CD28-targeted single-domain antibody, coding gene and application of CD28-targeted single-domain antibody

Abstract

The invention belongs to the technical field of biological medicines, and discloses a CD28-targeted single-domain antibody, a coding gene and application thereof. The CD28-targeted single-domain antibody contains complementary determining regions CDR1, CDR2 and CDR3 which are respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the single-domain antibody has an amino acid sequence as shown in SEQ ID NO.4. The CD28-targeted single-domain antibody has the advantages that the CDR1, the CDR2 and the CDR3 are respectively as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; the antibody is combined with Human-CD28 and has good reaction activity, a foundation is provided for research and development of CD28-targeted drugs, and the antibody has important significance in the fields of drug application, clinical detection and diagnosis and the like.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, specifically relating to a single-domain antibody targeting CD28, its encoding gene, and its applications. Background Technology

[0002] CD28 is an important co-stimulatory molecule, primarily expressed on the surface of T cells, and plays a crucial role in T cell activation and proliferation. It enhances T cell responses to antigens by binding to B7-1 (CD80) and B7-2 (CD86). Due to CD28's critical role in immune regulation, the development of specific antibodies against CD28 has significant biomedical implications.

[0003] Single-domain antibodies, also known as nanobodies, consist of only one heavy chain variable region (VHH) and are small single-domain antibodies derived from camel-like animals (such as camels and alpacas). They possess a small molecular weight (approximately 15 kDa), high stability, and good tissue penetration, making them highly sought after in targeted therapy and bioimaging. Due to their high affinity and specificity, CD28 nanobodies bind to CD28 molecules more effectively than traditional IgG antibodies. Nanobodies developed based on the CD28 protein can significantly enhance or inhibit T cell activity, achieving better biological effects, depending on different research and development needs.

[0004] CD28 nanobodies can serve as an adjunct tool in tumor immunotherapy, enhancing anti-tumor immune responses by boosting T-cell activity. In some autoimmune diseases, modulating the CD28 signaling pathway may be beneficial for treatment, suggesting CD28 nanobodies as a potential novel therapeutic strategy. Furthermore, CD28 nanobodies can be used as vaccine adjuvants, enhancing vaccine-induced immune responses and improving vaccine efficacy. Due to their small molecular weight and highly specific targeting properties, CD28 nanobodies can also be used in bioimaging and disease diagnosis, helping to identify specific immune states in the body. Therefore, CD28 nanobodies not only have applications in basic research but also hold broad promise for immunotherapy and precision medicine, warranting further research and development. Summary of the Invention

[0005] One objective of this invention is to provide a single-domain antibody targeting CD28, wherein the single-domain antibody comprises CDR1, CDR2 and CDR3; wherein CDR1 is the amino acid sequence shown in SEQ ID NO.1, CDR2 is the amino acid sequence shown in SEQ ID NO.2, and CDR3 is the amino acid sequence shown in SEQ ID NO.3.

[0006] Furthermore, the single-domain antibody targeting CD28 provided by the present invention has the amino acid sequence shown in SEQ ID NO.4.

[0007] In some embodiments of the present invention, the preparation steps of the single-domain antibody targeting CD28 include: immunizing alpacas with CD28 antigen, isolating PBMCs of immunized alpacas, extracting RNA from PBMCs and reverse transcribing it into cDNA, amplifying VHH using nested PCR, digesting VHH and phage particles with enzymes respectively, ligating VHH to a vector, and then constructing a phage library by electroporation; then using the constructed phage library for panning and screening, using Human CD28 as the antigen, enriching through panning, further selecting single clones for initial screening, obtaining positive clones for sequencing, sequencing analysis to select the correct antibody sequence for eukaryotic expression, verifying the expressed antibody by ELISA, and selecting antibodies with good specificity and high sensitivity to obtain the desired antibody.

[0008] A second objective of this invention is to provide a nucleic acid molecule that encodes the aforementioned single-domain antibody targeting CD28.

[0009] Furthermore, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.5.

[0010] A third objective of this invention is to provide an expression vector comprising a nucleic acid molecule encoding the aforementioned single-domain antibody targeting CD28.

[0011] A fourth objective of the present invention is to provide an engineered cell containing the above-mentioned single-domain antibody targeting CD28, or a nucleic acid molecule encoding the single-domain antibody targeting CD28, or the above-mentioned expression vector.

[0012] A fifth objective of this invention is to provide the application of a single-domain antibody, nucleic acid molecule, expression vector, or engineered cell targeting CD28 as described above in the preparation of a CD28 detection reagent.

