A nanobody against bcl6 protein, fusion protein and application thereof

Abstract

The application discloses a kind of anti-BCL6 protein nanobody, fusion protein and its application, belong to biotechnology field.The application is designed by using artificial intelligence technology to specifically target the nanobody of BCL6, the nanobody has good specificity, high affinity, can efficiently combine BCL6 antigen;Further, the higher affinity nanobody mutant of the rational design of nanobody is obtained by artificial intelligence;Further, by the fusion of nanobody or nanobody mutant with high affinity and good specificity and RBCC domain or its mutant, the fusion protein is obtained, the fusion protein can utilize its nanobody domain specific binding target protein, start protein degradation pathway, realize the targeted degradation of BCL6 protein, improve the expression of intracellular anticancer gene p53 and cell cycle protein p21, has very big clinical application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanobody, fusion protein, and its application against BCL6 protein. Background Technology

[0002] The 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 translocations. BCL6 belongs to the BTB / POZ family of transcriptional repressors and consists of a BTB / POZ domain (bric-a-brac, tramtrack, broad complex, BTB) at the N-terminus, a PEST motif (also known as repression domain 2, RD2) in the middle, and six zinc fingers (ZF) at the C-terminus. These domains regulate the transcription of target genes through various interactions. BCL6 primarily functions in the immune system, regulating B cell differentiation and maturation. In the germinal center (GC) response, BCL6 represses 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] Germinal centers are the primary sites for B cell differentiation and antibody affinity maturation. In the GC response, BCL6 helps B cells tolerate somatic mutations and oxidative stress generated during rapid proliferation by inhibiting genes associated with DNA damage responses, thus maintaining the normal progression of the GC response. This inhibitory effect makes BCL6 crucial in promoting the generation of high-affinity B cells, effector memory B cells, and plasma cell differentiation. Dysregulation of BCL6 expression may lead to abnormal GC responses, subsequently triggering various lymphomas and other malignancies. Besides lymphoma, BCL6 also plays an important role in breast cancer, lung cancer, and glioma. BCL6 plays a key role in the development and progression of these cancers by regulating the expression of different genes. Therefore, BCL6 has become an important target for cancer therapy.

[0004] Existing BCL6 inhibitors are primarily designed to target its BTB domain, exerting their inhibitory activity through competitive binding with co-repressors. However, research on BCL6 degraders is relatively limited. Among them, BCL6 PROTAC (Proteolysis Targeting Chimeras) exhibits poor protein degradation activity, while other degraders are BCL6 molecular glues, which were obtained incidentally during inhibitor screening and cannot be rationally designed. These molecular glues show better efficacy compared to similar inhibitors.

[0005] Nanobodies are single-domain antibodies derived from heavy-chain antibodies of camel species. They possess the specificity and affinity of traditional antibodies while exhibiting smaller molecular weight and higher stability. Nanobodies can penetrate tissues and cells and bind more easily to target proteins, thus holding broad application prospects in biomedical research and therapy.

[0006] Targeted protein degradation (TPD) technology utilizes the ubiquitin-proteasome and lysosomal degradation systems within cells to achieve specific and efficient degradation of disease-related proteins, thereby achieving therapeutic effects. Compared to traditional small molecule inhibitors, TPD has several advantages: it is event-driven, requires lower dosages, and only needs a catalytic amount to exert its effect; it can also target some undrugable proteins.

[0007] TRIM-away is an emerging TPD (transmitted protein degradation) technology that utilizes members of the TRIM (Tripartite motif-containing) protein family as E3 ubiquitin ligases. It specifically recognizes and binds to target proteins, promoting their ubiquitination and ultimately degradation in the proteasome. TRIM-away technology holds significant potential for BCL6 degradation, offering a more efficient and precise method that could potentially improve the treatment outcomes of BCL6-related cancers.

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

[0009] The purpose of this invention is to provide a nanobody, a fusion protein, and their applications against BCL6 protein. This addresses the problem in the prior art of lacking nanobodies with high specificity, high affinity, and the ability to efficiently bind to the BCL6 antigen.

[0010] In a first aspect, the present invention provides a nanobody against BCL6 protein, comprising a complementarity-determining region (CDR1), a complementarity-determining region (CDR2), and a 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 this invention, the term "nanobody" is also called a single-domain antibody (sdAb), which refers to an antibody composed of only one H chain V region, also known as a VHH antibody. The antigen-binding ability and stability of nanobodies are essentially consistent with those of complete antibodies. A nanobody is composed sequentially of a frame region FR1, a complementarity-determining region CDR1, a frame region FR2, a complementarity-determining region CDR2, a frame region FR3, a complementarity-determining region CDR3, and a frame region FR4. Nanobodies can be truncated at the N-terminus or C-terminus to contain only a portion of FR1 and / or FR4, or to lack one or both of the frame regions, as long as they substantially maintain antigen binding and specificity. The complementarity-determining region is primarily responsible for antigen binding.

