A VHH chain of an anti-APP nanobody and its application

Abstract

The present invention discloses a VHH chain of an anti-APP nanobody and its application. The anti-APP nanobody of the present invention has the following beneficial effects: (1) small molecule and drug delivery. (2) easy to manufacture and express. (3) high affinity and strong specificity. (4) stable performance and good plasticity. (5) low immunogenicity and good metabolic characteristics. The APP nanobody of the present invention retains the ability of the VVH chain antibody and avoids the introduction of non-human exogenous proteins as much as possible, taking into account the function of effectively binding to anti-APP and the lowest possible heterology. In addition, the reduction of heterology can not only reduce hypersensitivity reactions but also prolong its half-life in the body and improve metabolic characteristics.

Description

Technical Field

[0001] The present invention relates to the field of immunotechnology, in particular to a VHH chain of an anti-APP nanobody and applications thereof. Background Art

[0002] Approximately 50 million people worldwide suffer from Alzheimer's disease (AD). With the increasing global aging population, AD has become the fourth leading cause of death worldwide, severely threatening human health and quality of life. Research into the molecular mechanisms of AD began as early as the 1980s, with thousands of scientists dedicating themselves to this multifactorial disease in an effort to unravel its mysteries. Numerous studies have shown that abnormal enzymatic cleavage of the amyloid precursor protein (APP) is a key cause of AD.

[0003] APP is a typical type I transmembrane protein with multiple isoforms, the most prominent of which are APP770 / APP751 / APP695. APP is metabolized through both the amyloidogenic (β-pathway) and non-amyloidogenic (α-pathway). The β-secretase cleaves APPβ (secreted APP-N terminal fragment, sAPPβ) and C99, which is then activated by γ-secretase to produce amyloid peptide (β-amyloid peptide, Aβ) and the APP intracellular domain (AICD). The α-secretase cleaves APPα and C83, which is then activated by γ-secretase to produce AICD. Aβ peptides aggregate to form SP, which in turn induces the pathological characteristics of AD.

[0004] Camelid antibodies are unique, naturally occurring antibodies devoid of light chains. They consist solely of a single antigen-binding heavy chain variable domain (VHH) and two conventional CH2 and CH3 regions. The VHH heavy chain variable domain of camelid antibodies can exist independently and stably in vitro, resulting in the formation of nanobodies. Nanobody crystals are 2.5 nm wide and 4 nm long, with a molecular weight only one-tenth that of traditional intact antibodies (approximately 15 kDa), yet they retain full antigen recognition capabilities. Due to their tiny structure and intact antigen recognition capabilities, nanobodies exhibit advantages over traditional antibodies, including high affinity, high specificity, strong penetrating power, high stability, and ease of expression and modification. Most importantly, nanobodies are more susceptible to targeting smaller antigenic epitopes than traditional antibodies, resulting in a nanobody library with far greater diversity than traditional antibodies.

[0005] Currently, nanobodies are being applied primarily in three areas. First, in structural biology, nanobodies are used to specifically bind to unstable domains in certain proteins, enabling structural elucidation of these proteins. For example, Professor Kobilka, winner of the 2012 Nobel Prize in Chemistry, pioneered the use of nanobodies to solve the crystal structure of the adrenergic receptor in its active state (PDB: 3P0G). Second, expressing nanobodies in cells can serve as biosensors to specifically track target proteins in cells. Most importantly, in drug development, nanobodies can be used as therapeutic agents or to facilitate drug discovery by specifically maintaining the conformation of proteins associated with certain diseases. In February 2019, the FDA approved the first nanobody drug, caplacizumab, for the treatment of acquired thrombotic thrombocytic purpura (aTTP) in adults. Numerous pharmaceutical companies and research institutions worldwide are actively exploring the potential of nanobodies in tumors, inflammation, and metabolic diseases.

[0006] Therefore, the prior art has defects and needs to be improved. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a VHH chain of an anti-APP nanobody and its application to meet the application of anti-APP.

[0008] The technical solution of the present invention is as follows: a VHH chain of an anti-APP nanobody is provided, comprising a framework region FR and a complementary determining region CDR, the framework region FR comprising FR1, FR2, FR3 and FR4, and the complementary determining region CDR comprising: CDR1, CDR2 and CDR3; the specific amino acid sequence is as follows: FR1 is SEQ ID NO: 1, CDR1 is SEQ ID NO: 2, FR2 is SEQ ID NO: 3, CDR2 is SEQ ID NO: 4, FR3 is SEQ ID NO: 5, CDR3 is SEQ ID NO: 6, and FR4 is SEQ ID NO: 7.

