Anti-BCL6 protein nano antibody, fusion protein and application thereof

Abstract

The invention discloses an anti-BCL6 protein nano antibody, a fusion protein and application thereof, and belongs to the technical field of biology. According to the invention, the nano antibody specifically targeting BCL6 is designed by using an artificial intelligence technology, and the nano antibody has good specificity and high affinity, and can efficiently bind to a BCL6 antigen; furthermore, rational design of the nano-antibody is carried out through artificial intelligence, so that a nano-antibody mutant with higher affinity is obtained; furthermore, a nano antibody or a nano antibody mutant with high affinity and relatively good specificity is fused with an RBCC structural domain or a mutant thereof to obtain a fusion protein, and the fusion protein can utilize the specificity of the nano antibody structural domain to bind to a target protein and start a protein degradation pathway, so that targeted degradation of the BCL6 protein is realized, and the BCL6 protein can be degraded in a targeted manner. The expression of cancer suppressor genes p53 and cyclin p21 in cells is improved, and the method has a very great clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a nanobody against BCL6 protein, a fusion protein and their applications. Background Art

[0002] B-cell lymphoma 6 (BCL6) gene was initially discovered in diffuse large B-cell lymphoma (DLBCL) and is one of the genes affected by chromosomal translocation. BCL6 belongs to the transcriptional repressor of the BTB / POZ family, which consists of a BTB / POZ domain (bric-a-brac, tramtrack, broad complex, BTB) at the amino terminus, a PEST motif in the middle (also known as the second repression domain, RD2), and six zinc finger structures (Zinc finger, ZF) at the carboxyl terminus. These domains regulate the transcription of target genes through different interactions. BCL6 mainly functions in the immune system, regulating the differentiation and maturation of B cells. In the germinal center (GC) reaction, BCL6 inhibits gene transcription to protect B cells from adverse factors such as somatic mutations and oxidative stress. It mainly maintains genomic instability by inhibiting the expression of genes involved in DNA damage sensing or its downstream checkpoints, thereby promoting the rapid proliferation and differentiation of B cells.

[0003] The germinal center is the main site for B cell differentiation and antibody affinity maturation. In the GC reaction, BCL6 helps B cells tolerate somatic mutations and oxidative stress generated during rapid proliferation by inhibiting genes related to the DNA damage response, thus maintaining the normal progress of the GC reaction. This inhibitory effect makes BCL6 play a key role in promoting the generation of high-affinity B cells, the production of effector memory B cells, and plasma cell differentiation. Dysregulation of BCL6 expression may lead to abnormal GC reactions, which in turn trigger various lymphomas and other malignancies. In addition to lymphomas, BCL6 also plays an important role in breast cancer, lung cancer, and gliomas. BCL6 plays a key role in the development and progression of these cancers by regulating different gene expressions. Therefore, BCL6 has become an important target for cancer treatment.

[0004] Existing BCL6 inhibitors are mainly designed for its BTB domain, and exert inhibitory activity by competitively binding with corepressors. However, there are relatively few studies on BCL6 degraders. Among them, BCL6 PROTAC (Proteolysis Targeting Chimeras) protein degradation activity is poor, and other degraders are BCL6 molecular glues, which are obtained by chance when screening inhibitors and cannot be rationally designed. These molecular glues have better effects than inhibitors of the same type.

[0005] Nanobody is a single-domain antibody derived from the heavy chain antibody of camelids. It has the specificity and affinity of traditional antibodies, but has a smaller molecular weight and higher stability. Nanobody can penetrate tissues and cells and bind to target proteins more easily, so it has broad application prospects in biomedical research and treatment.

[0006] Targeted protein degradation (TPD) technology uses the ubiquitin proteasome system and lysosomal degradation system in cells to achieve specific and efficient degradation of disease-related proteins, thereby achieving the effect of disease treatment. Compared with traditional small molecule inhibitors, TPD has more advantages: event-driven, low dosage, only catalyst amount is needed to work; it can also target some undruggable proteins.

[0007] TRIM away is an emerging TPD technology that uses members of the TRIM (Tripartite motif-containing) protein family as E3 ubiquitin ligases to specifically recognize and bind to target proteins, promote their ubiquitination and ultimately degradation in the proteasome. The application of TRIM away technology in BCL6 degradation has great potential and can provide a more efficient and precise means of BCL6 degradation, which is expected to improve the therapeutic effect of BCL6-related cancers.

[0008] However, there are currently no reports on research on nanoantibodies targeting BCL6. Summary of the invention

[0009] The purpose of the present invention is to provide a nanobody, a fusion protein and an application thereof against BCL6 protein, so as to solve the problem that there is a lack of nanobodies with strong specificity, high affinity and high efficiency in binding to BCL6 antigen in the prior art.

[0010] In a first aspect, the present invention provides a nanobody against BCL6 protein, comprising complementarity determining region CDR1, complementarity determining region CDR2 and complementarity determining region CDR3; wherein, the complementarity determining region CDR1 has an amino acid sequence as shown in any one of SEQ ID NO: 1-2, the complementarity determining region CDR2 has an amino acid sequence as shown in any one of SEQ ID NO: 3-4, and the complementarity determining region CDR3 has an amino acid sequence as shown in any one of SEQ ID NO: 5-8.

