Recombinant C-type single-domain nano antibody combined with CDH17 and application of recombinant C-type single-domain nano antibody

Abstract

The invention belongs to the technical field of immunology, and provides a recombinant C-type single-domain nano antibody combined with CDH17 and application of the recombinant C-type single-domain nano antibody. According to the invention, an antibody with binding capacity to CDH17 is screened by using a phage display technology, then m01s is used as a skeleton recombinant antibody, and the C-type single-domain antibody with high affinity activity is obtained through antibody recombination. The obtained recombinant C-type single-domain nano antibody combined with CDH17 can be used as an indicator for diagnosis and prognosis of cancers, and is expected to become a candidate drug of CAR-T drugs for gastric cancer, breast cancer, colon cancer and the like due to high affinity to tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and in particular relates to a recombinant C-type single-domain nanobody binding to CDH17 and an application thereof. Background Art

[0002] Liver-intestinal cadherin (Cadherin-17, also known as CDH17; HPT-1; LI-Cadherin) is a newly discovered cadherin in recent years. The extracellular domain of CDH17 is composed of seven repeating sequences EC1-EC7, so it belongs to a subclass of the 7D-cadherin family. The CDH17 target plays an important role in tumor treatment. Some drugs targeting the CDH17 target have been developed for the treatment of certain types of cancer, such as gastric cancer, breast cancer and colon cancer. These drugs inhibit the growth and division of tumor cells by binding to the CDH17 target, thereby achieving the purpose of treating tumors. In addition, the study of the CDH17 target can also provide new markers for the diagnosis and prognosis of cancer.

[0003] Nanobodies (VHH) have been widely used as antigen-binding domains in CAR-T due to their small size, excellent stability, high affinity and strong preparation feasibility. Nanobody-based CAR structures have shown good functions on more than a dozen different tumor-specific targets. The resulting nanobody-based CAR-T or CAR-NK cells have shown anti-tumor effects both in vivo and in vitro. The application of nanobodies in CAR-T therapy has been well demonstrated from the bench to the bedside and has shown great potential in the formation of more challenging advanced CAR-T. Recently, a new framework based on the CH2 domain of the antibody Fc segment has been considered promising for the development of a new type of single-domain antibody, called a C-type single-domain antibody. Summary of the invention

[0004] In view of the problems in the background technology, the present invention provides a recombinant C-type single-domain nanobody binding to CDH17 and its application.

[0005] In the present invention, a nanobody with binding ability to CDH17 is screened out by utilizing phage display technology, and the CH2 domain mutant m01s of the constant region of human antibody IgG is used as the skeleton. The three complementary determining regions CDR1, CDR2, and CDR3 of the nanobody screened out by phage display technology replace LOOP-BC, LOOP-DE, and LOOP-FG of the m01s sequence to form a new antibody sequence with m01s as the skeleton (named m01s-E4, and the new sequence is shown in SEQ ID No. 1); the new sequence is used to construct a vector, and the recombinant C-type single-domain nanobody with high affinity activity binding to CDH17 of the present invention is obtained through a transfection, expression, and purification process.

[0006] The present invention also provides the use of the above-mentioned C-type single-domain nanoantibodies in the preparation of diagnostic reagents and therapeutic drugs for cancer. Studies have found that abnormal expression of CDH17 is closely related to the occurrence of various solid tumors, especially in gastrointestinal cancers (GICs) and neuroendocrine tumors (NETs), where its high expression phenomenon is particularly significant. Therefore, the above-mentioned C-type single-domain nanoantibodies can be used in the preparation of drugs and diagnostic reagents for cancer types such as gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer, and breast cancer.

[0007] The beneficial effects of the present invention are: Antibodies obtained through immune screening and recombination processes have higher specificity and affinity activity; because they contain the m01s skeleton, the recombinant antibodies have dual valence: they can bind to antigens and molecules such as FcRn (extending plasma half-life), thereby achieving (or partially achieving) the function of full-length antibodies; The recombinant C-type single-domain nanoantibody binding to CDH17 of the present invention can also be used as an indicator for the diagnosis and prognosis of cancer; due to the recombinant modification, the single-domain antibody of the present invention has a high affinity for tumor cells and is expected to become a candidate drug for CAR-T drugs such as gastric cancer, breast cancer and colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 This is a schematic diagram of the positive results of monoclonal phage flow screening obtained in an embodiment of the present invention.

[0009] Figure 2 This is the SDS-PAGE electrophoresis diagram of the m01s-E4 antibody obtained in the example of the present invention.

[0010] Figure 3 This is the EC50 flow cytometry result of the m01s-E4 antibody obtained in the example of the present invention binding to the tumor cell line.

[0011] Figure 4 These are the IP experimental results of the m01s-E4 antibody and tumor cell lines obtained in the examples of the present invention.

[0012] Figure 5 Flow cytometric comparison of the m01s-E4 antibody obtained in the present invention and the known antibody Nb289.

