Anti-pd-1 nanobodies and uses thereof

Abstract

The present application provides anti-PD-1 nanobodies and uses thereof. The anti-PD-1 nanobodies comprise a VHH chain. The present application obtains the nanobody sequences by screening, and provides humanized antibodies, and Fc fusion proteins containing the nanobody sequences. The anti-PD-1 nanobodies of the present disclosure are expected to be used as therapeutic antibodies for the treatment of various PD-1 high expression malignant tumors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to anti-PD-1 nanobodies and their applications. Background Technology

[0002] In the classical immune surveillance theory, the immune system can recognize tumor antigens and eliminate tumor cells. Activation of oncogenes causes tumor cells to alter themselves 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 PD-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, the tumor tissue could be eliminated, and the theory of immunotherapy has been widely proven in clinical treatment.

[0003] PD-1 (programmed death receptor 1), also known as CD279 (differentiation cluster 279), is an important immunosuppressive molecule. First reported in 1992, the human PD-1 encoding gene PDCD1 is located at 2q37.3, is 2097 bp in length, and consists 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 pancreatic islet cells), and immune-exempt sites (such as the placenta, testis, and eye). 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.

[0004] 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); 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, as demonstrated in esophageal cancer, gastric cancer, renal cancer, ovarian cancer, bladder cancer, pancreatic cancer, and melanoma. Summary of the Invention

[0005] The purpose of this invention is to provide anti-PD-1 nanobodies and their applications.

[0006] To achieve the above objectives, the present invention provides an anti-PD-1 nanobody comprising: a VHH chain, wherein the complementarity-determining region of the VHH chain includes CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9 or SEQ ID NO: 10.

[0007] Optionally, the amino acid sequence of the VHH chain includes sequences selected from any one of SEQ ID NO:5, 6 or SEQ ID NO:12-18, or sequences having at least 85%, 90%, 95%, 97% or 99% identity with them.

[0008] The present invention also provides a fusion protein comprising: the aforementioned anti-PD-1 nanobody.

[0009] Optionally, the fusion protein further includes an Fc segment; the Fc segment is fused to the N-terminus or C-terminus of the anti-PD-1 nanobody.

[0010] Optionally, the fusion protein further includes: other functional fragments besides the anti-PD-1 nanobody and the Fc segment; the Fc segment is fused with the anti-PD-1 nanobody, and / or the Fc segment is fused with the other functional fragments.

[0011] Optionally, the Fc region is selected from human IgG1, IgG2, IgG3, or IgG4 or their variants; preferably, the Fc region is selected from a variant of human IgG1; preferably, the Fc domain is selected to eliminate immune effector function, preferably including any of the following mutations, the following mutations being counted according to EU:

[0012]

[0013] The present invention also provides an anti-PD-1 antibody molecule, comprising: one or more of the aforementioned anti-PD-1 nanobodies; or comprising: the aforementioned fusion protein; wherein the anti-PD-1 antibody molecule is a monovalent antibody, a bivalent or multivalent antibody, a bispecific antibody, or a multispecific antibody.

[0014] The present invention also provides a chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain and an intracellular domain, wherein the extracellular antigen-binding domain comprises the aforementioned anti-PD-1 nanobody.

[0015] The present invention also provides a nucleic acid molecule encoding either the anti-PD-1 nanobody or the fusion protein.

[0016] The present invention also provides a carrier comprising the aforementioned nucleic acid molecule.

[0017] The present invention also provides a host cell obtained by transformation using the aforementioned vector.

[0018] The present invention also provides an antibody-drug conjugate comprising the aforementioned anti-PD-1 nanobody and a drug conjugated to the anti-PD-1 nanobody.

[0019] The present invention also provides a pharmaceutical composition comprising: the anti-PD-1 nanobody, or the fusion protein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0020] The present invention also provides the use of the aforementioned anti-PD-1 nanobody in the preparation of medicaments for inhibiting or treating diseases.

[0021] The present invention also provides the use of the aforementioned anti-PD-1 nanobody for the purpose of preventing or treating diseases by stimulating immune function, such as for the preparation of vaccines for prevention or treatment.

[0022] Optionally, the disease is a PD-1 mediated disease.

[0023] Optionally, the disease is cancer; preferably, the cancer includes breast cancer, lung cancer, stomach cancer, intestinal cancer, kidney cancer, melanoma, bladder cancer, head and neck cancer, lymphoma, malignant skin tumors, or non-small cell lung cancer.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The antibody sequence of this invention is novel and is expected to be used as a therapeutic antibody for the treatment of various malignant tumors with high PD-1 expression.

[0026] 2. The PD-1 nanobody of the present invention has high specificity and high affinity; in addition, the VHH chain of the nanobody can be combined with various molecular chains to form different antibody molecular forms, such as forming specific antibodies against two or more targets. Attached Figure Description

[0027] Figure 1 The image shows the results of ELISA detection of PD-1 nanobody binding to human PD-1 protein.

[0028] Figure 2 The figure shows the results of ELISA detection of the PD-1 antibody blocking the binding activity of PD-L1 / PD-1.

[0029] Figure 3 Figure showing the results of LISA detection of PD-1 nanobody binding to cynomolgus PD-1 protein.

[0030] Figure 4 The figure shows the results of ELISA detection of the binding of humanized PD-1 nanobody Fc fusion protein to human PD-1 protein.

[0031] Figure 5 The figure shows the results of ELISA detection of the humanized PD-1 nanobody Fc fusion protein blocking the binding of human PD-L1 protein to PD-1 protein.

[0032] Figure 6 The figure shows the results of ELISA detection of the binding of humanized PD-1 nanobody Fc fusion protein to human PD-1 protein.

[0033] Figure 7 The figure shows the results of ELISA detection of the humanized PD-1 nanobody Fc fusion protein blocking the binding of human PD-L1 protein to PD-1 protein.

