Anti-pd-l1 nanobody and uses thereof

Abstract

The application provides an anti-PD-L1 nanobody and a use thereof, and the anti-PD-L1 nanobody comprises at least one VHH fragment, and the VHH fragment comprises three amino acid fragments of CDR1, CDR2 and CDR3. The anti-PD-L1 nanobody and the Fc fusion protein thereof have high specificity and affinity, low immunogenicity to human, and significant anti-tumor effect.

Description

[0001] This application is a divisional application of the patent filed on November 20, 2020, with application number 202011309419.7 and invention title "An anti-PD-L1 nanobody and its use".

[0002] Cross-reference to related applications

[0003] This application claims priority to patent application No. CN201911235055.X, filed on December 5, 2019, entitled “A Trifunctional Fusion Protein Containing a TGF-β Inhibitor and Its Application Thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0004] This invention belongs to the field of biomedicine, and more specifically, this invention relates to an anti-PD-L1 nanobody and its Fc fusion protein, as well as the use of the anti-PD-L1 nanobody and its Fc fusion protein. Background Technology

[0005] In the classical immune surveillance theory, the immune system recognizes and eliminates tumor antigens. If the immune system can completely eliminate tumor cells, immune clearance can proceed stably. If tumor cells evade clearance through mutation, the immune system will rebalance. During this process, the immunogenicity of tumor cells gradually decreases. The proliferative capacity of tumor cells weakens under the pressure of the immune system, making tumor cell detection more difficult.

[0006] Activation of oncogenes leads to alterations in tumor cells and the tumor microenvironment, disrupting the balance between the immune system and tumor cells. When the immune system and tumor cells enter an escape phase, the malignancy of tumor cells increases, and the loss of MHC molecules prevents them from being recognized and eliminated by immune cells. The tumor microenvironment can also suppress the immune system by releasing immunosuppressive factors such as IL-10 and TGF-β. Tumor cells also highly express immunosuppressive proteins (such as programmed death-ligand-1 and PD-L1). When effector T cells bind to tumor cells, PD-L1 interacts with PD-1 and induces T cell apoptosis, which is one of the main reasons why tumors develop resistance to the immune system, leading to rapid tumor growth and metastasis. Theoretically, if the host's immune system were artificially activated and redirected to tumor cells, tumor tissue could be eliminated, and the theory of immunotherapy has been widely proven in clinical practice.

[0007] Immunotherapy can be divided into two categories: specific and non-specific treatments. Specific therapies within this category include the following treatment strategies: Tumor vaccines activate immune cells against the patient's antigens through injection of tumor-specific immunotherapies. Tumor vaccines include: inactivated tumor cell vaccines, tumor antigen vaccines, tumor DNA vaccines, dendritic cell (DC) vaccines, and bacterial vaccines. Specific ACT immunotherapy primarily includes three treatment methods:

[0008] a) Tumor-infiltrating lymphocytes (TILs): Lymphocytes are isolated from tumor tissue and cultured in vitro. TILs can secrete IL-2, which has specific anti-tumor properties.

[0009] b) T-cell receptor (TCR) therapy: T cells recognize tumor antigens via their single-chain antibody fragments (scFv) and clone the single-chain antibody fragment TCR into normal T cells using a viral vector. Thus, normal T cells are transformed into specific tumor-killing T cells.

[0010] c) CAR-T: T cells are genetically modified to obtain tumor-specific receptor T cells. Unlike conventional T cell recognition mechanisms, CAR-T cells are not restricted by MHC molecules to recognize tumor antigens. Therefore, CAR-T cells can overcome tumor immune escape mechanisms by increasing co-stimulatory signaling molecules, thereby enhancing the T cell's ability to kill tumor cells.

[0011] In nonspecific ACT immunotherapy, there are two main treatment methods: lymphokine-activated killer (LAK) cell therapy and cytokine-induced killer (CIK) cell therapy.

[0012] a) LAK cell therapy: LAK cells use IL-2 to stimulate immune cells in peripheral blood lymphocytes, including NK cells and T cells, and enhance their ability to recognize target cells by overexpressing FAS ligands, and kill tumor cells by releasing perforin and granzymes.

[0013] b) CIK cell therapy: CIK cells are derived from peripheral blood lymphocytes (PBLs) of patients or healthy individuals and are expanded in vitro under stimulation with anti-CD3 antibodies, IFN-γ, and IL-2. CIK cells mainly exert their anti-tumor effects through FasL and perforin.

[0014] Immune checkpoints are protective molecules in the human immune system, preventing inflammatory damage caused by excessive T cell activation in a healthy body. Tumor cells can exploit this property by overexpressing immune checkpoint molecules, suppressing the body's immune response, evading the surveillance and killing effects of the immune system, and thus promoting tumor cell growth. Immune checkpoint inhibitor therapy can inhibit the activity of immune checkpoints in the tumor microenvironment, reactivating the T cell immune response against the tumor and achieving an anti-tumor effect. Complete T cell activation is regulated by a "dual signaling" system: the first signal comes from the specific binding of the T cell's own TCR (T cell receptor) to the MHC of the antigen, i.e., T cell recognition of the antigen; the second signal comes from co-stimulatory molecules, which participate in the interaction between co-stimulatory molecules expressed by antigen-presenting cells (APCs) and corresponding receptors or ligands (such as CD28) on the surface of T cells. For example, CD28-B7 is a positive co-stimulatory signal, while negative co-stimulatory molecules are mainly the CTLA4-B7 pathway and the PD-1 / PD-L1 pathway. After tumor cell invasion, this inhibitory pathway is used by tumor cells to suppress T cell activation, thereby evading the clearance action of the immune system.

[0015] PD-1 (CD279) was first reported in 1992. The human PD-1 encoding gene, PDCD1, is located at 2q37.3, with a full length of 2097 bp, consisting of 6 exons. The translation product is a 288-amino acid precursor protein of PD-1, which is cleaved into a signal peptide consisting of the first 20 amino acids to yield the mature protein. PD-1 includes an extracellular immunoglobulin variable region (IgV domain), a hydrophobic transmembrane domain, and an intracellular domain. The N-terminal ITIM motif of the intracellular tail domain contains two phosphorylation sites, while the C-terminus contains an ITSM motif. PD-1 is a membrane protein belonging to the CD28 immunoglobulin superfamily. It is mainly expressed on the surface of activated T cells, and also shows low-abundance expression on CD4-CD8-T cells in the thymus, activated NK cells, and monocytes. PD-1 has two ligands: PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) from the B7 protein family. PD-L1 and PD-L2 share 40% of their amino acid sequences. The main difference lies in their expression patterns. PD-L1 is constitutively lowly expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells and islet cells), and immune-exempt sites (such as the placenta, testes, and eyes). Inflammatory cytokines such as type I and type II interferons, TNF-α, and VEGF can all induce PD-L1 expression. PD-L2, on the other hand, is only expressed in activated macrophages and dendritic cells. After PD-1 binds to activated T cells, the ITSM motif of PD-1 undergoes tyrosine phosphorylation, which in turn leads to the dephosphorylation of downstream protein kinases Syk and PI3K, inhibiting the activation of downstream pathways such as AKT and ERK. Ultimately, this inhibits the transcription and translation of genes and cytokines required for T cell activation, thus exerting a negative regulatory effect on T cell activity.

