50 μl/Kong Zhongzhi.
The results of ELISA detection of Fc fusion proteins of humanized PD-L1 nanobodies blocking binding of human PD-L1 protein to PD-1 protein are shown in FIG. 19.
Example 18: FACS detection of binding of PD-L1 nanobody Fc fusion protein QP3447 to human non-small lung cancer cell HCC827 naturally expressing human PD-L1
The human non-small cell lung cancer cell HCC827 naturally expresses PD-L1 at high levels. HCC827 cells (confluence 80%) grown in log phase were prepared, concentration adjusted and plated in 96 well plates, 1E5 cells/well, 1 x PBS washed once, added 3% bsa,250 μl/well, incubated for 1h at 37 ℃; adding antibodies to be detected with different concentrations, and incubating on ice for 1h at 50 μl/hole; plates were washed 2 times with 1 XPBS, PE-anti-hFc (1:200) was added, 50 μl/well and incubated on ice for 1h; plates were washed 3 times with 1 XPBS and read on the machine.
The results of FACS detection of the binding of PD-L1 nanobody FC fusion protein QP3447 to human non-small lung cancer cell HCC827, which naturally expresses human PD-L1, are shown in fig. 20.
Example 19: humanized nanobody Fc fusion proteins promote T cell proliferation in mixed lymphocyte reactions
In vitro Activity identification of PD-L1 function (Mixed lymphocyte reaction MLR): preparation of DC (donor 1) cells: resuscitates PBMC with EasySep TM Human monocyte isolation kit (STEMCELL, 19359) to isolate monocytes, adding rhGM-CSF (1000U/ml) and rhIL4 (500U/ml), culturing cells at 37deg.C for 6 days to induce iDC; half-changing liquid every 2-3 days, and simultaneously supplementing rhGM-CSF (1000U/ml) and rhIL4 (500U/ml); collecting cells 300x g, centrifuging for 5min, re-suspending with culture medium containing rhGM-CSF (1000U/ml) and rhIL4 (500U/ml), adding LPS (1 μg/ml), and culturing at 37deg.C for 1 day to induce mature DC; the cells were collected and counted for use. Preparation of T (donor 2) cells: resuscitates PBMC with EasySep TM The human cd4+ T cell isolation kit (stemcel, 17952) isolated cd4+ T cells.
Preparing an antibody: medium 1: the antibody (initial concentration 10. Mu.g/ml) was diluted in a 5-gradient for 6 concentrations. DC cells: t cells were 1:10, adding antibodies with different concentrations, mixing and culturing, detecting the expression of IL2 in the culture supernatant on the 2 nd day, and detecting the expression of IFNgamma in the culture supernatant on the 5 th day.
As a result, as shown in FIGS. 18 to 21, in the mixed lymphocyte reaction experiment, QP509 and QP3447 had a remarkable antibody concentration dependence on the concentrations of cytokines IFNγ and IL-2 generated after T cell activation.
Example 20: evaluation of inhibition of tumor growth by anti-PD-L1 nanobody in transgenic murine model
The experimental steps are as follows: since the PDL1 antibody was unable to recognize mouse PDL1, the growth inhibitory effect of the anti-PD-L1 nanobody on mouse colon cancer tumor cells MC38 was evaluated using an immune checkpoint humanized mouse model (C57 BL/6-hPDL 1). MC38-hPDL1 (tg) -mPL 1 (KO) cells in log phase were collected and PBS was resuspended to a suitable concentration for inoculation. Then, each experimental mouse was inoculated subcutaneously 1×10 on the right back 6 MC38-hPDL1 cells, periodically observing tumor growth, and keeping the average volume of tumor growth about 100mm 3 At this time, the administration was randomly grouped according to tumor size and mouse weight. The day of group dosing was defined as day 0. The dosing regimen is shown in table 6 below:
TABLE 6
Figure BDA0002789311500000211
The dosing volume was 10. Mu.l/g body weight.
