A high-affinity anti-SIRPγ monoclonal antibody and its use

Abstract

The present invention discloses a high-affinity anti-SIRPγ monoclonal antibody and its use, belonging to the field of biotechnology. Its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence of the nucleic acid molecule encoding the calcium anti-SIRPγ monoclonal antibody is shown in SEQ ID NO.2. The present invention uses gene editing technology and immunology methods to construct a SIRPγ phage natural antibody library, and obtains high-affinity SIRPγ-specific nano-monoclonal therapeutic antibodies through enrichment screening, and obtains its specific sequence. The obtained antibody can not only accurately target SIRPγ hi CSCs, and have low immunogenicity, high specificity and affinity, for further in-depth research on the immunotherapy of lung cancer and the strategy of targeting CSCs using SIRPγ monoclonal antibodies.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-SIRPγ monoclonal antibody with high affinity and a use thereof. Background Art

[0002] Malignant tumors, represented by lung cancer and liver cancer, have clinical characteristics such as insidious onset, high malignancy, high heterogeneity, and poor prognosis. In clinical practice, nearly 1 / 3 of patients develop treatment resistance within 2 years of immunotherapy, followed by recurrence and metastasis, resulting in poor prognosis. Immune escape is a key factor leading to treatment resistance, metastasis and recurrence in patients. Cancer stem cells (CSCs) in the tumor microenvironment play an important role in the immune escape process, which seriously restricts the expansion of precision immunotherapy strategies for tumors.

[0003] Cancer stem cells (CSCs) are a small group of seed cells in tumors with unlimited proliferation and differentiation potential. They are the root cause of tumor occurrence, recurrence, metastasis, drug resistance and immune escape. Therefore, targeting cancer stem cells can fundamentally destroy the origin of tumors, reshape the tumor immunosuppressive microenvironment, and inhibit tumor growth. Discovering new molecular markers to identify small cell groups of CSCs with immune escape characteristics will help develop new strategies for targeted cancer treatment by attacking CSCs.

[0004] SIRPγ (signal regulatory protein γ), as a member of the SIRPs family, is mainly expressed in T lymphocytes and activated natural killer (NK) cells. SIRPγ appears as an immunoglobulin-like cell surface receptor, which mediates cell-to-cell adhesion after binding to CD47. This interaction is essential for enhancing antigen-specific T cell proliferation and providing co-stimulatory signals for T cell activation. At the same time, SIRPγ is a CSCs marker found in non-small cell lung cancer, which determines the CSCs characteristics and immune escape of non-small cell lung cancer. Different from the classic "don't eat me" signal of SIRPα / CD47, high expression of SIRPγ (SIRPγ hi CSCs can promote the expression of IL-1β and GM-CSF cytokines in an autocrine and paracrine-dependent manner by activating Hippo / Yap signaling, maintaining the stable expression of CD47, activating the "don't eat me" signaling, thereby inhibiting the phagocytosis of tumor cells and achieving SIRPγ hi Therefore, targeting SIRPγ hi Immunotherapy strategies for CSCs are helpful for the treatment of lung cancer. Currently, there are studies on anti-SIRPγ specific antibody-related drugs, but there is no efficient anchoring of SIRPγ hiDevelopment of SIRPγ nano-monoclonal therapeutic antibodies for CSCs, low immunogenicity, high specificity and affinity. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides an anti-SIRPγ monoclonal antibody with high affinity and its use. The present invention uses gene editing technology and immunology methods to construct a SIRPγ phage natural antibody library, and obtains high-affinity SIRPγ-specific nano-monoclonal therapeutic antibodies through enrichment screening. By obtaining its specific sequence, the obtained antibody can not only accurately target SIRPγ hi CSCs, and have low immunogenicity, high specificity and affinity, for further in-depth research on the immunotherapy of lung cancer and the strategy of targeting CSCs using SIRPγ monoclonal antibodies.