[0013] Figure 6 This is a sequence alignment diagram of VHH (ie, E4) and m01s obtained in an embodiment of the present invention. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0015] 1. Preparation of recombinant C-type single-domain nanobodies binding to CDH17: (1) Immunization of alpacas with tumor cell lines Tumor cells AGS and MKN45 were fully mixed with Freund's complete adjuvant or Freund's incomplete adjuvant and used to immunize alpacas for a total of 4 times, with an interval of 2-3 weeks between each immunization.

[0016] (2) Construction and screening of phage display library after immunization S1: After immunization 4 times, blood was drawn from the jugular vein of the camel and lymphocytes were separated. Total RNA was extracted using the RNA extraction kit RC112 from Novagen. RNA was reverse transcribed into cDNA using the Novagen RNA reverse transcription kit (HiScript II Q Select RT SuperMixfor qPCR R232). Next, the cDNA was used as a template to amplify the VHH sequence using VHH-specific primers (synthesized by Qingke Biotech). The amplified nanobody sequence was cloned into the phagemid pComb3XSS (modified by our laboratory) using homologous recombination, and the recombinant product was electrotransferred to the TG1 competent state. The phages collected after amplification and culture were the initial nanobody phage library.

[0017] S2: The phage library was panned for three rounds using the tumor cell lines AGS and MKN45 for positive panning and Raji cells for negative panning (refer to Sabir JS, Atef A, El-Domyati FM, Edris S, Hajrah N, AlzohairyAM, Bahieldin A. Construction of naïve camelids VHH repertoire in phage display-based library. CR Biol. 2014 Apr;337(4):244-9. doi: 10.1016 / j.crvi.2014.02.004. Epub 2014 Mar 20. PMID: 24702893).

[0018] S3: Identification of positive clones by monoclonal phage cell ELISA: After three rounds of continuous panning, 96 individual colonies were picked into a 96-well round-bottom culture dish to prepare monoclonal phage displaying antibodies. Positive tumor cell line AGS was plated with ELISA plates for cell ELISA to screen positive clones.

[0019] S4: Monoclonal phage flow cytometry screening of positive clones: 50 μL of monoclonal phage from the 96-well plate was transferred to a new 96-well U-bottom culture dish and blocked with 3% BSA for 30 minutes. 5 Tumor cells AGS and MKN45 were added to each well and incubated at 4°C for 30 min; centrifuged at 200 g for 5 min, washed twice with PBSA, and the prepared anti-VHH fluorescent-labeled antibody (BSA dilution, 1:400) was added and incubated at 4°C for 30 min; centrifuged at 200 g for 5 min, washed twice with PBSA, and tested on the machine (see Stark Y, Venet S, Schmid A. Whole Cell Panning with Phage Display. Methods Mol Biol. 2017;1575:67-91. doi: 10.1007 / 978-1-4939-6857-2_5. PMID: 28255875.).

[0020] After three rounds of panning of the constructed phage display library, monoclonal phage screening was performed to obtain a candidate antibody with a strong signal. Table 1 shows that in the monoclonal phage cell ELISA identification experiment, 29 positives were initially obtained (background marked in gray).

[0021] Table 1 In the monoclonal phage cell ELISA identification experiment, 29 positive results were obtained.

[0022] Figure 1 The positive images determined by monoclonal phage flow cytometry screening are the binding of phage supernatant to AGS cells (A) and MKN45 cells (B). Based on the ELISA results, E4 was determined to be positive. The phage corresponding to the positive was sequenced to obtain the complete sequence of VHH (the sequence is shown in SEQ ID No. 5).

[0023] (3) Recombination of antibodies S1: Sequence alignment Bioinformatics tools (such as BLAST, Clustal Omega, etc.) were used to compare the CDR1, CDR2, and CDR3 sequences of the nanoantibodies and analyze their similarities and characteristics.

[0024] S2: Select target sequence Determine the LOOP-BC, LOOP-DE, and LOOP-FG portions of the m01s sequence to be replaced and ensure that these regions are functionally suitable to accept the new CDR sequences.

[0025] S3: Sequence replacement The three complementary determining regions CDR1, CDR2, and CDR3 (amino acid sequences are shown in SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4, respectively) of the nanobody E4 screened by phage display technology were replaced with the corresponding LOOP regions in m01s (CDR1 replaced LOOP-BC to obtain CDR1-IMGT of the recombinant antibody, CDR2 replaced LOOP-DE to obtain CDR2-IMGT of the recombinant antibody, and CDR3 replaced LOOP-FG to obtain CDR3-IMGT of the recombinant antibody), forming a new fusion sequence named m01s-E4 (the new fusion sequence is shown in SEQ ID No. 1). The sequence alignment of E4 and m01s is shown in Figure 6 shown.

[0026] S4: Add tags A His tag was added at the appropriate position of the new sequence to facilitate subsequent purification and detection.

[0027] S5: Cloning into vector The modified new sequence was cloned into the mammalian expression vector pTT5.

