Anti-cd22 nanobody and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to an anti-CD22 nanobody and a preparation method and application thereof. The anti-CD22 nanobody comprises a framework region and a complementarity determining region, wherein the complementarity determining region comprises CDR1 (SEQ ID NO: 1), CDR2 (SEQ ID NO: 2) and CDR3 (SEQ ID NO: 3). The preparation method comprises the following steps: immunizing a llama with a Human CD22 / His protein, extracting RNA from PBMC cells and reverse transcribing the RNA into cDNA, constructing a phage display library, obtaining a positive clone through CD22 antigen protein panning after sequencing analysis, and expressing the antibody through a mammalian cell expression system. The application adopts an optimized phage display technology, has a short screening cycle, and the obtained nanobody has a small molecular weight, a stable structure and high affinity to CD22. The antibody can be efficiently expressed through a mammalian cell, and retains natural modification activity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-CD22 nanobody, its preparation method, and its application. Background Technology

[0002] Monoclonal antibodies have been successfully applied to the diagnosis and treatment of various diseases (such as chimeric antibodies, humanized antibodies, and fully human antibodies, miniaturized genetically engineered antibodies, etc., with drawbacks including easy polymerization during expression, low affinity, and problems with large-scale production). However, the therapeutic effects of antibody-based treatments remain limited, urgently requiring further improvement. Nanobodies, consisting only of the variable region of heavy-chain antibodies, possess many unique properties, such as small size (~15 kDa), good solubility, strong stability, ease of expression, short development cycle, and various screening methods. Their use as a therapeutic tool is increasingly accepted, and they are considered the basis for chimeric antigen receptors and targeted drug delivery. Nanobodies exhibit stronger and faster tissue penetration capabilities, reaching dense tissues such as solid tumors to exert their effects. Their relatively short half-life in the blood also helps avoid the accumulation of toxicity in some cases. Compared to monoclonal antibodies, nanobodies demonstrate stronger antigen-binding ability. Phage display technology, due to its simplicity, efficiency, in vitro properties, speed, and low cost, has also become a powerful tool for selecting target-specific ligands.

[0003] CD22 protein is a single-transmembrane glycoprotein, 149 kDa in size, belonging to the immunoglobulin superfamily, and regulating the function and proliferation of B cells. Also known as Siglec-2, it is a member of the sialic acid-binding immunoglobulin-like lectins (Siglec) family. The extracellular domain (CD22 ECD) of CD22 protein consists of seven Ig-like domains (domain 1–domain 7), while the intracellular structure of CD22 includes the immunoreceptor tyrosine inhibitory motif (ITIM) and the immunoreceptor tyrosine activating motif (ITAM).

[0004] CD22 is one of the inhibitory co-receptors on the surface of B cells, and its main function is to inhibit BCR signaling. It is primarily expressed intracellularly in the early stages of B cell development, including in pro-B and pre-B cells, and its expression gradually shifts to the cell surface as B cells mature. CD22 is specifically expressed in the vast majority of mature peripheral B cells and is expressed in most B-cell malignancies, including non-Hodgkin's lymphoma (NHL), hairy cell leukemia (HCL), chronic lymphocytic leukemia, and acute lymphoblastic leukemia (B-ALL). Therefore, CD22 is also an effective target for the treatment of hematological malignancies.