[0016] In tumor cells, tumor cells and the tumor microenvironment negatively regulate T cell activity and suppress the immune response by upregulating PD-L1 expression and binding it to PD-1 on the surface of tumor-specific CD8+ T cells. Tumor cells can upregulate PD-L1 expression through the following four pathways: 1. Amplification of the gene encoding PD-L1 (9p24.1); 2. Activation of EGFR, MAPK, and PI3K-Akt signaling pathways, and HIF-1 transcription factor can upregulate PD-L1 expression at the transcriptional level; 3. Induction by EBV (EBV-positive gastric and nasopharyngeal carcinomas show high PD-L1 expression); and 4. Epigenetic regulation. In the tumor microenvironment, stimulation by inflammatory factors such as interferon-γ can also 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. Tumor-infiltrating T cells, capable of recognizing tumor antigens, can secrete interferon-γ, thereby inducing upregulation of PD-L1 expression. This process is known as "adaptive immune resistance," a mechanism through which tumor cells can protect themselves. Increasing evidence suggests that tumors utilize PD-1-dependent immunosuppression to evade the immune system. High expression of PD-L1 and PD-L2 has been found in various solid tumors and hematologic malignancies. Furthermore, PD-L expression is strongly correlated with poor prognosis in tumor cells, including esophageal cancer, gastric cancer, renal cancer, ovarian cancer, bladder cancer, pancreatic cancer, and melanoma.

[0017] Currently, the FDA has approved the following PD-1 therapeutic monoclonal antibodies: Nivolumab (Opdivo, September 2014), Pembrolizumab (Keytruda, December 2014), and Cemiplimab (Libtayo, September 2018). The FDA has also approved the following PD-L1 therapeutic monoclonal antibodies: Atezolizumab (Tecentriq, September 2014), Avelumab (Bavencio, May 2016), and Duravirumab (Imfinzi, May 2017). The approved indications are shown in the table below.

[0018]

[0019] In addition, there are PD-1 monoclonal antibodies such as Pidilizumab, AMP-224, AMP-514 and PDR001, as well as PD-L1 monoclonal antibodies such as BMS-936559 and CK-301 that are under development and in clinical trials.

[0020] However, among the existing monoclonal antibodies, the affinity is not ideal, and due to their large size, they are highly immunogenic. Summary of the Invention

[0021] The purpose of this invention is to provide a novel anti-PD-L1 nanobody and its Fc fusion protein, as well as the uses of the anti-PD-L1 nanobody and its Fc fusion protein, in accordance with the existing needs in the art.

[0022] In one aspect, the present invention provides an anti-PD-L1 nanobody and its derivatives, characterized in that 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 CDR1, CDR2, and CDR3 respectively have the following amino acid sequences:

[0023]

[0024]

[0025] Furthermore, the anti-PD-L1 nanobody according to the present invention is characterized in that the amino acid sequence of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 1 to SEQ ID NO: 20.

[0026] Furthermore, the anti-PD-L1 nanobody according to the present invention is characterized in that, in the amino acid sequence of the anti-PD-L1 nanobody, except for CDR1, CDR2 and CDR3, at least 80% (e.g. 90%, 95%, 98%, 99% or 99.9%) of the amino acid sequence is the same as the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 20.

[0027] Furthermore, the anti-PD-L1 nanobody according to the present invention is characterized in that the derivative is derived from fusion with a polypeptide, including serum albumin and fragments, or serum albumin-binding protein; fusion with FCRn-binding protein; and chemical coupling with a polymer such as polyethylene glycol.

[0028] On the other hand, the present invention also provides an Fc fusion protein of an anti-PD-L1 nanobody, characterized in that the Fc fusion protein of the anti-PD-L1 nanobody comprises the anti-PD-L1 nanobody as described above and an Fc segment, wherein the Fc segment is selected from the group consisting of human IgG1, IgG2, IgG3, IgG4 and their mutants, such as those shown in SEQ ID NO:61 or SEQ ID NO:62.

[0029] On the other hand, the present invention also provides a humanized anti-PD-L1 nanobody and its derivatives, 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 sequences shown in SEQ ID NO: 43 to SEQ ID NO: 57.

[0030] On the other hand, the present invention also provides an Fc fusion protein of a humanized anti-PD-L1 nanobody, characterized in that the Fc fusion protein of the humanized anti-PD-L1 nanobody comprises the anti-PD-L1 nanobody as described above and an Fc segment, wherein the Fc segment is selected from the group consisting of human IgG1, IgG2, IgG3, IgG4 and their mutants, such as those shown in SEQ ID NO:61 or SEQ ID NO:62.

[0031] Furthermore, the humanized anti-PD-L1 nanobody according to the present invention is characterized in that the derivative is derived from fusion with a polypeptide, including serum albumin and fragments, or serum albumin-binding protein; fusion with FCRn-binding protein; and chemical coupling with a polymer such as polyethylene glycol.

[0032] On the other hand, the present invention also provides the use of the anti-PD-L1 nanobody or its Fc fusion protein as described above, and the humanized anti-PD-L1 nanobody or its Fc fusion protein as described above, in the preparation of a kit for blocking the binding of PD-L1 and PD-1.

[0033] On the other hand, the present invention also provides the use of the anti-PD-L1 nanobody or its Fc fusion protein as described above, and the humanized anti-PD-L1 nanobody or its Fc fusion protein as described above, in the preparation of a medicament for inhibiting tumor growth.

[0034] On the other hand, the present invention also provides the use of the anti-PD-L1 nanobody or its Fc fusion protein as described above, and the humanized anti-PD-L1 nanobody or its Fc fusion protein as described above, in the preparation of medicaments for treating cancer, infection or immunomodulatory diseases.

[0035] Furthermore, according to the use of the present invention, the cancer or tumor is characterized as colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, esophageal cancer, prostate cancer, kidney 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 carcinoma, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, or myelodysplastic syndrome.

[0036] Beneficial effects of the present invention

[0037] The anti-PD-L1 nanobody and its Fc fusion protein of the present invention have high specificity, high affinity, weak immunogenicity in humans, and significant anti-tumor effects. Attached Figure Description

[0038] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0039] Figure 1 The results of ELISA detection of the binding of PD-L1 nanobody Fc fusion protein QP326-QP333 to human PD-L1 protein are shown.