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

[0013] Understandably, the framework region is highly conservative. Therefore, conventional framework regions in existing technologies can be selected according to actual needs, as long as they can improve the stability of the nanobody.

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

[0015] In this invention, the inventors used artificial intelligence technology to design a nanobody that specifically targets BCL6. 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, the nanobody mutant being selected from any of the following: B1) having an amino acid sequence as shown in SEQ ID NO:21; B2) having an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence defined in B1); B3) having an amino acid sequence with more than 80% sequence identity compared to the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by linking a tag or 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 an amino acid sequence as 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 this invention, the inventors further used artificial intelligence to rationally design nanobodies, resulting in nanobodies with higher affinity mutants.

[0018] In some embodiments, in addition to the mutation occurring at one site at position 106, the aforementioned nanobody mutants may further have conserved amino acid substitutions at other sites, resulting in the mutated amino acids, such as those in the nanobody shown in SEQ ID NO:18, exhibiting higher affinity. Preferably, the conserved substitution of amino acids preserves the higher affinity of the nanobody of the present invention. It will be apparent to those skilled in the art that such substitutions can occur in regions other than the aforementioned sites while still retaining the corresponding affinity. Preferably, the conserved substitution variant has at least one conserved amino acid substitution. Examples of conserved substitutions are substitutions occurring within the following amino acid groups: 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 and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecule 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 linking any of the above-mentioned nanobodies or the above-mentioned nanobodies mutants with an RBCC domain or a mutant thereof through a second adapter; wherein, the amino acid sequence of the second adapter 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.

[0020] In this invention, the inventors fuse nanobodies or nanobodies mutants with high affinity and good specificity with RBCC domains or their mutants (truncated TRIM family proteins) to obtain a fusion protein. This fusion protein can utilize the specific binding of its nanobodies' domains to target proteins, initiate protein degradation pathways, achieve targeted degradation of BCL6 protein, and increase the expression of intracellular tumor suppressor gene p53 and cell cycle protein p21. This protects B cells from cell suicide caused by DNA damage and inhibits the proliferation rate of cancer cells, thus having great clinical application value.

[0021] Understandably, the linker peptide can be selected from conventional linker peptides in existing technologies according to actual needs, as long as it enables the two proteins to fuse well.

[0022] In some embodiments, the fusion protein is selected from any of the following: C1) having an amino acid sequence as shown in any of SEQ ID NO:35-44; C2) having an amino acid sequence with one or more amino acid substitutions, deletions, or additions compared to the amino acid sequence defined by C1); C3) having an amino acid sequence with more than 80% sequence identity compared to the amino acid sequence defined by C1) or C2); C4) an amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined by C1) or C2) or C3).

[0023] The nanobodies, nanobodies mutants, and fusion proteins provided by this invention can be natural, recombinant, or synthetic active polypeptides. These active polypeptides can be naturally purified products, chemically synthesized products, or products produced from prokaryotic hosts (e.g., Escherichia coli) or eukaryotic hosts (e.g., yeast, higher plants) using recombinant technology.

[0024] In A4), B4), or C4) of this invention, the linkage can be achieved through direct peptide bonds or through a linker, using methods conventional in the art. The tags include, but are not limited to: GST (glutathione thioredoxin) tag protein, Trx (thioredoxin) tag protein, His tag protein (His-tag), Flag tag protein, LacZ tag protein, GFP (green fluorescent protein), sfGFP (hyperfolded green fluorescent protein), and HA tag (hemagglutinin tag). Those skilled in the art can select appropriate tag proteins according to actual needs. The use of tags does not alter the function of the target protein (nanobody, nanobody mutant, or fusion protein); its purpose is for separation, purification, detection, or tracing. Tags can be separated from the target protein (nanobody, nanobody mutant, or fusion protein) using chemical cleavage methods or enzymatic methods (such as introducing protease cleavage sites and using TEV protease to remove the tag) known in the art.

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

[0026] In some embodiments, the nucleic acid molecule defined in D1) is selected from any of the following: E1) a nucleic acid molecule having a nucleotide sequence as shown in any 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 of the above-mentioned nanobodies, nanobodies mutants, or fusion proteins; E3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in E1) or E2) and encodes any of the above-mentioned nanobodies, nanobodies mutants, or fusion proteins.

[0027] The nucleic acid molecules provided by this invention can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or RNA, such as mRNA or hnRNA; and these nucleic acid molecules can usually be obtained by PCR amplification or artificial synthesis.

[0028] The recombinant vector provided by the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate the relevant sequence, and the expression vector is used to express the relevant gene. The vector used to construct the expression vector can be at least one of pET23a, pET28a, and pcDNA3.1 vector.