[0005] Besides inhibiting BCR signaling, CD22 molecules also play a role in regulating B cell proliferation, differentiation, and migration in the bone marrow. Current therapies for B-cell lymphoma include monoclonal antibodies, antibody-drug conjugates, radioimmunoassay conjugates, CD22 chimeric antigen receptor T (CAR-T) cells, and dual-targeting CAR-T cell immunotherapy using a combination of CD19 and CD22. Epratuzumab, a humanized IgG antibody against CD22, has been approved by the FDA for the treatment of systemic lupus erythematosus. In the absence of BCR activation, epratuzumab can directly induce CD22 phosphorylation, thereby recruiting SHP-1 and reducing calcium channel blockers (CCPs). 2+ It influxes and inhibits BCR signaling. It has shown good clinical efficacy in non-Hodgkin's lymphoma (NHL) patients as a monotherapy or in combination with rituximab, and is currently also used to treat acute myeloid leukemia. The CD22 antibody-drug conjugate innotuzumab ozogamicin, an antibody-drug conjugate of CD22 antibody (inotuzumab) and enediyne toxin (ozogamicin), is used clinically to treat adult relapsed / refractory B-cell ALL. The bispecific antibody JNJ-75348780, composed of CD3 and CD22, is also currently in clinical trials for the treatment of relapsed and refractory non-Hodgkin's lymphoma patients. Several other CD22-related CAR-T cell therapies are also undergoing clinical trials. Exploring CD22 as a novel therapeutic target for B-cell lymphoma and developing novel immunotherapies related to anti-CD22 nanobodies has become a research hotspot.

[0006] Based on the fact that CD22 is specifically expressed on the surface of mature B lymphocytes and possesses functional properties, and combined with the advantages of nanobodies, we screened CD22-specific nanobodies using phage display technology, studied the binding activity of nanobodies to CD22, and provided a new approach for the future development of CD22-targeted detection and treatment of B lymphocyte malignancies. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide an anti-CD22 nanobody, its preparation method, and its application.

[0008] In a first aspect of the present invention, an anti-CD22 nanobody is provided, the anti-CD22 nanobody comprising a framework region and a complementarity-determining region; the complementarity-determining region comprises CDR1, CDR2, and CDR3, the sequence of the complementarity-determining region CDR1 is shown in SEQ ID NO: 1; the sequence of the complementarity-determining region CDR2 is shown in SEQ ID NO: 2; and the sequence of the complementarity-determining region CDR3 is shown in SEQ ID NO: 3.

[0009] Furthermore, the full-length amino acid sequence of the anti-CD22 nanobody is shown in SEQ ID NO: 4.

[0010] In a second aspect of the invention, a nucleic acid molecule encoding the above-described anti-CD22 nanobody is provided.

[0011] In a third aspect of the present invention, a recombinant expression vector containing the above-mentioned nucleic acid molecules is provided.

[0012] Furthermore, the carrier is -pComb3XSS.

[0013] In a fourth aspect of the invention, a host cell containing the above-described carrier is provided.

[0014] Furthermore, the host cells are TG1 or SS320; SS320 host cells are preferred.

[0015] In a fifth aspect of the present invention, a method for preparing anti-CD22 nanobodies is provided, comprising the following steps:

[0016] S1. Construct a human CD22 / His antigen protein and use it to immunize alpacas;

[0017] S2. RNA was extracted from immunized alpaca PBMC cells and reverse transcribed into cDNA;

[0018] S3. Amplify nanobody gene fragments using cDNA as templates to construct a phage display antibody library;

[0019] S4. Single clones were selected by human CD22 / his protein panning and subjected to phage-ELISA to obtain positive clones that bind to CD22 protein;

[0020] S5. After sequencing analysis of positive clones, nanobody sequences are selected;

[0021] S6. High-throughput expression was performed using a mammalian cell high-throughput expression system to obtain antibody protein. FACS detection was performed using a CD22 high-expression cell line to finally obtain an antibody that binds to Human CD22.

[0022] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising the above-described anti-CD22 nanobody and a pharmaceutically acceptable carrier.

[0023] In a seventh aspect of the invention, the use of the anti-CD22 nanobody in the preparation of a medicament for treating B-cell malignancies such as non-Hodgkin's lymphoma (NHL), hairy cell leukemia (HCL), chronic lymphocytic leukemia, and acute lymphoblastic leukemia (B-ALL) is provided.

[0024] In an eighth aspect of the invention, the use of the anti-CD22 nanobody in the preparation of reagents or kits for detecting CD22 is provided.

[0025] In a ninth aspect of the invention, the use of the anti-CD22 nanobody in the preparation of a product that specifically binds to CD22 is provided.