[0006] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0007] The purpose of the present invention is to provide an anti-SIRPγ monoclonal antibody with high affinity, the sequence of which is shown in SEQ ID NO.1.

[0008] QVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAISRNGIFTNYVDSVKGRFTISRDDNAKNTVYLQMNSLKPEDTAVYFCTLRNMWGLNHTPFWGQGTQVTVSS (SEQ ID NO. 1).

[0009] Another object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned anti-SIRPγ monoclonal antibody, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] CAGGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGGGGCGGCAGCCTGCGCCTGAGCTGCGCGGCGAGCGGCCGCACCTTTAACAACTATGCGATGGGCTGGTTTCGCCAGGCGCCGGGCAAAGAACGCGAATTTGTGGCGGCGATTAGCCGCAACGGCATTTTTACCAACTATGT GGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTTTTGCACCCTGCGCAACATGTGGGGCCTGAACCATACCCCGTTTTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGC (SEQ IDNO.2).

[0011] Another object of the present invention is to provide an expression vector comprising the above nucleic acid molecule.

[0012] Another object of the present invention is to provide a recombinant cell, comprising the above nucleic acid molecule or expression vector. The cell can be a bacterium, a fungus, a plant cell, an insect cell or a mammalian cell.

[0013] Another object of the present invention is to provide an engineered bacterium, comprising the above-mentioned nucleic acid molecule or expression vector.

[0014] Another object of the present invention is to provide a method for producing the above-mentioned anti-SIRPγ monoclonal antibody, comprising culturing the above-mentioned recombinant cells or engineered bacteria to produce antibodies.

[0015] Another object of the present invention is to provide an antibody composition, comprising the above-mentioned anti-SIRPγ monoclonal antibody and a pharmaceutically acceptable carrier, buffer and excipient thereof.

[0016] Another object of the present invention is to provide a kit comprising the above-mentioned anti-SIRPγ monoclonal antibody.

[0017] Use of the above-mentioned antibody, antibody composition or kit in basic medical research for non-diagnosis / treatment purposes, wherein the basic medical research is Western Blot, immunohistochemistry and flow cytometry analysis.

[0018] Use of the above-mentioned antibody, antibody composition or kit in preparing a reagent for treating lung cancer and / or determining prognosis.

[0019] Beneficial effects of the present invention:

[0020] The present invention uses immunology and genetic engineering techniques to immunize alpacas, obtain total RNA, and reverse transcribe it into cDNA; and uses phage display technology to amplify the heavy chain variable region (VHH) of anti-SIRPγ monoclonal antibodies in two steps to construct a SIRPγ phage natural antibody library, and screen out specific antibodies with high affinity, thereby realizing the development of efficient SIRPγ anchoring. hi CSCs, low immunogenicity, high specificity and affinity nano-monoclonal therapeutic antibodies provide new information and ideas for the immune and CSCs targeting strategies for lung cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the electrophoresis result of the first step of nested PCR;

[0022] Figure 2 This is the electrophoresis result of the second step of nested PCR;

[0023] Figure 3 This is the result of random sequencing of phage library Lib 671-11. DETAILED DESCRIPTION

[0024] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0025] Example 1 Construction of phage natural antibody library

[0026] 1. Alpaca Immunity

[0027] This application uses 4 forms of antigens to immunize an alpaca. Specifically, the alpaca is immunized 11 times with different forms of antigens targeting the SIRPγ target to obtain alpacas that meet the ELISA titer of the antigen>32000 (the secondary antibody uses anti-alpaca / camel Fc antibody) and enter the next stage of the experiment. The specific process is as follows:

[0028] (1) Based on the sequence structure of SIRPγ protein, different forms of SIRPγ target antigens were constructed to obtain SIRP-γ-His, Nhfc-SIRPγ-D1, Nhfc-SIRPγ-D1-S79 antigens and H1975-SIRP-γ cells overexpressing SIRPγ antigen.