[0040] Figure 2 The results of ELISA detection of the binding of the PD-L1 nanobody Fc fusion protein QP334-QP341 to human PD-L1 protein are shown.

[0041] Figure 3 The results of ELISA detection of the binding of PD-L1 nanobody Fc fusion protein QP342-QP345 to human PD-L1 protein are shown.

[0042] Figure 4 The results of FACS detection of the binding of the PD-L1 nanobody Fc fusion protein to HCC827 human non-small lung cancer cells naturally expressing human PD-L1 are shown.

[0043] Figure 5 The results of ELISA detection show that the PD-L1 nanobody Fc fusion protein QP326-QP335 blocks the binding of human PD-L1 and PD-1 proteins;

[0044] Figure 6 The results of ELISA detection of PD-L1 nanobody Fc fusion protein QP336-QP345 blocking the binding of human PD-L1 and PD-1 proteins are shown.

[0045] Figure 7 The results of ELISA detection of PD-L1 nanobody Fc fusion proteins QP326-QP329 and QP331-QP333 blocking the binding of human PD-L1 and CD80 proteins are shown.

[0046] Figure 8 The results of ELISA detection of PD-L1 nanobody Fc fusion proteins QP334, QP341, and QP343-QP345 blocking the binding of human PD-L1 and CD80 proteins are shown.

[0047] Figure 9 The results of ELISA detection of the binding of PD-L1 nanobody Fc fusion proteins QP326-QP329 and QP331-QP333 to cynomolgus monkey PD-L1 protein are shown.

[0048] Figure 10 The results of ELISA detection of the binding of PD-L1 nanobody Fc fusion proteins QP334, QP341, and QP343-QP345 to cynomolgus monkey PD-L1 protein are shown.

[0049] Figure 11 The results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP341 and QP508-QP512 binding to human PD-L1 protein are shown.

[0050] Figure 12 The results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP344 and QP513-QP517 binding to human PD-L1 protein are shown.

[0051] Figure 13 The results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP332 and QP518-QP522 binding to human PD-L1 protein are shown.

[0052] Figure 14 The results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP341 and QP508-QP512 blocking the binding of human PD-L1 and PD-1 proteins are shown.

[0053] Figure 15 The results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP344 and QP513-QP517 blocking the binding of human PD-L1 and PD-1 proteins are shown.

[0054] Figure 16 The results of ELISA detection of humanized PD-L1 nanobody Fc fusion protein QP332 and QP518-QP522 blocking the binding of human PD-L1 and PD-1 proteins are shown.

[0055] Figure 17 The results of ELISA detection of the binding of the Fc fusion protein of the humanized PD-L1 nanobody to the cynomolgus PD-L1 protein are shown.

[0056] Figure 18The results of ELISA detection of the binding of human PD-L1 protein to the Fc fusion proteins QP509 and QP3447 of the humanized PD-L1 nanobody are shown.

[0057] Figure 19 The results of ELISA detection of the Fc fusion protein of humanized PD-L1 nanobody blocking the binding of human PD-L1 protein to PD-1 protein are shown.

[0058] Figure 20 The results of FACS detection of PD-L1 nanobody FC fusion protein QP3447 binding to HCC827 human non-small lung cancer cells naturally expressing human PD-L1 are shown.

[0059] Figures 21 to 24 The results show that humanized nanobody Fc fusion proteins QP509 and QP3447 promote T cell proliferation in mixed lymphocyte responses;

[0060] Figure 25 and Figure 26 The results show the effects of anti-PD-L1 nanobodies on tumor growth in a transgenic mouse model. Detailed Implementation

[0061] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0062] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0063] Experimental methods not specifying specific conditions in this embodiment are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Examples include molecular cloning, laboratory manuals, Cold Spring Harbor Laboratory, contemporary molecular biology methods, cell biology, etc. Reagents not specifying their origin are commercially available, standard reagents.

[0064] Example 1: Alpaca Immunization and Construction of a Phage Immunobank

[0065] Alpaca Immunization: One healthy alpaca (vicugnapacos, alpaca, lamapacos) was immunized with recombinant protein of the extracellular region of PD-L1 (Essential China, 10084-H08H). Each immunization was 0.11 mg of protein, for a total of 5 times. The first immunization used Freund's complete adjuvant CFA, and the second to fifth immunizations used Freund's incomplete adjuvant IFA. The mixture was emulsified with the antigen and injected subcutaneously at multiple sites.

[0066] Library construction: Peripheral blood mononuclear cells (PBMCs) were isolated from 50 ml of peripheral blood. Total RNA was extracted from PBMCs, reverse transcribed into cDNA by RT-PCR, and primers were designed for PCR amplification of the VHH gene to construct an immunological library. Peripheral blood lymphocyte isolation: 50 ml of peripheral blood was collected, and PBMCs were isolated according to the instructions for lymphocyte separation solution. Total RNA was extracted from PBMCs using TRIzol reagent. Reverse transcription was performed (see [link to instructions]). The instructions for the First-Strand Synthesis System for RT-PCR (III) state that a total of 8 μg of RNA was transcribed. Nested PCR was performed in the first round, and the approximately 750 bp VHH fragment was recovered from the gel for the second round of nested PCR. The phage library vector pComb3XSS was constructed. The pComb3XSS phage vector was digested with SfiI to separate it into two large fragments: a 1672 bp fragment (SSstuffer) and a 3301 bp fragment (target fragment). This vector contains His and HA tags for easy purification and detection. The vector and target fragment were ligated by digesting both the vector and the target fragment with SfiI, incubating overnight at 50°C, and then recovering the target fragment from the gel. The ligation molar ratio was Vector:VHH = 1:3. Ten electroporations were performed. Immediately after each electroporation, 1 mL of 2YT medium was added for resuscitation, totaling 100 mL of the resuscitation product. The product was incubated at 37°C and 180 rpm for 45 min. 100 μL of the product was used to determine the storage volume, and the remainder was centrifuged. The remaining product was resuspended in 5 mL of 2YT and spread onto 8 200 mm agar plates. The next day at 10:00 AM... -5 There are 73 clones in total, therefore the library size is 7.3 × 10⁻⁶. 8 (73*100*10 5 ).

[0067] Example 2: Screening and Identification of Nanobody Immunotherapy Library

[0068] Two rounds of screening were conducted using recombinant proteins from the extracellular region of PDL1.