The body weight and tumor volume of the mice were observed 3 times a week, the major and minor diameters of the tumors were measured with a vernier caliper, and the tumor volume (mm) was calculated as 3 )＝0.5×(a×b 2 ) Calculating and recording the tumor growth condition, and drawing a tumor growth curve. The results are shown in FIGS. 22-23 and Table 7 below:
TABLE 7
Figure BDA0002789311500000221
According to statistical analysis of tumor volume and tumor weight at day D24 at the end of the experiment, compared with the control group, the tumor volume (TGItv% = 62.97%) and tumor weight (TGItw% = 62.77%) of QP3447 in group G2 all have significant differences (P < 0.05); the tumor volume (TGI% = 65.08%) and tumor weight (TGI tw% = 58.36%) of the G3 group control molecule alemtuzumab were significantly different (P < 0.05). QP3447 and alemtuzumab (Atezolizumab) both showed strong antitumor activity in this model.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Sequence listing
<110> Qu Xiangdong
<120> an anti-PD-L1 nanobody and use thereof
<130> PD200223N
<160> 62
<170> SIPOSequenceListing 1.0
<210> 1
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 1
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Leu Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 2
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 2
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Ala Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Leu Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 3
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 3
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 4
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 4
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Ser Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 5
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 5
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Asp Gly Gly Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Thr Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 6
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 6
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ala Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 7
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 7
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Thr Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 8
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 8
Gln Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Thr Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 9
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 9
Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Asn Leu Asp Leu His
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 10
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 10
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Asp Gly Gly Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Thr Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 11
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 11
Gln Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Asp Gly Gly Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Thr Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 12
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 12
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Leu Asp Ser His
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Asp Gly Gly Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Thr Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 13
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 13
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Asp Asn Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Asp Gly Gly Thr Thr Asn Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Thr Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Gln Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 14
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 14
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asn Lys Ser Gly Glu Thr Thr Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Val Asp
65 70 75 80
Leu Gln Thr Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 15
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 15
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Cys Ile Asn Lys Ser Gly Glu Thr Thr Tyr Tyr Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Val His
65 70 75 80
Leu Gln Thr Asn Asn Leu Thr Pro Ala Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Phe
100 105 110
Arg Gln Phe Phe Pro Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 16
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 16
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 17
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 17
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 18
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 18
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Thr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Gln Ala Thr Val Ser
115 120 125
Ser
<210> 19
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 19
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Asn Phe
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 20
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 20
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Thr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Ser
65 70 75 80
Leu Gln Met Asn Asn Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser
<210> 21
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 21
Gly Phe Thr Leu Asp Asp Tyr Ala Ile Gly
1 5 10
<210> 22
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 22
Gly Phe Thr Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 23
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 23
Gly Phe Asn Leu Asp Leu His Ala Val Gly
1 5 10
<210> 24
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 24
Gly Arg Thr Leu Asp Ser His Ala Ile Gly
1 5 10
<210> 25
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 25
Gly Phe Thr Ser Asp Asn Tyr Ala Ile Gly
1 5 10
<210> 26
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 26
Gly Phe Thr Leu Asp Thr Tyr Ala Ile Gly
1 5 10
<210> 27
<211> 10
<212> PRT
<213> Artificial sequence ()
<400> 27
Gly Phe Ser Leu Asp Asn Phe Ala Ile Gly
1 5 10
<210> 28
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 28
Cys Ile Ser Lys Ser Gly Glu Thr Thr Asn Tyr Val Asp Ser Val Lys
1 5 10 15
Gly
<210> 29
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 29
Cys Ile Ser Lys Asp Gly Gly Thr Thr Asn Tyr Val Asp Ser Val Lys
1 5 10 15
Gly
<210> 30
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 30
Cys Ile Ser Lys Ser Gly Glu Thr Ala Asn Tyr Val Asp Ser Val Lys
1 5 10 15
Gly
<210> 31
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 31
Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val Lys
1 5 10 15
Gly
<210> 32
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 32
Cys Ile Asn Lys Ser Gly Glu Thr Thr Tyr Tyr Val Asp Ser Val Lys
1 5 10 15
Gly
<210> 33
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 33
Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val Lys
1 5 10 15
Asp
<210> 34
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 34
Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val Lys
1 5 10 15
Gly
<210> 35
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 35
Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val Lys
1 5 10 15
Gly
<210> 36
<211> 22
<212> PRT
<213> Artificial sequence ()
<400> 36
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
1 5 10 15
Leu Gln Phe Phe Arg Ser
20
<210> 37
<211> 22
<212> PRT
<213> Artificial sequence ()
<400> 37
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
1 5 10 15
Arg Gln Phe Phe Arg Ser
20
<210> 38
<211> 22
<212> PRT
<213> Artificial sequence ()
<400> 38
Ala Ala Gly Ser Trp Cys Thr Val Gly Thr Met Ser Arg Gln Phe Phe
1 5 10 15
Arg Gln Phe Phe Gln Ser
20
<210> 39
<211> 22
<212> PRT
<213> Artificial sequence ()
<400> 39
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Phe
1 5 10 15
Arg Gln Phe Phe Arg Ser
20
<210> 40
<211> 22
<212> PRT
<213> Artificial sequence ()
<400> 40
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Phe
1 5 10 15
Arg Gln Phe Phe Pro Ser
20
<210> 41
<211> 22
<212> PRT
<213> Artificial sequence ()
<400> 41
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
1 5 10 15
Arg Gln Phe Phe His Ser
20
<210> 42
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 42
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 43
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 43
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 44
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 44
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 45
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 45
Gln Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 46
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 46
Gln Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Glu Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 47
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 47
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Asn Phe
20 25 30
Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 48
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 48
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Asn Phe
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 49
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 49
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Asn Phe
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 50
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 50
Gln Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Asn Phe
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 51
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 51
Gln Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Asn Phe
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Thr Phe Phe Val Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe His Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 52
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 52
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 53
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 53
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 54
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 54
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 55
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 55
Gln Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 56
<211> 129
<212> PRT
<213> Artificial sequence ()
<400> 56
Gln Leu Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val
35 40 45
Ser Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Trp Cys Thr Val Gly Ser Met Ser Arg Gln Phe Tyr
100 105 110
Arg Gln Phe Phe Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 57
<211> 214
<212> PRT
<213> Artificial sequence ()
<400> 57
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 58
<211> 448
<212> PRT
<213> Artificial sequence ()
<400> 58
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser
20 25 30
Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
130 135 140
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
145 150 155 160
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser
180 185 190
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
225 230 235 240
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
260 265 270
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
290 295 300
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
305 310 315 320
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
325 330 335
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
385 390 395 400
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
435 440 445
<210> 59
<211> 379
<212> PRT
<213> Artificial sequence ()
<400> 59
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Ala Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu
245 250 255
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
260 265 270
Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr Ala Ile Gly Trp Phe Arg
275 280 285
Gln Ala Pro Gly Lys Gly Arg Glu Gly Val Ser Cys Ile Ser Lys Ser
290 295 300
Gly Glu Thr Thr Phe Phe Val Glu Ser Val Lys Asp Arg Phe Thr Ile
305 310 315 320
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu
325 330 335
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Gly Ser Trp Cys
340 345 350
Thr Val Gly Ser Met Ser Arg Gln Phe Tyr Arg Gln Phe Phe His Ser
355 360 365
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
370 375
<210> 60
<211> 232
<212> PRT
<213> Artificial sequence ()
<400> 60
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 61
<211> 232
<212> PRT
<213> Artificial sequence ()
<400> 61
Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Ala Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 62
<211> 17
<212> PRT
<213> Artificial sequence ()
<400> 62
Cys Ile Ser Lys Ser Gly Glu Thr Ser Tyr Tyr Val Asp Ser Val Arg
1 5 10 15
Gly

Claims (16)

1. An anti-PD-L1 nano antibody and a derivative thereof are characterized in that,
the anti-PD-L1 nanobody comprises at least one VHH fragment in which three amino acid fragments of CDR1, CDR2, and CDR3 are included, and the amino acid sequences of CDR1, CDR2, and CDR3 are as follows:
1) SEQ ID NO:21, the sequence of CDR1 shown in seq id no: GFTLDDYAIG;
2) SEQ ID NO:34, and the sequence of CDR2 is: CISKSGETTFFVESVKD;
3) SEQ ID NO:42, and the sequence of CDR3 is: ATGSWCTVGSMSRQFYRQFFHS.