[0029] In some embodiments, the method for preparing the recombinant cells includes the step of converting the recombinant vector into expression host cells.

[0030] In this invention, the expression host cell is a conventional host cell in the art, as long as the recombinant vector can stably replicate itself and the gene it carries can be effectively expressed. It can be a prokaryotic cell or a eukaryotic cell, such as Escherichia coli or yeast. For example, Escherichia coli can be E. coli BL21(DE3), Rosetta(DE3), or BL21(DE3)plysS. In this invention, E. coli BL21(DE3) is preferred as the expression host.

[0031] In a fifth aspect, the present invention provides the use of any of the above-described nanobodies, the above-described nanobodies mutants, any of the above-described fusion proteins, or the above-described biomaterials in any of the following: F1) use in the preparation of products for the prevention and / or treatment of BCL6 target-related diseases; F2) use in the preparation of products for the screening, diagnosis, or auxiliary diagnosis of BCL6 target-related diseases; wherein, BCL6 target-related diseases are BCL6-positive tumors.

[0032] In this invention, BCL6-positive tumors refer to tumors in which BCL6 expression is detected in tumor cells. Such tumors can be common types such as follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, and Hodgkin's lymphoma.

[0033] In a sixth aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of BCL6 target-related diseases, the pharmaceutical composition comprising any of the above-described nanobodies, the above-described nanobodies mutants, any of the above-described fusion proteins or the above-described biomaterials, and a pharmaceutically acceptable carrier.

[0034] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for enabling the active ingredient to dissolve at a desired rate after administration to a subject, or to promote the effective absorption of the active ingredient after administration of the composition to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient in the composition.

[0035] The pharmaceutical compositions provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

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

[0037] The pharmaceutical compositions provided by this invention can be administered in any form, including injection (intravenous), mucosal membrane, oral (solid and liquid formulations), inhalation, ocular, rectal, local, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be in controlled-release or sustained-release formulations (e.g., liposomes or microspheres).

[0038] In a seventh aspect, the present invention provides a method for preparing any of the above-described nanobodies, nanobodies mutants, or fusion proteins, comprising the following steps: culturing the above-described recombinant cells, inducing expression to obtain a culture; and isolating the nanobodies, nanobodies mutants, or fusion proteins from the culture.

[0039] In this invention, there are no special requirements for the culture method, culture conditions, and culture medium, as long as the recombinant cells grow normally. Furthermore, the methods for isolating any of the aforementioned nanobodies, nanobody mutants, or fusion proteins from the culture are all conventional methods in the art.

[0040] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention utilizes artificial intelligence technology to design nanobodies that specifically target BCL6. These nanobodies exhibit good specificity and high affinity, enabling them to efficiently bind to the BCL6 antigen. Furthermore, through the rational design of nanobodies using artificial intelligence, nanobodies with even higher affinity mutants are obtained. Even further, by fusing nanobodies or nanobodies with high affinity and good specificity with the RBCC domain or its mutant, a fusion protein is obtained. This fusion protein can utilize its nanobodies' domains to specifically bind to target proteins, initiating protein degradation pathways and achieving targeted degradation of BCL6 protein. This increases the expression of intracellular tumor suppressor gene p53 and cell cycle protein p21, thereby protecting B cells from cell suicide caused by DNA damage and inhibiting the proliferation rate of cancer cells, demonstrating significant clinical application value. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the element arrangement structure of the recombinant expression vector constructed in this invention;

[0042] Figure 2The figures shown are SDS-PAGE detection results of the nanobody in Example 2 of this invention. In Figure (a), lane M is the marker, and lanes 0-6 are sfGFP-BN0, sfGFP-BN1, sfGFP-BN2, sfGFP-BN3, sfGFP-BN4, sfGFP-BN5, and sfGFP-BN6, respectively. In Figure (b), lane M is the marker, and lanes 0-6 are sfGFP-BNC0, sfGFP-BNC1, sfGFP-BNC2, sfGFP-BNC3, sfGFP-BNC4, sfGFP-BNC5, and sfGFP-BNC6, respectively.

[0043] Figure 3 This is a graph showing the SDS-PAGE detection results of the BCL6 antigen in Example 2 of the present invention;

[0044] Figure 4 This is an ELISA test result of the affinity between the nanobody and the BCL6 antigen in Example 3 of the present invention;

[0045] Figure 5 The graph shows the BLI test results of the affinity of nanobodies BN1, BN2, BN4, BNC2 and BCL6 antigen in Example 4 of this invention.

[0046] Figure 6 The figures shown are (a) and (b) graphs of the binding energy and total score of BN2-WT and mutant BN2M with BCL6 antigen calculated by Rosetta in Example 5 of the present invention, and (c) graph of the binding energy and total score of BN2-WT and mutant BN2M with BCL6 antigen calculated by Amber.