[0011] In the present invention, the term "nanobody" is also referred to as single domain antibody (sdAb), which refers to an antibody composed of only one H chain V region, and can also be referred to as VHH antibody. The ability of the nanobody to bind to the antigen and its stability are basically the same as those of a complete antibody. The nanobody is sequentially composed of framework region FR1, complementarity determining region CDR1, framework region FR2, complementarity determining region CDR2, framework region FR3, complementarity determining region CDR3 and framework region FR4. The nanobody can be truncated at the N-terminus or C-terminus so that it only contains part of FR1 and / or FR4, or lacks one or two of the framework regions, as long as it substantially maintains antigen binding and specificity. Among them, the complementarity determining region is mainly responsible for binding to the antigen.

[0012] In some embodiments, the nanobody further comprises framework region FR1, framework region FR2, framework region FR3 and framework region FR4; wherein, the framework region FR1 has an amino acid sequence as shown in any one of SEQ ID NO: 9-10, the framework region FR2 has an amino acid sequence as shown in any one of SEQ ID NO: 11-12, the framework region FR3 has an amino acid sequence as shown in any one of SEQ ID NO: 13-14, and the framework region FR4 has an amino acid sequence as shown in any one of SEQ ID NO: 15-16.

[0013] It can be understood that the framework region has a high degree of conservation. Therefore, conventional framework regions in the prior art can be selected according to actual use needs, as long as the stability of the nanobody can be improved.

[0014] In some embodiments, the nanobody is selected from any one of the following: A1) having an amino acid sequence as shown in any one of SEQ ID NO: 17-20; A2) an amino acid sequence having one or several amino acid substitutions, deletions or additions compared with the amino acid sequence defined in A1); A3) an amino acid sequence having more than 80% sequence identity compared with the amino acid sequence defined in A1) or A2); A4) an amino acid sequence obtained by linking a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

[0015] In the present invention, the inventors designed a nanobody specifically targeting BCL6 using artificial intelligence technology. This nanobody has good specificity, high affinity, and can efficiently bind to the BCL6 antigen.

[0016] In a second aspect, the present invention provides a nanobody mutant against BCL6 protein, and the nanobody mutant is selected from any one of the following: B1) having the amino acid sequence shown in SEQ ID NO: 21; B2) an amino acid sequence having one or several amino acid substitutions, deletions or additions as compared with the amino acid sequence defined in B1); B3) an amino acid sequence having a sequence identity of more than 80% as compared with the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in B1), B2) or B3); wherein, in B1), having the amino acid sequence shown in SEQ ID NO: 21 is obtained by mutating the amino acid sequence shown in SEQ ID NO: 18 at the following site: T106Q.

[0017] In the present invention, the inventors further performed rational design of nanobodies through artificial intelligence to obtain nanobody mutants with higher affinity.

[0018] In some embodiments, in the above-mentioned nanobody mutant, in addition to having the above-mentioned mutation at one site in the 106th position, it can further have conservative substitutions of amino acids at other sites, so that the mutated amino acid has higher affinity than the nanobody shown in SEQ ID NO: 18. Preferably, the conservative substitution of amino acids retains the high affinity of the nanobody of the present invention. It is obvious to those skilled in the art that such substitutions can occur in regions other than the above-mentioned sites while still retaining the corresponding affinity. Preferably, the conservative substitution variant has at least one position of amino acid conservative substitution. Examples of conservative substitutions are substitutions that occur within the following groups of amino acids: basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine, asparagine), hydrophobic amino acids (such as leucine, isoleucine and valine), aromatic amino acids (such as phenylalanine, tryptophan and tyrosine), and small amino acids (such as glycine, alanine, serine, threonine and methionine).

[0019] In a third aspect, the present invention provides a fusion protein, which is obtained by connecting any one of the above-mentioned nanobodies or the above-mentioned nanobody mutants with an RBCC domain or its mutant through a second linker; wherein, the amino acid sequence of the second linker is as shown in SEQ ID NO:31, the amino acid sequence of the RBCC domain is as shown in SEQ ID NO:29, and the amino acid sequence of the RBCC domain mutant is as shown in SEQ ID NO:30.

[0020] In the present invention, the inventors fused a nanobody or a nanobody mutant with high affinity and good specificity with an RBCC domain or its mutant (truncated TRIM family protein) to obtain a fusion protein. This fusion protein can specifically bind to the target protein using its nanobody domain, initiate the protein degradation pathway, achieve targeted degradation of the BCL6 protein, increase the expression of the tumor suppressor gene p53 and the cyclin p21 in cells, thereby protecting B cells from cell suicide caused by DNA damage, and inhibiting the amplification rate of cancer cells, which has great clinical application value.

[0021] It can be understood that the linker peptide can be selected from conventional linker peptides in the prior art according to actual usage needs, as long as it can enable better fusion of the two proteins.

[0022] In some embodiments, the fusion protein is selected from any one of the following: C1) having an amino acid sequence as shown in any one of SEQ ID NOs: 35-44; C2) an amino acid sequence having one or several amino acid substitutions, deletions or additions compared with the amino acid sequence defined in C1); C3) an amino acid sequence having more than 80% sequence identity compared with the amino acid sequence defined in C1) or C2); C4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in C1) or C2) or C3).