[0026] The present invention has the following technical effects:

[0027] 1) This invention uses optimized phage display technology, which is easy to operate and can complete antibody screening within 7-10 days, with a short screening time; the obtained antibodies have small molecular weight, strong penetrability, simple and stable structure, and high affinity and strong specificity for CD22 antigen.

[0028] 2) The antibody preparation method of the present invention is simple and fast; it uses a mammalian cell expression system to induce efficient antibody expression, and can perform correct folding and post-translational modification, so that its activity is closer to that of natural antibodies.

[0029] 3) The antibody of the present invention has a wide range of applications, including treatment (without Fc segment side effects), diagnosis, and easy engineering modification. Attached Figure Description

[0030] Figure 1 The serum titer for CD22 protein immunity.

[0031] Figure 2 This is an image of RNA agarose gel electrophoresis.

[0032] Figure 3 This is an electrophoresis image of one round of PCR products.

[0033] Figure 4 This is an electrophoresis image of the second round of PCR products.

[0034] Figure 5 CD22 enrichment results

[0035] Figure 6 The results are for Antibody Protein ELISA Binding.

[0036] Figure 7 Cell binding experiment results Detailed Implementation

[0037] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention.

[0038] For any techniques or conditions not specified in this embodiment, the operation shall be carried out in accordance with conventional technical methods and instrument manuals in this field; for reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0039] Example 1

[0040] In this embodiment, the method for preparing CD22 nanobody includes the following steps:

[0041] 1. Immunogen preparation

[0042] Based on the gene and protein sequence information of Human CD22 (UniProt Reference Sequence: P20273-1), the immunogen Human CD22 was expressed, and a His-tag was linked to its C-terminus to obtain modified amino acids for subsequent purification and detection.

[0043] 2. Animal immunization

[0044] Alpaca were immunized three times with the modified antigen and Freund's adjuvant mixture obtained in step 1 to obtain alpaca PBMC cells: alpaca were primarily immunized with an emulsion mixture of human CD22 / His protein (i.e., the modified amino acids obtained in step 1) and Freund's complete adjuvant. On days 21 and 42, alpaca were boosted with human CD22 / His protein and Freund's incomplete adjuvant. One week after each immunization, blood was collected to detect the serum titer of Anti-CD22 / His. One week after the third immunization, 50 mL of blood was collected for screening and library construction.

[0045] 3. Serum titer detection and PBMC cell isolation

[0046] Anti-CD22 / His serum titer was detected by ELISA. The detection procedure was as follows: CD22 / His protein at a concentration of 2 μg / mL was coated onto an ELISA plate overnight at 4°C. 3% BSA blocking solution was added, and the plate was blocked for 1 hour. After washing 3 times, the first well was diluted 100-fold, and each subsequent well was diluted 2-fold (i.e., each well was diluted to twice the concentration of the previous well). Serum obtained after three immunizations (control was alpaca serum before immunization) was added. The plate was incubated at room temperature for 1 hour, washed 3 times, and horseradish peroxidase-labeled Goatanti-Alpaca IgG (H+L) secondary antibody diluted 1:10000 was added to each well. The plate was incubated at room temperature for 45 minutes, washed 6 times, and TMB chromogenic solution was added. The plate was incubated at 37°C for 5-10 minutes. The reaction was terminated by adding stop solution, and the OD450 was measured.

[0047] When the OD450 value of the sample to be tested is more than three times that of the negative control, the antiserum titer is considered positive. The result is as follows: Figure 1 As shown, Figure 1 The antiserum titer after three immunizations is shown to be 102400. This demonstrates that the antigen can induce alpacas to produce high-titer antiserum specifically targeting the CD22 protein, and antibody libraries can be constructed using 3-immunized PBMCs.

[0048] The collected blood was diluted twice with PBS, and PBMCs were isolated using lymphocyte separation medium (Ficoll reagent). Trizol was then added to dissolve the cells, resulting in a cell concentration of 10⁻⁶. 7 / ml of PBMC cells, stored at -80 degrees Celsius.

