Anti-CCL1 protein nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an anti-CCL1 protein nano antibody and application thereof. Six nano antibodies capable of being specifically bound with CCL1 are screened through a phage display technology, the nano antibodies are high in binding specificity for CCL1 antigens and capable of recognizing hidden epitopes which are difficult to bind with traditional antibodies, and due to the fact that the molecular weight is small, the nano antibodies have higher tissue penetrating power and meanwhile show excellent stability. Therefore, the nano antibody disclosed by the invention can be used for treating tumors taking a CCL1-CCR8 signal channel as a treatment target, the anti-tumor immune response is enhanced by effectively blocking the CCL1-CCR8 axis in the immunotherapy of malignant tumors, and the nano antibody shows high sensitivity and specificity in the CCL1 protein detection process, and can be applied to the immunotherapy of the malignant tumors. Therefore, the probe has remarkable advantages in the aspects of tumor immunotherapy, CCL1 protein detection and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a nanobody against CCL1 protein and its application. Background Art

[0002] Regulatory T cells (Tregs) in tumors play a key role in maintaining self-antigen tolerance and suppressing anti-tumor immunity. These Tregs weaken the anti-tumor activity of effector T cells through multiple mechanisms, resulting in a significant immunosuppressive feature in the tumor microenvironment (TME), thus promoting tumor immune escape. Therefore, depleting Tregs in the TME is an important strategy to enhance the anti-tumor immune response and is also a research hotspot in current tumor immunotherapy.

[0003] Chemokines are a class of cytokines that are highly expressed locally at the site of inflammation and can regulate the directional migration of leukocytes and lymphocytes, playing an important role in immune response regulation and tissue homeostasis maintenance. Chemokine (C-C motif) ligand 1 (CCL1) mediates the migration of immune cells to the site of inflammation or damaged tissue by binding to its specific receptor CCR8. Studies have shown that the CCL1-CCR8 axis not only plays a key role in inflammatory diseases but also plays an important function in the pathological process of tumor immune escape. In the tumor microenvironment, the high expression of CCL1 significantly promotes the recruitment and aggregation of CCR8+ Tregs. These CCR8+ Tregs exhibit stronger immunosuppressive ability and can inhibit the proliferation and anti-tumor activity of effector T cells. At the same time, CCL1 further enhances the stability and inhibitory function of Tregs by activating the CCR8 signaling pathway, exacerbating tumor immune escape. Therefore, the CCL1-CCR8 axis has been identified as a key tumor immunotherapy target. Blocking the CCL1-CCR8 axis has been shown to significantly reduce the accumulation of Tregs in the tumor microenvironment and inhibit their immunosuppressive function without significantly affecting the functional activity of effector T cells. This provides a scientific basis for the development of neutralizing antibodies against CCL1. By neutralizing CCL1, the recruitment and function of Tregs can be effectively blocked, enhancing the anti-tumor immune response and providing a new potential strategy for the immunotherapy of malignant tumors. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a nanobody against CCL1 protein with strong binding specificity, small molecular weight and excellent stability, providing a new strategy for tumor immunotherapy.

[0005] To achieve the above object, the technical solutions adopted by the present invention include:

[0006] In a first aspect, the present invention provides a nanobody against CCL1 protein, and the amino acid sequence of the nanobody is as shown in (a) or (b):

[0007] (a) Any one of the amino acid sequences shown in SEQ ID NO: 1-6;

[0008] (b) An amino acid sequence having more than 90% homology with the amino acid sequence in (a) and capable of resisting CCL1 protein, which is obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence in (a).

[0009] VHH (nanobody) is a special antibody derived from camelid heavy chain antibodies, which consists of only a single variable heavy chain region (VHH) and two conventional CH2 and CH3 regions, and the CH1 region is absent in the structure. Compared with traditional antibodies, nanobodies are small in size (the crystal size is about 2.5nm×4nm, and the molecular weight is only 15kD, which is 1 / 10 of traditional antibodies), but still maintain the complete antigen recognition ability.