[0028] S6: Expression and purification of recombinant antibodies The constructed vector was transfected into HEK293F (ATCC, CBP60437) using polyethyleneimine (PEI), and the transfected mammalian cells were suspended and cultured for 5 days; then the cell culture supernatant was collected by centrifugation at 2000 rpm for 10 minutes. The nanobody was purified using an affinity chromatography column and analyzed by SDS-PAGE electrophoresis. The results are as follows Figure 2 shown.

[0029] (4) IP identification of recombinant antibodies and tumor cell lines Preparation of cell lysate: Take 5×10 6 The cells were washed three times with PBS, resuspended in cell lysis buffer, and lysed for 2 minutes; centrifuged at 18000g and 4°C for 15 minutes to remove the precipitate, and the supernatant was the cell lysis buffer.

[0030] IP experiment: Take 50μl of His-tag antibody-coupled magnetic beads into a 1.5ml centrifuge tube, place the centrifuge tube on a magnetic stand for 2 minutes, discard the supernatant, wash 3 times with 1ml PBS, add 10 ug of recombinant antibody, and incubate at 4°C with rotation overnight. Next, place the centrifuge tube on a magnetic stand for 2 minutes, discard the supernatant, wash 3 times with 1ml PBS, add 200μl of cell lysis buffer, and incubate at 4°C with rotation for 1 hour. Next, place the centrifuge tube on a magnetic stand for 2 minutes, discard the supernatant, wash 5 times with 1ml PBS, add 20μl PBS to resuspend the precipitate, add 20μl reducing buffer to mix, incubate at 100°C for 10 minutes, centrifuge at 8000g for 1 minute, take 5μl and load it on SDS-PAGE gel. The IP experiment results are as follows: Figure 4 As shown, a single band appeared after the antibody bound to the cell lysate (AGS), indicating that the antibody specifically bound to the cell surface CDH17 protein.

[0031] (5) Flow cytometry detection of recombinant antibodies binding to tumor cells EC 50 A PBS solution with a concentration gradient of 0.1 nM to 10 μM was established for human CDH17 protein. 0.5 μM of m01s-E4 recombinant protein was added to each gradient to make a total volume of 100 μL. After incubation at room temperature for 30 minutes, 5 × 10 5 Tumor cells AGS and MKN45 were combined and incubated at 4°C for 30 minutes. The prepared anti-His fluorescent labeled antibody (BSA dilution, 1:400) was added and incubated at 4°C for 30 minutes. Centrifugation was performed at 200g for 5 minutes, and PBSA was washed twice before detection. Flow cytometry was used to detect the binding of the recombinant antibodies to the tumor cell lines AGS and MKN45. Figure 3 As shown, the antibodies can specifically bind to tumor cells, and their EC50 are 20.44nM (A: AGS) and 24.3nM (B: MKN45), respectively.

[0032] 2. Comparison between recombinant antibody and Nb289 antibody: 0.2 μg of m01s-E4 and control antibody Nb289 were mixed with 5×10 5 Tumor cells AGS and MKN45 were combined and incubated at 4°C for 30 minutes. The prepared anti-His fluorescent labeled antibody (BSA dilution, 1:400) was added and incubated at 4°C for 30 minutes. Centrifuged at 200g for 5 minutes, washed twice with PBSA, and tested on the microscope. Figure 5 This is a flow cytometry comparison of the recombinant antibody and the known antibody Nb289 reported in the literature on different tumor cells. A is AGS cells and B is MKN45 cells (refer to Xu X, Ding Y, Dong Y, Yuan H, XiaP, Qu C, Ma J, Wang H, Zhang X, Zhao L, Li Z, Liang Z, Wang J. Nanobody-Engineered Biohybrid Bacteria Targeting Gastrointestinal Cancers InduceRobust STING-Mediated Anti-Tumor Immunity. Adv Sci (Weinh). 2024 Aug;11(31):e2401905. doi: 10.1002 / advs.202401905. Epub 2024 Jun 18. PMID: 38888519;PMCID: PMC11336900.). The results showed that the antibody had better binding activity and is expected to become a new generation of candidate antibodies for tumor treatment and detection.

Claims

1. A recombinant C-type single-domain nanobody that binds to CDH17, characterized in that: The antibody is based on the CH2 domain mutant m01s of the human antibody IgG constant region as the skeleton, and comprises three complementary determining regions CDR1-IMGT, CDR2-IMGT and CDR3-IMGT, and the amino acid sequences of the three complementary determining regions CDR1-IMGT, CDR2-IMGT and CDR3-IMGT are shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, respectively.

2. The recombinant C-type single-domain nanobody binding to CDH17 according to claim 1, characterized in that The amino acid sequence of the antibody is shown in SEQ ID No.

1.

3. Use of the recombinant C-type single-domain nanobody binding to CDH17 according to any one of claims 1 or 2 in the preparation of diagnostic reagents and therapeutic drugs for cancer.

4. The use of the recombinant C-type single-domain nanobody binding to CDH17 according to claim 3 in the preparation of diagnostic reagents and therapeutic drugs for cancer, characterized in that: The cancer is gastric cancer, esophageal cancer, pancreatic cancer, colorectal cancer or breast cancer.

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