[0034] Figure 8 , Figure 9 The image shows the results of IL-2 secretion in the supernatant after 48 hours of mixed lymphocyte reaction.

[0035] Figure 10 , Figure 11 The image shows the results of IFN-γ secretion in the supernatant after 120 hours of mixed lymphocyte reaction.

[0036] Figure 12 The image shows the detection results of IL-2 secretion in PBMCs stimulated by SEB for 48 hours after the antibody-anti-PD-1 antibody was used to detect PBMC proliferation.

[0037] Figure 13 The figure shows the results of the efficacy test of the antibody against the mouse colon cancer CDX model MC38-hPDL1-mClaudin18.2.

[0038] Figure 14 This is a graph showing the tumor growth curves for each group after grouping.

[0039] Figure 15 The image shows the D17 tumor weight of mice in each group that have had the antibodies of this invention added and the control antibody added.

[0040] Figure 16 The graph shows the weight gain curves of mice in each group. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] Experimental methods not specifying particular conditions in this experiment are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their origin are commercially available, standard reagents.

[0043] the term:

[0044] "Antibody" (Ab) refers to an immunoglobulin molecule (Ig) that contains at least one antigen-binding site and can specifically bind to antigens.

[0045] An antigen is a substance in the body that can induce an immune response and specifically binds to an antibody. The binding of an antibody to an antigen is mediated by interactions between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the antigen surface where the antibody binds is called an "antigenic determinant" or "epitope." Generally, each antigen has multiple determinants.

[0046] The term "antibody" as used in this invention is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, multispecific antibodies (e.g., bispecific antibodies) containing at least two distinct antigen-binding domains, and single-domain antibodies or nanobodies. Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. The antibodies of this invention can be derived from any animal, including but not limited to immunoglobulin molecules from humans, non-human primates, mice, rats, cattle, horses, chickens, camels, and alpacas. Antibodies may contain additional modifications, such as non-natural amino acids, Fc effector functional mutations, and glycosylation site mutations. Antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided that these antibodies exhibit the desired biological activity.

[0047] The basic structure of a conventional antibody consists of a Y-shaped monomer formed by two identical heavy chains (H) and two identical light chains (L) linked by disulfide bonds. Each chain comprises 2–5 domains (also called functional regions) containing approximately 110 amino acids each, with similar sequences but different functions. The amino acid sequences near the N-terminus of both the light and heavy chains vary considerably, forming a domain called the variable region (V region); the region near the C-terminus with a relatively constant amino acid sequence is called the constant region (C region). The V regions of the heavy and light chains are called VH and VL, respectively. Each of the VH and VL regions has three highly variable regions in terms of amino acid composition and sequence, called hypervariable regions (HVR); these regions form a spatial conformation complementary to the antigenic epitope and are also known as complementarity determining regions (CDRs). The three CDRs of VH can be represented by VHCDR1, VHCDR2, and VHCDR3, respectively, and the three CDRs of VL can be represented by VLCDR1, VLCDR2, and VLCDR3, respectively. The six CDRs of VH and VL together constitute the antigen-binding site. The diversity of amino acids in the CDR region is the molecular basis for the specific binding of antibodies to a large number of different antigens. The amino acid composition and sequence outside the CDRs in the V region are relatively stable and are called the framework region (FR). VH and VL each have four framework regions, represented by FR1, FR2, FR3, and FR4, respectively. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are called CH and CL, respectively. The heavy chain constant regions of IgG, IgA, and IgD have three domains: CH1, CH2, and CH3, while those of IgM and IgE have four domains: CH1, CH2, CH3, and CH4. The region between CH1 and CH2 is the hinge region, which is rich in proline. Therefore, it is easily stretched and bent, and can change the distance between the two arms of the Y-shape, which is conducive to the simultaneous binding of antigenic epitopes by both arms.

[0048] The "antigen-binding fragment" of a conventional antibody refers to Fab fragments, F(ab')2 fragments, Fv fragments, ScFv fragments, etc., which possess antigen-binding activity. A "Fab fragment" (fragment of antigen binding, Fab) refers to an antibody fragment composed of VL, VH, CL, and CH1 domains, which binds to a single antigenic epitope (monovalent). Those skilled in the art will know that papain hydrolyzes IgG to form two identical Fab fragments and one Fc fragment; pepsin hydrolyzes IgG to form one F(ab')2 fragment and several polypeptide fragments (pFc'). If the disulfide bonds between the F(ab')2 heavy chains break, two Fab' fragments can be formed, which can be further enzymatically hydrolyzed into Fv fragments. Fv fragments contain variable regions of the antibody heavy chain and light chain, but no constant regions. Single-chain variable fragments (scFv), or single-chain antibodies, are composed of variable regions of the antibody heavy chain and light chain linked by a linker.

[0049] In 1993, Hamers' laboratory discovered that camel serum, in addition to the conventional tetravalent antibodies, also contained a large number of molecules similar to immunoglobulins (IgG). These molecules are called heavy chain antibodies (HCAbs), which naturally lack the light chain and CH1 constant region of the heavy chain of traditional antibodies, but still possess strong antigen-binding ability. Hamers' laboratory also analyzed and identified the structure and sequence of heavy chain antibodies in camel serum, finding that the antigen-binding region of heavy chain antibodies consists only of variable domain fragments, which are functionally equivalent to the antigen-binding fragment (Fab) of traditional antibodies. Therefore, the antigen recognition region fragment of heavy chain antibodies is called VHH (variable domain of the heavy chain of heavy-chain antibody), and based on this, nanobodies containing only the VHH domain were developed. Nanobodies are also known as single-domain antibodies (sdAbs).