[0010] The present invention has screened 6 nanobodies (P6-A10, P5-H4, P3-B10, P3-D9, P4-G1 and P5-G4) that can specifically bind to CCL1 through phage display technology (the amino acid sequences are successively shown in SEQ ID NO: 1-6). The nanobodies have strong binding specificity for the CCL1 antigen, can recognize cryptic epitopes that are difficult to bind by traditional antibodies; and their small molecular weight makes them have stronger tissue penetration ability; at the same time, they show excellent stability and can tolerate high temperature and acid-base environments. In addition, the production process of the nanobodies is relatively simple, suitable for large-scale industrial production, with low cost, and their structure is convenient for optimization and modification through genetic engineering, such as being designed as a fusion protein. Due to their low immunogenicity, the nanobodies of the present invention are more easily humanized, significantly improving their safety and effectiveness in clinical applications.

[0011] The VHH sequence of the nanobody contains three regions with relatively high variability, which are the regions that interact with the antigen and are called antigen - complementary determining regions; the sequences of the non - CDR regions are relatively conserved and are called framework regions. Among them, the amino acid sequences of the antigen - complementary determining regions CDR1, CDR2, and CDR3 of the P6 - A10 nanobody (SEQ ID NO: 1) are shown in SEQ ID NO: 7 - 9 in sequence; the amino acid sequences of the antigen - complementary determining regions CDR1, CDR2, and CDR3 of the P5 - H4 nanobody (SEQ ID NO: 2) are shown in SEQ ID NO: 10 - 12 in sequence; the amino acid sequences of the antigen - complementary determining regions CDR1, CDR2, and CDR3 of the P3 - B10 nanobody (SEQ ID NO: 3) are shown in SEQ ID NO: 10 - 12 in sequence; the amino acid sequences of the antigen - complementary determining regions CDR1, CDR2, and CDR3 of the P3 - D9 nanobody (SEQ ID NO: 4) are shown in SEQ ID NO: 13 - 14 and SEQ ID NO: 12 in sequence; the amino acid sequences of the antigen - complementary determining regions CDR1, CDR2, and CDR3 of the P4 - G1 nanobody (SEQ ID NO: 5) are shown in SEQ ID NO: 10, SEQ ID NO: 15, and SEQ ID NO: 12 in sequence; the amino acid sequences of the antigen - complementary determining regions CDR1, CDR2, and CDR3 of the P5 - G4 nanobody (SEQ ID NO: 6) are shown in SEQ ID NO: 13 - 14 and SEQ ID NO: 12 in sequence. The composition of the nanobodies of the present invention is all fr - cdr1 - fr - cdr2 - cdr3, where fr is the framework sequence, and the framework sequences are not completely the same. The specific framework sequences can be referred to the amino acid sequence information in SEQ ID NO.1 - 6.

[0012] Preferably, the amino acid sequence of the nanobody is as shown in SEQ ID NO: 1.

[0013] When the present invention verified the effectiveness of the 6 screened nanobodies by double - antibody sandwich ELISA, it was found that the P6 - A10 nanobody with the amino acid sequence as shown in SEQ ID NO: 1 has a better affinity for human CCL1 antigen. Therefore, it can significantly improve the sensitivity and specificity in the process of detecting CCL1 protein, and can effectively block the CCL1 - CCR8 axis in the immunotherapy of malignant tumors, effectively prevent the recruitment and function of Tregs, and enhance the anti - tumor immune response.

[0014] In a second aspect, the present invention provides a nucleic acid molecule, which is a nucleic acid molecule encoding the above - mentioned nanobody.

[0015] Preferably, the nucleotide sequence of the nucleic acid molecule is any one of those shown in SEQ ID NO: 16-21.

