Single-domain antibody targeting human CD28 and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and discloses a single-domain antibody targeting human CD28 and application thereof. The CD28-targeting single-domain antibody has an amino acid sequence as shown in SEQ ID NO.1. The antibody can be specifically combined with Human-CD28 and has good activity, a basis is provided for research and development of CD28-targeting drugs, the CD28-targeting single-domain antibody can be used for detecting / diagnosing CD28 and treating related diseases with abnormal CD28 expression, and the CD28-targeting single-domain antibody has good application prospects in immunotherapy and precision medicine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically relates to a single-domain antibody capable of binding to CD28, and a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its therapeutic use. Background Technology

[0002] CD28, as a co-stimulatory molecule expressed on the surface of T lymphocytes, plays a crucial role in T cell activation. It primarily functions as a "second signal" (binding to B7-1 (CD80) and B7-2 (CD86)) to lower the threshold required for effective T cell activation, thereby enhancing T cell responses to antigens. Given CD28's critical role in immune regulation, the development of specific antibodies against CD28 is of great significance.

[0003] Single-domain antibodies, also known as nanobodies, are small, single-domain antibodies containing only one heavy chain variable region (VHH) domain, derived from camel-like animals (such as camels and alpacas). Nanobodies have an elliptical structure, are small in size, and have a molecular weight about 1 / 10 that of monoclonal antibodies (approximately 15 kDa). Compared to ordinary antibodies, they are chemically more reactive, exhibiting high affinity, good stability, and strong permeability, enabling them to bind to antigens more effectively. They can be highly expressed in prokaryotic or eukaryotic systems and are easy to produce. Furthermore, their small size allows them to bind securely to solid-phase carriers at high density to capture trace amounts of antigens, fully leveraging nanobody-based immunoassay methods to detect and identify clinically difficult-to-detect targets. Therefore, nanobodies have significant value in disease treatment and diagnosis, and hold great promise for antibody-targeted diagnosis and treatment of tumors.

[0004] The applications of CD28-based nanobodies are mainly reflected in the following aspects: (1) Tumor immunotherapy: enhancing anti-tumor immune response by enhancing T cell activity. (2) Combined use with immune checkpoint inhibitors: CD28 nanobodies are used in combination with immune checkpoint inhibitors such as PD-1 / PD-L1 to enhance T cell activity and overcome the immune escape mechanism of tumor cells. (3) Vaccine development: as an auxiliary factor, enhancing the immune response effect of anti-tumor vaccines and promoting the synergistic effect of B cells and T cells. (4) Regulation of autoimmune diseases: CD28 nanobodies can be used to regulate autoimmune responses or may play a role in the treatment of autoimmune diseases (such as rheumatoid arthritis, systemic lupus erythematosus, etc.). (5) Treatment of allergic diseases: CD28 nanobodies can help induce immune tolerance to specific allergens, thus playing a potential medicinal value in the treatment of allergic diseases. Based on the small molecular weight and high specificity of nanobodies, CD28 nanobodies can also be used for bioimaging and disease diagnosis to help identify specific immune states of the body. Therefore, CD28 nanobodies not only have applications in basic research, but also have broad prospects in immunotherapy and precision medicine. Currently, there is a lack of single-domain antibody products targeting CD28 with strong affinity and pharmaceutical value, which urgently require further research and development. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides a single-domain antibody that specifically targets CD28, which can be used in the fields of detection and pharmaceuticals.

[0006] In order to solve the above problems, one of the technical solutions provided by the present invention is: to provide a single-domain antibody targeting human CD28, wherein the single-domain antibody targeting human CD28 has an amino acid sequence as shown in SEQ ID NO.1.

[0007] In some specific embodiments of the present invention, the method for obtaining the single-domain antibody targeting CD28 includes the following steps:

[0008] (1) Antibody immunization: First, the alpaca was immunized with CD28 antigen, and the titer of the serum separated from the immunized alpaca was detected.

[0009] (2) Phage library construction: When the serum titer meets the requirements for library construction, PBMCs after alpaca immunization are isolated, RNA is extracted from PBMCs and quality is tested, RNA is reverse transcribed into cDNA, VHH is amplified by nested PCR, VHH and phage particles are digested with enzymes respectively, VHH is ligated to the vector, and then the phage library is constructed by electroporation.

[0010] (3) Antibody screening: The constructed phage library was used for panning and screening. Human CD28 was used as the antigen. After panning and enrichment, single clones were selected for initial screening. Positive clones were sequenced, and the correct antibody sequence was selected for eukaryotic expression. The expressed antibody was verified by ELISA. Antibodies with good specificity and high sensitivity were selected.

[0011] To address the aforementioned problems, the second technical solution provided by this invention is: to provide a nucleic acid molecule that encodes the aforementioned single-domain antibody targeting human CD28.

[0012] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2.

[0013] To address the aforementioned problems, the third technical solution provided by this invention is: to provide an expression vector comprising a nucleic acid molecule encoding the aforementioned single-domain antibody targeting human CD28.

[0014] Preferably, the vector is a eukaryotic expression vector or a prokaryotic expression vector. The eukaryotic expression vector is the pcDNA3.4 vector.