[0023] The above-mentioned nanobody, the above-mentioned nanobody mutant, and the above-mentioned fusion protein provided by the present invention can be natural, recombinant or synthetic active polypeptides, and the active polypeptides can be natural purified products, chemically synthesized products, or products produced using recombinant techniques from prokaryotic hosts (such as Escherichia coli) or eukaryotic hosts (such as yeast, higher plants).

[0024] In the present invention, in A4), B4) or C4), the connection can be directly connected by a peptide bond or through a linker, and the connection method is a conventional method in the art. Among them, the tags include but are not limited to: GST (glutathione S-transferase) tagged protein, Trx (thioredoxin) tagged protein, His-tagged protein (His-tag), Flag-tagged protein, LacZ-tagged protein, GFP (green fluorescent protein), sfGFP (superfolder green fluorescent protein), HA tag (hemagglutinin tag). Those skilled in the art can select a suitable tagged protein according to actual usage needs. The use of the tag does not change the function of the target protein (nanobody, nanobody mutant or fusion protein), and its purpose is to isolate, purify, detect or trace. The tag can be separated from the target protein (nanobody, nanobody mutant or fusion protein) by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site and using TEV protease to cleave and remove the tag).

[0025] In a fourth aspect, the present invention provides a biological material, and the biological material is selected from any one of the following: D1) a nucleic acid molecule encoding any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one of the above-mentioned fusion proteins; D2) a recombinant vector containing the nucleic acid molecule of D1); D3) a recombinant cell containing the nucleic acid molecule of D1) or the recombinant vector of D2).

[0026] In some embodiments, the nucleic acid molecule defined in D1) is selected from any one of the following: E1) a nucleic acid molecule having a nucleotide sequence shown in any one of SEQ ID NO: 22-26; E2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in E1) under stringent conditions and encodes any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one of the above-mentioned fusion proteins; E3) a nucleic acid molecule having a sequence identity of more than 90% with the nucleic acid molecule defined in E1) or E2) and encoding any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one of the above-mentioned fusion proteins.

[0027] The above nucleic acid molecule provided by the present invention can be DNA, such as cDNA, genomic DNA or recombinant DNA; it can also be RNA, such as mRNA or hnRNA, etc.; and this nucleic acid molecule can usually be obtained by PCR amplification or artificial synthesis methods.

[0028] The above recombinant vector provided by the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related genes. Among them, the vectors used when constructing the expression vector can be at least one of pET23a, pET28a, and pcDNA3.1 vectors.

[0029] In some embodiments, the preparation method of the above recombinant cell includes the step of transforming the above recombinant vector into an expression host cell.

[0030] In the present invention, the expression host cell is a conventional host cell in the art, as long as it can satisfy that the recombinant vector can stably replicate by itself and the genes carried by it can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc. Escherichia coli can be, for example, E. coli BL21(DE3), Rosetta(DE3), BL21(DE3)plysS. In the present invention, the Escherichia coli expression host E. coli BL21(DE3) is preferably used.

[0031] In the fifth aspect, the present invention provides the use of any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants, any one of the above-mentioned fusion proteins or the above-mentioned biomaterials in any one of the following: F1) Use in the preparation of a product for preventing and / or treating BCL6 target-related diseases; F2) Use in the preparation of a product for screening, diagnosing or assisting in the diagnosis of BCL6 target-related diseases; wherein, the BCL6 target-related disease is a BCL6-positive tumor.

[0032] In the present invention, a BCL6-positive tumor refers to a tumor in which BCL6 expression is detected in tumor cells. Such tumors can be, for example, common tumors such as follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, Hodgkin lymphoma, etc.

[0033] In the sixth aspect, the present invention provides a pharmaceutical composition for preventing and / or treating BCL6 target-related diseases. The pharmaceutical composition includes any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants, any one of the above-mentioned fusion proteins or the above-mentioned biomaterials, and a pharmaceutically acceptable carrier.

[0034] In the present invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide methods to enable the active ingredient to dissolve at the desired rate after the subject receives the administration, or to promote the effective absorption of the active ingredient after the subject receives the administration of the composition. Pharmaceutical excipients can be inert fillers or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition.

[0035] The pharmaceutical composition provided by the present invention can be prepared by any method known to those skilled in the art according to the disclosed content. For example, it includes but is not limited to conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0036] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid preparations, semi-solid preparations, and liquid preparations.

[0037] The pharmaceutical composition provided by the present invention can be administered in any form, including injection (intravenous), mucosal membrane, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be in a controlled-release or sustained-release dosage form (such as liposomes or microspheres).

[0038] In a seventh aspect, the present invention provides a method for preparing any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one of the above-mentioned fusion proteins, comprising the following steps: culturing the above-mentioned recombinant cells, and obtaining a culture after induced expression; separating the nanobody, the nanobody mutant or the fusion protein from the culture.

[0039] In the present invention, there are no special requirements for the culture method, culture conditions and culture medium, as long as the normal growth of the recombinant cells is ensured. And the methods for separating any one of the above-mentioned nanobodies, the above-mentioned nanobody mutants or any one of the above-mentioned fusion proteins from the culture are all conventional methods in the art.