[0016] Among the 6 nanobodies screened and obtained in the present invention, the nucleotide sequence corresponding to P6-A10 is SEQ ID NO: 16, the nucleotide sequence corresponding to P5-H4 is SEQ ID NO.17, the nucleotide sequence corresponding to P3-B10 is SEQ ID NO.18, the nucleotide sequence corresponding to P3-D9 is SEQ ID NO.19, the nucleotide sequence corresponding to P4-G1 is SEQ ID NO.20, and the nucleotide sequence corresponding to P5-G4 is SEQ ID NO.21.

[0017] In a third aspect, the present invention provides an expression vector, which contains the nucleic acid molecule described above.

[0018] In a fourth aspect, the present invention provides a host cell, which contains the expression vector described above.

[0019] Preferably, the host cell includes at least one of mammalian cells, Escherichia coli, and yeast.

[0020] Preferably, the host cell is a mammalian cell.

[0021] In a fifth aspect, the present invention provides the use of the nanobody described above, and / or the nucleic acid molecule described above, and / or the expression vector described above, and / or the host cell described above in the preparation of a drug for treating a disease targeted at the CCL1-CCR8 signaling pathway.

[0022] Preferably, the disease includes tumors.

[0023] In a sixth aspect, the present invention provides the use of the nanobody described above, and / or the nucleic acid molecule described above in the preparation of a reagent for detecting CCL1 protein.

[0024] In a seventh aspect, the present invention further provides a method for preparing the nanobody, including the following steps:

[0025] S1. Take the peripheral blood of the alpaca immunized with CCL1 antigen, isolate the peripheral blood lymphocytes, extract the lymphocyte RNA, and then reverse transcribe and amplify the cDNA;

[0026] S2. Use the cDNA as a template to amplify the VHH gene fragment of the alpaca antibody, ligate the VHH gene fragment with the vector and then transform it into competent cells to obtain a nanobody phage library;

[0027] S3. Perform panning on the nanobody phage library using the CCL1 antigen, and then screen the positive phages against CCL1 to obtain the nanobody against CCL1.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] Six nanobodies (P6-A10, P5-H4, P3-B10, P3-D9, P4-G1, and P5-G4) that can specifically bind to CCL1 were obtained by screening using the phage display technique in the present invention. The nanobodies have strong binding specificity for the CCL1 antigen, can recognize cryptic epitopes that are difficult to bind by traditional antibodies; and their small molecular weight gives them stronger tissue penetration ability; at the same time, they show excellent stability and can tolerate high temperature and acid-base environments. In addition, the production process of the nanobodies is relatively simple, suitable for large-scale industrial production, with low cost, and their structure is convenient for optimization and modification through genetic engineering, such as being designed as a fusion protein. Due to their small immunogenicity, the nanobodies of the present invention are more easily humanized, significantly improving their safety and effectiveness in clinical applications. Description of the Drawings

[0030] Figure 1 It is the result graph of the plasma titer after immunization injection of alpaca;

[0031] Figure 2 It is the result graph of the total RNA agarose gel electrophoresis of alpaca PBMC;

[0032] Figure 3 It is the result graph of the colony PCR agarose gel electrophoresis;

[0033] Figure 4 It is the result graph of the sequence diversity alignment;

[0034] Figure 5 It is the standard curve graph of the ELSIA detection of six nanobodies. Detailed Embodiments

[0035] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and the test materials used in the following embodiments are all commercially available through conventional commercial channels unless otherwise specified.

[0037] Example 1. Preparation method of a nanobody against CCL1 protein

[0038] 1. Immunization injection of alpaca

[0039] This experimental study immunized a non-immunized healthy alpaca. The immunization process included antigen detection, immunization, blood collection, etc. According to the specific target protein and immunization effect, at least 7 immunizations were arranged, and additional immunizations were judged as needed. The specific immunization method is as follows:

[0040] (1) Before immunization, complete Freund's adjuvant and CCL1 antigen (the amino acid sequence of the CCL1 antigen is shown in SEQ ID NO: 22) were mixed at a volume ratio of 1:1, placed on an injection emulsifier for emulsification to form a uniform mixture, and then evenly divided into 7 portions;

[0041] (2) A healthy adult alpaca was immunized subcutaneously at multiple points every two weeks for a total of seven times. Blood was collected before each antigen immunization for immunization evaluation ( Figure 1 ), and 5 mL of blood was taken each time;

[0042] (3) 50 mL of blood was collected from the jugular vein of the alpaca 5 - 7 days after the 7th immunization.