[0047] Figure 7 The figures shown are (a) and (b) the results of energy changes before and after the mutation site change of the mutant BN2M-T106Q calculated by Amber in Example 5 of the present invention, and (c) and (d) the results of ELISA test on the affinity of the nanobody mutant to the BCL6 antigen.

[0048] Figure 8 This is a diagram showing the results of electroporation of the GFP plasmid into Raji cells in Example 6 of the present invention;

[0049] Figure 9 The image shows the Western blot results of the fusion protein targeting and degrading BCL6 protein in Example 6 of this invention, where 1 is RBCC-BN1, 2 is RBCC-BN2M, 3 is RBCC-BN2, 4 is RBCC-BNC2, and 5 is the control.

[0050] Figure 10The image shows the Western blot results of the fusion protein targeting and degrading BCL6 protein in Example 6 of this invention. In the image, 1 is RS79E-BN1, 2 is RS79E-BN2M, 3 is RS79E-BN2, 4 is RS79E-BNC2, and 5 is the control.

[0051] Figure 11 This is a Western blot (WB) image showing the targeted degradation of BCL6 protein by the fusion protein RS79E-BNC2 in Example 6 of this invention.

[0052] Figure 12 This is a graph showing the changes in p53 and p21 levels during the degradation of BCL6 protein by the fusion proteins RBCC-BN2 and RS79E-BNC2 in Example 6 of the present invention. In this graph, a and b correspond to RBCC-BN2, and c and d correspond to RS79E-BNC2.

[0053] Figure 13 The image shows the Western blot results of the fusion protein targeting the degradation of BCL6 protein in Example 6 of this invention. In the image, 1 is the control, 2 is RBCC-BNC2, 3 is RBCC-BNC2+MG132, and 4 is RBCC-BNC2+BafA1. Detailed Implementation

[0054] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] Experimental methods not specifically described in the examples are generally performed according to conventional experimental methods in the field of molecular biology, including but not limited to those described in *Molecular Cloning: A Laboratory Manual* by M.R. Green and *Molecular Biology* by Robert F. Weaver, or according to the experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, all reagents and biological materials used in the examples are commercially available.

[0056] Example 1: Artificial Intelligence Design of Nanobodies

[0057] First, artificial intelligence technology combined with bioinformatics tools (such as SEPPA and MetMHCpan) was used to predict hotspot epitopes of the BCL6 antigen, and these epitopes were linearly fragmented to generate fragments containing at least four consecutive residues. Then, CDR-like fragments matching these fragments were screened using the AbAg database. These CDR-like fragments were then structurally superimposed and optimized with epitope fragments to construct CDR fragments that specifically bind to the BCL6 antigen. Next, suitable frame region fragments were selected (the amino acid sequences of the CDR fragments and frame region fragments are shown in Table 1 below). Finally, a series of nanobodies targeting specific epitopes of the BCL6 antigen were obtained, and these were named nanobodies BN1-6 and BNC1-6, respectively.

[0058] Table 1. Amino acid sequences of CDR fragments and framework fragments

[0059]

[0060] For example, the amino acid sequence of nanobody BN1 is shown below:

[0061] MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSGITTIKEMGRSIHEIPREEFDY WGQGTLVTVSS(SEQ ID NO:17);

[0062] The underscores represent the complementary determinant regions CDR1, CDR2, and CDR3, respectively.

[0063] The amino acid sequence of nanobody BN2 is shown below:

[0064] MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSGLVATVKEAGRSIHEIPREELEEFDY WGQGTLVTVSS(SEQ IDNO:18);

[0065] The underscores represent the complementary determinant regions CDR1, CDR2, and CDR3, respectively.

[0066] The amino acid sequence of nanobody BN4 is shown below:

[0067] MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDTYIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSVGASTLYGSAYEEFDY WGQGTLVTVSS(SEQ ID NO:19);

[0068] The underscores represent the complementary determinant regions CDR1, CDR2, and CDR3, respectively.

[0069] The amino acid sequence of the nanobody BNC2 is shown below:

[0070] EFQVQLVESGGGSVQAGGSLRLSCTAS GGSEYSYSTF SLGWFRQAPGQEREAVAAI ASMGGL TYYADSVKGRFTISRDNAKNTVTLQMNNLKPEDTAIYYCAA GLVATVKEAGRSIHEIPREEL WGQGTQVTVSS(SEQ IDNO:20);

[0071] The underscores represent the complementary determinant regions CDR1, CDR2, and CDR3, respectively.

[0072] The amino acid sequence of the nanobody BN0 framework is obtained by removing the complementarity-determining regions CDR1, CDR2 and CDR3 from the nanobody BN1 (amino acid sequence as shown in SEQ ID NO:17) (only the framework region sequence is included).