[0049] 4. Antibody gene extraction and phage antibody library construction

[0050] 1) RNA extraction: RNA was extracted from PBMCs using the Trizol method, and the RNA concentration was determined and identified by agarose gel electrophoresis. Figure 2 If 28S, 18S, and 5S small RNA bands are visible, it indicates good RNA integrity; a 28S to 18S RNA ratio of 2:1 indicates no RNA degradation. Use the amount required for reverse transcription, and store any remaining amount at -80℃.

[0051] 2) Obtain cDNA: RNA is reverse transcribed into cDNA using oligo(dT) (the reverse transcription kit is TaKaRa-SMARTcribe Reverse Transcript).

[0052] 3) Antibody gene amplification: Nested PCR was used to perform the first and second rounds of PCR to obtain the target gene fragment.

[0053] Figure 3 These are the results of the first round of PCR; the target fragment is around 750bp. Figure 4 The results of the second round of PCR show that the target band is approximately 400 bp. The obtained target gene fragment was cloned into a phage expression vector to obtain a phage vector containing the antibody fragment. This vector was then electroporated into SS320 electroporation competent cells, and the cells were collected to obtain a CD22 immune phage antibody library with a volume of 1.367E+09cfu.

[0054] 5. Selection of CD22 phage antibody library

[0055] 1) The obtained Cumulus bacteria were packaged into phages and their titer was determined to be 2E+13cfu / ml.

[0056] 2) Human CD22 / His antigen protein was coated in 96-well microplates, with 4 wells coated to form a positive sieve plate; 4 wells of a negative sieve plate were also coated with 3% BSA and incubated overnight at 4°C. The antigen coating solution was removed from the positive sieve wells, and the plates were washed 3 times with 0.1% PBST and then blocked with 3% BSA blocking solution. The coating solution was removed from the negative sieve wells and the plates were directly blocked with 3% BSA. At the same time, the CD22 phage library 1E+12pfu was blocked with 3% BSA for 1 hour.

[0057] 3) After the blocking time is complete, remove the blocking solution from the negative sieve wells, wash with 0.1% PBST, transfer the blocked phage to the negative sieve plate, and incubate at room temperature for 1 hour; remove the blocking solution from the positive sieve wells, wash with 0.1% PBST, transfer the sample from the negative sieve wells to the positive sieve antigen wells, incubate for 1 hour, remove the supernatant, wash with 0.1% PBST, add trypsin elution buffer, incubate at room temperature for 10 minutes, and collect the eluent together; this is the output phage; add glycerol to the collected eluent to a final concentration of 20%.

[0058] 4) Add half of the elution buffer to 2 ml of SS320 with an OD600 of 0.5, let it stand for 30 min to infect, centrifuge, keep a portion of the supernatant, mix well, plate, and incubate overnight at 37 degrees Celsius; take another 10 μl of elution buffer and perform serial dilution (SS320 culture medium dilution), let it stand for 30 min to infect, spot on titer plates, and incubate overnight at 37 degrees Celsius; calculate titer the next day, collect the plates, package the phages, and then perform the second round of panning, the process is the same as the first round, but the amount of antigen coated and the amount of phage added are reduced accordingly.

[0059] The selection results are as follows Figure 5 As shown, based on the ratio of the output phage obtained after screening to the input phage, the second round showed a significant enrichment effect. Therefore, the phage obtained in the second round was selected, infected with SS320, plated, and single clones were selected for culture and clone ELISA.

[0060] 6. Clone Phage ELISA

[0061] Human CD22 / His protein was coated onto 96-well microplates and incubated overnight at 4°C. The next day, 1% BSA was added for blocking at room temperature for 1 hour, followed by washing three times with 0.05% PBST. The supernatant of the cultured single-clone bacterial culture was centrifuged and added to the plate, incubated at room temperature for 1 hour, washed three times with 0.05% PBST, and then diluted secondary antibody anti-M13 (1:10000) was added. The plate was incubated at room temperature for 40 minutes, washed six times with 0.05% PBST, and then TMB chromogenic solution was added. The plate was incubated for 5-10 minutes, and then stop the reaction by adding stop solution. The OD450 value was measured using a microplate reader. Clones with an OD450 value greater than 3 times that of the negative control were considered positive. The positive clones were sent for sequencing analysis. The clones were then transferred to a human FC eukaryotic expression vector for purification and expression. Finally, the nanobody CD22-01, which binds to human CD22 / His protein, was obtained. Its CDR region and full-length sequence are as follows:

[0062] CDR1: Has the amino acid sequence shown in SEQ ID NO:1;

[0063] CDR2: Has the amino acid sequence shown in SEQ ID NO:2;

[0064] CDR3: Has the amino acid sequence shown in SEQ ID NO:3;

[0065] The full-length amino acid sequence is shown in SEQ ID NO: 4.

[0066] 7.Antibody Protein ELISABinding:

[0067] 96-well ELISA plates were coated with CD22 antigen (2 μg / ml, 100 μl / well) and incubated overnight at 4°C. The plates were washed three times with 0.05% PBST, and then blocked for 2 hours at room temperature with 200 μl of 1% BSA. After washing three times with 0.05% PBST, CD22-01 antibody (100 μl / well, initial concentration 200 nM) was added, followed by 4-fold serial dilutions for a total of 12 wells. The plates were incubated for 1 hour at room temperature. After washing three times with 0.05% PBST, secondary antibody was added and incubated for 30 minutes at room temperature. The plates were washed six times with 0.05% PBST, and TMB chromogenic buffer was added. The plates were incubated for 5-10 minutes, and then stop the reaction with stop solution. The OD450 values ​​were measured using an ELISA reader, and the data were analyzed.

[0068] The results are as follows Figure 6 As shown in the figure, the results indicate that the CD22-01 antibody binds to the human CD22 / His antigen protein.

[0069] 8. Cell function assay: cell binding (FACS)

[0070] Cultured CD22 overexpressing cells were prepared to a cell count of 2E+06 cells / ml using MACS buffer. 50 μL of cell suspension was added to each well of a 96-well plate. The prepared antibody was serially diluted 4-fold with MACS buffer, and 50 μL of antibody dilution was added to each well of the plate. Positive and negative controls were added. The plates were incubated at 4°C in the dark for 60 min. The cells were washed three times with MACS buffer, and then resuspended in 100 μL of fluorescent secondary antibody (1:1000). The plates were incubated at 4°C in the dark for 30 min, washed three times with MACS buffer, and then resuspended in 200 μL of MACS buffer. The cells were then analyzed using flow cytometry. Figure 7 The results showed that the CD22-01 antibody bound to cells overexpressing CD22.

[0071] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-CD22 nanobody, characterized in that, The anti-CD22 nanobody includes a framework region and a complementarity-determining region (CDR). The CDR includes CDR1, CDR2, and CDR3. The sequence of the CDR1 is shown in SEQ ID NO:

1. The sequence of the CDR2 is shown in SEQ ID NO:

2. The sequence of the CDR3 is shown in SEQ ID NO:

3.

2. The anti-CD22 nanobody according to claim 1, characterized in that, Its full-length amino acid sequence is shown in SEQ ID NO:

4.

3. A nucleic acid molecule encoding the anti-CD22 nanobody of claim 1 or 2.

4. A recombinant expression vector containing the nucleic acid molecule of claim 3.

5. A host cell containing the vector of claim 4.

6. A pharmaceutical composition, characterized in that, It includes the anti-CD22 nanobody and pharmaceutically acceptable carrier as described in claim 1 or 2.

7. The use of the anti-CD22 nanobody as described in claim 1 or 2 in the preparation of a medicament for treating B-cell malignant tumors, wherein the B-cell malignant tumor is selected from non-Hodgkin lymphoma (NHL), hairy cell leukemia (HCL), chronic lymphocytic leukemia, and acute lymphoblastic leukemia (B-ALL).

8. The use of the anti-CD22 nanobody as described in claim 1 or 2 in the preparation of reagents or kits for detecting CD22.

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