[0043] 2. Phage library construction

[0044] Using the alpaca peripheral venous blood collected above as raw material, a phage library with high diversity was constructed. The specific construction method is as follows:

[0045] (1) Using density gradient centrifugation, lymphocytes were isolated from alpaca peripheral venous blood, and then total mRNA of lymphocytes was extracted. The results of agarose gel electrophoresis of alpaca PBMC total RNA are shown as Figure 2 shown;

[0046] (2) The obtained total RNA was reverse transcribed using a Takara reverse transcription kit to obtain cDNA;

[0047] (3) A specific nanobody VHH gene fragment was amplified from the reverse transcribed cDNA. PCR amplification was performed using Taq DNA Polymerase Hot Start enzyme. This operation was completed by Shenzhen Kangti Life Technology Co., Ltd. The PCR system was prepared as shown in Table 1 below, and the amplification program was as shown in Table 2 below;

[0048] (4) The amplified VHH target fragment was cloned into the pComb3XSS vector, and then the vector containing the target fragment was transformed into TG1 competent cells. After recovery, it was diluted 10 - fold in gradient and plated to calculate the number of transformed colonies. It was detected that the library capacity of the bacterial library was 1.29×10 8 pfu. 51 monoclonal bacteria were randomly selected from the gradient dilution plate for colony PCR to verify that the cloning positive rate of the bacterial library was 98%. The results are shown as Figure 3as shown

[0049] (5) Culture the bacterial library to an appropriate OD value, add helper phage and incubate to obtain the Anti-CCL1-VHH phage library. To further identify whether the Anti-CCL1-VHH phage library was successfully constructed, 48 monoclonal clones were randomly selected, sequenced, and then translated into protein sequences using GENtle software. Sequence diversity alignment showed that all 48 sequences were independent sequences with 100% diversity, and the diversity of the bacterial library met the requirements. The results are as Figure 4 shown

[0050] Table 1 PCR Amplification System

[0051] Reagent Dosage cDNA 0.5 - 4 μL 1st - for / 1st - rev 2 μL / 2 μL dNTP Mix 4 μL 10×ExTaq Buffer 5 μL HS Ex Taq 0.25 μL <![CDATA[ddH2O]]> Up to 50 μL

[0052] Table 2 Amplification Program

[0053]

[0054] 3. Magnetic Bead Screening of Specific Nanobodies

[0055] (1) Phage Screening and Elution:

[0056] First round of screening: After activating the magnetic beads (washing with Buffer 2), bind them to the CCL1 antigen. After washing, add the immune library to screen for phages. After washing the magnetic beads 10 times, elute with trypsin solution to obtain the phage eluate;

[0057] Second round of screening: The input phage is the phage sub-library from the first round of screening. Repeat the screening steps of the first round to obtain the second-round eluate;

[0058] Third round of screening: The input phage is the phage sub-library from the second round of screening. Repeat the screening steps of the previous two rounds to obtain the third-round eluate.

[0059] (2) Phage Amplification and Purification:

[0060] Amplification: Infect the bacterial liquid with phages by inoculating the TG1 strain, spread and culture, and then collect the bacterial liquid to amplify the bacterial sub-library;

[0061] Purification: Purify the phages using the PEG / NaCl precipitation method and store them as the phage sub-library.

[0062] The titer of the phage library was detected after each round of amplification to confirm the phage library titer. The results are shown in Table 3 below.