[0050] Nanobodies are easily modified and can be formed into multivalent forms. Due to their small molecular weight and single-gene encoding, nanobodies are easily genetically engineered. Multiple nanobodies can be aggregated using short linker sequences, and they can even be linked and combined with Fab, Fv, and ScFv fragments of conventional antibodies to form multivalent or multispecific antibody structures. Bivalent or multivalent antibodies can recognize the same epitope but have a higher affinity for antigens than monovalent antibodies. Bispecific or multispecific antibodies can bind to different targets or different binding regions on the same target, exhibiting stronger antigen recognition capabilities than monovalent antibodies.

[0051] Nanobodies readily form new fusion molecules with other structures (such as BSA, IgG-Fc, etc.). In these new fusion molecules, the nanobody binds directionally to its target antigen, and the portion fused with the nanobody performs the corresponding function. Therefore, they can be used in combination with other drugs or applied in diagnostics and as experimental research tools in various fields. Nanobody screening can be divided into steps including alpaca immunization, lymphocyte extraction, nanobody library construction, phage library construction, specific phage screening, E. coli expression, and antibody purification.

[0052] The term "fusion" refers to the connection of components by direct peptide bonds, the connection via linker fragments, or fusion through intermolecular interactions. In a single peptide chain, fusion refers to the connection by direct peptide bonds or the connection via linker fragments.

[0053] The terms "Fc," "Fc fragment," or "Fc segment" refer to crystallizable fragments that lack antigen-binding activity and are the sites where antibodies interact with effector molecules or cell surface Fc receptors (FcRs). Fc fragments contain the constant region polypeptide of the antibody, excluding the CH1 region of the heavy chain. Fc fragments bind to cells with corresponding Fc receptors on their surface, producing different biological effects. In ADCC (antibody-dependent cell-mediated cytotoxicity), the Fab fragment of the antibody binds to the antigenic epitopes of virus-infected cells or tumor cells, while its Fc fragment binds to FcRs on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells. ADCP, or antibody-dependent cellular phagocytosis, operates on the mechanism of antibody-mediated phagocytosis. The target cells activate the FcγR mechanism on the surface of macrophages, inducing phagocytosis, leading to internalization of the target cells and acidification and degradation of the phagosomes. Because heavy chain antibodies (HCAbs) naturally lack the CH1 constant region of the light chain and heavy chain of conventional antibodies, the Fc segment of heavy chain antibodies only has the CH2 and CH3 domains.

[0054] Those skilled in the art will recognize that Fc variants or mutations can take many forms, and different Fc variants can be selected in different embodiments. The Fc segment of an antibody can selectively eliminate immune effector function, including, but not limited to, combinations of the following mutations (based on EU counts):

[0055]

[0056] The CDR amino acid residues of the antibody or antigen-binding fragment described in this invention conform to the known Kabat numbering rules in both number and position.

[0057] "Humanized antibodies" refer to antibodies obtained by transplanting the CDR sequence of a non-human antibody into the variable region framework of a human antibody. These framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0058] "Sequence identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides, which is the degree to which two polynucleotides or two polypeptides have the same bases or amino acids.

[0059] The term "vector" refers to a polynucleotide molecule capable of transporting another polynucleotide linked to it. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be attached. Another type of vector is a viral vector, in which the additional DNA segment can be attached to the viral genome. Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Typically, expression vectors useful in recombinant DNA technologies are in the form of plasmids.

[0060] Chimeric antigen receptor T-cells (CAR-T) are genetically modified T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and expand. The chimeric antigen receptor (CAR) is the core component of CAR-T, consisting of three main structural parts: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain. From the N-terminus to the C-terminus, the structure of a chimeric antigen receptor (CAR) contains a signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain. Antibodies or antibody antigen-binding fragments can serve as the extracellular antigen-binding domain and are an important component of CARs. Nanobodies, with their unique properties such as low molecular weight, good water solubility, and high stability, have recently been used to develop CAR-T cells for targeted delivery.

[0061] Antibody-drug conjugates (ADCs) are binding proteins that are linked to one or more chemical drugs (optionally therapeutic agents or cytotoxic agents). Antibody-drug conjugates can be obtained by linking cytotoxic small molecules (cytotoxins) and antibodies through permanent or unstable chemical linkers. Fully human single-domain antibody-drug conjugates (UdADCs), which conjugate human nanobodies with drugs, have demonstrated significant advantages over traditional antibody-drug conjugates in tumor organoids, tumor microspheres, and mouse tumor models.

[0062] Example 1: Preparation of antigen and control antibody

[0063] PD-1-His recombinant protein (protein code QPP11) was purchased from Sinocare Biotechnology Co., Ltd., catalog number: 10377-H08H.

[0064] The PD-1-hFc recombinant protein (protein code QP1138), produced internally by the company, is a PD-1 extracellular region sequence (selected from UNIPROT, sequence number Q15116, amino acids 24-170) fused with the human IgG1 FC, as shown below. It was loaded into the eukaryotic expression vector pTargeT to construct an expression clone, stably transfected into CHOS cells, and purified to obtain the protein.

[0065] SEQ ID NO: 1QP1138

[0066] FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*.

[0067] The PD-L1-hFc recombinant protein (protein code QP004), produced internally by the company, is a PD-L1 extracellular region sequence (selected from UNIPROT, sequence number Q9NZQ7, amino acids 1-238) fused with the human IgG1 FC, as shown below. It was loaded into the eukaryotic expression vector pQD to construct an expression clone, transiently transfected into 293E cells to express the protein, and then purified to obtain the protein.

[0068] SEQ ID NO: 2QP004

[0069] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISY GGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPN EREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*.

[0070] The control antibody was the anti-PD-1 antibody Nivolumab (5C4), which was obtained by fusing the following two sequences, inserted into the eukaryotic expression vector pQD, constructed an expression clone, transiently expressed the protein in 293E cells, and purified to obtain the protein QP32103211.