[0013] A sixth object of the present invention is to provide a kit comprising the above-described single-domain antibody targeting CD28.

[0014] A seventh objective of this invention is to provide a pharmaceutical composition comprising the above-described single-domain antibody targeting CD28, nucleic acid molecule, expression vector, or engineered cell, and one or more pharmaceutically acceptable excipients or vectors.

[0015] The present invention also provides the use of the above-mentioned single-domain antibody, nucleic acid molecule, expression vector, engineered cell, or pharmaceutical composition targeting CD28 in the preparation of a medicament for the diagnosis, prevention, and / or treatment of diseases or conditions related to abnormal CD28 expression.

[0016] In the above-described applications of the present invention, the drug has at least one of the following effects:

[0017] 1) Tumor immunotherapy: CD28 is a co-stimulatory factor for T cells. CD28 nanobodies can enhance the activity of T cells and help with immunotherapy, especially in tumor treatment, by specifically recognizing and killing tumor antigens that target CD28.

[0018] 2) Regulating autoimmune diseases: By regulating the CD28 signaling pathway, CD28 nanobodies have the potential to be used to treat autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.

[0019] 3) Vaccine enhancers: CD28 nanobodies can be used as vaccine enhancers to enhance the immune response triggered by the vaccine and improve its effectiveness.

[0020] Compared with the prior art, the beneficial effects of this application are as follows:

[0021] This invention provides a single-domain antibody targeting CD28, its encoding gene, and its applications. The single-domain antibody targeting CD28 has the amino acid sequence shown in SEQ ID NO.4, and its encoding gene has the nucleotide sequence shown in SEQ ID NO.5. ELISA gradient verification shows that the single-domain antibody can specifically bind to Human-CD28 and has good reactivity, providing a new option for drug preparation for detecting or diagnosing CD28 and for treating diseases related to abnormal CD28 expression. It also provides a research foundation for exploring the mechanisms of T cell activation and immune regulation. Attached Figure Description

[0022] Figure 1 Image showing the results of the long stripe measurement of the storage capacity of alpaca M137.

[0023] Figure 2 The image shows the results of a three-round solid-phase panning phage pool ELISA.

[0024] Figure 3 This is a graph showing the results of ELISA antigen-antibody detection. Detailed Implementation

[0025] 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. Where specific techniques or conditions are not specified in the embodiments of the present invention, they shall be operated in accordance with the conventional technical methods and instrument manuals in the art; the materials and reagents used, unless otherwise specified, are commercially available reagents and materials.

[0026] The method for preparing antibodies according to this invention can be found in patent document CN117534761 A. This invention first uses CD28 protein to immunize alpacas to obtain alpaca PBMC cells; RNA is extracted from PBMCs and reverse transcribed to obtain cDNA; using this as a template, the VHH gene fragment is amplified by nested PCR; then, the target gene fragment is cloned into a phage vector, transformed into competent cells, and a phage library is constructed. Using Human CD28 / His as the antigen, the phage library is enriched through panning, and single clones are selected for initial screening. Positive clones are sent for testing, and sequencing analysis is used to select the correct antibody sequence for eukaryotic expression. Subsequently, ELISA detection is performed to finally screen for antibodies with high specificity and sensitivity.

[0027] Example 1: Construction and Screening Method of Antibody Phage Library

[0028] (1) Antibody immunization: Alpacas were immunized with Human CD28 protein every 2 weeks. After each immunization, blood was collected and the upper serum was separated and preserved for subsequent antibody titer detection. Then, alpaca PBMC cells were separated.

[0029] Alpaca immunization and the use of specific antibodies to detect the strength of the immune response of alpacas to immunoantigens are detailed in Table 1.

[0030] Table 1

[0031]

[0032] Analysis of Immunization Results: Serum titers of alpacas against Human CD28 / His after the third immunization: The results are shown in Table 2. Table 2 shows that the immunogenicity of alpaca M137 reached over 16000. This indicates that the antigen can induce alpacas to produce high-titer antiserum specifically targeting the CD28 protein. The serum titer met the requirements for library construction, indicating that this immunization was successful and subsequent phage library construction and antibody screening can proceed.

