Anti-CD38 nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of antibody preparation, and particularly relates to an anti-CD38 nano antibody as well as a preparation method and application thereof. The method comprises the following steps: constructing and expressing CD38 antibody protein, immunizing alpaca, obtaining immune alpaca PBMC (peripheral blood mononuclear cells), extracting RNA (ribonucleic acid) of the cells, carrying out reverse transcription to obtain cDNA (complementary deoxyribonucleic acid), carrying out PCR (polymerase chain reaction) to obtain antibody gene segments, constructing the antibody gene segments on a phage expression vector to construct a CD38 immune antibody library, carrying out two rounds of panning, carrying out Clone ELISA (enzyme-linked immuno sorbent assay) detection on clones to obtain an antibody combined with the CD38 protein, carrying out sequencing analysis, and selecting a unique sequence. High-throughput expression is carried out through a mammalian cell high-throughput expression system, antibody protein is obtained, FACS detection is carried out through a CD38 high-expression cell line, and finally the antibody combined with CD38 is obtained. The method is simple and rapid to operate, a mammalian cell expression system induces efficient expression of the antibody, processing modification can be performed after translation, and the activity of the antibody is closer to that of a natural antibody.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody preparation, and specifically relates to an anti-CD38 nano antibody and a preparation method and application thereof. Background Art

[0002] Nanobodies, also known as single-domain antibodies and VHH antibodies, are heavy chain antibodies that are naturally devoid of light chains and are found in camelids such as alpacas and cartilaginous fish. They include two constant regions (CH2 and CH3), a hinge region, and a heavy chain variable region VHH. Nanobodies have a molecular weight of only 10% of traditional antibodies and retain the complete antigen-binding ability of HCAbs. They have strong specificity, good affinity, high stability, strong targeting and tissue penetration, low immunogenicity, and are easy to humanize. They are widely used in research fields such as molecular imaging and diagnosis, drug delivery, tumor immunotherapy, and cell therapy. It is worth mentioning that nanobodies have shown excellent application value and broad prospects in central nervous system diseases, circulatory system diseases, infectious diseases, tumors, and inflammatory diseases. Their continuous research and development has brought new hope to patients.

[0003] CD38 is a single-chain, transmembrane type II glycoprotein, 45 kDa in size, consisting of a short cytoplasmic tail, a single transmembrane domain, and a longer extracellular C-terminal domain. CD38 is a bifunctional enzyme with both NADase and cyclase activities, regulating cell recruitment, cytokine release, and NAD availability. It plays a key role in the inflammatory process during autoimmunity. CD38 is expressed not only in human immune cells such as lymphocytes and plasma cells, but also aberrantly in various tumor cells, closely implicated in tumor development and progression. CD38 is expressed in primary tumor cells such as B cells, T cells, and high-grade B cell lymphomas, as well as in malignant plasma cell neoplasms such as MGUS, MM, extramedullary plasmacytomas, and plasmacytoma. Within the bone marrow, CD38 expression inhibits cell growth and induces apoptosis in human B cell precursors. In immature human B cell lines, CD38 inhibits cell proliferation and induces apoptosis in heavy chain remodeling cells by activating spleen tyrosine kinase and phosphatidylinositol 3-kinase signaling pathways. CD38 and its ligand CD31 interact with each other, functioning as cell adhesion molecules and regulating the flow of calcium ions in the cytoplasm. They can also regulate the migration of leukemia cells and hematological malignant tumor cells such as CD38+MM through the endothelial cell wall. Therefore, CD38 immunotherapy has opened up a new research direction for the treatment of multiple myeloma (MM), especially for relapsed / refractory MM. Both Sanofi's Sarclisa and Johnson & Johnson's Darzalex are CD38 inhibitors. Darzalex is the world's first drug targeting CD38, and it is far ahead in the number of indications and sales. Daratumumab is the world's first CD38 monoclonal antibody approved for the treatment of multiple myeloma. So far, the drug has been approved for multiple indications. In March 2020, the U.S. FDA approved the CD38 antibody (isatuximab-irfc) developed by Sanofi for marketing in combination with pomalidomide and dexamethasone for the treatment of adult patients with multiple myeloma (MM). MOR202 is a fully human anti-CD38 monoclonal antibody. In addition to its ADCC mechanism of action, the antibody can also induce killing of myeloma cells in MM patients through ADCP. In 1994, Goldmacher et al. conjugated ricin toxin molecules to the anti-CD38 monoclonal antibody HB7 to form an immunotoxin. This conjugated antibody can effectively kill human CD38+ MM cells, lymphoma cells, and other primary tumor-derived tumor cells in vitro. The AMG 424 bispecific antibody was jointly developed by Amgen and Xencor, targeting CD38 and CD3, for the treatment of multiple myeloma (MM).CD38 is one of the most likely effective targets for CAR-T therapy in the treatment of MM. The research results of Mihara et al. demonstrated for the first time that CD38-specific CAR-T has a therapeutic effect on CD38+ hematological malignancies both in vitro and in vivo. CD38-specific CAR-T can effectively eliminate myeloma cell lines and tumor cells derived from MM patients.