[0029] SIRPγ protein sequence:

[0030] MPVPASWPHPPGPFLLLTLLGLTEVAGEEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEM ALGAKPSAPVVLGPAARTTPEHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPTGQSVAYSIRSTARVVLDPWDVRSQVICEVAHVTLQGDPLRGTANLSEAIRGPASSLTALLLIAVLLGPIYVPWKQKT(SEQ ID NO.3).

[0031] Nhfc-SIRPγ-D1 antigen sequence:

[0032] EEELQMIQPEKLLLVTVGKTATLHCTVTSLLPVGPVLWFRGVGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPENVEFKSGPGTEMALGAKPS(SEQ ID NO.4).

[0033] Nhfc-SIRPγ-D1-S79 antigen sequence:

[0034] EPKSADKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGSGGGGSEEELQMIQPEKLLLVTGETATLHCTVTSLWPVGPVLWFRGVGPGRELIYRTGTGRFPRVTTVSDLTKRNNMDFSIRISSITPADVGTYYCVKFRKGSPEDVEFKSGPGTEVALGAKPS (SEQ ID NO. 5).

[0035] (2) SIRP-γ-His, Nhfc-SIRPγ-D1, Nhfc-SIRPγ-D1-S79 and H1975-SIRP-γ cells were injected into the left and right sides of the alpaca's cervical lymph nodes in multiple steps, and the alpaca (No. 671) was immunized 11 times. After the 8th and 11th immunizations, serum was collected for antibody titer determination, the effect was evaluated, and peripheral blood was collected. The specific immunization steps are shown in Table 1.

[0036] Table 1 Immunization steps

[0037]

[0038] Note: If using frozen cells for immunization, store the cells in liquid nitrogen and use one cell (2×10 7 Before immunization, the frozen cells were thawed in a 37 ℃ water bath, one cell was added to 8 mL DMEM + 10% FBS, washed once, centrifuged (300g, 6 min), the supernatant was discarded, and then resuspended with 500 μL saline / PBS buffer, the cell survival rate was examined under a microscope (survival rate>90%), and subcutaneous immunization was performed.

[0039] (3) Evaluation of immune effects

[0040] The ELISA method was used to evaluate the titer of the antigen SIRP-γ-His in alpacas at the end of 8 and 11 immunizations. The specific steps are shown in Table 2, and the results are shown in Table 3.

[0041] Table 2 ELISA serum titer evaluation steps

[0042]

[0043] Table 3 Antibody titer of alpaca serum No. 671

[0044]

[0045] According to the test results in Table 3, the titer of the eleven rabbit serum antibodies to SIRP-γ-His is >32000, the absolute value is relatively low, reaching the expected serum titer, and can be used for the next step of antibody library construction.

[0046] 2. Construction of phage natural antibody library

[0047] (1) After 11 days of immunization, peripheral blood was collected from alpacas and PBMCs were isolated by Ficoll density gradient centrifugation.

[0048] (2) Extract total RNA using the Omega EANA TOTAL RNA Kit;

[0049] (3) Reverse transcription into cDNA using a kit;

[0050] (4) Nested PCR (two-step method) was used to amplify the VHH3 gene. Through the first step of PCR amplification, the gene fragment of the Leader-CH2 region was obtained. The DNA electrophoresis results (see Figure 1 , bands 1 to 5 are the PCR results of the Leader-CH2 region gene fragment).

[0051] like Figure 1 As shown in Figure 2, two gene fragments were obtained, of which the 700 bp fragment was the Leader-VHH-Hinge-CH2 region of the IgG2 antibody subtype; and the 900 bp fragment was the Leader-VH-CH1-Hinge-CH2 region of the IgG1 antibody subtype. The 700 bp IgG2 gene fragment was recovered by gel excision, and then a second PCR amplification was performed to obtain a VHH3 gene fragment of about 400 bp (see Figure 2 ).