[0063] Table 3 Phage Titer

[0064] Number of screening rounds Input titer Screening titer Enrichment degree The 1st round <![CDATA[1.0×10 12 pfu]]> <![CDATA[6.0×10 8 pfu]]> <![CDATA[6.0×10 -4 > The 2nd round <![CDATA[2.0×10 13 pfu]]> <![CDATA[8.0×10 9 pfu]]> <![CDATA[4.0×10 -4 > The 3rd round <![CDATA[2.0×10 13 pfu]]> <![CDATA[2.8×10 11 pfu]]> <![CDATA[1.4×10 -2 >

[0065] (3) Monoclonal ELISA detection:

[0066] 1. Dilution of phage eluate and infection of bacteria: Take 10 μL of the phage eluate after the second and third rounds of screening, and perform 10-fold serial dilutions for a total of 12 gradients. Mix the phage solutions of each gradient with the revived TG1 strain and culture them.

[0067] 2. Screening of successfully transfected bacteria: Spread the cultured bacterial solution evenly on a solid medium plate containing 100 μg / mL Amp (apramycin) to screen out the successfully transfected bacteria.

[0068] 3. Selection of monoclonal colonies and transfer to 96-well plates: Randomly select 576 monoclonal colonies from the overnight culture plate and transfer them to 6 sterile 96-well cell culture plates for further screening (P1, P2, P3 for the second round of screening, and P4, P5, P6 for the third round of screening). Add 200 μL of 2×YT medium (containing 100 μg / mL Amp) to each well for culture.

[0069] 4. Addition of helper phage: Add helper phage to the bacterial culture medium in each 96-well plate to ensure that the helper phage can effectively infect the bacteria and display the target protein.

[0070] 5. Centrifugation to collect the supernatant: Centrifuge the bacteria in the 96-well culture plates and collect the supernatant for the subsequent first-round ELISA detection. The results are shown in Table 4-5 below;

[0071] Table 4 First-round ELISA of the second-round screening

[0072]

[0073]

[0074]

[0075] Table 5 First-round ELSIA of the third-round screening

[0076]

[0077]

[0078] 6. Secondary verification: Detect the specificity of the target protein displayed by the phage by ELISA method. To exclude false-positive results, perform secondary ELISA verification to ensure the accuracy of the screening results. The results are shown in Table 6 below.

[0079] Table 6 Secondary ELISA verification of the second round

[0080]

[0081] 4. Positive clone sequencing and analysis

[0082] A total of 26 positive clones were obtained after screening in this study and sequenced. Among them, P5-A1 had no forward and reverse primer sequences, P4-F5 was a double peak, and 24 were normal. According to the sequences before and after the nanobody, the nanobody sequences could be obtained from the sequencing results. After translating the 24 sequences into amino acids, they were sorted and subjected to multiple sequence alignment, and a total of 6 different nanobody sequences were obtained. The alignment results are shown in Table 7 below:

[0083] Table 7 Sequence alignment results

[0084]

[0085]

[0086] 5. Expression and purification of nanobodies

[0087] The above sequences were optimized for the human system. The kozak sequence GCCGCCACC and signal peptide were added to the N-terminus of the target sequence, and the Flag tag and 6x His tag were added to the C-terminus. After gene synthesis of the DNA sequence, it was subcloned into the pcDNA3.1(+) vector, and the restriction enzyme sites were selected as Hindm-EcORI. Secretory expression was carried out using the added signal peptide IL10, and protein purification was performed by affinity chromatography using the His tag.

[0088] Example 2. Exploration of the effectiveness of the nanobody

[0089] 1. The double antibody sandwich ELISA method was used to verify the effectiveness of the antibody. The specific method is as follows:

[0090] (1) Coating antibody: The 6 obtained nanobodies were diluted to 1 μg / mL with phosphate buffer. 100 μL of the diluted antibody was added to each well. After overnight incubation at room temperature, the coating solution was poured out, and the plate was washed three times with detergent for 1 minute each time;