[0040] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention designs a nanobody specifically targeting BCL6 by using artificial intelligence technology. This nanobody has good specificity and high affinity, and can efficiently bind to the BCL6 antigen; further, through the rational design of the nanobody by artificial intelligence, a nanobody mutant with higher affinity is obtained; furthermore, by fusing the nanobody or nanobody mutant with high affinity and good specificity with the RBCC domain or its mutant, a fusion protein is obtained. This fusion protein can specifically bind to the target protein using its nanobody domain, initiate the protein degradation pathway, achieve the targeted degradation of the BCL6 protein, increase the expression of the tumor suppressor gene p53 and the cyclin p21 in cells, thereby protecting B cells from cell suicide caused by DNA damage, and inhibiting the amplification rate of cancer cells, and has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a schematic diagram of the element arrangement structure of the recombinant expression vector constructed in the present invention; Figure 2This is the SDS-PAGE detection result graph of the nanobody in Example 2 of the present invention. Among them, in Figure (a), lane M is Marker, and lanes 0-6 are sfGFP-BN0, sfGFP-BN1, sfGFP-BN2, sfGFP-BN3, sfGFP-BN4, sfGFP-BN5, sfGFP-BN6 respectively; in Figure (b), lane M is Marker, and lanes 0-6 are sfGFP-BNC0, sfGFP-BNC1, sfGFP-BNC2, sfGFP-BNC3, sfGFP-BNC4, sfGFP-BNC5, sfGFP-BNC6 respectively; Figure 3 This is the SDS-PAGE detection result graph of the BCL6 antigen in Example 2 of the present invention; Figure 4 This is the ELISA test result graph of the affinity between the nanobody and the BCL6 antigen in Example 3 of the present invention; Figure 5 This is the BLI test result graph of the affinity between the nanobodies BN1, BN2, BN4, BNC2 and the BCL6 antigen in Example 4 of the present invention; Figure 6 They are respectively the graphs (a and b) of the binding energy and total scoring results of BN2-WT and the mutant BN2M with the BCL6 antigen calculated by Rosetta in Example 5 of the present invention and the graphs (c) of the binding energy and total scoring results of BN2-WT and the mutant BN2M with the BCL6 antigen calculated by Amber; Figure 7 They are respectively the graphs (a and b) of the energy change results before and after the mutation site change of the mutant BN2M-T106Q calculated by Amber in Example 5 of the present invention and the ELISA test result graphs (c and d) of the affinity between the nanobody mutant and the BCL6 antigen; Figure 8 This is the result graph of GFP plasmid electrotransferred into Raji cells in Example 6 of the present invention; Figure 9 This is the WB result graph of the fusion protein targeting the degradation of the BCL6 protein in Example 6 of the present invention. Among them, 1 is RBCC-BN1, 2 is RBCC-BN2M, 3 is RBCC-BN2, 4 is RBCC-BNC2, and 5 is the control; Figure 10 This is the WB result graph of the fusion protein targeting the degradation of the BCL6 protein in Example 6 of the present invention. Among them, 1 is RS80E-BN1, 2 is RS80E-BN2M, 3 is RS80E-BN2, 4 is RS80E-BNC2, and 5 is the control; Figure 11This is the WB result graph of the fusion protein RS80E-BNC2 targeting the degradation of BCL6 protein in Example 6 of the present invention; Figure 12 This is the result graph of the changes in the levels of p53 and p21 in the degradation of BCL6 protein by the fusion proteins RBCC-BN2 and RS80E-BNC2 in Example 6 of the present invention, where a and b correspond to RBCC-BN2, and c and d correspond to RS80E-BNC2; Figure 13 This is the WB result graph for verifying the pathway of the fusion protein targeting the degradation of BCL6 protein in Example 6 of the present invention, where 1 is the control, 2 is RBCC-BNC2, 3 is RBCC-BNC2 + MG132, and 4 is RBCC-BNC2 + BafA1. Detailed implementation manners

[0042] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0043] For the experimental methods without specific conditions indicated in the embodiments, they are usually carried out according to the conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in "Molecular Cloning: A Laboratory Manual" written by M.R. Green, "Molecular Biology" written by Robert·F·Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the embodiments can be obtained from commercial channels without special instructions.

[0044] Example 1 Design of nanobodies by artificial intelligence First, use artificial intelligence technology combined with bioinformatics tools (such as SEPPA, MetMHCpan, etc.) to predict the hot spots of the BCL6 antigen epitope, and perform linear fragmentation processing on it to generate fragments containing at least four consecutive residues. Subsequently, screen for CDR-like fragments matching the above fragments through the AbAg database, and perform structural superposition and optimization on the above CDR-like fragments and epitope fragments to construct CDR fragments that can specifically bind to the BCL6 antigen. Then, select appropriate framework region fragments (the amino acid sequences of the CDR fragments and framework region fragments are shown in Table 1 below). Finally, obtain a series of nanobodies against specific epitopes of the BCL6 antigen, and name them nanobodies BN1-6 and BNC1-6 respectively.

[0045] Table 1 Amino acid sequences of CDR fragments and framework region fragments

[0046] Among them, by way of example, the amino acid sequence of nanobody BN1 is shown as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSGITTIKEMGRSIHEIPREEFDY WGQGTLVTVSS(SEQ ID NO:17); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0047] The amino acid sequence of nanobody BN2 is shown as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSGLVATVKEAGRSIHEIPREELEEFDY WGQGTLVTVSS(SEQ IDNO:18); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0048] The amino acid sequence of nanobody BN4 is shown as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSVGASTLYGSAYEEFDY WGQGTLVTVSS(SEQ ID NO:19); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in sequence.