[0071] SEQ ID NO: 3>QD3210:

[0072] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVT VSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0073] SEQ ID NO: 4>QD3211:

[0074] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0075] Example 2: Alpaca Immunization and Construction of a Phage Immunobank

[0076] Alpaca immunization: Using recombinant PD-1 extracellular region protein (Essential Biotech, 10377-H08H) as the antigen, a healthy alpaca (vicugna pacos, alpaca, lama pacos) was immunized. For the first immunization, Freund's complete adjuvant (CFA) was used, and 0.3 mg of antigen protein was administered. For the second to fifth immunizations, Freund's incomplete adjuvant (IFA) was used to emulsify and mix the antigen with the protein before subcutaneous injection at multiple sites, and 0.2 mg of antigen protein was administered.

[0077] 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]). III. First-Strand Synthesis System for RT-PCR Instructions: Transcribe 8 μg of RNA. Nested PCR first round: After gel extraction and recovery of the approximately 750 bp VHH fragment, perform nested PCR second round. Construct the phage library vector pComb3XSS. The pComb3XSS phage vector is digested with SfiI to separate it into two large fragments: a 1672 bp fragment (SSstuffer) and a 3301 bp fragment (vector target fragment). This vector contains His and HA tags for easy purification and detection. Ligation of the vector and target fragment: Both the vector and target fragment are digested with SfiI and incubated overnight at 50°C. The target fragment is then recovered from the gel. The ligation molar ratio is 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 ).

[0078] Example 3: Screening and Identification of Nanobody Immunotherapy Library

[0079] Two rounds of screening were performed using recombinant proteins from the extracellular region of PD1.

[0080] First round (1st) screening: In an immunoassay tube, coat the tube with antigen at 5 ng / ul, add 1 ml, and incubate overnight at 4°C. Block: Block the immunoassay tube with 2% MPBS, incubate at 37°C for 1 hour. Diminishing: Add 1800ul of 2% MPBS, then add 200ul of input phage, add the antigen at a final concentration of 50 ng / ul, and rotate at room temperature for 1 hour. Binding: Transfer the supernatant to the antigen-coated immunoassay tube and rotate at room temperature for 1 hour. Wash: Wash the immunoassay tube 8 times with 1xPBST, then 4 times with 1xPBS. Elution: Add 800ul of 100mM TEA, incubate at room temperature for 10 minutes. Neutralization: Transfer the eluent to a 1.5 ml EP tube and add 400ul of 1M pH 7.4 Tris. Infection: Add the neutralized elution phage to 10 ml of TG1 with OD600 = 0.5. Incubate at 37°C for 4 minutes. Measure the titer, and plate the remaining bacterial culture on a large plate. Incubate overnight at 37°C.

[0081] Packaging 2nd input phage: Scrape bacteria from 2xTY medium, inoculate with 50ml of 2xTY medium + amp + 1% glucose until OD ~0.1, incubate at 37°C 200rpm for ~1 hour 20 mins until OD ~0.4-0.6. Add 500ul M 13KO7, incubate at 37°C for 40 mins, centrifuge and discard the supernatant, resuspend the pellet in 100ml of 2xTY medium + amp + kanamycin. Incubate overnight at 30°C 200rpm. 2nd input phage precipitation: Centrifuge the overnight culture at 4200rpm for 15 min. Take 40ml of supernatant, add 10ml of PEG / NaCl, mix well, and place on ice for 20 min. Centrifuge at 4200rpm for 15 min. Discard the supernatant. Briefly centrifuge and discard the supernatant. Resuspend the pellet in 1ml of 1xPBS. Centrifuge at 13000rpm for 10 min. Transfer the supernatant to a new 1.5EP tube. Use for subsequent rinsing, or add 0.5 ml of 50% glycerin and freeze at -80°C.

[0082] Second round (2) ndScreening: In an immunoassay tube, coat the tube with antigen at a concentration of 2 ng / ul, add 1 ml, and incubate overnight at 4°C. Blocking: Block the immunoassay tube with 2% MPBS, incubate at 37°C for 1 hour. Subtraction: Add 1800ul of 2% MPBS, then add 200ul of input phage, add the subtraction antigen to a final concentration of 50 ng / ul, and rotate at room temperature for 1 hour. Binding: Transfer the supernatant to the antigen-coated immunoassay tube and rotate at room temperature for 1 hour. Wash: Wash the immunoassay tube 8 times with 1xPBST, then 4 times with 1xPBS. Elution: Add 800ul of 100mM TEA, incubate at room temperature for 10 minutes. Neutralization: Transfer the eluent to a 1.5 ml EP tube and add 400ul of 1M pH 7.4 Tris. Infection: Add the neutralized elution phage to 10 ml of TG1 with an OD600 of 0.5. Incubate at 37°C for 4 minutes. Measure the titer, and plate the remaining bacterial culture onto a large plate. Incubate overnight at 37°C.

[0083] Packaging 2nd output phage: Scrape bacteria from 2xTY medium, inoculate with 50ml of 2xTY medium + amp + 1% glucose until OD ~0.1, incubate at 37°C 200rpm for ~1 hour 20 mins until OD ~0.4-0.6. Add 500ul M 13KO7, incubate at 37°C for 40 mins, centrifuge and discard the supernatant, resuspend the pellet in 100ml of 2xTY medium + amp + kanamycin. Incubate overnight at 30°C 200rpm. 2nd output phage precipitation: Centrifuge the overnight culture at 4200rpm for 15 min. Take 40ml of supernatant, add 10ml of PEG / NaCl, mix well, and place on ice for 20 min. Centrifuge at 4200rpm for 15 min. Discard the supernatant. Briefly centrifuge and discard the supernatant. Resuspend the pellet in 1ml of 1xPBS. Centrifuge at 13000rpm for 10 min. Transfer the supernatant to a new 1.5EP tube. Use for subsequent rinsing, or add 0.5 ml of 50% glycerin and freeze at -80°C.