[0009] In a preferred embodiment, the VHH chain of the anti-APP Nanobody comprises a framework region FR and a complementarity determining region CDR, and its amino acid sequence is shown in SEQ ID NO:8.

[0010] The present invention also provides an anti-APP nanobody, comprising: the VHH chain of the aforementioned anti-APP nanobody. The variable region of the nanobody has three complementary determining regions: CDR1, CDR2, and CDR3, wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.5, the amino acid sequence of CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of CDR3 is shown in SEQ ID NO.7. In a preferred technical solution, a preferred embodiment of the nanobody having this variable region sequence in the present invention is nanobody P2-10C.

[0011] The present invention also provides a gene sequence encoding the VHH chain of the aforementioned anti-APP Nanobody or encoding the aforementioned anti-APP Nanobody. The gene sequence has the nucleotide sequence shown in SEQ ID NO:9.

[0012] The present invention also provides a nucleotide construct comprising the aforementioned gene sequence.

[0013] The present invention also provides a recombinant expression vector comprising the aforementioned nucleotide construct. The expression vector is pET22b.

[0014] The present invention also provides a recombinant host cell comprising the aforementioned nucleotide construct or the aforementioned recombinant expression vector. The host cell is Escherichia coli BL21 (DE3).

[0015] The present invention also provides a method for producing anti-APP nanobodies, which is prepared by culturing the aforementioned recombinant host cells and inducing the recombinant host cells to express anti-APP nanobodies.

[0016] The present invention also provides a use of a VHH chain of an anti-APP nanobody in preparing a diagnostic reagent for detecting APP, using the aforementioned VHH chain of the anti-APP nanobody or the aforementioned anti-APP nanobody.

[0017] Using the above scheme, the present invention provides a VHM chain of an anti-APP nanobody and its application. The anti-APP nanobody of the present invention has the following beneficial effects:

[0018] (1) Small molecules and drug delivery. The anti-APP nanoantibody of the present invention is small in size. When used as a targeting molecule, it has less conformational influence and steric hindrance on the active site (effector molecule), resulting in higher activity of the effector molecule and can be used in drug delivery systems.

[0019] (2) Easy to manufacture and express: The anti-APP nanobody of the present invention can be efficiently expressed using Escherichia coli.

[0020] (3) High affinity and strong specificity: The anti-APP nanoantibody of the present invention has a VHH chain, which has a high affinity for the APP protein.

[0021] (4) Stable performance and good plasticity: The anti-APP nanoantibody of the present invention can be coupled to other molecules and can maintain a stable binding ability against APP.

[0022] (5) Low immunogenicity and good metabolic profile: The APP-targeting nanobodies of the present invention retain the capabilities of VVH chain antibodies while minimizing the introduction of non-human exogenous proteins, achieving both effective anti-APP binding and minimal heterogeneity. Furthermore, reduced heterogeneity not only reduces hypersensitivity reactions but also prolongs their half-life in vivo and improves their metabolic profile.

[0023] The anti-APP nanobody provided by the present invention has a unique antigenic determinant recognition site and has specific recognition and binding capabilities for APP antigens. The nanobody has a high antigen affinity, which can reach 7×10 -8 M, showing excellent detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a titer detection of APP-immunized alpacas according to an embodiment of the present invention;

[0025] Figure 2 The amino acid sequence diagram of the APP nanobody according to the embodiment of the present invention;

[0026] Figure 3 This is the SDS-PAGE image of the expression and purification of nanobody P2-10C;

[0027] Figure 4The affinity test curve of nanobody P2-10C (KD = 7 × 10 -8 M). DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection of the present invention.

[0029] Construction of anti-APP single domain heavy chain antibody phage library

[0030] Alpacas (Lama pacos) were immunized subcutaneously at multiple sites with 800 μg of APP protein emulsified in complete Freund's adjuvant. Booster immunizations were performed every two weeks with 800 μg of APP protein emulsified in incomplete Freund's adjuvant. Venous blood was collected seven days after each immunization. Serum titers were determined by indirect ELISA. Lymphocytes were isolated from the sample with the highest serum titer, and total RNA was extracted and reverse transcribed into cDNA. After two rounds of PCR amplification, antibody sequence amplification products were obtained. The selected vectors were digested with enzymes and ligated. Finally, the amplified products were transformed into TG1 competent Escherichia coli cells to generate a bacterial library. Phage libraries were prepared after infection and induction with helper phage (M13KO7).