[0069] First round of screening: PDL1-hFc (protein code QP004) and hFc protein were coated separately in immunotubes at 50 nM, 1 ml, and incubated overnight at 4°C. Blocking: Immunotubes were blocked with 2% Milk / PBS at 37°C for 1 hour. Subtraction: The blocking solution was discarded. 900 μl of 2% Milk / PBS was added to the hFc-coated immunotube, followed by 100 μl of phage immunotherapy library. The solution was incubated at room temperature for 1 hour. Binding: The phage immunotherapy library was transferred to the PDL1-hFc-coated immunotube and incubated at room temperature for 1 hour. Washing: Immunotubes were washed 5 times with 1×PBST and 5 times with 1×PBS. Elution: 800 μl of 100 mM TEA was added at room temperature for 10 min. Neutralization: The elution buffer was transferred to a 1.5 ml EP tube, and 400 μl of 1 M pH 7.4 Tris was added. Infection: Neutralized eluted phages were added to 10 ml of TG1 with OD600 = 0.5. Incubation was performed at 37°C for 4 min. The titer was measured, and the remaining bacterial suspension was plated on large plates. Incubation was carried out overnight at 37°C. First round of screening output: 3.7E+06.

[0070] Packaging the phage library: Scrape bacteria from 2×TY medium and inoculate them into 50ml of 2×TY medium + Amp + 1% glucose until the OD is approximately 0.1. Incubate at 37℃ for 200rpm for approximately 1 hour and 20 minutes until the OD is approximately 0.4-0.6. Add 500μl of M13KO7, infect at 37℃ for 40min, centrifuge, discard the supernatant, and resuspend the pellet in 100ml of 2×TY medium + Amp + Kana, incubate overnight at 30℃ for 200rpm. Collecting the phage library: Centrifuge the overnight culture, collect the supernatant, and add 1 / 4 volume of 5×PEG / NaCl. Centrifuge at 4℃ for 20min, discard the supernatant, centrifuge again, discard excess supernatant, add 1ml of PBS to every 50ml of supernatant to resuspend the phage pellet, centrifuge at 13000g for 10min, and transfer the supernatant to a new EP tube. This is the second round of phage input for the phage library, used for subsequent panning.

[0071] Second round (2nd) screening: In immunotubes, 10 nM PDL1-hFc (protein code QP004) and 50 nM hFc protein were coated separately, 2 ng / μl, 1 ml, and incubated overnight at 4°C. Blocking: Immunotubes were blocked with 2% Milk / PBS at 37°C for 1 hour. Subtraction: The blocking solution was discarded. In the hFc-coated immunotube, 2 ml of 2% Milk / PBS was added, followed by 400 μl of phage, and the mixture was rotated at room temperature for 1 hour. Binding: The supernatant was transferred to the PDL1-hFc-coated immunotube and rotated at room temperature for 1 hour. Washing: The immunotubes were washed 10 times with 1×PBST, followed by 10 times with 1×PBS. Elution: 800 μl of 100 mM TEA was added, and the mixture was incubated at room temperature for 10 min. Neutralization: The elution buffer was transferred to a 1.5 ml EP tube, and 400 μl of 1 M pH 7.4 Tris was added. Infection: Neutralized eluted phages were added to 10 ml of TG1 with OD600 = 0.5. Incubation was performed at 37°C for 4 min. The titer was measured, and the remaining bacterial suspension was plated on large plates. Incubation was carried out overnight at 37°C. Second round of screening results: 8.5E+08.

[0072] Immunological library screening and identification: After two rounds of panning, single clones were selected for phage ELISA to screen for positive clones that bind to PDL1. The positive clones were then sequenced to obtain the VHH sequence of the PD-L1 antibody.

[0073] VHH antibody secretion expression: In a 96-well plate, 150 μl of 2×YT + ampicillin + 1% glucose medium was added to each well, and the clones selected in the second round were inoculated and cultured at 37°C for 3 hours. 50 μl of the culture was transferred to a new 96-well plate, and 25 μl of 50% glycerol was added. The plate was then frozen. In the original plate, 50 μl of 2×YT + ampicillin + 3mM IPTG was added to each well. The plate was incubated overnight at 30°C and 220 rpm.

[0074] For ELISA: Coat PDL1-hFc (protein code QP004) 2 μg / ml, 50 μl / well, overnight at 4°C. Block the ELISA plate with 3% BSA, 37°C, 1 hour. Centrifuge the culture overnight and transfer the supernatant to a new 96-well plate. In the PDL1-hFc plate, incubate 25 μl / well of supernatant + 25 μl / well of 5% BSA, RT, 1 hour. PBST 3 times, PBS 6 times. Incubate with secondary antibody: HRP-anti-HA (1:5000), 50 μl / well, RT, 1 hour. PBST 6 times, PBS 3 times. Develop with TMB, stop with H2SO4. Select clones for sequencing verification.

[0075] Blocking ELISA: Coat PD1-hFc protein (protein ID QP1138) 5 μg / ml, 50 μl / well, overnight at 4°C. Block the ELISA plate with 3% BSA, 37°C, 1 hour. Centrifuge the culture overnight and transfer the supernatant to a new 96-well plate. Mix the supernatant with Biotin-PDL1-FC (protein ID Biotin-QP004.3) protein 0.3 μg / ml at a 1:1 ratio, 50 μl / well, RT, 1 hour. PBST 3 times, PBS 6 times. Incubate with secondary antibody: HRP-streptavidin (1:5000), 50 μl / well, RT, 1 hour. PBST 6 times, PBS 3 times. TMB staining, stop with H2SO4.

[0076] The ELISA results of positive clones are shown in Table 1 below:

[0077] Table 1

[0078]

[0079] Clones that are positive for binding to PDL1-hFc (protein number QP004) and block PD1 / PD-L1 binding are selected as positive clones that specifically bind to PD-L1 protein. Sequencing is performed to obtain the VHH sequence of the PD-L1 antibody. Sequences with identical CDR1, 2, and 3 are considered as the same clone, thus obtaining 20 unique nanobody sequences. The amino acid sequences are shown in SEQ ID NO: 1-20.

[0080] Example 3: Construction of FC fusion protein using nanobodies, cloning, expression, and purification of the protein.

[0081] Cloning Design and Construction: Twenty clones were converted into PD-L1-FC fusion proteins with a C-terminus of human IgG1 Fc. The reconstructed plasmids were expressed in HEK293 cells and purified by protein A affinity chromatography, yielding 20 candidate PD-L1VHH-FC fusion proteins QP326-QP345, with sequences as shown in SEQ ID NO: 1-20. These nanobodies are linked to a human IgG1 Fc fragment as shown in SEQ ID NO: 61. The Fc fusion proteins of the corresponding nanobodies are numbered by adding the suffix "Fc" to the corresponding nanobodies.