[0073] The amino acid sequence of the BNC0 nanobody framework is obtained by removing the complementarity-determining regions CDR1, CDR2 and CDR3 from the BNC2 nanobody (amino acid sequence as shown in SEQ ID NO:20) (containing only the framework region sequence).

[0074] The nucleotide sequences of the genes encoding nanobodies BN1, BN2, BN4, and BNC2 are shown in SEQ ID NO:22-25, respectively.

[0075] Example 2: Expression and purification of nanobodies and BCL6 antigen

[0076] To facilitate the isolation and purification of nanobodies, in this invention, the nanobodies from Example 1 are displayed on the surface of *E. coli* using superfolded green fluorescent protein (sfGFP, the nucleotide sequence of the gene encoding sfGFP is shown in SEQ ID NO:27). Specifically, using pET23a as the base vector, the gene encoding the aforementioned nanobodies is fused with the sfGFP gene via a first adapter (the nucleotide sequence of which is shown in SEQ ID NO:28), and an expression vector is constructed through homologous recombination. The element arrangement structure of this expression vector is as follows: Figure 1 As shown. For example, the recombinant plasmids are pET23a-sfGFP-BN1, pET23a-sfGFP-BN2, pET23a-sfGFP-BN4, and pET23a-sfGFP-BNC2.

[0077] The constructed recombinant plasmids were transformed into *E. coli* competent cells BL21(DE3) and cultured overnight at 37°C to obtain a series of recombinant strains. Single colonies were then picked and inoculated into 100 mL of LB broth (ampicillin concentration 50 μg / mL) and cultured with shaking at 37°C. OD 600 When the pH reached approximately 0.6, IPTG was added to a final concentration of 0.5 mM, and the mixture was incubated at 18°C ​​with shaking for 18 hours to induce incubation. After incubation, the bacterial cells and culture supernatant were collected separately by centrifugation at 12000 rpm and 4°C. The bacterial cells were washed 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 the secreted protein expressed by the strain was directly obtained from the TEN buffer. The secretion and expression of the nanobody were analyzed using SDS-PAGE, and the results are as follows: Figure 2 As shown.

[0078] from Figure 2 As can be seen, both nanobodies BN1-6 and BNC1-6 were successfully expressed.

[0079] For example, the purified nanobodies BN1, BN2, BN4, and BNC2 obtained by washing the outer membrane are stored for subsequent experiments.

[0080] Furthermore, the BCL6 antigen sequence (amino acids 6-129 in NCBI accession number P41182) was cloned into the pET28a vector via homologous recombination to obtain the recombinant plasmid pET28a-BCL6 (vector structure shown in [link to pET28a-BCL6]). Figure 1The constructed recombinant plasmids were transformed into E. coli competent cells BL21(DE3). Single colonies were picked and inoculated into 100 mL of LB liquid medium (kanamycin concentration of 100 μg / mL), and incubated overnight at 37°C until OD was reached. 600 When the pH reached approximately 0.6, 0.5 mM IPTG was added to the final concentration. The cells were incubated at 18°C ​​with shaking for 18 hours. After centrifugation at 6000 rpm for 10 min, the cells were collected and washed with Buffer A (20 mM Tris-HCl pH 8.0, 200 mM NaCl). The cells were then resuspended in Buffer A, and 1 mM PMSF was added. The cells were then autoclaved. After centrifugation at 18000 rpm for 30 min, the supernatant was collected, filtered, and purified using Ni-NTA. The Ni-NTA-purified protein was then purified using a molecular sieve (Superdex 75 increase 10 / 300, GL), pre-equilibrated with Buffer A. The purified protein was collected and analyzed by SDS-PAGE. The protein was aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C. The SDS-PAGE analysis results are shown below. Figure 3 As shown, the expected size of BCL6, calculated using http: / / www.expasy.org / , is 15.9 kDa, indicating that the BCL6 antigen was successfully expressed.

[0081] Example 3: ELISA experiment with nanobodies

[0082] This embodiment is used to verify whether the purified nanobodies BN1, BN2, BN4, and BNC2 from Example 2 can directly interact with the purified BCL6 antigen from Example 2. Specifically, the steps are as follows:

[0083] a) Dilute the BCL6 antigen to 1 μg / mL with 1×ELSIA coating buffer, and then spread the plate at 100 μL / well. Coat the plate and incubate overnight at 4°C.

[0084] b) Wash the plate with PBST, block with 1% BSA at room temperature for 2 hours, 100 μL / well;

[0085] c) Prepare nanobodies of different concentrations (BN1, BN2, BN4, BNC2) using 1% BSA, 100 μL / well, and incubate at room temperature for 1 h;

[0086] d) Incubate with HRP-conjugated mouse anti-HA-Tag mAb secondary antibody at room temperature for 1 h;

[0087] e) The reaction is terminated by TMB color development and the stop solution.