[0084] The selection strategy and results are shown below:

[0085] Table 1 Selection Strategy

[0086] <![CDATA[1 st ]]> <![CDATA[2 nd –QP1138]]> <![CDATA[2 nd –QPP11]]> antigen QP1138 5ng / ul QP1138 2ng / ul QPP11 2ng / ul

[0087] Table 2 Selection Results

[0088] <![CDATA[1 st ]]> <![CDATA[2 nd –QP1138]]> <![CDATA[2 nd –QPP11]]> NB089 1E6 1.5E8 2.3E6

[0089] Immunotherapy library screening and identification: After two rounds of panning, single clones were selected for phage ELISA to screen for PD-1-binding positive clones. Simultaneously, clones that were positive for PD-1 binding and those that blocked PD-1 and PD-L1 binding were also screened. Positive clones were sequenced to obtain the VHH sequence of the PD-1 antibody. The ELISA screening method is shown below:

[0090] Packaging phage and soluble expression: Clones selected: P1-P4, a total of 4 96-well plates. Single clones were inoculated into 180 μL of 2YT + amp and incubated at 37°C for 2 hours. Packaging phage: 30 μL of phage was added to 100 μL of 2YT + M13K07, incubated at 37°C for 1 hour, then 50 μL of 2YT + amp + kan was added, and the mixture was shaken overnight at 30°C. The supernatant was collected by centrifugation for ELISA. Soluble expression: 120 μL of bacterial culture was incubated with shaking for 3 hours, then 60 μL of 2YT was added, followed by 1 M IPTG to a final concentration of 1 mM. The mixture was shaken overnight at 30°C. The supernatant was collected by centrifugation for ELISA.

[0091] Phage ELISA: Coated with QP1138, negative control human IgG (protein number QP11851186), 2 ng / ul of PDL1.mFc (self-produced), 60ul / well, 4 plates per well. Coated overnight at 4°C, washed 3 times with PBS. Blocked with 200ul / well of 5% milk at room temperature for 1 hour. Add 1 ng / ul of QP1138 to PDL1.mFc, 60ul / well, incubated at room temperature for 1 hour. Add 20ul of phage supernatant and 40ul of 2% MPBS, mix well, and incubate at room temperature for 1 hour. Wash 3 times with PBST, add 60ul of anti-M13 HRP (Sinosure, 11973-MM05T-200), incubate at room temperature for 1 hour. Wash 5 times with PBST, develop with 100ul / well of TMB, incubate at room temperature for 10 minutes, and terminate the reaction with 100ul / well of 2M H2SO4. Clones that were positive for QP1138 binding and those that were not bound for QP11851186 were PD-1 specific binding clones and were sent for sequencing.

[0092] Blocking phage ELISA procedure: Coating protein QP004 (PD-L1-hFC) 4 ng / ul 60ul / well, add QPP11 (PD1.His, Sinocare) 1 ng / ul 60ul / well, add 60ul MPBS + 20ul phage supernatant, add 60ul anti-M13 HRP (Sinocare, 11973-MM05T-200) and incubate for 1 hour, wash 5 times with PBST, develop color with TMB, and terminate with H2SO4.

[0093] Soluble blocking ELISA procedure: Coating protein QP004 (PD-L1-hFC) 2 ng / ul 60ul / well, incubate overnight at 4°C, wash 3 times with PBS, block with 200ul / well of 5% milk for 1 hour. Wash 3 times with PBS. Add 0.1 ng / ul of biontin-QP1138 + 30ul of expression supernatant, incubate for 1 hour, wash 3 times with PBST, add Strep-HRP 1:5000, incubate 60ul / well for 1 hour, wash 5 times with PBST, develop with TMB, and stop with H2SO4.

[0094] Clones that are positive for PD-1 and can simultaneously block the binding of PD-1 and PD-L1 were selected. Positive clones were sequenced to obtain the VHH sequence of the anti-PD-1 antibody. This invention provides two unique nanobodies, numbered QP3120 and QP3126, with amino acid sequences shown in SEQ ID NO: 5 and 6, respectively.

[0095] SEQ ID NO: 5QP3120

[0096] The bolded and underlined parts represent the nanobody sequences CDR1, CDR2, and CDR3, with sequence numbers SEQ ID NO: 7, 8, and 9, respectively.

[0097] SEQ ID NO: 6QP3126

[0098] The bolded and underlined parts represent the nanobody sequences CDR1, CDR2, and CDR3, with sequence numbers SEQ ID NO: 7, 8, and 10, respectively.

[0099] As can be seen from the above sequences, QP3120 and QP3126 differ only in CDR3, which is a single amino acid. Therefore, the sequences of QP3120 and QP3126 are highly similar.

[0100] Example 4: Construction of FC fusion protein using nanobodies, cloning, expression, and purification of the protein.

[0101] Cloning design and construction: The C-terminus of the anti-PD-1 antibody nanobody VHH was fused with human IgG1 FC to construct the nanobody FC fusion protein antiPD-1VHH-FC. Eukaryotic expression plasmids were constructed and transiently transfected into HEK293 cells. The proteins were purified by protein A affinity chromatography and other methods. The two PD-1VHH-FC fusion proteins were renumbered as QP3120 and QP3126. The human IgG1 Fc fragment sequence is shown in SEQ ID NO: 11.

[0102] 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 minutes, not exceeding 20 minutes. 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 minutes.

[0103] Protein A 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 in the instrument are consistent with 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 in the instrument are consistent with 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 PAC-EP to neutral using pH adjustment buffer.

[0104] Example 5: ELISA detection of the binding of PD-1 nanobody Fc fusion protein to human PD-1 protein

[0105] Experimental Procedure: Plate Coating: Coat with QP1138 (PD-1–hFC) 1 μg / ml, 60 μl / well, overnight at 4℃, PBS*3; Blocking: 5% milk / PBS, 200 μl / well, 25℃, incubate for 1 h; Antigen: Incubate PD-1 nanobody PD-1VHH-FC QP3120 and QP3126 separately, starting at 25 μg / ml, 5-fold dilution, 8 gradients, 60 μl / well, 25℃, 1 h, PBST*5; Secondary Antibody: Anti-Fab-HRP, 1:8000 dilution, 60 μl / well, 25℃, 1 h, PBST*5; Color Development: TMB 100 μl / well, 5-10 min, stop reaction with 2M H2SO4, read at 450 nm. Results are as follows. Figure 1 As shown, the PD-1 nanobody Fc fusion protein binds to human PD-1 protein.