[0033] Table 2

[0034]

[0035] (2) Library construction: An alpaca immunized phage library ANb1338-M137-3M was constructed using isolated PBMCs after alpaca immunization. The standard technical route was followed: ① Isolation of PBMCs after alpaca immunization; ② Extraction and quality control of RNA from PBMCs; ③ Reverse transcription of RNA into cDNA; ④ Amplification of VHH using nested PCR; ⑤ Enzyme digestion of VHH and phage particles, followed by ligation of VHH into the vector and then electroporation for library construction; ⑥ Library quality evaluation.

[0036] Results analysis:

[0037] 1) VHH storage capacity determination

[0038] 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 .

[0039] 2) Diversity analysis

[0040] Forty-eight single clones were randomly selected for first-generation sequencing, and sequence analysis was performed on CDR1, 2, and 3 regions. It was found that among the 37 valid sequences obtained from alpaca M137, there were 5 sequences with repetition in CDR1 region, 1 sequence with repetition in CDR2 region, and 1 sequence with repetition in CDR3 region.

[0041] The ANb1338-M137-3M phage library constructed in this invention has a volume of 6.8 × 10⁻⁶. 9 PFU. Library sequence analysis showed that the correct insertion rate of VHH sequences was greater than 90%, indicating that the VHH sequences in this library have good diversity.

[0042] (3) Library screening: The ANb1338-M137-3M alpaca immune library was used for screening, with Human CD28 / His as the antigen, and positive clones were screened through screening and other methods.

[0043] The selection scheme is shown in Table 3, and the results of the three rounds of selection are shown in Table 4 and 5. Figure 2 As shown, the output / input and phase pool ELISA results of the three rounds of solid-phase panning indicate that specific enrichment occurred during the panning process.

[0044] Table 3

[0045]

[0046] Table 4

[0047] 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

[0048] This invention uses Human CD28 as the screening antigen and selects positive clones by ELISA. A total of 528 single clones were picked, and 415 positive clones were obtained, with a positive rate of 78.6%. All positive clones were sent for sequencing, and 46 unique sequences were obtained after sequencing. Simultaneously, 20 positive clones were randomly selected for gradient validation, and all of them were true positives after validation.

[0049] (4) ELISA antigen and antibody detection: The positive clones obtained in the above steps are sent for sequencing and the correct sequences obtained from sequencing analysis are constructed and expressed in large quantities. The expressed antibodies are verified by ELISA detection.

[0050] like Figure 3 As shown, the single-domain antibody (3SP-24) can specifically bind to Human CD28, exhibiting good reactivity with an EC50 of 0.1930 nM. The amino acid sequence of the single-domain antibody 3SP-24 is shown below:

[0051] QVQLVESGGGLVQAGGSLLRLSCAASGRTFS GYTMG WFRQAPGKERSFVA GVTGSDSATYYPNSVKG RFDISRDNAKNTMYLQMNGLKPEDTAVYYCAA SARYGDMRAMVREYDY WGQGTQVTVSS

[0052] The underlined area represents the CDR region of a single-domain antibody, and the nucleotide sequence encoding the antibody gene is shown in SEQ ID NO.5.

[0053] 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. 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 CD28, characterized in that, The single-domain antibody targeting CD28 contains complementarity-determining regions CDR1, CDR2, and CDR3 as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

2. The single-domain antibody targeting CD28 according to claim 1, characterized in that, The single-domain antibody targeting CD28 has the amino acid sequence shown in SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the single-domain antibody targeting CD28 as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

5.

5. An expression carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 3 or 4.

6. An engineered cell, characterized in that, Contains a single-domain antibody targeting CD28 as described in any one of claims 1-2, a nucleic acid molecule as described in any one of claims 3-4, or an expression vector as described in claim 5.

7. The use of the single-domain antibody targeting CD28 as described in any one of claims 1-2, the nucleic acid molecule as described in any one of claims 3-4, the expression vector as described in claim 5, or the engineered cells as described in claim 6 in the preparation of CD28 detection reagents.

8. A kit for detecting CD28, characterized in that, Contains a single-domain antibody targeting CD28 as described in claim 1 or 2.

9. A pharmaceutical composition, characterized in that, The invention comprises a single-domain antibody targeting CD28 as described in any of claims 1-2, a nucleic acid molecule as described in any of claims 3-4, an expression vector as described in claim 5, or an engineered cell as described in claim 6, and one or more pharmaceutically acceptable excipients or vectors.

10. Use of a single-domain antibody targeting CD28 as described in any one of claims 1-2, a nucleic acid molecule as described in any one of claims 3-4, an expression vector as described in claim 5, an engineered cell as described in claim 6, or a pharmaceutical composition as described 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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