[0088] f) Measure the absorbance at 450 nm using an ELISA reader.

[0089] Test results are as follows Figure 4 As shown.

[0090] from Figure 4 As can be seen, nanobodies BN1, BN2, BN4, and BNC2 have good affinity for the BCL6 antigen.

[0091] Example 4: Experiment with Nanobody Biomembrane Interference (BLI) Technology

[0092] This embodiment is used to further verify the binding of the purified nanobodies BN1, BN2, BN4, and BNC2 from Example 2 to the purified BCL6 antigen from Example 2, and to calculate the affinity between the two.

[0093] Specifically, the BCL6 antigen was immobilized on a chip, and nanobodies of different concentration gradients (BN1, BN2, BN4, and BNC2) were sequentially added to analyze their affinity for the antigen protein. The increase in optical thickness at the biosensor tip was recorded over 600 seconds, and the affinity for BCL6 was calculated based on the binding and dissociation rates of different antibody concentrations. The results are as follows: Figure 5 As shown.

[0094] from Figure 5 As can be seen, nanobodies BN1, BN2, BN4, and BNC2 have good affinity for the BCL6 antigen.

[0095] Example 5: Experiment to enhance the affinity of nanobodies

[0096] To further improve the affinity of nanobodies, this invention uses artificial intelligence for the rational design of nanobodies and mutates the nanobodies BN2 to obtain the following mutants BN2M-T106Q (denoted as T106Q) and BN2M-T106E (denoted as T106E).

[0097] Furthermore, the binding energies and total scores of BN2-WT and the mutant BN2M to the BCL6 antigen were calculated using Rosetta. Then, the energy changes of BN2-WT and T106Q and T106E were calculated using Amber. The results are as follows: Figure 6 As shown.

[0098] from Figure 6 As can be seen, both the 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), which is expected to have lower affinity.

[0099] Further calculations using Amber to determine the individual energy changes at the BN2M-T106Q mutation site revealed that the energy value decreased after the mutation from T to Q at position 106 of BN2M-T106Q, indicating better stability. Figure 7 (b) For the calculation of complex pairing energy, it was found that the Q after mutation also has a lower overall energy value compared with the T before mutation ( Figure 7 a).

[0100] Using the method described in Example 2, an expression vector for the nanobody mutant BN2M was constructed (see Example 2). Figure 1 Among them, the nanobody mutant BN2M includes BN2M-T106Q, the nucleotide sequence of BN2M-T106Q is obtained by replacing the nucleotide "ACC" at positions 316-318 of SEQ ID NO:18 with "CAG".

[0101] Then, the nanobody mutant BN2M-T106Q was obtained by inducing expression and purification.

[0102] Using the method described in Example 3, the affinity of the above-mentioned BN2 and its mutant BN2M for the BCL6 antigen was tested by ELISA. The results are as follows: Figure 7 As shown in c-7d.

[0103] from Figure 7 As can be seen in c-7d, BN2M-T106Q has a better affinity for BCL6 antigen than BN2 nanobody has an affinity for BCL6 antigen, and the affinity is increased by about 2.2 times.

[0104] Ultimately, a nanobody mutant with significantly enhanced affinity, BN2M-T106Q, was obtained and named nanobody mutant BN2M.

[0105] The amino acid sequence of the nanobody mutant BN2M is shown below:

[0106] MEVQLEESGGGLVQPGGSLRLSCAAS GFNIKDT YIGWVRQAPGKGEEWVASI YPTSGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAA GSGLVAQVKEAGRSIHEIPREELEEFDY WGQGTLVTVSS(SEQ IDNO:21);

[0107] The underscores represent the complementary determinant regions CDR1, CDR2, and CDR3, respectively.

[0108] The nucleotide sequence of the gene encoding the nanobody mutant BN2M is shown in SEQ ID NO:26.

[0109] Example 6: Experiment on the targeted degradation of BCL6 protein by fusion protein

[0110] To achieve targeted degradation of BCL6 protein, in this invention, the above-mentioned nanobodies BN1, BN2, BN4, BNC2 and the nanobodies mutant BN2M were fused with the RBCC domain or its mutant RS79E to obtain fusion proteins. The fusion proteins were then transformed into Raji human Burkitt's lymphoma cells, and the targeted degradation efficiency of the fusion proteins on BCL6 protein was detected, with the BN0 frame containing only the frame region sequence as a control.