[0049] The amino acid sequence of nanobody BNC2 is shown as follows: EFQVQLVESGGGSVQAGGSLRLSCTAS GGSEYSYSTF SLGWFRQAPGQEREAVAAI ASMGGL TYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAA GLVATVKEAGRSIHEIPREELWGQGTQVTVSS (SEQ ID NO:20); The underscores represent Complementary Determining Region 1 (CDR1), Complementary Determining Region 2 (CDR2) and Complementary Determining Region 3 (CDR3) in sequence.

[0050] The amino acid sequence of the framework of nanobody BN0 is obtained by removing CDR1, CDR2 and CDR3 from nanobody BN1 (the amino acid sequence is shown in SEQ ID NO:17) (only the framework region sequence is included).

[0051] The amino acid sequence of the framework of nanobody BNC0 is obtained by removing CDR1, CDR2 and CDR3 from nanobody BNC2 (the amino acid sequence is shown in SEQ ID NO:20) (only the framework region sequence is included).

[0052] The nucleotide sequences of the genes encoding nanobodies BN1, BN2, BN4, BNC2 are shown in SEQ ID NO:22 - 25 respectively.

[0053] Example 2 Expression and Purification of Nanobody and BCL6 Antigen For the convenience of separating and purifying the nanobody, in the present invention, the nanobodies in Example 1 are displayed on the surface of Escherichia coli through superfolder green fluorescent protein (sfGFP, the nucleotide sequence of the gene encoding sfGFP protein is shown in SEQ ID NO:27). Specifically, based on the pET23a vector, the gene encoding the above-mentioned nanobody is ligated and fused with the sfGFP gene through a first linker (its nucleotide sequence is shown in SEQ ID NO:28), and an expression vector is constructed by homologous recombination. The element arrangement structure of this expression vector is as Figure 1 shown. Exemplarily, the recombinant plasmids are pET23a - sfGFP - BN1, pET23a - sfGFP - BN2, pET23a - sfGFP - BN4, pET23a - sfGFP - BNC2 respectively.

[0054] The above - constructed recombinant plasmids are respectively transformed into Escherichia coli competent cell strain BL21(DE3), and cultured statically overnight at 37°C to obtain a series of recombinant strains. Then, single colonies are respectively picked and inoculated into 100 mL of LB liquid medium (the concentration of ampicillin is 50 μg / mL), and cultured with shaking at 37°C. OD 600When it is about 0.6, add IPTG with a final concentration of 0.5 mM, and induce it by shaking culture at 18 °C for 18 hours. After the culture is completed, centrifuge at 12,000 rpm and 4 °C to collect the bacterial cells and the culture medium supernatant respectively. For the bacterial cells, wash them with TEN buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, pH 8.0) at 4 °C for more than 4 hours, and directly obtain the protein secreted and expressed by the strain from the TEN buffer. Use SDS-PAGE to detect and analyze the secretion and expression of the nanobody, and the results are as Figure 2 shown.

[0055] From Figure 2 it can be seen that the nanobodies BN1-6 and BNC1-6 are both successfully expressed.

[0056] Exemplarily, the purified nanobodies BN1, BN2, BN4, and BNC2 obtained by washing the outer membrane are stored for subsequent experiments.

[0057] Furthermore, the BCL6 antigen sequence (the 6th - 129th amino acids in NCBI accession number: P41182) is cloned into the pET28a vector by homologous recombination to obtain the recombinant plasmid pET28a-BCL6 (the vector structure is shown in Figure 1 ). The constructed recombinant plasmid is transformed into the Escherichia coli competent cell BL21(DE3) strain respectively. Pick a single colony and inoculate it into 100 mL of LB liquid medium (the concentration of kanamycin is 100 μg / mL), and culture it statically overnight at 37 °C. When the OD 600 is about 0.6, add IPTG with a final concentration of 0.5 mM, and induce it by shaking culture at 18 °C for 18 hours. Centrifuge at 6,000 rpm for 10 min to collect the bacterial cells. After washing the bacterial cells with Buffer A (20 mM Tris-HCl pH 8.0, 200 mM NaCl), resuspend the bacterial cells in Buffer A, add PMSF with a final concentration of 1 mM, and disrupt them by high pressure. Centrifuge at 18,000 rpm for 30 min, collect the supernatant. After filtering the supernatant, perform Ni-NTA purification. The protein purified by Ni-NTA is purified by a molecular sieve (Superdex 75 increase 10 / 300, GL), and the molecular sieve is pre-equilibrated with Buffer A. Collect the purified protein, detect it by SDS-PAGE, divide the protein into small portions, and store it at -80 °C after quick freezing in liquid nitrogen. The SDS-PAGE identification and analysis results are as Figure 3 shown. Calculated by http: / / www.expasy.org / , the expected size of BCL6 is 15.9 kDa. The results show that the BCL6 antigen is successfully expressed.

