Single domain antibody targeting CD28 molecule and application thereof

Abstract

The invention belongs to the technical field of immunology, and discloses a single-domain antibody targeting CD28 molecules and application, and the amino acid sequence of the single-domain antibody is as shown in SEQ ID No.1. The antibody has good reaction activity with human CD28, can be used for detecting or diagnosing CD28 and treating related diseases with abnormal CD28 expression, provides a basis for research and development of CD28-targeted drugs, and has very wide prospects and important significance in the fields of drug application, clinical detection and diagnosis and the like.

Description

Technical Field

[0001] This invention belongs to the field of immunology technology, specifically relating to single-domain antibodies targeting the CD28 molecule and their applications. Background Technology

[0002] CD28 is an important co-stimulatory molecule, primarily expressed on the surface of T cells, and participates 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 crucial role in immune regulation, developing specific antibodies against CD28 has significant biomedical implications.

[0003] Nanobodies 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 permeability, making them a promising candidate for targeted therapy and bioimaging.

[0004] Nanobodies have high affinity and specificity. CD28 nanobodies can bind to CD28 molecules more effectively than traditional IgG antibodies. Nanobodies developed based on CD28 protein can significantly enhance or inhibit T cell activity and achieve better biological effects depending on different research and development needs.

[0005] CD28 nanobodies not only have potential applications in basic research, but also in immunotherapy and precision medicine, making them worthy of further research and development. Summary of the Invention

[0006] One of the objectives of this invention is to provide a single-domain antibody targeting CD28, which demonstrates good specificity through specific enrichment and affinity assays.

[0007] The above technical objectives are achieved through the following solutions:

[0008] A single-domain antibody targeting CD28, the amino acid sequence of which is shown in SEQ ID No. 1. Any of the above amino acid sequences may further include a derived sequence that optionally incorporates, deletes, modifies, and / or substitutes at least one amino acid, and is capable of retaining CD28 binding affinity.

[0009] A second objective of this invention is to provide a nucleic acid molecule that encodes the aforementioned single-domain antibody targeting CD28.

[0010] Furthermore, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.1.

[0011] 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.

[0012] A fourth objective of the present invention is to provide an engineered cell containing the above-described single-domain antibody targeting CD28, a nucleic acid molecule encoding the single-domain antibody targeting CD28, or the above-described expression vector.

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

[0014] A sixth objective of this invention is to provide the use of the above-mentioned CD28-targeting single-domain antibody, nucleic acid molecule, expression vector, engineered cell, or pharmaceutical composition in the preparation of a medicament for the diagnosis, prevention, and / or treatment of diseases or conditions related to CD28 expression.

[0015] Furthermore, the preparation steps of the single-domain antibody targeting CD28 include: firstly, constructing CD28 antigen to immunize alpacas, and then detecting the titer of serum isolated from immunized alpacas; once the serum titer meets the requirements for library construction, then isolating PBMCs from immunized alpacas, extracting RNA from PBMCs and performing quality control, reverse transcribing the RNA into cDNA, amplifying VHH using nested PCR, digesting VHH and phage particles with enzymes respectively, then 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, specific enrichment is performed through panning, further selecting single clones for initial screening, obtaining positive clones for sequencing, and selecting unique sequence positive clones for ELISA gradient verification to screen for antibodies with good specificity and high sensitivity.

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

[0017] Based on the advantages of small molecular weight, strong penetration, and low immunogenicity, CD28 nanobodies have broad application prospects:

[0018] 1) Treatment of hematologic malignancies: CD28 nanobodies have shown significant efficacy in the treatment of B-cell-related cancers (such as acute lymphoblastic leukemia and non-Hodgkin's lymphoma).

[0019] 2) Targeted drug delivery: Nanobodies can be combined with cytotoxic drugs or radioisotopes to achieve targeted drug delivery, enhance efficacy and reduce side effects.

[0020] 3) Immunotherapy: As part of immunotherapy, nanobodies can enhance anti-tumor immune responses and improve patient survival rates.

[0021] 4) Diagnosis and imaging: Nanobodies can be used for early diagnosis and imaging of tumors, helping to monitor disease progression.

[0022] 5) Potential applications for other diseases: In addition to tumors, CD28 nanobodies may also play a role in the treatment of autoimmune diseases and infectious diseases.