[0004] In aging tissues, chronic inflammation caused by senescent cells induces CD38 expression in M1 macrophages and leads to a subsequent decrease in NAD+. There is a large amount of CD38 in aging tissues. Treatment with CD38 antibodies can increase the NAD+ level in the white adipose tissue of elderly mice to a level similar to that of young mice. CD38 is a very potential target for "treating aging."

[0005] CD38 antibody technology has made significant progress in tumor therapy (especially multiple myeloma) and autoimmune diseases, but its development and application still face some shortcomings. For example, some CD38 antibodies have limited targeting and binding ability to tumor cells, and are prone to off-target effects, especially in complex microenvironments. Nanobodies, approximately 15 kDa, have a smaller size that may reduce binding to non-target cells, or reduce cross-reactivity through epitope selection optimization, making them more easily able to penetrate tissue barriers and enhance their targeting and binding ability to tumor cells. Summary of the Invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and to provide an anti-CD38 nanobody and a preparation method and application thereof.

[0007] In the first aspect of the present invention, an anti-CD38 nanobody is provided, wherein the anti-CD38 nanobody comprises a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2 and CDR3, and the amino acid sequence of the complementarity determining region is as follows:

[0008] 1) The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO.1; the amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO.2; and the amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO.3.

[0009] 2) The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 are at least 90% identical to the amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3, respectively.

[0010] Furthermore, the amino acid sequence of the anti-CD38 nanobody is shown in SEQ ID NO.4.

[0011] In a second aspect of the present invention, a nucleic acid encoding the anti-CD38 nanobody as described in the first aspect is provided.

[0012] Furthermore, the nucleic acid sequence of the anti-CD38 nanobody is shown in SEQ ID NO.5.

[0013] In a third aspect of the present invention, a vector containing the nucleic acid described in the second aspect is provided. Further, the vector is pComb3XSS.

[0014] In a fourth aspect of the present invention, a host cell containing the vector described in the third aspect is provided. Furthermore, the host cell is TG1 or SS320, and SS320 host cell is preferably selected.

[0015] In a fifth aspect of the present invention, a method for preparing an anti-CD38 nanobody as described in the first aspect is provided, comprising the following steps: constructing and expressing CD38 protein, immunizing alpacas to obtain immune alpaca PBMC cells, extracting RNA from the PBMC cells, reverse transcribing into cDNA, obtaining antibody gene fragments by PCR, and constructing them into a phage expression vector; electroporating the obtained vector carrying the antibody gene fragment into an SS320 electroporation competent medium to obtain a CD38 immune antibody library, performing two rounds of CD38 protein panning, and detecting antibodies that bind to the CD38 protein by Clone ELISA. After sequencing and analysis, unique sequences are selected, and high-throughput expression is performed by a mammalian cell high-throughput expression system to obtain antibody proteins, which are detected by FACS using a CD38 high-expressing cell line to finally obtain antibodies that bind to CD38.

[0016] In the present invention, the alpaca immunization, the primers used in the step of amplifying the antibody gene fragment, the panning method, and the construction of the CD38 high-expressing cell line are obtained after optimization in conventional operations.

[0017] In a sixth aspect of the present invention, a product or kit is provided containing the Nanobody as described in the first aspect or the biomaterial as described in the second, third or fourth aspect.

[0018] In the seventh aspect of the present invention, there is provided the use of the anti-CD38 nanobody as described in the first aspect in the preparation of drugs for treating various inflammatory injuries, autoimmune diseases, anti-aging treatments, and tumor immunotherapy.

[0019] In an eighth aspect of the present invention, there is provided use of the anti-CD38 nanobody as described in the first aspect in the preparation of a product for detecting CD38.

[0020] In the ninth aspect of the present invention, there is provided use of the anti-CD38 nanobody as described in the first aspect in the preparation of a product that binds to CD38.