[0031] Panning and identification of anti-APP single domain heavy chain antibodies

[0032] Solid phase affinity panning was used to select single domain heavy chain antibodies against APP from an anti-APP single domain heavy chain antibody phage library. 120 μL of APP diluted with PBS was added to each enzyme-labeled well and coated overnight at 4°C. The coating concentrations for each round of selection were 100, 75, and 50 μg / mL, respectively. The coating solution was aspirated, the plate was washed 5 times with PBS, 300 μL of 3% BSA-PBS was added to each well, and the plate was blocked at 37°C for 2 hours. The plate was washed 5 times with PBS, 100 μL of phage antibody library (containing approximately 1× 1011 CFU) was added, and the plate was incubated at 37°C for 2 hours. Unbound phages were aspirated, and the plate was washed 3-5 times (increased by 5 times per round) with PBST (containing 0.5% Tween-20), and then washed 15-25 times with PBS. The phages adsorbed on the enzyme-labeled wells were eluted with 100 μL of elution buffer (glycine-HCl, pH 2.2), and 35 μL of elution buffer was used. The eluate was neutralized with Tris-HCl (1 mol / L, pH 8.0), and 10 μL was taken for titer determination. The remaining 125 μL of the eluate was amplified and used for the next round of panning. After four rounds of panning, helper phage KM13 was used to rescue randomly selected monoclonal clones, and phage particles displaying the antibody variable region were obtained. The binding activity and specificity of the phage particles were then determined by indirect phage-ELISA. ELISA-positive clones were sent to a biotechnology service company for sequencing, and the DNA sequence of the insert was obtained, which encodes a single-domain heavy chain antibody against APP. Please refer to Figure 2 The amino acid sequence of the obtained monovalent Nanobody P2-10C (SEQ ID NO.8) is:

[0033] DVQLQESGGGLVQPGGSLRLSCAASGIIFSGYYMSWVRQAPGKGPEWVSSINPSGSSTRYADSVKGRFTISRDNAKNTVYLQMYSLKPEDTALYYCSRSRDGLGDVRGQGTQVTVSS, wherein the amino acid sequence at positions 1-25 is FR1, the amino acid sequence at positions 26-33 is CDR1, the amino acid sequence at positions 34-50 is FR2, the amino acid sequence at positions 51-57 is CDR2, the amino acid sequence at positions 58-95 is FR3, the amino acid sequence at positions 96-111 is CDR3, and the amino acid sequence at positions 112-122 is FR4.

[0034] Preparation of anti-APP single domain heavy chain antibody

[0035] The anti-APP single domain heavy chain antibody gene fragment (SEQ ID NO.9)

[0036] atgtgcagctgcaagagtccggcgggggcctggtgcaacctggcggcagcctgagactgagctgcgccgctagcggcatcatcttcagcggctactacatgagctgggtgagacaagcccccggcaagggccccgagtgggtgagcagcatcaaccctagcggcagcagcacaagatacgccgacagcgtgaagggcagattcaccatcagcagagacaacgccaagaacaccgtgtacctgcagatgtacagcctgaagcccgaggacaccgccctgtactactgcagcagaagcagagacggcctgggcgacgtgagaggccaaggcacccaagtgaccgtgagcagc was cloned into the expression vector pET22b, and the expression vector of the anti-APP single domain heavy chain antibody was constructed and identified by PCR and enzyme digestion. The expression vector was transformed into Escherichia coli BL21, and the single clone colony was picked into 5 ml LB containing antibiotics. Incubate the cells in the culture medium at 37°C for 5 h; add 5 ml of the bacterial solution to 500 ml of LB medium containing antibiotics to expand the culture; when the OD value of the bacterial solution reaches 0.6-0.8, add 0.5 mM IPTG and induce at 18°C overnight. The bacterial pellet was collected by centrifugation at 4000 rpm for 5 min; the pellet was resuspended in a solution containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM PMSF, and 5 mM imidazole and sonicated at 300 W for 20 min; the supernatant was collected by centrifugation at 18000 rpm for 15 min; the supernatant was mixed with 1 ml Ni resin and incubated for 30 min; the supernatant was discarded after centrifugation at 1000 rpm for 2 min, and the pellet was resuspended in a buffer containing 20 mM imidazole and loaded into a gravity column; impurities were washed with 20 mM, 40 mM, and 60 mM imidazole solutions in sequence, and the target protein was eluted with 6 ml of a solution containing 500 mM imidazole and directly transferred to a concentrator tube. Samples were taken for gel analysis and concentrated to 1 ml at 3000 rpm for 10 min / time; the pellet was passed through a molecular sieve and frozen after gel analysis.