[0015] To address the aforementioned problems, the present invention provides a fourth technical solution: providing a host cell that can express the aforementioned single-domain antibody targeting human CD28, or contains the aforementioned expression vector.

[0016] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell. The prokaryotic cell is Escherichia coli.

[0017] To address the aforementioned problems, the fifth technical solution provided by this invention is: to provide an application of a single-domain antibody targeting human CD28 as described above in the preparation of a human CD28 detection reagent.

[0018] To address the aforementioned problems, the sixth technical solution provided by this invention is: to provide a kit for detecting human CD28, which contains the aforementioned single-domain antibody targeting human CD28.

[0019] To address the aforementioned problems, the seventh technical solution provided by the present invention is: to provide a pharmaceutical composition or agent comprising the aforementioned single-domain antibody targeting human CD28, and / or pharmaceutically acceptable excipients.

[0020] In order to solve the above problems, the eighth technical solution provided by the present invention is: to provide the use of the above-mentioned single-domain antibody or pharmaceutical composition or agent targeting human CD28 in the preparation of a medicament for diagnosing, preventing and / or treating diseases or conditions related to abnormal expression of CD28.

[0021] Preferably, the disease is a tumor or an autoimmune disease, such as rheumatoid arthritis or systemic lupus erythematosus.

[0022] Compared with the prior art, the beneficial effects of this application are as follows:

[0023] This invention provides a single-domain antibody targeting human CD28 and its applications. The single-domain antibody targeting human CD28 is obtained by screening alpacas after immunizing them with CD28 protein. It has the amino acid sequence shown in SEQ ID NO.1, and its encoding gene has the nucleotide sequence shown in SEQ ID NO.2. ELISA detection verifies that the single-domain antibody can specifically bind to Human-CD28 and has good reactivity. It can be used to prepare drugs for detecting / diagnosing CD28 and for treating diseases related to abnormal CD28 expression, showing promising applications in immunotherapy and precision medicine. Attached Figure Description

[0024] Figure 1 Image showing the results of the long stripe measurement of the storage capacity of alpaca M137.

[0025] Figure 2 The image shows the results of a three-round solid-phase panning phage pool ELISA.

[0026] Figure 3 This is a graph showing the results of ELISA antigen-antibody detection. Detailed Implementation

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0029] Example 1: Screening of Monoclonal Antibodies

[0030] This invention uses Human CD28 protein to immunize M137 alpacas three times, collect peripheral blood to obtain PBMC cells, extract RNA and reverse transcribe it to obtain cDNA, obtain antibody gene fragments by nested PCR, digest and ligate the vector and gene fragments with enzymes, transform them into E. coli for amplification, amplify the VHH gene fragment, clone the target gene fragment into a phage vector, electroporate into competent cells, and construct an alpaca immune library; then, using Human CD28 / His fusion protein as antigen, enrich it through panning and screening, select single clones for initial screening, screen positive clones for testing, sequence analysis to select the correct antibody sequence for eukaryotic expression, perform ELISA detection, and finally screen for antibody 3SP-168 with good specificity and high sensitivity.

[0031] The amino acid sequence of the single-domain antibody 3SP-168 is shown below (SEQ ID NO.1):

[0032] QLQLVESGGGLVQPGGSLRLSCVASGRTFS SSGLG WFRQAPGKEREFVA RIRWSGNSPYYAESVKA RFTISRDNAKNTVYLQMNSLNPEDTAVYYCAA DYIGRPDNWSDRTSYDY WGQGTQVTVSS

[0033] The underlined region is the CDR region of the single-domain antibody, and the gene sequence encoding the antibody is shown in SEQ ID NO.2.

[0034] The specific steps for antibody screening are as follows:

[0035] 1.1 Antibody Immunization

[0036] The specific process is as follows: Healthy #M137 alpacas were selected as the immunization subjects. Day 0: 5 mL of peripheral blood was collected before immunization to monitor the immune response and serve as a negative serum control. Day 10: Antigen quality control (concentration, purity, endotoxin, Binding ELISA with control antibody, and Binding ELISA with negative serum) was performed. Day 14: 400 μg of Human CD28 / hFc was mixed with GERBU adjuvant at a 1:1 ratio (volume ratio), emulsified, and injected subcutaneously at multiple sites. One week later, on Day 21, 50 mL of peripheral blood was collected to monitor the immune response and PBMC separation. Day 28: 400 μg of Human CD28 / His was mixed with GERBU adjuvant at a 1:1 ratio, emulsified, and injected subcutaneously at multiple sites. One week after each immunization, 50 mL of peripheral blood was collected to monitor the immune response and PBMC separation. One week after the third immunization (Day 42), alpaca PBMCs were separated from the blood.

[0037] The serum titer of Human CD28 / His after the third immunization of alpaca M137 showed that the alpaca's immune titer reached over 16,000, meeting the requirements for library construction. This indicates that the immunization was effective. The antigen can induce alpacas to produce high-titer antiserum that specifically targets the CD28 protein, which can be used for subsequent phage library construction and antibody screening.