[0021] The present invention has the following technical effects:

[0022] 1) The antibody of the present invention has a small molecular weight, strong specificity, a simple structure, is easy to engineer, and has the characteristic of efficiently binding to CD38.

[0023] 2) The antibody preparation method of the present invention is simple and rapid to operate; and the mammalian cell expression system induces efficient expression of antibodies, which can be processed and modified after translation, and their activity is closer to that of natural antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 RNA extraction results for alpaca PBMC.

[0025] Figure 2 These are the PCR results after two amplifications of alpaca PBMC.

[0026] Figure 3 This is the Elisa binding experiment result of antibody CD38.

[0027] Figure 4 Cellbinding experiment results of antibody CD38. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0029] The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0030] Example 1

[0031] In this embodiment, the method for preparing CD38 nanobody comprises the following steps:

[0032] 1) Based on the CD38 gene and protein sequence information (NCBI reference sequence: NW_021964157.1), the immune antigen CD38 was expressed and a His-tag was linked to its C-terminus to obtain modified amino acids for subsequent purification and detection;

[0033] 2) Immunize alpacas three times with a mixture of the modified antigen obtained in step 1 and Freund's adjuvant to obtain alpaca PBMC cells: prime the alpacas with an emulsified mixture of human CD38 / His protein and Freund's adjuvant, and boost the immunizations twice on days 21 and 42 with human CD38 / His protein and Freund's adjuvant. One week after each immunization, collect blood for human CD38 / His serum titer; one week after the third immunization, collect 50 ml of blood for screening and library construction.

[0034] 3) ELISA assay for anti-CD38 / His serum titer: Coat the plate with 2 μg / ml CD38 / His protein overnight and block with 3% BSA blocking buffer for 1 hour. Wash three times, then add 100 ml of serum obtained after three immunizations, serially diluted 2-fold, to each well and incubate at 37°C for 1.5 hours. Wash twice, then add a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-Alpaca IgG (H+L) secondary antibody to each well and incubate at 37°C for 1 hour. Wash five times, then add TMB substrate and incubate at 37°C for 5-10 minutes. The reaction is terminated by adding 0.1 M H₂SO₄ and the OD₄450 nm is measured. Antiserum titer is considered positive when the OD₄450 value of the test sample is at least three times that of the negative control (pre-immune alpaca serum). The titer of antiserum after three immunizations is 12,800. These results indicate that the antigen can induce alpacas to produce high-titer antiserum specific for CD38 protein, and that antibody library can be constructed using PBMC from 3 rabbits.

[0035] 4) Dilute the collected blood twice with PBS, separate PBMCs with Ficoll, extract RNA from PBMCs with Trizol, measure RNA concentration with nanodrop, and identify RNA quality by agarose gel electrophoresis. Figure 1 It can be seen that the RNA integrity is good.

[0036] 5) RNA was reverse transcribed into cDNA using oligo(dT), and the first and second rounds of PCR were performed using nested PCR to obtain the target gene fragment. Figure 2 The target gene fragment was cloned into a phage expression vector and electroporated into an SS320 electroporation competent cell to obtain a CD38 immune phage antibody library.

[0037] 6) Panning of the CD38 phage antibody library: The positive screening plate was coated with human CD38 / His protein in a 96-well ELISA plate; the negative screening plate was coated with 3% BSA, both at 4°C overnight. The next day, the phage library, the positive screening plate, and the negative screening plate were blocked with 3% BSA for 1 hour. The blocking buffer from the negative screening plate was removed, and the blocked phage library was transferred to the negative screening plate and incubated at room temperature for 1 hour. The blocking buffer from the positive screening plate was removed, and the phage from the negative screening plate was transferred to the positive screening plate and incubated at room temperature for 1 hour. The immunoplate was washed with 0.1% PBST, trypsinized for 10 minutes, and then eluted and collected to obtain the output phage. The output phage was used as the input phage for the second round of panning, following the same panning procedure as the first round. After two rounds of panning, the phage was enriched and infected with SS320 cells, plated, and single colonies were selected for culture.