[0082] Additionally, QP322 is used as a reference in the embodiments, and patent CN201910567277.5 can be consulted. Its anti-PD-L1VHH sequence is as follows:

[0083] EVQLLESGGGLVQPGGSLRLSCAASGFTYGTYAMSWFRQAPGKGREGVACIDIYGRASYTDPVKGRFTISQDNSKNTLYLQMNSLKAEDTAVYYCAARDFGYCTASWVHEGFSRYWGQGTLVTVSS

[0084] In addition, atezolizumab was used as a positive control in the examples. The protein number was QP11801181, and the light and heavy chain sequences were composed of SEQ ID NO:58 and SEQ ID NO:59, respectively.

[0085] Protein expression: 293E cell culture density was maintained at 0.2-3 × 10⁻⁶. 6 The cells were cultured in maintenance medium (GIBCO Freestyle 293 expression medium) between 0.5 and 0.8 × 10⁶ / ml. One day before transfection, the cells to be transfected were centrifuged and the medium changed, adjusting the cell density to 0.5-0.8 × 10⁶ / ml. 6 / ml. On the day of transfection, the density of 293E cells was 1-1.5×10⁻⁶. 6 / ml. Prepare plasmid and transfection reagent PEI. The required amount of plasmid for transfection is 100 μg / 100 ml of cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI thoroughly and let stand for 15 min, not exceeding 20 min. Slowly add the plasmid and PEI mixture to 293E cells and incubate in a shaker at 37°C, 120 rpm, with 8% CO2. On the fifth day after transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 min.

[0086] Protein Affinity Chromatography Purification: Pass the column through equilibration buffer at least 3 CV (20 mL actual volume), ensuring the pH and conductivity of the final effluent match the equilibration buffer at a flow rate of 1 mL / min. Pass 40 mL of the centrifuged culture supernatant through the column at a flow rate of 0.33 mL / min. Pass the column through equilibration buffer again at at least 3 CV (20 mL actual volume), ensuring the pH and conductivity of the final effluent match the equilibration buffer at a flow rate of 0.33 mL / min. Pass the column through elution buffer. Begin collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU and stop collecting when UV280 falls to 15 mAU at a flow rate of 1 mL / min. After sample collection, adjust the pH of PAC-EP to neutral using pH adjustment buffer.

[0087] Example 4: Experimental steps for ELISA detection of the binding of PD-L1 nanobody Fc fusion protein to human PD-L1 protein:

[0088] Plate coating: Coating with positive antibody atezolizumab 2 μg / ml, PD-L1 VHH-FC 1 μg / ml (13.3 nM), 60 μl / well, incubated overnight at 4℃, PBS*3;

[0089] Blocking: 5% milk / PBS, 200 μl / well, 25℃, incubate for 1 h;

[0090] Antigen: Incubate PD-L1-his protein QP003, starting at 5 μg / ml, 4-fold dilution, 8 gradients, 60 μl / well, 25℃, 1h, PBST*5;

[0091] Secondary antibody: anti-his HRP, 1:8000 dilution, 60 μl / well, 25℃, 1h, PBST*5;

[0092] Color development: 100 μl TMB / well, 5-10 min, 2 M H2SO4 to terminate the reaction, 450 nm reading.

[0093] The results are as follows Figure 1-3 As shown, the PD-L1 nanobody Fc fusion protein binds to human PD-L1 protein.

[0094] Example 5: SPR detection of the binding affinity of PD-L1 nanobody Fc fusion protein to human PD-L1 protein

[0095] Affinity was detected by surface plasmon resonance (SPR), and the affinity of the test molecule for human PD-L1 protein was determined by Biacore T200 (GE).

[0096] The information on the antigen and control antibody is as follows:

[0097] Antigen: Human PD-L1 recombinant protein (Sino-Chinese, 10084-H08H), code QPJ08;

[0098] Control antibody: Atezolizumab, code QP11801181.

[0099] The SPR affinity results are shown in Table 2 below:

[0100] Table 2: Biacore detection of the binding affinity between PD-L1 nanobody Fc fusion protein and human PD-L1 protein.

[0101] serial number Abs Ka(1 / Ms) Kd(1 / s) KD(M) 1 Atezolizumab 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

[0102] Results: All the PD-L1 nanobody Fc fusion proteins obtained by screening bound to human PD-L1 protein.

[0103] Example 6: FACS detection of binding of PD-L1 nanobody Fc fusion protein to HCC827 human non-small lung cancer cells naturally expressing human PD-L1.

[0104] Human non-small cell lung cancer cells (HCC827) naturally overexpress PD-L1. HCC827 cells (80% confluence) were prepared in logarithmic growth phase, seeded at 1E5 cells / well in COSTA 96-well plates after adjusting the concentration. The plates were washed once with 1×PBS, and 250 μl of 3% BSA was added to each well, incubated at 37°C for 1 h. Then, a 4-fold serial dilution (33.33 nM to 0.008 nM, corresponding to a mass concentration of 5 μg / mL to 0.001 μg / mL) of the target antibody was added to each well, 50 μl, and incubated on ice for 1 h. The plates were washed twice with 1×PBS, and 50 μl of PE-anti-hFc (1:200) was added to each well, incubated on ice for 1 h. The plates were washed three times with 1×PBS, and the readings were obtained.

[0105] The results are as follows Figure 4 As shown, FACS detection showed that the PD-L1 nanobody Fc fusion protein bound to HCC827 human non-small lung cancer cells that naturally express human PD-L1.

[0106] Example 7: ELISA detection of PD-L1 nanobody Fc fusion protein blocking the binding of human PD-L1 and PD-1 proteins

[0107] Coating protein QP1138 (PD1-FC) 2 μg / ml 50 μl / well, incubated overnight at 4℃. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Prepare 2 μg / ml PDL1-mouse FC and different concentrations of antibody separately, mix in equal volumes, and incubate at room temperature for 1 h. Wash 3 times with PBST, then 3 times with PBS. Secondary antibody incubation: HRP-mouse IgG (1:5000) 50 μl / well, wash 6 times with PBST, then 3 times with PBS. Color development: TMB 100 μl / well, color development for 10 min. Stop with 2M H2SO4 50 μl / well.

[0108] The results are as follows Figure 5 and Figure 6 As shown, the PD-L1 nanobody Fc fusion protein can block the binding of human PD-L1 and PD-1 proteins.

[0109] Example 8: ELISA detection of PD-L1 nanobody Fc fusion protein blocking the binding of human PD-L1 and CD80 proteins

[0110] Coating protein CD80-hFC 2 μg / ml 50 μl / well, incubate overnight at 4℃. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubate at room temperature for 1 h. Prepare 2 μg / ml PDL1-mouse FC and different concentrations of antibody, dilute 3-fold, mix in equal volumes, and incubate at room temperature for 1 h. Wash 3 times with PBST, then 3 times with PBS. Secondary antibody incubation: HRP-mouse IgG (1:5000) 50 μl / well, wash 6 times with PBST, then 3 times with PBS. Color development: TMB 100 μl / well, develop for 10 min. Stop treatment with 2M H2SO4 50 μl / well.