[0052] 3. Antibody library quality data

[0053] The VHH3 gene fragment was cloned into the New Lib VHH XE vector using the GA reaction kit, and the GA reaction product was electroporated into the super-state cell SR320 containing helper phage. The culture supernatant was collected to obtain the antibody library (Phage). After expansion and culture, the antibody library was aliquoted and frozen at -80°C. The immune library of this application includes two small libraries: R1 (IgG2a VHH antibody containing a long hinge region) and R2 (IgG2c VHH antibody containing a short hinge region). The two small libraries were combined to form the immune library Lib 671-11. The quality data of the immune library is shown in Table 4.

[0054] Table 4 Immune library indicators

[0055]

[0056] According to the data in Table 4, the capacity of the immune library constructed by the present invention is 3.75×10 9 , GA recombination efficiency (clone PCR positive clone number / total clone number) is 90%. It can fully meet the relevant indicators of high-quality immune library and is named Lib 671-11.

[0057] 4. Random clone sequencing

[0058] The monoclones with successful GA reaction were sequenced, and the monoclones with sequencing results that met the standards (complete sequencing, no overlapping peaks, no terminator mutations, etc.) were sequenced. The results were as follows: Figure 3 As shown: the unique sequence ratio is 29 / 29, which meets the requirements of high-quality immune library and can be used for downstream screening.

[0059] Example 2 Screening for high affinity antibodies

[0060] SIRPγ VHH-specific antibodies were enriched by protein panning technology; then, high-affinity SIRPγ VHH-specific antibodies were screened by phage ELISA. Finally, clones obtained by solid phase screening were sequenced and affinity sorted, and the target SIRPγ VHH-specific antibody sequence was finally obtained through RT-PCR and gene-specific PCR. The specific process is as follows:

[0061] 1. Enrichment

[0062] (1) Coating: On Day 1, coat the antigen SIRP-γ-His, 500 ng / well, on the Maxi-sorp plate at 4°C overnight.

[0063] (2) Blocking: On Day 2, block the ELISA plate with 1% PVA at room temperature for 2 h

[0064] (3) Binding: Add antibody library, mix, and incubate at room temperature for 2 h.

[0065] (4) Cleaning: Use PT to clean the Maxi-sorp board.

[0066] (5) Elution: Add 100 μL of 100 mM HCl to the Maxi-sorp plate to elute the bound phage.

[0067] (6) Neutralization: Use 1 M Tris-HCl for neutralization.

[0068] (7) Amplification: Use 100 μL of neutralization solution to infect 1 mL OD 600 =0.8 NEBalpha5F' cells, shake at 37℃ for 1 h; add M13K07 helper phage, shake at 37℃ for 1 h; transfer to 40 mL 2YT / Carb / Kan medium, at 37℃, expand culture overnight.

[0069] (8) Enrichment identification: Drop plate coating (conducted simultaneously with step (7)), and count the degree of enrichment on Day 3, using PVA wells as negative controls; if the phage clones enriched in the antigen wells are more than 10 times the phage clones enriched in the PVA wells, the enrichment is defined as successful.

[0070] (9) Harvest: On Day 3, the expanded phages are separated and purified using the PEG8000 / NaCl precipitation method, and then enter the next round of screening until they are successfully enriched.

[0071] (10) Phage ELISA: After successful enrichment, single clones are randomly picked and sequenced.

[0072] In a maxi-sorp 96-well plate, the immune alpaca library Lib671-11 was solid-phase screened using SIRP-γ-His antigen as bait; SIRP-His was significantly enriched in the R4 round and the screening was stopped (Table 5).

[0073] Table 5 Statistics of Drop clones screened by protein panning

[0074]

[0075] 2. From the clones enriched in round 4 of SIRP-His, 96 single clones were randomly selected for the first phageELISA. The specific process is as follows:

[0076] (1) Coating: On Day 1, coat the antigen recombinant protein SIRP-γ-His (100 ng / well) on the ELISA plate at 4°C overnight.