[0037] Affinity determination of anti-APP nanobodies

[0038] The affinity of the nanobody prepared in Example 3 was determined using the intermolecular interaction test method SPR (surface plasmon resonance).

[0039] Affinity KD(M) = kdis(1 / s) / kon(1 / Ms). The results of the test were as follows: kdis(1 / s) = 0.009213; kon(1 / Ms) = 129500; KD(M) = kdis(1 / s) / kon(1 / Ms) = 7× 10 -8 M.

[0040] See also Figure 1 , Figure 1 This is a titer test of APP-immunized alpacas according to an embodiment of the present invention; it can be seen from the figure that the antigen immunization of alpacas of the present invention is effective, which is the key to obtaining high-affinity and specific anti-APP nanoantibodies, and provides a basis for obtaining effective anti-APP nanoantibodies.

[0041] Figure 3 This is the SDS-PAGE diagram of the expression and purification of nanobody P2-10C; Figure 3 As can be seen from the figure, the nanobody has a small molecular weight.

[0042] Figure 4 This is the affinity test curve of nanobody P2-10C (KD = 7×10 -8 M). It can be seen from the figure that the nanobody has high affinity and good specificity.

[0043] In summary, the present invention provides a VHM chain of an anti-APP nanobody and its application. The anti-APP nanobody of the present invention has the following beneficial effects:

[0044] (1) Small molecules and drug delivery. The anti-APP nanoantibody of the present invention is small in size. When used as a targeting molecule, it has less conformational influence and steric hindrance on the active site (effector molecule), resulting in higher activity of the effector molecule and can be used in drug delivery systems.

[0045] (2) Easy to manufacture and express: The anti-APP nanobody of the present invention can be efficiently expressed using Escherichia coli.

[0046] (3) High affinity and strong specificity: The anti-APP nanoantibody of the present invention has a VHH chain, which has a high affinity for the APP protein.

[0047] (4) Stable performance and good plasticity: The anti-APP nanoantibody of the present invention can be coupled to other molecules and can maintain a stable binding ability against APP.

[0048] (5) Low immunogenicity and good metabolic profile: The APP-targeting nanobodies of the present invention retain the capabilities of VVH chain antibodies while minimizing the introduction of non-human exogenous proteins, achieving both effective anti-APP binding and minimal heterogeneity. Furthermore, reduced heterogeneity not only reduces hypersensitivity reactions but also prolongs their half-life in vivo and improves their metabolic profile.

[0049] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A VHH chain of an anti-APP nanobody, comprising a framework region FR and a complementarity determining region CDR, characterized in that: The framework regions FR include FR1, FR2, FR3 and FR4, and the complementarity determining regions CDR include: CDR1, CDR2 and CDR3; the specific amino acid sequences are as follows: FR1 is SEQ ID NO: 1, CDR1 is SEQ ID NO: 2, FR2 is SEQ ID NO: 3, CDR2 is SEQ ID NO: 4, FR3 is SEQ ID NO: 5, CDR3 is SEQ ID NO: 6, and FR4 is SEQ ID NO:

7.

2. The VHH chain of an anti-APP nanobody according to claim 1, characterized in that Its amino acid sequence is shown in SEQ ID NO:

8.

3. An anti-APP nanobody, characterized in that include: The VHH chain of the anti-APP Nanobody according to claim 1 or 2.

4. A gene, characterized in that It encodes the VHH chain of the anti-APP Nanobody according to claim 1 or 2 or it encodes the anti-APP Nanobody according to claim 3.

5. A gene according to claim 4, characterized in that It has the nucleotide sequence shown in SEQ ID NO:

9.

6. A nucleotide construct, characterized in that It comprises the gene according to claim 4 or 5.

7. A recombinant expression vector, characterized in that: It comprises the nucleotide construct according to claim 6.

8. A recombinant host cell, characterized in that It comprises the nucleotide construct according to claim 6 or the recombinant expression vector according to claim 7.

9. A method for producing an anti-APP nanobody, characterized in that: The method is prepared by culturing the recombinant host cell as claimed in claim 8 and inducing the recombinant host cell to express the anti-APP nanobody.

10. Use of a VHH chain of an anti-APP nanobody in the preparation of a diagnostic reagent for detecting APP, characterized in that: The VHH chain of the anti-APP Nanobody according to claim 1 or 2 or the anti-APP Nanobody according to claim 3 is used.

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