[0111] The results are as follows Figure 7 and Figure 8 As shown, the PD-L1 nanobody Fc fusion protein can block the binding of human PD-L1 and CD80 proteins.

[0112] Example 9: ELISA detection of PD-L1 nanobody Fc fusion protein binding to cynomolgus monkey PD-L1 protein

[0113] Rabbit His antibody (Genscript, A00174) 2 μg / ml, 50 μl / well, incubated overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Incubate monkey PDL1-his (sinobio 90251-C08H) 1 μg / ml, 50 μl / well, incubated at 25°C for 1 h, washed 3 times with PBS. Incubate antibody QP11801181 (starting concentration 20 μg / ml), QP322, QP326… (starting concentration 10 μg / ml), 8 gradients, 1:4 dilution. 50 μl / well, incubated at 25°C for 1 h, washed 6 times with PBS. Incubate secondary antibody HRP-anti-human FC, 1:5000 dilution, 60 μl / well, incubated at 25°C for 1 h, washed 6 times with PBST. Develop with TMB, stop the reaction with H2SO4. Set the microplate reader to 450nm for reading.

[0114] The results are as follows Figure 9 and Figure 10 As shown, the PD-L1 nanobody Fc fusion protein binds to cynomolgus monkey PD-L1 protein.

[0115] Example 10: Humanization design of nanobodies, cloning, expression, and purification of proteins

[0116] Humanization design of nanobodies: By comparing the germline gene database of heavy and light chain variable regions of human antibodies with IMGT and MOE software, germline genes of heavy and light chain variable regions with high homology to QP341, QP344, and QP332 were selected as templates. The CDRs of mouse antibodies were transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then, some important amino acid residues were selected for reverse mutation combinations. The amino acid residues were identified and annotated using the Kabat numbering system. Primers were designed for PCR to construct the VH gene fragments of each humanized antibody, which were then homologously recombinated with the expression vector pQD containing the signal peptide and constant region gene (FC) fragments to construct the full-length antibody expression vector VH-FC-pQD. Multiple primers were designed using the online software DNAWorks (v3.2.4) (http: / / helixweb.nih.gov / dnaworks / ) to synthesize the VH / VK gene fragment containing the required recombination gene: 5'-30bp signal peptide + VH + 30bp FC-3'. Following the instructions for TaKaRa Primer STARGXL DNA polymerase, the designed primers were used for two-step PCR amplification to obtain the VH / VK gene fragment containing the required recombination gene. The expression vector pQD containing the signal peptide and constant region gene (FC) fragment was constructed and digested using restriction endonucleases, such as BsmBI, which recognize sequences different from their digestion sites. The vector was digested with BsmBI, and the gel was recovered for later use. The recombinant expression vector VH-FC-pQD was then constructed. VH containing the gene fragment required for recombination and the expression vector pQD (with signal peptide and constant region gene (FC) fragment) recovered by BsmBI restriction enzyme digestion were added to DH5H competent cells at a ratio of 3:1. The cells were incubated at 0°C on ice for 30 min, followed by heat shock at 42°C for 90 s. Five volumes of LB medium were added, and the cells were incubated at 37°C for 45 min. The cells were then plated on LB-Amp plates and cultured overnight at 37°C. Single clones were picked and sent for sequencing to obtain the target clones.

[0117] The light and heavy chain variable region sequences and protein expression numbers of each clone humanized design are shown in Table 3 below, where all antibodies are fused with the human IgG1-FC constant region at their C-terminus.

[0118] Table 3 Human-centered design of QP341, QP344, and QP332

[0119]

[0120]

[0121] Cloning Construction: The humanized clones designed above were converted into PD-L1-FC fusion proteins with a C-terminus of human IgG1 FC. The reconstructed plasmids were expressed in HEK293 cells and purified by protein A affinity chromatography, yielding a total of 15 humanized PD-L1 VHH-FC fusion proteins QP509-QP522, with sequences as shown in SEQ ID NO: 43-57. The nanobody was linked to a human IgG1 FC fragment, as shown in SEQ ID NO: 61.

[0122] Protein expression: 293E cell culture density was maintained at 0.2-3 × 10⁻⁶. 6 The cells were cultured in maintenance medium (GIBCO Freestyle 293 expression medium) between 0.5 and 0.8 × 10⁶ / ml. One day before transfection, the cells to be transfected were centrifuged and the medium changed, adjusting the cell density to 0.5-0.8 × 10⁶ / ml. 6 / ml. On the day of transfection, the density of 293E cells was 1-1.5×10⁻⁶. 6 / ml. Prepare plasmid and transfection reagent PEI. The required plasmid amount is 100μg / 100ml of cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI thoroughly and let stand for 15 min, not exceeding 20 min. Slowly add the plasmid and PEI mixture to 293E cells and incubate in a shaker at 37℃ and 120 rpm in 8% CO2. On the fifth day after transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 min.

[0123] Protein Affinity Chromatography Purification: Pass the column through equilibration buffer at least 3 CV (20 mL actual volume), ensuring the pH and conductivity of the final effluent match the equilibration buffer at a flow rate of 1 mL / min. Pass 40 mL of the centrifuged culture supernatant through the column at a flow rate of 0.33 mL / min. Pass the column through equilibration buffer again at at least 3 CV (20 mL actual volume), ensuring the pH and conductivity of the final effluent match the equilibration buffer at a flow rate of 0.33 mL / min. Pass the column through elution buffer, starting to collect the elution peak (PAC-EP) when UV280 rises to 15 mAU and stopping collection when UV280 falls to 15 mAU at a flow rate of 1 mL / min. After sample collection, adjust the pH of PAC-EP to neutral using pH adjustment buffer.

[0124] Example 11: ELISA detection of binding of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein

[0125] The sample was coated with humanized nanobody QP509 (1 μg / ml) and the positive control atezolizumab (2 μg / ml). Incubation was performed overnight at 4°C, followed by three washes with PBS. Blocking was done with 5% milk and incubated at RT for 1 h. Biotin-QP004 (Biotin-PD-L1 hFC) was incubated at an initial concentration of 0.5 μg / ml, diluted 5-fold, and incubated at RT for 1 h, followed by three washes with PBST. HRP-streptavidin was diluted 5000-fold and washed 5 times with PBST. TMB was developed for 10 min. The reading was taken at 450 nm.

[0126] The results are as follows Figure 11-13 As shown, ELISA was used to detect the binding of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein, and molecules with low EC50 values ​​were selected for further validation.