[0023] Compared with the prior art, the beneficial effects of this application are as follows:

[0024] This invention provides a single-domain antibody targeting CD28, which has the amino acid sequence shown in SEQ ID NO. 1. Affinity measurements show that it possesses high affinity, indicating broad application prospects in immunotherapy and precision medicine. Attached Figure Description

[0025] Figure 1 Results of three-round solid-phase panning phage pool ELISA;

[0026] Figure 2 A gradient validation plot for ELISA of unique sequences obtained through library screening (partial results). 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: The preparation steps of the single-domain antibody targeting CD28 include:

[0030] First, CD28 antigen was constructed to immunize alpacas. Serum from the immunized alpacas was then analyzed for titer. Once the serum titer met the requirements for library construction, PBMCs (Peripherally Borary Cell Matrix) were isolated from the immunized alpacas. RNA was extracted from the PBMCs and subjected to quality control. The RNA was reverse transcribed into cDNA, and VHH (viral phage antigen) was amplified using nested PCR. Both VHH and phage particles were digested with enzymes, and VHH was ligated to a vector. A phage library was then constructed via electroporation. The constructed phage library was then panned and screened using Human CD28 as the antigen. Specific enrichment was performed through panning, and single clones were further selected for initial screening. Positive clones were sequenced to obtain single-domain antibodies, the amino acid sequences of which are shown below:

[0031] QVQLVESGGGLVQAGGSLRLSCAISGQTIS IYDMGLG WFRQAPGKEREFVA AISRSGSSTDYVDLAKG RFTISRDNAKDTVYLQMNSLKPEDTAVYTCAA RPLALWSTTERDGDGYTY WGQGTQVTVSS

[0032] The underlined lines represent the CDRs of single-domain antibodies, numbered according to IMGT rules, and their encoded nucleotide sequences are shown in SEQ ID NO.2.

[0033] The selection scheme mentioned above is as follows:

[0034]

[0035] The phage library mentioned above was added and labeled as Input; the phages obtained through panning were labeled as Output. After panning, the output / input ratio was used to determine whether specific enrichment had occurred. The results of the three rounds of panning are shown in Table 1 and... Figure 1 As shown:

[0036] Table 1 Output / Input of Three Rounds of Solid Phase Sorting

[0037] Input Output Output / Input bacteriophage titer 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

[0038] As can be seen from Table 1, the Output / Input samples obtained through three rounds of solid-phase panning show specific enrichment.

[0039] In summary, solid-phase panning was performed using Human CD28 as the antigen. Output / input and phase pool ELISA results showed that specific enrichment occurred during panning. Using Human CD28 as the screening antigen, 528 single clones were selected by ELISA, resulting in 415 positive clones. Sequencing yielded 46 unique sequences. Simultaneously, 20 positive clones were randomly selected for gradient validation, and all were confirmed as true positives.

[0040] Identification of antibody affinity:

[0041] Unique sequences obtained from library screening were validated using ELISA gradient spectroscopy. Some results are shown below. Figure 2 As shown, the antibody (3SP-10) exhibited good reactivity against CD28, with an EC50 of 1.3570 nM.

[0042] 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 the CD28 molecule, characterized in that, The amino acid sequence of the single-domain antibody is shown in SEQ ID No.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the single-domain antibody of claim 1.

3. A molecular expression vector, characterized in that, The molecular expression vector contains the nucleic acid molecule of claim 2.

4. A method for preparing a single-domain antibody against the human CD28 receptor as described in claim 1, characterized in that, Includes the following steps: S1. Based on the protein and gene sequence information of CD28, analyze and design an immune antigen, and link a Histag to its C-terminus to obtain a modified antigen. S2. Immunize alpacas with the antigen obtained in step S1, test the titer of the serum separated from the immunized alpacas, and then extract effector B cells from the peripheral blood to obtain alpaca PBMC cells. S3. Using the alpaca PBMC cells obtained in step S2 as raw materials, total RNA was extracted, and cDNA fragments were obtained by reverse transcription PCR. The VHH gene fragment was amplified using this as a template. The target gene fragment was then cloned into a phage vector and transformed into competent cells to construct a bacterial library. S4. The phage library constructed in step S3 is used for bacterial packaging, enriched by panning, and single clones are further selected for initial screening. Positive clones are sent for testing, and sequencing analysis is used to select the correct antibody sequence for eukaryotic expression. S5. Perform cell function tests using the antibody expressed in step S4, and screen for antibodies with good specificity to obtain the final product.

5. The use of the single-domain antibody of claim 1 in the preparation of a kit for detecting human CD28.

6. A kit for detecting human CD28, characterized in that, The kit contains the single-domain antibody as described in claim 1.

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