[0058] Example 3 ELISA Experiment of Nanobody This example is used to verify whether the purified nanobodies BN1, BN2, BN4, and BNC2 in Example 2 can directly interact with the purified BCL6 antigen in Example 2. Specifically, the steps are as follows: a) Dilute the BCL6 antigen to 1 μg / mL with 1×ELSIA coating buffer, and plate 100 μL per well to coat the microplate, then leave it at 4°C overnight; b) Wash the microplate with PBST, and block it with 1% BSA at room temperature for 2 h, 100 μL per well; c) Prepare different concentrations of nanobodies BN1, BN2, BN4, and BNC2 with 1% BSA, 100 μL per well, and incubate at room temperature for 1 h; d) Incubate with HRP-conjugated Mouse anti HA-Tag mAb secondary antibody at room temperature for 1 h; e) Develop color with TMB, and terminate the reaction with the termination solution; f) Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay reader.

[0059] The test results are as Figure 4 shown.

[0060] It can be seen from Figure 4 that the nanobodies BN1, BN2, BN4, and BNC2 have good affinity with the BCL6 antigen.

[0061] Example 4 Biolayer Interferometry (BLI) Experiment of Nanobody This example is used to further verify the binding of the purified nanobodies BN1, BN2, BN4, and BNC2 in Example 2 to the purified BCL6 antigen in Example 2, and calculate the affinity between the two.

[0062] Specifically, the BCL6 antigen is immobilized on the chip, and different concentration gradients of nanobodies BN1, BN2, BN4, and BNC2 are sequentially added to analyze the affinity with the antigen protein. Record the increase in the optical thickness at the tip of the biosensor within 600 seconds, and calculate the affinity with BCL6 according to the binding and dissociation rates of different concentrations of antibodies. The results are as Figure 5 shown.

[0063] It can be seen from Figure 5 that the nanobodies BN1, BN2, BN4, and BNC2 have good affinity with the BCL6 antigen.

[0064] Example 5 Experiment on Improving the Affinity of Nanobody To further improve the affinity of nanobodies, the present invention uses artificial intelligence for the rational design of nanobodies, mutates the nanobody BN2, and obtains the following mutants BN2M-T106Q (denoted as T106Q), BN2M-T106E (denoted as T106E).

[0065] Furthermore, the binding energy and total score of BN2-WT and the mutant BN2M with the BCL6 antigen were calculated by Rosetta, and then the energy changes of BN2-WT and T106Q, T106E were calculated using Amber. The results are as Figure 6 shown.

[0066] As can be seen from Figure 6 , both T106Q and T106E have higher scores ( Figure 6 b) and lower energy values ( Figure 6 a). T106Q has a lower energy value ( Figure 6 c), and is expected to have a lower affinity.

[0067] Furthermore, the energy change of the mutation site of BN2M-T106Q alone was calculated using Amber. It was found that after the 106th position of BN2M-T106Q was mutated from T to Q, the energy value became lower, indicating better stability ( Figure 7 b). For the calculation of the complex pairing energy, it was found that compared with the T before mutation, the paired amino acids of the mutated Q generally also had a lower energy value ( Figure 7 a).

[0068] Using the method in Example 2, an expression vector of the nanobody mutant BN2M was constructed (see Figure 1 ). Among them, the nanobody mutant BN2M includes BN2M-T106Q, and the nucleotide sequence of BN2M-T106Q is obtained by replacing the nucleotide "ACC" at positions 316-318 in SEQ ID NO:18 with "CAG".

[0069] Then, induction expression and purification were carried out to obtain the nanobody mutant BN2M-T106Q.

[0070] Using the method in Example 3, an ELISA experiment was carried out on the affinity of the above-mentioned BN2 and its mutant BN2M with the BCL6 antigen. The results are as Figure 7 shown in c-7d.

[0071] As can be seen from Figure 7 c-7d, compared with the affinity of the nanobody BN2 with the BCL6 antigen, BN2M-T106Q has a better affinity with the BCL6 antigen, and the affinity is increased by about 2.2 times.

[0072] Finally, a nanobody mutant BN2M-T106Q with significantly improved affinity was obtained and named as nanobody mutant BN2M.

[0073] Among them, the amino acid sequence of the nanobody mutant BN2M is shown as follows: MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSGLVAQVKEAGRSIHEIPREELEEFDY WGQGTLVTVSS(SEQ IDNO:21); The underlines represent complementary determining regions CDR1, CDR2, and CDR3 in turn.

[0074] The nucleotide sequence of the gene encoding the nanobody mutant BN2M is shown as SEQ ID NO:26.

[0075] Example 6 Experiment on Targeted Degradation of BCL6 Protein by Fusion Protein In order to achieve the targeted degradation of BCL6 protein, in the present invention, the above-mentioned nanobodies BN1, BN2, BN4, BNC2, and the nanobody mutant BN2M were respectively fused with the RBCC domain or its mutant RS80E to obtain fusion proteins, and the fusion proteins were transformed into Raji human Burkitt's lymphoma cells to detect the targeted degradation efficiency of the fusion proteins on BCL6 protein, with the BN0 framework containing only the framework region sequence as a control.