[0038] 1.2 Library Construction

[0039] Alpaca were immunized and then used isolated PBMCs to construct an alpaca immunization phage library ANb1338-M137-3M.

[0040] The library of this invention was constructed using conventional methods, and the experimental methods can be found in patent document CN117534761A. The technical route mainly includes: (1) isolating PBMCs after immunization of alpacas; (2) extracting RNA from PBMCs and reverse transcribing it into cDNA; (3) amplifying VHH using nested PCR; (4) digesting VHH and phage particles with enzymes respectively, then ligating VHH to the vector, followed by electroporation for library construction; (5) evaluating the quality of the library.

[0041] Results Analysis

[0042] 1) VHH storage capacity determination

[0043] The formula for calculating storage capacity is: Storage capacity = Bacterial culture volume × Dilution factor × Number of spots × 10 × Positive rate, where the bacterial culture volume is 6 mL and the dilution factor is 10. 6 The number of long spots was 123, the insertion rate was 93%, and the total storage capacity was 6.8 × 10⁻⁶. 9 pfu.

[0044] 2) Diversity analysis

[0045] Forty-eight single clones were randomly selected for first-generation sequencing, and sequence analysis was performed on CDR1, 2, and 3 regions. Among the 37 valid sequences obtained from alpaca M137, 5 sequences were duplicated in CDR1, 1 in CDR2, and 1 in CDR3. Library sequence analysis showed a VHH correct insertion rate greater than 90%, indicating good VHH sequence diversity in the constructed ANb1338-M137-3M phage library.

[0046] 1.3 Screening for single-domain antibodies targeting CD

[0047] The ANb1338-M137-3M alpaca immune bank was used for panning and screening, with Human CD28 / His as the antigen, and positive clones were screened through panning and screening methods.

[0048] The selection scheme is shown in Table 1. After selection, the output / input is used to determine whether specific enrichment has occurred.

[0049] Table 1

[0050]

[0051] The results of the three rounds of selection are shown in Table 2 and... Figure 2 As shown, the Output / Input of the three rounds of solid-phase panning exhibits specific enrichment.

[0052] Table 2

[0053] ANb1338-M137-3M Input Output Output / Input Phagetiter 1SP 2.00E+12 1.35E+07 6.75E-06 1.30E+13 2SP 2.00E+11 7.50E+06 3.75E-05 8.00E+12 3SP 2.00E+10 3.60E+05 1.80E-05 2.00E+11

[0054] Therefore, this invention uses the ANb1338-M137-3M alpaca immunological library as the source and Human CD28 as the antigen for solid-phase panning. Output / Input and phase pool ELISA results show that specific enrichment occurred during panning. Then, using Human CD28 as the screening antigen, positive clones were screened by ELISA, resulting in 528 single clones and 415 positive clones, a positive rate of 78.6%. All positive clones were sequenced, yielding 46 unique sequences. Simultaneously, 20 positive clones were randomly selected for gradient validation, and all were confirmed as true positives.

[0055] 1.4 ELISA Detection Test

[0056] Positive clones were selected and sequenced. The correct alpaca VHH gene obtained from sequencing analysis was constructed into the eukaryotic expression vector pcDNA3.4 and subjected to large-scale expression and purification. The expression antibody was validated by ELISA, and the results are as follows: Figure 3 As shown in the figure. The results showed that the antibody (3SP-168) could specifically bind to CD28 and had good reactivity, with an EC50 of 0.4466 nM.

[0057] Obviously, the above embodiments of the present invention are merely examples to clearly illustrate the technical solution of the present invention, and are not intended to limit the specific implementation of the present invention. For those skilled in the art, the present invention can have various modifications and variations, but any modifications, equivalent substitutions, and improvements made within the spirit and principles of the claims of the present invention should be included within the protection scope of the claims of the present invention.

Claims

1. A single-domain antibody targeting human CD28, characterized in that, The amino acid sequence of the single-domain antibody is shown in SEQ ID NO.

1.

2. A nucleic acid encoding a single-domain antibody targeting human CD28 as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

2.

3. An expression vector comprising the nucleic acid of claim 2.

4. The expression vector according to claim 3, characterized in that, The vector is a eukaryotic expression vector or a prokaryotic expression vector.

5. A host cell, characterized in that, It can express the nucleic acid as described in claim 2, or the expression vector comprising any one of claims 3-4.

6. The host cell according to claim 5, characterized in that, The host cell is a eukaryotic cell or a prokaryotic cell.

7. The use of the single-domain antibody as described in claim 1, the nucleic acid as described in claim 2, the vector as described in any one of claims 3-4, or the host cell as described in any one of claims 5-6 in the preparation of a human CD28 detection reagent.

8. A kit for detecting human CD28, characterized in that, It includes the single-domain antibody as described in claim 1.

9. A pharmaceutical composition or preparation, characterized in that, It comprises the single-domain antibody as described in claim 1, and / or pharmaceutically acceptable excipients.

10. The use of a single-domain antibody as claimed in claim 1, or a pharmaceutical composition or agent as claimed in claim 9, in the preparation of a medicament for the diagnosis, prevention, and / or treatment of diseases or conditions associated with abnormal CD28 expression.

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