[0091] (2) Blocking: 5 g of bovine serum albumin (BSA) was added to 100 mL of washing buffer (PBST) to prepare a blocking solution. The blocking solution was diluted 50 times to obtain a dilution solution. 300 μL was added to each well, incubated at 37 °C for 2 hours, and then the blocking solution was discarded. The plate was washed 3 times with PBST for 1 minute each time;

[0092] (3) Sample addition: Dilute the human CCL1 standard product from RD Company in gradients to 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.3 pg / mL, and 15.6 pg / mL. Add 100 μL to each well, set up three replicates for each concentration, cover with a sealing film, incubate at 37 °C for 120 minutes, then discard the liquid, wash the plate three times, one minute each time;

[0093] (4) Add detection antibody: Dilute the CCL1 detection antibody from RD Company to 100 ng / mL using a diluent, incubate at 37 °C for one hour, then discard the antibody solution, wash the plate 3 times with PBST, one minute each time;

[0094] (5) Add enzyme-labeled secondary antibody: Dilute the enzyme-labeled secondary antibody from RD Company 200 times, add 100 μL to each well, incubate at 37 °C for 20 minutes; wash the plate 3 times with PBST, one minute each time;

[0095] (6) Add substrate: Add 100 μL of substrate solution (tetramethylbenzidine) to each well, incubate at 37 °C for 20 minutes, and avoid placing the plate under direct light;

[0096] (7) Terminate the reaction: Add 50 μL of termination solution to each well, gently tap the plate to ensure thorough mixing;

[0097] (8) Result judgment: Immediately use an enzyme-labeled instrument set at 450 nm to read the optical density of each well, draw a curve as Figure 5 , and it is found that the antibody P6-A10 has a better binding ability to the human CCL1 antigen.

[0098] 2. Detection and exploration of the content of CCL1 in human blood by the P6-A10 nanobody

[0099] In this study, the P6-A10 nanobody and the polyclonal antibody from RD Company were used to detect the content of CCL1 in human blood to explore the capture ability of the P6-A10 nanobody described in the present invention. The specific method is as follows:

[0100] Collect blood samples using blood collection tubes or centrifuge tubes containing anticoagulants. Within 30 minutes after collection, centrifuge at 1000 × g for 15 minutes at 2 - 8 °C, and carefully collect the upper plasma. Use the P6-A10 nanobody and the polyclonal antibody from RD Company as capture antibodies respectively, and explore using the above double-antibody sandwich ELISA detection method. The results show that the concentration of CCL1 in the wells with the P6-A10 nanobody is 240.6 pg / mL, and the concentration of CCL1 in the wells with the polyclonal antibody from RD Company is 239.2 pg / mL, indicating that the capture ability of the nanobody described in the present invention is comparable to that of the commercial polyclonal antibody.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nanobody against CCL1 protein, characterized in that: The amino acid sequence of the Nanobody is as shown in (a) or (b): (a) an amino acid sequence as shown in any one of SEQ ID NOs: 1-6; (b) An amino acid sequence in (a) obtained by substitution and / or deletion and / or addition of amino acid residues, which has more than 90% homology with the amino acid sequence described in (a) and is capable of resisting CCL1 protein.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the nanobody is shown in SEQ ID NO:

1.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleic acid molecule encoding the Nanobody according to claim 1.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule is any one of SEQ ID NOs: 16-21.

5. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 3.

6. A host cell, characterized in that The host cell comprises the expression vector according to claim 5.

7. The host cell according to claim 6, characterized in that The host cell includes at least one of a mammalian cell, Escherichia coli and a yeast.

8. Use of the nanobody according to claim 1 or 2 in the preparation of a reagent for detecting CCL1 protein.

9. Use of the Nanobody according to claim 1 or 2, and / or the nucleic acid molecule according to claim 3 or 4, and / or the expression vector according to claim 5, and / or the host cell according to claims 6-7 in the preparation of a drug for treating a disease with the CCL1-CCR8 signaling pathway as a therapeutic target.

10. The use according to claim 9, characterized in that The disease includes a tumor.

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