[0106] Example 6: ELISA detection of PD-1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein binding

[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. Terminate with 2M H2SO4 50 μl / well. Results as shown. Figure 2 As shown, the PD-1 nanobody Fc fusion protein can block the binding of human PD-L1 and PD-1 protein.

[0108] Example 7: ELISA detection of PD-1 nanobody Fc fusion protein binding to cynomolgus monkey PD-1 protein

[0109] Rabbit His antibody (GenScript, A00174) 2 μg / ml, 50 μl / well, incubated overnight at 4℃. Washed 3 times with PBS. Blocking: 3% BSA 250 μl / well, incubated at room temperature for 1 h. Incubated monkey PD1-his (sinobio 90251-C08H) 1 μg / ml, 50 μl / well, incubated at 25℃ for 1 h, washed 3 times with PBS. Incubated antibody starting concentration 10 μg / ml, diluted 8 times. 50 μl / well, incubated at 25℃ for 1 h, washed 6 times with PBS. Incubated secondary antibody HRP-anti-human FC, 1:5000 dilution, 60 μl / well, incubated at 25℃ for 1 h, washed 6 times with PBST. TMB color development, reaction terminated with H2SO4. Microplate reader set to 450 nm for reading. Results as follows. Figure 3 As shown, the PD-1 nanobody Fc fusion protein binds to cynomolgus monkey PD-1 protein.

[0110] Example 8: Humanization design of nanobodies, cloning, expression, and purification of proteins

[0111] Humanization design of nanobodies: By comparing the germline gene database of heavy and light chain variable regions of human antibodies (IMGT) with MOE software, germline genes of heavy and light chain variable regions with high homology to QP3120 and QP3126 were used as templates. The CDRs of alpaca nanobodies 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 STAR GXL 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 DH5a 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.

[0112] 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.

[0113] Table 3 Human-centered design of QP3120 and QP3126

[0114]

[0115] Cloning Construction: The humanized clone designed above was converted into a PD-L1-FC fusion protein with a human IgG1 FC terminal at the C-terminus. The reconstructed plasmid was expressed in HEK293 cells and purified by protein A affinity chromatography, yielding a total of 7 humanized PD-1VHH-FC fusion proteins QP629-QP635, with sequences as shown in SEQ ID NO: 12-18. The nanobody is linked to a human IgG1 FC fragment at the rear end, as shown in SEQ ID NO: 11.

[0116] 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 minutes, not exceeding 20 minutes. 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 minutes.

[0117] Protein A 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 in the instrument are consistent with 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 in the instrument are consistent with 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.

[0118] Example 9: ELISA detection of binding of humanized PD-1 nanobody Fc fusion protein to human PD-1 protein

[0119] For plate preparation, use anti-his antibody (1ug / ml, 60ul / well), incubate overnight at 4℃, and wash twice with PBST. Block with 5% skimmil (200ul / well), incubate at room temperature for 1 hour, and wash twice with PBST. Incubate with antigen PD1-his (1ug / ml, 60ul / well), incubate at room temperature for 1 hour, and wash five times with PBST. Incubate with antibody starting at 133.3nM, 4-fold dilution, and the last one a 100-fold dilution (60ul / well), incubate at room temperature for 1 hour, and wash five times with PBST. Incubate with secondary antibody anti-hFc HRP (1:5000), 60ul / well, incubate at room temperature for 1 hour, and wash five times with PBST. Develop with TMB for 10 minutes. Read the value at 450nm.

[0120] The results are as follows Figure 4 As shown, ELISA was used to detect the binding of humanized PD-1 nanobody Fc fusion protein to human PD-1 protein, and molecules with low EC50 values ​​were selected for further validation.

[0121] Example 10: ELISA detection of humanized PD-1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein binding combine

[0122] 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 30 μg / ml, diluted 1:3, mixed in equal volumes, and incubated 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, washed 6 times with PBST, then 3 times with PBS. Colorimetric development: TMB 100 μl / well, development for 10 min. Stop treatment with 2M H2SO4 50 μl / well.

[0123] The results are as follows Figure 5 As shown, ELISA detection of humanized PD-1 nanobody Fc fusion protein can block the binding of human PD-L1 protein to PD-1 protein.

[0124] To evaluate the functional activity of the anti-PD-1 nanobody, two forms of humanized nanobody Fc fusion protein were designed. Specifically, the first form is to fuse anti-PD-1VHH to the N-terminus of human IgG1 FC segment mutation eliminating FCγR function (EU count L234A / L235A), as shown in SEQ ID NO: 19; the second form is to fuse anti-PD-1VHH to the C-terminus of human IgG1 FC segment mutation eliminating FCγR function (EU count L234A / L235A). The protein number and sequence number are shown in Table 4.

[0125] Table 4. Design of two forms of humanized nanobody Fc fusion protein

[0126]

[0127] Example 11: ELISA detection of binding of humanized PD-1 nanobody Fc fusion protein to human PD-1 protein

[0128] For plate preparation, use anti-His antibody 1ug / ml, 60ul / well, incubate overnight at 4℃, and wash twice with PBST. Block with 5% skimmilk, 200ul / well, incubate at room temperature for 1 hour, and wash twice with PBST. Incubate with antigen PD1-His, 1ug / ml, 60ul / well, incubate at room temperature for 1 hour, and wash five times with PBST. Incubate with antibody starting at 133.3nM, 4-fold dilution, the last one 100-fold dilution, 60ul / well, incubate at room temperature for 1 hour, and wash five times with PBST. Incubate with secondary antibody anti-hFc HRP, 1:5000, 60ul / well, incubate at room temperature for 1 hour, and wash five times with PBST. Develop with TMB for 10 minutes. Read the value at 450nm. Results are as follows. Figure 6 As shown, the humanized PD-1 nanobody fused to both the N-terminus and C-terminus of the Fc layer binds to human PD-1 protein.