[0111] Specifically, using pcDNA3.1 as the base vector, the genes encoding the frameworks of the aforementioned nanobodies BN1, BN2, BN4, BNC2, and the nanobodies mutants BN2M and BN0 were fused with the genes encoding the RBCC domain (the amino acid sequence of which is shown in SEQ ID NO:29, and the nucleotide sequence of which is shown in SEQ ID NO:32) or its mutant RS79E (the amino acid sequence of which is shown in SEQ ID NO:30, and the nucleotide sequence of which is shown in SEQ ID NO:33) through a second linker (the amino acid sequence of which is shown in SEQ ID NO:31, and the nucleotide sequence of which is shown in SEQ ID NO:34). An expression vector was then constructed via homologous recombination. The element arrangement structure of the expression vector is as follows: Figure 1 As 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-RS79E-BN1, pcDNA3.1-RS79E-BN2, pcDNA3.1-RS79E-BN4, pcDNA3.1-RS79E-BNC2, and pcDNA3.1-RS79E-BN2M.

[0112] The amino acid sequences of the fusion proteins RBCC-BN1, RBCC-BN2, RBCC-BN4, RBCC-BNC2, RBCC-BN2M, RS79E-BN1, RS79E-BN2, RS79E-BN4, RS79E-BNC2, and RS79E-BN2M are shown in SEQ ID NO:35-44, respectively.

[0113] Using a Lonza 4D electroporator, the GFP plasmid was electroporated into healthy Raji cells. After electroporation, the electroporation efficiency and protein expression were observed using the GFP plasmid. The results are as follows: Figure 8 As shown.

[0114] from Figure 8 As can be seen, the above transfection method has high transfection efficiency and can achieve efficient plasmid transfection to the greatest extent.

[0115] Following the above method, for example, the recombinant plasmids pcDNA3.1-RBCC-BN1, pcDNA3.1-RBCC-BN2, pcDNA3.1-RBCC-BNC2, pcDNA3.1-RBCC-BN2M, pcDNA3.1-RBCC-BN0 and GFP plasmid were electroporated into healthy Raji cells.

[0116] Cells were collected at 48h and 72h after transfection, and then subjected to Western blot analysis. Based on the Western blot results, the efficiency of protein-targeted degradation was calculated after 48h and 72h of transfection. The results are as follows: Figure 9 As shown.

[0117] from Figure 9 As can be seen, 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 BCL6 protein. Among them, the fusion protein RBCC-BN2 has the most obvious degradation effect on BCL6 protein, with a protein-targeted degradation efficiency of about 50% after 48 hours and about 70% after 72 hours.

[0118] Similarly, following the above method, for example, the recombinant plasmids pcDNA3.1-RS79E-BN1, pcDNA3.1-RS79E-BN2, pcDNA3.1-RS79E-BNC2, pcDNA3.1-RS79E-BN2M, pcDNA3.1-RBCC-BN0 and GFP plasmid were electroporated into healthy Raji cells.

[0119] Cells were collected 72 hours after electroporation, and then Western blot analysis was performed. Based on the Western blot results, the efficiency of protein-targeted degradation 72 hours after transfection was calculated. The results are as follows: Figure 10 As shown.

[0120] from Figure 10 As can be seen, the fusion protein RS79E-BN2 exhibits the most significant and stable degradation effect after electroporation.

[0121] Cells were further selected after electroporation of the recombinant plasmid pcDNA3.1-RS79E-BNC2 for 48 h and 72 h, and then subjected to Western blot analysis. Based on the Western blot results, the efficiency of protein-targeted degradation after 48 h and 72 h of transfection was calculated. The results are as follows: Figure 11 As shown.

[0122] from Figure 11 As can be seen, the fusion protein RS79E-BNC2 showed the most significant effect after electroporation. At 48 hours, the protein-targeted degradation efficiency was about 40% (lanes D2-4), and at 72 hours, the protein-targeted degradation efficiency was about 80% (lanes D3-4).

[0123] Cells electroporated with recombinant plasmids pcDNA3.1-RS79E-BNC2 and pcDNA3.1-RBCC-BN2 were further selected and subjected to Western blot analysis. Based on the Western blot results, the expression levels of the tumor suppressor gene p53 and the cell cycle protein p21 in the cells were obtained. The results are as follows: Figure 12 As shown.

[0124] from Figure 12 As can be seen, the degradation of the fusion proteins RS79E-BNC2 and RBCC-BN2 is accompanied by an increase in the expression levels of the intracellular tumor suppressor gene p53 and the cell cycle protein p21.

[0125] Finally, the degradation pathway of BCL6 protein targeted by the fusion protein was investigated.