[0127] Example 12: ELISA detection of humanized PD-L1 nanobody Fc fusion protein blocking the binding of human PD-L1 and PD-1 proteins

[0128] Coating protein QP1138 (PD1-FC) 2 μg / ml 50 μl / well, incubated overnight at 4℃. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Prepare 2 μg / ml PDL1-mouse FC and different concentrations of QP509 and other humanized nanobodies 15 μg / ml, positive control atezolizumab 30 μg / ml, diluted 1:3, mix in equal volumes, and incubate at room temperature for 1 h. Wash 3 times with PBST, then 3 times with PBS. Secondary antibody incubation: HRP-mouse IgG (1:5000) 50 μl / well, wash 6 times with PBST, then 3 times with PBS. Color development: TMB 100 μl / well, color development for 10 min. Stop treatment with 2M H2SO4 50 μl / well.

[0129] The results are as follows Figure 14-16 As shown, ELISA detection of the humanized PD-L1 nanobody Fc fusion protein blocked the binding of human PD-L1 protein to PD-1 protein, further verifying this finding.

[0130] Example 13: SPR detection of the binding affinity of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein

[0131] Affinity was detected by surface plasmon resonance (SPR), and the affinity of the test molecule for human PD-L1 protein was determined by Biacore T200 (GE).

[0132] The information on the antigen and control antibody is as follows:

[0133] Antigen: Human PD-L1 recombinant protein (sinobiologic, 10084-H08H);

[0134] Control antibody: Atezolizumab.

[0135] The SPR affinity results are shown in Table 4 below:

[0136] Table 4

[0137] Abs Ka(1 / Ms) Kd(1 / s) KD(M) Atezolizumab 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

[0138] Humanized PD-L1 nanobody Fc fusion protein binds to human PD-L1 protein.

[0139] Example 14: ELISA detection of binding of humanized PD-L1 nanobody Fc fusion protein to cynomolgus monkey PD-L1 protein

[0140] Rabbit His antibody (Genscript, A00174) 2 μg / ml, 50 μl / well, incubated overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Incubate monkey PDL1-his (sinobio 90251-C08H) 1 μg / ml, 50 μl / well, incubated at 25°C for 1 h, washed 3 times with PBS. Incubate positive control atezolizumab (starting concentration 20 μg / ml, QP322, QP326… starting concentration 10 μg / ml, 1:4 dilution in 8 gradients). 50 μl / well, incubated at 25°C for 1 h, washed 6 times with PBS. Incubate secondary antibody HRP-anti-human FC, 1:5000 dilution, 60 μl / well, incubated at 25°C for 1 h, washed 6 times with PBST. Develop with TMB, stop the reaction with H2SO4. Set the microplate reader to 450nm for reading.

[0141] The results are as follows Figure 17 As shown, the humanized PD-L1 nanobody Fc fusion protein binds to cynomolgus monkey PD-L1 protein.

[0142] To evaluate the in vitro functional activity of PD-L1, two forms of humanized nanobody Fc fusion proteins were designed. Specifically, the first form involves fusing QP509 VHH to the N-terminus of the FC protein, and the protein is designated as QP509(QP509 VHH-hFC). The second form involves fusing QP509 VHH to the C-terminus of human IgG1 FC segment mutation that eliminates FCγR function (EU counting L234A / L235A / K338A), and the protein is designated as QP3447(hFC(L234A / L235A / K338A)-QP509 VHH). The amino acid sequence of QP3447 is shown in SEQ ID NO: 60.

[0143] Example 15: SPR detection of the binding affinity of humanized PD-L1 nanobody Fc fusion protein to human PD-L1 protein

[0144] Affinity was detected by surface plasmon resonance (SPR), and the affinity of the test molecule for human PD-L1 protein was determined by Biacore T200 (GE).

[0145] The information on the antigen and control antibody is as follows:

[0146] Antigen: Recombinant human PD-L1 protein (SinoBio, 10084-H08H)

[0147] Control antibody: Atezolizumab (ROCHE).

[0148] The experimental results are shown in Table 5.

[0149] Table 5: Binding affinity of QP3447 and other molecules to human PD-L1.

[0150] protein 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 Atezolizumab 2.07E+05 2.87E-04 1.39E-09

[0151] Example 16: ELISA detection of QP3447 binding to human PD-L1 protein

[0152] Rabbit His antibody (Genscript, A00174) 2 μg / ml, 50 μl / well, incubated overnight at 4°C. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Incubate human PDL1-his (sinobio 10084-H08H) 1 μg / ml, 50 μl / well, at 25°C for 1 h, washed 3 times with PBS. Incubate positive controls atezolizumab, QP509, and QP3447 at an initial concentration of 67 nM, 5-fold dilutions in 7 gradients, with the last well diluted 100-fold. 50 μl / well, incubated at 25°C for 1 h, washed 6 times with PBS. Incubate secondary antibody HRP-anti-human FC, 1:5000 dilution, 60 μl / well, at 25°C for 1 h, washed 6 times with PBST. Develop with TMB, stop the reaction with H2SO4. Set the microplate reader to 450nm for reading.

[0153] The results of ELISA detection of the binding of human PD-L1 nanobody Fc fusion proteins QP509 and QP3447 to human PD-L1 protein are shown in the figure. Figure 18 middle.

[0154] Example 17: ELISA detection of humanized PD-L1 nanobody Fc fusion protein blocking the binding of human PD-L1 and PD-1 proteins

[0155] Coating protein QP1138 (PD1-FC) 2 μg / ml 50 μl / well, incubated overnight at 4℃. Wash 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Prepare 2 μg / ml PDL1-mouse FC fusion protein, 50 μl / well, incubated at room temperature for 1 h, washed 3 times with PBST, washed 3 times with PBS. Incubate with different concentrations of QP509, QP3447 and positive control atezolizumab 333 nM starting concentration, 5-fold dilution in 8 concentration gradients, incubated at room temperature for 1 h. Wash 3 times with PBST, washed 3 times with PBS. Secondary antibody incubation: HRP-mouse IgG (1:5000) 50 μl / well, washed 6 times with PBST, washed 3 times with PBS. Colorimetric development: TMB 100 μl / well, color development for 10 min. Stop with 2M H2SO4 50 μl / well.

[0156] The results of ELISA detection of the Fc fusion protein of humanized PD-L1 nanobody blocking the binding of human PD-L1 protein to PD-1 protein are shown in the figure. Figure 19 middle.

[0157] Example 18: FACS detection of binding of PD-L1 nanobody Fc fusion protein QP3447 to HCC827 human non-small lung cancer cells naturally expressing human PD-L1.

[0158] Human non-small cell lung cancer cells (HCC827) naturally overexpress PD-L1. HCC827 cells in logarithmic growth phase (80% confluence) were prepared, adjusted to the desired concentration, and seeded in 96-well plates at 1E5 cells / well. The plates were washed once with 1×PBS, and 250 μl of 3% BSA was added to each well. The plates were incubated at 37°C for 1 h. Different concentrations of the target antibody were added, 50 μl per well, and incubated on ice for 1 h. The plates were washed twice with 1×PBS, and 50 μl of PE-anti-hFc (1:200) was added to each well. The plates were incubated on ice for 1 h. The plates were washed three times with 1×PBS, and the readings were obtained.