[0129] Example 12: ELISA detection of humanized PD-1 nanobody Fc fusion protein blocking human PD-L1 and PD-1 protein binding combine

[0130] 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 30 μg / ml, diluted 1:3, mixed in equal volumes, and incubated 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, washed 6 times with PBST, then 3 times with PBS. Colorimetric development: TMB 100 μl / well, development for 10 min. Stop treatment with 2M H2SO4 50 μl / well.

[0131] The results are as follows Figure 7 As shown, the humanized PD-1 nanobody fused to both the N-terminus and C-terminus of Fc can block the binding of human PD-L1 protein to PD-1 protein.

[0132] Example 13: Humanized nanobody Fc fusion protein promotes T cell proliferation in mixed lymphocyte reactions

[0133] Mixed lymphocyte reaction refers to the co-culture of human T cells and allogeneic dendritic cells. The lymphocytes are activated and proliferate upon stimulation by allogeneic antigens, producing a wide variety of cytokines. Anti-PD-1 antibodies, in a concentration-dependent manner, block the immunosuppressive signaling pathway of PD-1 / PD-L1 binding, stimulating T cell proliferation and the release of cytokines such as IL-2 / IFN-γ. The release of IL-2 / IFN-γ was detected by ELISA to investigate the in vitro proliferative bioactivity of anti-PD-1 antibodies in mixed lymphocyte reactions.

[0134] Monocytes were isolated from PBMCs and induced into dendritic cells (DCs) by adding rhGM-CSF and rhIL-4. CD4+ T cells were also isolated from another donor PBMC. DCs and T cells were mixed at a 1:10 ratio, and different concentrations of anti-PD-1 antibody were added. The mixtures were cultured for 2-5 days, and the expression of IL-2 and IFN-γ in the culture supernatant was detected. Results are as follows: Figure 8 , Figure 9 , Figure 10 , Figure 11 As shown, humanized PD-1 nanobody fused to both the N-terminus and C-terminus of Fc can stimulate T cell proliferation and enhance the production of IL-2 and IFN-γ in mixed lymphocyte reaction (MLR).

[0135] In summary, the various forms of humanized PD-1 nanobodies fused to the N-terminus and C-terminus of Fc, namely QP916, QP3517, QP3519, QP917, QP918, QP919, and QP3518, all stimulated T cell proliferation and enhanced the production of IL-2 and IFN-γ in the mixed lymphocyte reaction (MLR), which was superior to the control Novilumab analog (QP32103211).

[0136] Example 14: Bioactivity of PD-1 nanobody in stimulating in vitro proliferation of human PBMCs

[0137] Human peripheral blood mononuclear cells (PBMCs) are composed of various leukocytes, mainly including monocytes, B cells, T cells, NK cells, dendritic cells, and macrophages. In vitro stimulation of PBMCs with the superantigen SEB leads to the presentation and activation of APC cells, resulting in lymphocyte activation and proliferation, and the production of numerous cytokines. PD-L1 antibody enhances T cell proliferation and the release of cytokines such as IL-2 and IFN-γ by blocking the immunosuppressive signal of PD-1 / PD-L1 binding. The release of IL-2 and IFN-γ was detected by ELISA to further investigate the bioactivity of Q-1801 in the in vitro proliferation assay of PBMCs. PBMCs were seeded in 96-well plates, and different concentrations of SEB were added to the wells, followed by the addition of anti-PD-1 antibody. The cells were gently mixed and cultured for 2-5 days. IL-2 and IFN-γ secretion in the cell culture supernatant were detected by ELISA. Results are as follows: Figure 12 The results showed that QP3517, QP918, QP3519, and QP919 significantly enhanced the activation and proliferation of PBMCs and increased IL-2 production in SEB-stimulated PBMCs in vitro, demonstrating non-inferiority to the control antibody novilumab. Humanized PD-1 nanobody fused to both the N-terminus and C-terminus of the Fc region stimulated T cell proliferation and enhanced IL-2 production in SEB-stimulated PBMCs.

[0138] Example 15: Anti-human PD1 antibody inhibits tumor growth in animals in vivo.

[0139] Experimental Objective: To verify the inhibitory effect of anti-human PD1 antibodies on tumor growth through CDX animal efficacy studies.

[0140] Experimental procedure: Collect cultured MC38-hPDL1 cells by centrifugation, disperse the cells with 1×PBS, and prepare a cell density of 5×10⁶ cells / year. 6 Cells / ml suspension. 0.1 ml of cell suspension was subcutaneously injected into the right rib area of ​​C57BL / 6-hPD1 mice to establish an MC38-hPDL1 tumor-bearing mouse model. When the average tumor volume of C57BL / 6-hPD1 mice reached approximately 73 mm²... 3 Mice were randomly divided into groups of seven. The weight of all animals was measured, and tumor volume was measured using calipers. Grouping was based on tumor volume, ensuring similar tumor volumes across different groups. Grouping was designated Day 0, and medication was initiated on that day. Detailed administration methods, dosages, and routes are shown in the table below.

[0141] Table 5: Dosing parameters for animal efficacy studies

[0142]

[0143] The administration volume is 10 μL / g.

[0144] After drug administration began, mouse body weight and tumor volume were measured three times per week. Tumor volume was calculated using the formula: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). The experiment was terminated three days after the last administration of the drug. The mice were euthanized, and the tumors were harvested, weighed, and photographed.