[0038] 7) Clone ELISA: Human CD38 / His protein was coated onto a 96-well microtiter plate overnight at 4°C. The next day, the plate was blocked with 1% BSA at room temperature for 1 hour. The plate was then washed three times with 0.05% PBST. The cultured monoclonal cell suspension was centrifuged and the supernatant was added to the microtiter plate. A positive control (PC) (anti-human CD38, Daraomuab) and an isotype control were also added. The plate was incubated at room temperature for 1 hour, washed three times with 0.05% PBST, and diluted secondary antibody anti-M13 (1:10,000) was added for 40 minutes at room temperature. The plate was washed six times with 0.05% PBST, and TMB colorimetric solution was added for 5-10 minutes. The reaction was stopped by adding stop solution. The OD450 value was measured using a microtiter plate reader. Positive clones were identified if their OD450 value was more than three times that of the isotype control. Monoclonal clones that bound to the CD38 protein were selected for next-generation sequencing. These clones were then expressed in a human FC eukaryotic expression vector and purified. Three sequences were successfully expressed.

[0039] 8) Cell function assay: cell binding (FACS). The cultured CD38-overexpressing cells were prepared into a cell suspension with MACS buffer at a cell count of 3E+08 cells / ml, and 50ul / well of the cell suspension was added to a 96-well plate. The prepared antibody was diluted 4-fold with MACS buffer, and 50ul of the antibody dilution was added to the designated wells of the 96-well plate. The mixture was mixed thoroughly, and the positive control PC (Anti-Human CD38, Daraumuab) and isotype control (Isotype control) were added at the same time. The cells were incubated at 4°C in the dark for 60min. The cells were washed three times with MACS buffer, and 100ul of fluorescent secondary antibody (1:1000) was added to resuspend the cells. The cells were incubated at 4°C in the dark for 30min. The cells were washed three times with MACS buffer, and 200ul of MACS buffer was added to resuspend the cells. The cells were measured on a flow cytometer and the data were analyzed. The results showed that the three antibodies bound to the CD38-overexpressing cells.

[0040] Antibody Elisa binding test results are as follows Figure 3 As shown, the results of the antibody Cellbinding experiment are as follows Figure 4 As shown in the figure, the ELISA binding and cell binding results of CD38-12 were better than those of the positive control, demonstrating excellent antibody binding activity.

[0041] The amino acid sequence of the anti-CD38 nanobody numbered CD38-12 is shown in SEQ ID NO. 4, and the sequence of the nucleic acid encoding the anti-CD38 nanobody is shown in SEQ ID NO. 5. In the anti-CD38 nanobody with the amino acid sequence shown in SEQ ID NO: 4, the framework region and the complementary determining regions CDR1-3 are included; the complementary determining region CDR1 sequence is shown in SEQ ID NO. 1; the complementary determining region CDR2 sequence is shown in SEQ ID NO. 2; and the complementary determining region CDR3 sequence is shown in SEQ ID NO. 3.

[0042] The above are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or any direct or indirect application in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-CD38 nanobody, characterized in that The anti-CD38 nanobody includes a framework region and a complementary determining region; the complementary determining region includes CDR1, CDR2 and CDR3, and the amino acid sequence of the complementary determining region is as follows: The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO.1; The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO.2; The amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO.

3.

2. The anti-CD38 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-CD38 nanobody is shown in SEQ ID NO.

4.

3. A nucleic acid encoding the anti-CD38 nanobody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid sequence of the anti-CD38 nanobody is shown in SEQ ID NO.

5.

5. A vector containing the nucleic acid according to claim 3.

6. A host cell containing the vector according to claim 5.

7. The method for preparing the anti-CD38 nanobody according to claim 2, wherein: The method comprises the following steps: constructing a CD38 protein expression system, immunizing alpacas to obtain immune alpaca PBMC cells, extracting RNA from the PBMC cells, reverse transcribing the RNA into cDNA, obtaining antibody gene fragments through PCR and constructing the fragments into a phage expression vector, electroporating the obtained vector carrying the antibody gene fragments into an SS320 competent cell to obtain a CD38 immune antibody library, performing two rounds of CD38 protein panning and CloneELISA detection to obtain antibodies binding to the CD38 protein, selecting unique sequences after sequencing analysis, performing high-throughput expression through a mammalian cell high-throughput expression system to obtain antibody proteins, performing FACS detection using a CD38 high-expressing cell line, and finally obtaining antibodies binding to CD38.

8. A product or kit comprising the anti-CD38 nanobody according to claim 1 or 2 or the biomaterial according to any one of claims 4 to 6.

9. Use of the anti-CD38 nanobody according to claim 1 or 2 in the preparation of drugs for treating various inflammatory injuries, autoimmune diseases, tumor immunotherapy and anti-aging treatment drugs.

10. Use of the anti-CD38 nanobody according to claim 1 or 2 in the preparation of a product for detecting CD38.

11. Use of the anti-CD38 nanobody according to claim 1 or 2 in the preparation of a product that binds to CD38.

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