2. The anti-PD-L1 nanobody and the derivative thereof according to claim 1,
the amino acid sequence of the anti-PD-L1 nano antibody is shown in SEQ ID NO: shown at 16.
3. The anti-PD-L1 nanobody and the derivative thereof according to claim 1,
In the amino acid sequence of the anti-PD-L1 nanobody, at least 90% of the amino acid sequence, except CDR1, CDR2, and CDR3, is identical to SEQ ID NO:16, and the amino acid sequence shown in 16 is identical.
4. The anti-PD-L1 nanobody and its derivatives according to claim 1, wherein the derivatives are derived from fusion with a polypeptide; or chemical coupling with a polymer.
5. The anti-PD-L1 nanobody and its derivatives according to claim 4, wherein the polypeptide comprises serum albumin and fragments, or serum albumin binding proteins, and the polymer comprises polyethylene glycol.
6. An Fc fusion protein of an anti-PD-L1 nanobody, which is characterized in that,
the Fc fusion protein of an anti-PD-L1 nanobody comprises an anti-PD-L1 nanobody according to any one of claims 1 to 3 and an Fc fragment.
7. The Fc fusion protein of an anti-PD-L1 nanobody according to claim 6, wherein the Fc fragment is set forth in SEQ ID No. 61 or SEQ ID No. 62.
8. A humanized anti-PD-L1 nano antibody and its derivative are characterized in that,
the humanized anti-PD-L1 nanobody is obtained by modifying the anti-PD-L1 nanobody according to claim 1 and has the amino acid sequence of SEQ ID NO:44 to SEQ ID NO: 47.
9. The humanized anti-PD-L1 nanobody and its derivatives according to claim 8, wherein the derivatives are derived from fusion with a polypeptide; or chemical coupling with a polymer.
10. The anti-PD-L1 nanobody and its derivatives according to claim 9, wherein the polypeptide comprises serum albumin and fragments, or serum albumin binding proteins; the polymer comprises polyethylene glycol.
11. A humanized anti-PD-L1 nanobody Fc fusion protein is characterized in that,
the Fc fusion protein of a humanized anti-PD-L1 nanobody comprises an anti-PD-L1 nanobody according to claim 8, and an Fc fragment.
12. The Fc fusion protein of a humanized anti-PD-L1 nanobody according to claim 11, wherein the Fc fragment is set forth in SEQ ID No. 61 or SEQ ID No. 62.
13. Use of an anti-PD-L1 nanobody and its derivatives according to any one of claims 1 to 5, an Fc fusion protein of an anti-PD-L1 nanobody according to claim 6 or 7, a humanized anti-PD-L1 nanobody and its derivatives according to any one of claims 8 to 10, or an Fc fusion protein of a humanized anti-PD-L1 nanobody according to claim 11 or 12 in the preparation of a kit for blocking PD-L1 and PD-1 binding.
14. Use of the anti-PD-L1 nanobody and its derivatives according to any one of claims 1 to 5, the Fc fusion protein of the anti-PD-L1 nanobody according to claim 6 or 7, the humanized anti-PD-L1 nanobody and its derivatives according to any one of claims 8 to 10, or the Fc fusion protein of the humanized anti-PD-L1 nanobody according to claim 11 or 12 in the manufacture of a medicament for inhibiting tumor growth.
15. Use of the anti-PD-L1 nanobody and its derivatives according to any one of claims 1 to 5, the Fc fusion protein of the anti-PD-L1 nanobody and its derivatives according to claim 6 or 7, the humanized anti-PD-L1 nanobody and its derivatives according to any one of claims 8 to 10, or the Fc fusion protein of the humanized anti-PD-L1 nanobody according to claim 11 or 12 in the manufacture of a medicament for the treatment of cancer, tumor or immunomodulatory disease.
16. The use according to claim 15, characterized in that,
the cancer or tumor is colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, renal cancer, cervical cancer, bone marrow cancer, lymphoma, leukemia, thyroid cancer, endometrial hyperplasia, uterine cancer, bladder cancer, neuroendocrine tumor, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, long-noded skin fibrosarcoma, merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.
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