[0145] The following analytical methods were selected for data analysis:

[0146] Tumor proliferation rate, T / C (%) = (T-T0) / (C-C0) × 100% (T0 and C0 are the tumor volumes of the treatment group and the control group at the time of grouping, respectively, and T and C are the tumor volumes of the treatment group and the control group at a specific time point after drug administration, respectively).

[0147] Tumor inhibition rate, TGI (%), is calculated using the formula: TGI% = (1 - T / C) × 100%

[0148] Experimental results:

[0149] The mean tumor volume in the vehicle control group mice was 705.60±105.85 mm3 on day 17 after drug administration. The mean tumor volumes on day 17 of the nanobody molecule groups QP918 (5 mg / kg), QP918 (10 mg / kg), QP3517 (5 mg / kg), and QP3517 (10 mg / kg) were 23.24±16.14 mm3, 26.28±13.27 mm3, 14.63±13.32 mm3, and 11.59±8.42 mm3, respectively, with TGIs of 107.95%, 107.41%, 109.29%, and 109.68%, respectively. The mean tumor volume on day 17 of the control molecule QP32103211 (10 mg / kg) was 47.99±27.75 mm3, with a TGI of 104.01% (see table below). Compared with the Vehicle group, both the control molecule QP32103211 and the two nanobody molecules significantly inhibited tumor growth (p<0.001), and QP918 and QP3517 showed a slightly better trend than the control molecule QP32103211. Moreover, the mice did not experience a significant decrease in body weight during the administration process, indicating that the antibody molecules had no significant toxic side effects on the mice.

[0150] Table 6. Tumor volume analysis results of PD-1 antibody inhibition of tumor growth.

[0151]

[0152]

[0153] The results of tumor weight analysis were similar to those of tumor volume analysis. In the Vehicle control group, the mean tumor weight on day 17 after administration was 741.07 ± 318.89 mg. In the antibody molecule QP918-5mpk, QP918-10mpk, QP3517-5mpk, and QP3517-10mpk treatment groups, the mean tumor weights on day 17 after the end of administration were 9.73 ± 16.38 mg, 23.37 ± 38.99 mg, 9.76 ± 22.33 mg, and 11.66 ± 27.04 mg, respectively. In the control molecule QP3210 / 3211-10mpk treatment group, the mean tumor weight on day 17 after administration was 35.57 ± 57.34 mg. (See table below)

[0154] Table 7. Tumor weight analysis results of PD-1 antibody inhibition of tumor growth.

[0155]

[0156] Therefore, through CDX animal efficacy trials, we found that the screened antibody molecule has a better in vivo tumor-inhibiting ability than the control molecule QP32103211.

[0157] Experimental results are as follows Figures 13 to 16 As shown, the results of the efficacy test of the antibody against the mouse colon cancer CDX model MC38-hPDL1-mClaudin18.2 are as follows: Figure 13 As shown. Figure 14 These are the tumor growth curves for each group after grouping. Figure 15 The weight of tumor D17 in mice in each group containing the antibodies of this invention and the control antibody is [not specified]. Figure 16 The figures show the body weight curves for each group of mice. The experimental results demonstrate that the PD-1 nanobodies QP918 and QP3517 of this invention exhibit better tumor growth inhibition effects in vivo compared to the control antibody Novilumab analog (QP32103211).

[0158] 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.

[0159] 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.

[0160] 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-1 nanobody, characterized in that, include: The VHH chain, wherein the complementarity-determining regions of the VHH chain include CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9 or SEQ ID NO:

10.

2. The anti-PD-1 nanobody according to claim 1, characterized in that, The amino acid sequence of the VHH chain has at least 85% identity with the sequence shown in any one of SEQ ID NO:5, 6 or SEQ ID NO:12-18.

3. The anti-PD-1 nanobody according to claim 1, characterized in that, The amino acid sequence of the VHH chain is shown in any one of SEQ ID NO:5, 6 or SEQ ID NO:12-18.

4. A fusion protein, characterized in that, The fusion protein is formed by fusing the anti-PD-1 nanobody of any one of claims 1 to 3 with the Fc segment.

5. The fusion protein according to claim 4, characterized in that, The Fc segment is fused to the N-terminus or C-terminus of the anti-PD-1 nanobody.

6. The fusion protein according to claim 4, characterized in that, The Fc segment is selected from human IgG1, IgG2, IgG3, or IgG4 or their variants.

7. The fusion protein according to claim 6, characterized in that, The Fc segment is selected from a variant of human IgG1.

8. The fusion protein according to claim 6, characterized in that, The Fc segment domain is selected to eliminate immune effector function.

9. The fusion protein according to claim 6, characterized in that, The Fc segment contains any of the following mutation types, which are counted according to EU: 。 10. An anti-PD-1 antibody molecule, characterized in that, include: One or more anti-PD-1 nanobodies according to any one of claims 1 to 3; wherein the anti-PD-1 antibody molecule is a monovalent antibody, a bivalent or multivalent antibody, a bispecific antibody, or a multispecific antibody.

11. A chimeric antigen receptor, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, characterized in that, The extracellular antigen-binding domain includes the anti-PD-1 nanobody according to any one of claims 1 to 3.

12. A nucleic acid molecule, characterized in that, Its encoding is: the anti-PD-1 nanobody according to any one of claims 1 to 3, or the fusion protein according to any one of claims 4 to 9.

13. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 12.

14. A host cell, characterized in that, It is obtained by conversion using the carrier described in claim 13.

15. A pharmaceutical composition, characterized in that, It contains: an anti-PD-1 nanobody according to any one of claims 1 to 3, or a fusion protein according to any one of claims 4 to 9, and one or more pharmaceutically acceptable carriers.

16. Use of the anti-PD-1 nanobody according to any one of claims 1 to 3 in the preparation of a medicament for inhibiting or treating a disease, wherein the disease is breast cancer, lung cancer, gastric cancer, intestinal cancer, kidney cancer, melanoma, bladder cancer, head and neck cancer, lymphoma, or malignant skin tumor.

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