[0076] Specifically, based on the pcDNA3.1 vector, the genes encoding the above-mentioned nanobodies BN1, BN2, BN4, BNC2, and the nanobody mutant BN2M, BN0 framework were ligated and fused with the gene encoding the RBCC domain (its amino acid sequence is shown as SEQ ID NO:29, and the nucleotide sequence is shown as SEQ ID NO:32) or its mutant RS80E (its amino acid sequence is shown as SEQ ID NO:30, and the nucleotide sequence is shown as SEQ ID NO:33)) through the second linker (its amino acid sequence is shown as SEQ ID NO:31, and the nucleotide sequence is shown as SEQ ID NO:34)), and expression vectors were constructed by homologous recombination. The element arrangement structure of the expression vectors is as Figure 1As shown. The recombinant plasmids are pcDNA3.1-RBCC-BN1, pcDNA3.1-RBCC-BN2, pcDNA3.1-RBCC-BN4, pcDNA3.1-RBCC-BNC2, pcDNA3.1-RBCC-BN2M, pcDNA3.1-RBCC-BN0, pcDNA3.1-RS80E-BN1, pcDNA3.1-RS80E-BN2, pcDNA3.1-RS80E-BN4, pcDNA3.1-RS80E-BNC2, pcDNA3.1-RS80E-BN2M respectively.

[0077] Among them, the amino acid sequences of the fusion proteins RBCC-BN1, RBCC-BN2, RBCC-BN4, RBCC-BNC2, RBCC-BN2M, RS80E-BN1, RS80E-BN2, RS80E-BN4, RS80E-BNC2, RS80E-BN2M are respectively as shown in SEQ ID NO: 35 - 44.

[0078] Using the Lonza 4D electroporator, the GFP plasmid was electroporated into Raji cells in good condition. After electroporation, the electroporation efficiency and protein expression were observed through the GFP plasmid. The results are as Figure 8 shown.

[0079] It can be seen from Figure 8 that the above transfection method has a high transfection efficiency and can achieve the efficient transfer of plasmids to the greatest extent.

[0080] According to the above method, exemplarily, the above recombinant plasmids pcDNA3.1-RBCC-BN1, pcDNA3.1-RBCC-BN2, pcDNA3.1-RBCC-BNC2, pcDNA3.1-RBCC-BN2M, pcDNA3.1-RBCC-BN0 and the GFP plasmid were respectively electroporated into Raji cells in good condition.

[0081] The cells transfected for 48 h and 72 h were collected, and then Western blot detection was performed respectively. According to the Western blot results, the efficiency of protein targeted degradation after transfection for 48 h and 72 h was calculated. The results are as Figure 9 shown.

[0082] It can be seen from Figure 9It can be seen that, compared with the control, the fusion proteins RBCC-BN1, RBCC-BN2, RBCC-BN4, RBCC-BNC2, and RBCC-BN2M all have a certain degradation effect on the BCL6 protein, and the fusion protein RBCC-BN2 has the most obvious degradation effect on the BCL6 protein. The efficiency of protein targeted degradation is about 50% after 48 h, and about 70% after 72 h.

[0083] Similarly, according to the above method, for example, the above recombinant plasmids pcDNA3.1-RS80E-BN1, pcDNA3.1-RS80E-BN2, pcDNA3.1-RS80E-BNC2, pcDNA3.1-RS80E-BN2M, pcDNA3.1-RBCC-BN0, and the GFP plasmid were respectively electrotransfected into Raji cells in good condition.

[0084] Cells were selected for collection 72 h after electrotransfection, and then Western blot detection was performed respectively. According to the Western blot results, the efficiency of protein targeted degradation was calculated 72 h after transfection. The results are as Figure 10 shown.

[0085] From Figure 10 it can be seen that the fusion protein RS80E-BN2 has the most obvious and stable degradation effect after electrotransfection.

[0086] Cells transfected with the recombinant plasmid pcDNA3.1-RS80E-BNC2 for 48 h and 72 h were further selected for collection, and then Western blot detection was performed respectively. According to the Western blot results, the efficiency of protein targeted degradation was calculated 48 h and 72 h after transfection. The results are as Figure 11 shown.

[0087] From Figure 11 it can be seen that the fusion protein RS80E-BNC2 has the most obvious effect after electrotransfection. The efficiency of protein targeted degradation is about 40% at 48 h (lanes D2-4), and about 80% at 72 h (lanes D3-4).

[0088] Cells transfected with the recombinant plasmids pcDNA3.1-RS80E-BNC2 and pcDNA3.1-RBCC-BN2 were further selected for collection, and then Western blot detection was performed respectively. According to the Western blot results, the expression levels of the tumor suppressor gene p53 and the cyclin p21 in the cells were obtained. The results are as Figure 12 shown.

[0089] From Figure 12It can be seen that the degradation of the fusion protein RS80E-BNC2 and RBCC-BN2 is accompanied by an increase in the expression levels of the tumor suppressor gene p53 and the cyclin p21 in cells.

[0090] Finally, the degradation pathway of the fusion protein targeting the degradation of BCL6 protein was explored.

[0091] Specifically, the proteasome inhibitor MG132 with a final concentration of 10 μM or the autophagy inhibitor BafA1 with a final concentration of 100 nM was selected for use. Among them, MG132 can inhibit the ubiquitin-proteasome pathway, and BafA1 can inhibit the lysosomal pathway. Three copies of the pcDNA3.1-RBCC-BNC2 recombinant plasmid and the GFP plasmid were respectively electrotransfected into Raji cells in good condition. After 24 h of transfection, the corresponding MG132 and BafA1 were added to 2 of the cells carrying the pcDNA3.1-RBCC-BNC2 recombinant plasmid, and after continuous culture, Western blot detection was performed. The results are as Figure 13 shown.