[0159] FACS analysis results of PD-L1 nanobody FC fusion protein QP3447 bound to naturally expressing human PD-L1 in human non-small lung cancer cells HCC827 showed... Figure 20 middle.

[0160] Example 19: Humanized nanobody Fc fusion protein promotes T cell proliferation in mixed lymphocyte responses

[0161] PD-L1 functional activity assay (mixed lymphocyte reaction MLR): Preparation of DC (donor 1) cells: resuscitate PBMCs and use EasySep TMHuman monocytes were isolated using a STEMCELL, 19359 cytokine isolation kit. rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml) were added, and cells were cultured at 37°C for 6 days to induce iDCs. The medium was changed halfway every 2-3 days, with additional rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml). Cells were collected, centrifuged at 300 x g for 5 min, resuspended in medium containing rhGM-CSF (1000 U / ml) and rhIL4 (500 U / ml), and LPS (1 μg / ml) was added. Cells were cultured at 37°C for another day to induce mature DCs. Cells were collected, counted, and prepared for use. T (donor 2) cells were prepared by resuscitating PBMCs and using EasySep... TM Human CD4+ T cell isolation kit (STEMCELL, 17952) for isolating CD4+ T cells.

[0162] Antibody preparation: Dilute the antibody (initial concentration 10 μg / ml) 1:5 in culture medium to obtain 6 concentrations. Mix DC cells and T cells at a ratio of 1:10, add different concentrations of antibody, and culture. Detect IL2 expression in the culture supernatant on day 2 and IFNγ expression in the culture supernatant on day 5.

[0163] The results are as follows Figure 18-21 As shown, in the mixed lymphocyte reaction experiment, the concentrations of cytokines IFNγ and IL-2 produced after T cell activation by QP509 and QP3447 were significantly antibody concentration-dependent.

[0164] Example 20: Evaluation of the inhibitory effect of anti-PD-L1 nanobody on tumor growth in a transgenic mouse model

[0165] Experimental Procedure: Since PDL1 antibodies cannot recognize mouse PDL1, an immune checkpoint humanized mouse model (C57BL / 6-hPDL1) was used to evaluate the growth inhibitory effect of anti-PD-L1 nanobodies on mouse colon cancer tumor cells MC38. MC38-hPDL1(tg)-mPDL1(KO) cells in logarithmic growth phase were collected, resuspended in PBS to an appropriate concentration, and then seeded. Each experimental mouse was then subcutaneously seeded with 1 × 102 cells on its right back. 6 MC38-hPDL1 cells were used to monitor tumor growth regularly until the tumor reached an average volume of approximately 100 mm. 3 Mice were randomly assigned to groups based on tumor size and body weight for drug administration. The day of administration was defined as day 0. The administration regimen is shown in Table 6 below:

[0166] Table 6

[0167]

[0168] The administration volume is 10 μl / g body weight.

[0169] Mouse body weight and tumor volume were observed three times a week. The long and short axes of the tumor were measured using calipers, and the tumor volume (mm) was calculated according to the formula. 3 )=0.5×(a×b 2 Calculate and record tumor growth, and plot a tumor growth curve. Results are as follows: Figure 22-23 And as shown in Table 7 below:

[0170] Table 7

[0171]

[0172] Based on the statistical analysis of tumor volume and weight at the experimental endpoint (day 24), compared with the control group, the G2 group showed significant differences in tumor volume (TGItv%) and tumor weight (TGItw%) of QP3447 (P < 0.05); the G3 group, using the control molecule atezolizumab, showed significant differences in tumor volume (TGI%) (TGI%) (TGI tw%) (TGI tw%) (P < 0.05). Both QP3447 and atezolizumab demonstrated strong antitumor activity in this model.

[0173] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0174] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0175] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. An anti-PD-L1 nanobody, characterized in that, The anti-PD-L1 nanobody comprises three amino acid fragments: CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are shown in any of the following sets: (1) CDR1: GFTLDDYAIG (SEQ ID NO: 21), CDR2: CISKSGETSYYVDSVRG(SEQ ID NO:31)、 CDR3:AAGSWCTVGSMSRQFYRQFFRS(SEQ ID NO:38); (2) CDR1: GFTLDDYAIG (SEQ ID NO: 21), CDR2: CISKSGETTNYVDSVKG (SEQ ID NO:28), CDR3:AAGSWCTVGSMSRQFYRQFFRS(SEQ ID NO:38); (3) CDR1: GFTLDDYAIG (SEQ ID NO: 21), CDR2:CISKSGETANYVDSVKG(SEQ ID NO:30)、 CDR3:AAGSWCTVGSMSRQFYRQFFRS(SEQ ID NO:38); (4) CDR1: GFTLDDYAIG (SEQ ID NO: 21), CDR2: CISKSGETSYYVDSVKG (SEQ ID NO:32), CDR3: AAGSWCTVGSMSRQFYRQFFRS (SEQ ID NO:38).

2. The anti-PD-L1 nanobody according to claim 1, characterized in that, The amino acid sequence of the anti-PD-L1 nanobody is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO: 6 to 8 or SEQ ID NO:

3.

3. The anti-PD-L1 nanobody according to claim 1, characterized in that, The amino acid sequence of the anti-PD-L1 nanobody is shown in any one of SEQ ID NO: 6 to 8 or SEQ ID NO:

3.

4. The anti-PD-L1 nanobody according to claim 1, characterized in that, The anti-PD-L1 nanobody is a humanized anti-PD-L1 nanobody.

5. The anti-PD-L1 nanobody according to claim 1, characterized in that, The anti-PD-L1 nanobody has an amino acid sequence shown in any one of SEQ ID NO: 53 to SEQ ID NO:

57.

6. An Fc fusion protein of an anti-PD-L1 nanobody, characterized in that, The Fc fusion protein of the anti-PD-L1 nanobody comprises: the anti-PD-L1 nanobody according to any one of claims 1 to 5 and the Fc segment, wherein the Fc segment is selected from the group consisting of human IgG1, IgG2, IgG3, IgG4 and their mutants.

7. The Fc fusion protein of the anti-PD-L1 nanobody as described in claim 6, characterized in that, The Fc segment is shown as SEQ ID NO:61 or SEQ ID NO:

62.

8. Use of the anti-PD-L1 nanobody of any one of claims 1 to 5, or the Fc fusion protein of the anti-PD-L1 nanobody of claim 7, in the preparation of a medicament for the treatment of colorectal cancer or non-small cell lung cancer.

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