[0126] Specifically, a final concentration of 10 μM of the MG132 proteasome inhibitor or a final concentration of 100 nM of the BafA1 autophagy inhibitor was selected. MG132 inhibits the ubiquitin-proteasome pathway, while BafA1 inhibits the lysosomal pathway. Three copies of the pcDNA3.1-RBCC-BNC2 recombinant plasmid and GFP plasmid were electroporated into healthy Raji cells. 24 h after transfection, the corresponding MG132 and BafA1 were added to two of the cells carrying the pcDNA3.1-RBCC-BNC2 recombinant plasmid. After further culture, Western blot analysis was performed, and the results are as follows: Figure 13 As shown.

[0127] from Figure 13 As can be seen from this, the fusion protein in this invention targets and degrades BCL6 protein via the ubiquitin-proteasome pathway.

[0128] In summary, this invention utilizes artificial intelligence technology to design nanobodies that specifically target BCL6. These nanobodies exhibit good specificity, high affinity, and efficient binding to the BCL6 antigen. Furthermore, through the rational design of nanobodies using artificial intelligence, mutant nanobodies with even higher affinity are obtained. Even further, by fusing nanobodies or nanobodies with high affinity and good specificity with the RBCC domain or its mutant, a fusion protein is obtained. This fusion protein can utilize its nanobodies' domains to specifically bind to the target protein, initiating a protein degradation pathway and achieving targeted degradation of the BCL6 protein.

[0129] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A nanobody against BCL6 protein, characterized in that, Including complementarity determination region CDR1, complementarity determination region CDR2 and complementarity determination region CDR3; The amino acid sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO:1, the amino acid sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of the complementarity-determining region CDR3 is shown in SEQ ID NO:

5. Alternatively, the amino acid sequence of the complementarity-determining region CDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the complementarity-determining region CDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of the complementarity-determining region CDR3 is as shown in SEQ ID NO:

6. Alternatively, the amino acid sequence of the complementarity-determining region CDR1 is as shown in SEQ ID NO:1, the amino acid sequence of the complementarity-determining region CDR2 is as shown in SEQ ID NO:3, and the amino acid sequence of the complementarity-determining region CDR3 is as shown in SEQ ID NO:

7. Alternatively, the amino acid sequence of the complementarity-determining region CDR1 is as shown in SEQ ID NO:2, the amino acid sequence of the complementarity-determining region CDR2 is as shown in SEQ ID NO:4, and the amino acid sequence of the complementarity-determining region CDR3 is as shown in SEQ ID NO:

8.

2. The anti-BCL6 protein nanobody according to claim 1, characterized in that, The nanobody also includes frame regions FR1, FR2, FR3, and FR4; The amino acid sequence of the frame region FR1 is shown in SEQ ID NO:9, the amino acid sequence of the frame region FR2 is shown in SEQ ID NO:11, the amino acid sequence of the frame region FR3 is shown in SEQ ID NO:13, and the amino acid sequence of the frame region FR4 is shown in SEQ ID NO:

15. Alternatively, the amino acid sequence of frame region FR1 is as shown in SEQ ID NO:10, the amino acid sequence of frame region FR2 is as shown in SEQ ID NO:12, the amino acid sequence of frame region FR3 is as shown in SEQ ID NO:14, and the amino acid sequence of frame region FR4 is as shown in SEQ ID NO:

16.

3. The anti-BCL6 protein nanobody according to claim 2, characterized in that, The nanobody is selected from any of the following: A1) The amino acid sequence is shown in any one of SEQ ID NO:17-20; A2) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in A1).

4. A mutant nanobody against BCL6 protein, characterized in that, The nanobody mutant is selected from any one of the following: B1) The amino acid sequence is shown in SEQ ID NO:21; B2) An amino acid sequence obtained by attaching a tag or signal peptide to the N-terminus and / or C-terminus of the amino acid sequence defined in B1).

5. A fusion protein, characterized in that, The fusion protein is obtained by linking the nanobody of any one of claims 1-3 or the nanobody mutant of claim 4 with the RBCC domain or a mutant thereof through a second connector; 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. The amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 35, 36, 38, 39, 43.

6. A biomaterial, characterized in that, The biomaterial is selected from any one of the following: D1) A nucleic acid molecule encoding the nanobody of any one of claims 1-3, the nanobody mutant of claim 4, or the fusion protein of claim 5; D2) A recombinant vector containing the nucleic acid molecules described in D1); D3) Recombinant cells containing the nucleic acid molecule described in D1) or the recombinant vector described in D2).

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises any one of the nanobodies of claims 1-3, the nanobodies mutant of claim 4, the fusion protein of claim 5, or the biomaterial of claim 6, and a pharmaceutically acceptable carrier.

8. A method for preparing a nanobody according to any one of claims 1-3, a nanobody mutant according to claim 4, or a fusion protein according to claim 5, characterized in that, Includes the following steps: The recombinant cells of claim 6 are cultured and induced to express the desired expression to obtain a culture. Isolate the nanobody, the nanobody mutant, or the fusion protein from the culture.

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