[0092] As can be seen from Figure 13 it, the fusion protein in the present invention targets the degradation of BCL6 protein through the ubiquitin-proteasome pathway.

[0093] In summary, the present invention designs a nanobody specifically targeting BCL6 by using artificial intelligence technology. This nanobody has good specificity and high affinity, and can efficiently bind to the BCL6 antigen. Further, through the rational design of the nanobody by artificial intelligence, a nanobody mutant with higher affinity is obtained. Furthermore, by fusing the nanobody or nanobody mutant with high affinity and good specificity with the RBCC domain or its mutant, a fusion protein is obtained. This fusion protein can specifically bind to the target protein using its nanobody domain, initiate the protein degradation pathway, and achieve the targeted degradation of BCL6 protein.

[0094] The above-described embodiments only express the implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A nanobody against BCL6 protein, characterized in that including complementarity determining region CDR1, complementarity determining region CDR2 and complementarity determining region CDR3; Wherein, the complementary determining region CDR1 has an amino acid sequence as shown in any one of SEQ ID NOs: 1-2, the complementary determining region CDR2 has an amino acid sequence as shown in any one of SEQ ID NOs: 3-4, and the complementary determining region CDR3 has an amino acid sequence as shown in any one of SEQ ID NOs: 5-8.

2. The anti-BCL6 protein nanobody according to claim 1, characterized in that The Nanobody also includes a framework region FR1, a framework region FR2, a framework region FR3 and a framework region FR4; Wherein, the framework region FR1 has an amino acid sequence as shown in any one of SEQ ID NOs: 9-10, the framework region FR2 has an amino acid sequence as shown in any one of SEQ ID NOs: 11-12, the framework region FR3 has an amino acid sequence as shown in any one of SEQ ID NOs: 13-14, and the framework region FR4 has an amino acid sequence as shown in any one of SEQ ID NOs: 15-16.

3. The anti-BCL6 protein nanobody according to claim 2, characterized in that The Nanobody is selected from any one of the following: A1) has an amino acid sequence as shown in any one of SEQ ID NOs: 17-20; A2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in A1); A3) an amino acid sequence having a sequence identity of more than 80% with the amino acid sequence defined in A1) or A2); A4) An amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1) or A2) or A3).

4. A nanobody mutant against BCL6 protein, characterized in that: The Nanobody mutant is selected from any one of the following: B1) has the amino acid sequence shown in SEQ ID NO:21; B2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in B1); B3) an amino acid sequence having a sequence identity of more than 80% with the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in B1) or B2) or B3); Wherein, in B1), the amino acid sequence shown in SEQ ID NO: 21 is obtained by subjecting the amino acid sequence shown in SEQ ID NO: 18 to the following mutation: T106Q.

5. A fusion protein, characterized in that The fusion protein is obtained by connecting the Nanobody according to any one of claims 1 to 3 or the Nanobody mutant according to claim 4 to the RBCC domain or its mutant via a second linker; Among them, the amino acid sequence of the second linker is shown in SEQ ID NO:31, the amino acid sequence of the RBCC domain is shown in SEQ ID NO:29, and the amino acid sequence of the RBCC domain mutant is shown in SEQ ID NO:

30.

6. The fusion protein according to claim 5, characterized in that The fusion protein is selected from any one of the following: C1) has an amino acid sequence as shown in any one of SEQ ID NOs: 35-44; C2) an amino acid sequence having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in C1); C3) an amino acid sequence having a sequence identity of more than 80% with the amino acid sequence defined in C1) or C2); C4) An amino acid sequence obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in C1) or C2) or C3).

7. A biomaterial, characterized in that: The biological material is selected from any one of the following: D1) A nucleic acid molecule encoding the Nanobody according to any one of claims 1 to 3, the Nanobody mutant according to claim 4, or the fusion protein according to any one of claims 5 to 6; D2) a recombinant vector containing the nucleic acid molecule described in D1); D3) A recombinant cell containing the nucleic acid molecule described in D1) or the recombinant vector described in D2).

8. Use of the Nanobody according to any one of claims 1 to 3, the Nanobody mutant according to claim 4, the fusion protein according to any one of claims 5 to 6, or the biomaterial according to claim 7 in any of the following: F1) Use in the preparation of products for preventing and / or treating BCL6 target related diseases; F2) Application in the preparation of products for screening, diagnosis or auxiliary diagnosis of BCL6 target related diseases; in, The BCL6 target-related disease is a BCL6-positive tumor.

9. A pharmaceutical composition for preventing and / or treating BCL6 target-related diseases, characterized in that: The pharmaceutical composition comprises the Nanobody according to any one of claims 1-3, the Nanobody mutant according to claim 4, the fusion protein according to any one of claims 5-6, or the biomaterial according to claim 7, and a pharmaceutically acceptable carrier.

10. A method for preparing the Nanobody according to any one of claims 1 to 3, the Nanobody mutant according to claim 4, or the fusion protein according to any one of claims 5 to 6, characterized in that: The steps include: Cultivating the recombinant cell according to claim 7, and obtaining a culture after inducing expression; Said Nanobody, said Nanobody mutant or said fusion protein is isolated from said culture.

Images