[0077] (2) Shaking: On Day 1, select the positive phage clones and shake them in a deep-well plate at 37°C overnight.

[0078] (3) Blocking: On Day 2, use 1% PVA to block the ELISA plate at room temperature for 2 h.

[0079] (4) Binding: On Day 2, centrifuge the deep-well plate and the supernatant is the phage; transfer 50 μL of the supernatant to the ELISA plate and incubate at room temperature for 2 h.

[0080] (5) Cleaning: Clean the ELISA plate with PT and add 100 μL of Anti-M13 HRP antibody at room temperature for 1 h.

[0081] (6) Color development: Wash the ELISA plate with PT and then with PBS, add 100 μL TMB, and incubate at 37 °C for 5-10 min.

[0082] (7) Termination: Add 50 μL of 1 M phosphoric acid to terminate the reaction.

[0083] (8) Detection: Measure the absorbance at 450 nm using an enzyme-labeled instrument. The statistical results of the positive rate are shown in Table 6.

[0084] Table 6 R4 round monoclonal phage ELISA

[0085]

[0086] Note: The clones with OD value of SIRP-γ-His (100 ng / well) recombinant protein binding and OD value of 1% BSA binding ≥ 2 were defined as positive clones.

[0087] 3. Second phage ELISA

[0088] Referring to the process of step 2, the 96 monoclonal clones selected in step 2 were subjected to a second phage ELISA, and the statistical results of the positive rates are shown in Table 7.

[0089] Table 7 Second phage ELISA

[0090]

[0091] Note: The clones with OD value of binding to SIRP-γ-His (200 ng / well) recombinant protein and OD value of binding to 1% BSA ≥ 2 were defined as positive clones.

[0092] 4. The monoclonal antibody obtained by screening was cloned and sequenced, and its amino acid sequence is as follows:

[0093] QVQLVESGGGLVQAGGSLRLSCAASGRTFNNYAMGWFRQAPGKEREFVAAISRNGIFTNYVDSVKGRFTISRDDNAKNTVYLQMNSLKPEDTAVYFCTLRNMWGLNHTPFWGQGTQVTVSS (SEQ ID NO. 1).

[0094] Then, the affinity of the monoclonal antibody to different antigens was tested by ELISA. There were three groups of experiments. 450 The numerical values ​​and affinity test results are shown in Table 8, indicating that it has excellent affinity for the target protein.

[0095] Table 8 OD 450 Numerical and affinity test results

[0096]

[0097] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A high-affinity anti-SIRPγ monoclonal antibody, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. A nucleic acid molecule encoding the anti-SIRPγ monoclonal antibody according to claim 1, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

2.

3. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to claim 2, or the expression vector according to claim 3.

5. An engineered bacterium, characterized in that: Comprising the nucleic acid molecule according to claim 2, or the expression vector according to claim 3.

6. A method for producing the anti-SIRPγ monoclonal antibody according to claim 1, characterized in that: The method comprises culturing the recombinant cell according to claim 4 or the engineered bacteria according to claim 5 to produce antibodies.

7. An antibody composition, characterized in that The invention comprises the anti-SIRPγ monoclonal antibody according to claim 1 and a pharmaceutically acceptable carrier, buffer and excipient thereof.

8. A kit, characterized in that: The invention comprises the anti-SIRPγ monoclonal antibody according to claim 1.

9. Use of the antibody according to claim 1, the antibody composition according to claim 7 or the kit according to claim 8 in basic medical research for non-diagnosis / treatment purposes, wherein: The basic medical research is Western Blot, immunohistochemistry and flow cytometry analysis.

10. Use of the antibody according to claim 1, the antibody composition according to claim 7 or the kit according to claim 8 in preparing a reagent for treating lung cancer and / or determining prognosis.

Images