Anti-human CD117 nanobody and use thereof

Abstract

Provided are an anti-human CD117 nanobody and use thereof. The nanobody comprises at least one VHH chain. The VHH chain comprises a CDR1, a CDR2, and a CDR3. The amino acid sequence of the CDR1 is set forth in SEQ ID NO: 4, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 5, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 6; or the amino acid sequence of the CDR1 is set forth in SEQ ID NO: 7, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 8, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 9; or the amino acid sequence of the CDR1 is set forth in SEQ ID NO: 10, the amino acid sequence of the CDR2 is set forth in SEQ ID NO: 11, and the amino acid sequence of the CDR3 is set forth in SEQ ID NO: 12. The use is use of the nanobody and a formulation thereof in the preparation of a drug for treating thalassemia. The nanobody has a good binding ability to CD117, and has the advantages of small molecular weight, high binding activity, low immunogenicity, easy modification, etc.

Description

A nanobody against human CD117 and its application Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an anti-human CD117 nanobody and its application. Background Technology

[0002] Thalassemia syndrome (also known as thalassemia) is a single-gene inherited hemoglobin disorder caused by a defect in the globin gene. It is characterized by impaired or absent production of one globin chain in adult hemoglobin, leading to an imbalance in the ratio of α-globin to β-globin. Unbound free α-globin chains precipitate in erythrocyte precursors, resulting in ineffective erythropoiesis or premature death of erythrocyte precursors. The main manifestations include chronic hemolytic anemia, decreased oxygen-carrying capacity, and iron overload. Based on the type of peptide chain damage, thalassemia can be classified into α, β, δ, and δβ types, with α-thalassemia and β-thalassemia being the most common. Based on the degree of clinical transfusion, it can be divided into transfusion-dependent and transfusion-independent thalassemia. Clinical symptoms can present as mild, moderate, or severe. Thalassemia not only affects the patient's quality of life but also places a heavy economic burden on their family.

[0003] Current treatments for thalassemia include blood transfusions combined with iron chelation therapy, drug therapy, hematopoietic stem cell transplantation, gene therapy, and hepcidin-related therapies. Blood transfusions combined with iron chelation therapy are a common treatment for thalassemia, but this approach has limitations. Long-term transfusions can lead to iron overload and carry the risk of infectious diseases. Drug therapy includes both Western and traditional Chinese medicine. Commonly used Western medicines include hydroxyurea, thalidomide, butyrates, and erythrocyte maturation agents; however, follow-up studies have revealed various risks associated with these drugs. For example, long-term use of hydroxyurea carries a risk of cancer, and thalidomide poses a teratogenic risk to infants. Therefore, the safety and efficacy of these Western medicines in treating thalassemia require further investigation. Regarding traditional Chinese medicine, studies have reported that a formula using Codonopsis pilosula, Astragalus membranaceus, tortoise shell, and Angelica sinensis to replenish qi, nourish essence, and generate blood can significantly increase Hb and HbF levels in thalassemia patients and improve anemia symptoms. However, the mechanisms, treatment courses, dosages, and safety of traditional Chinese medicine in treating thalassemia require further in-depth research. Hepcidin is a key regulator of iron homeostasis produced by hepatocytes and regulates intestinal iron absorption, and is considered a promising new direction for thalassemia treatment. However, research on hepcidin has primarily been conducted in mice, and more data is needed to support its clinical application in thalassemia treatment. With the deepening of research in recent years, clinical treatment of thalassemia has made some progress in gene therapy and hematopoietic stem cell transplantation (HSCT). The main goal of gene therapy is to transfer normal HBB gene copies or reconstruct γ-globin expression, thereby reconstructing HbF. Currently, clinical applications mainly utilize CRISPR / Cas9 technology, and CTX001, an investigational autologous cell therapy already in clinical trials, is another example. In 2019, Zynteglo, the world's first approved lentiviral gene therapy for the treatment of β-thalassemia, received EU approval for marketing. However, it was halted in February 2021 due to two patients being diagnosed with acute myeloid leukemia and myelodysplastic syndrome. The reason for this is currently unclear, but this does not negate the therapy; on the contrary, this case highlights the necessity of choosing safer and more effective vectors and candidate antibodies. Currently, the only treatment for severe β-thalassemia is hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation can restore normal hematopoietic function in patients to achieve the goal of treatment. However, this treatment is expensive and bone marrow matching is difficult. Therefore, a more reasonable approach is needed to improve the efficiency of hematopoietic stem cell transplantation, thereby reducing treatment costs and solving the problem of bone marrow matching.

[0004] The primary challenge in hematopoietic stem cell transplantation is obtaining large quantities of purified hematopoietic stem cells to remove interference from impurity cells. Therefore, eliminating or enriching specific cells in the graft is a current focus in the transplantation field. Since HSCs lack distinct morphological characteristics, primarily appearing as lymphocyte-like mononuclear blasts, they can only be identified by certain proteins on their cell surface.

[0005] CD117 (also known as c-Kit or stem cell factor receptor [SCFR]) is an ancient membrane receptor tyrosine kinase (RTK) that plays a crucial role in regulating the self-renewal, survival, proliferation, and differentiation of hematopoietic stem cells, as well as the overall stemness of progenitor cells. Nanobodies, first reported by Belgian scientists in Nature in 1993, offer numerous advantages over traditional antibodies. The unique structure of VHH single-domain antibodies based on alpaca heavy chain antibodies combines the advantages of both traditional antibodies and small molecule drugs, almost perfectly overcoming the drawbacks of traditional antibodies such as long development cycles, low stability, and stringent storage conditions. They are gradually becoming an emerging force in next-generation therapeutic biomedical and clinical diagnostic reagents. Compared to conventional antibodies, nanobodies offer advantages such as: small molecular weight, enabling them to penetrate the blood-brain barrier; high expression in prokaryotic or eukaryotic systems; high specificity and affinity; ease of engineering modification; and low immunogenicity in humans. The development of nanobodies primarily employs phage display technology. This biotechnology inserts the DNA sequence of a foreign protein or peptide into an appropriate position in the structural gene of the phage coat protein, allowing the foreign gene to be expressed along with the coat protein. Simultaneously, the foreign protein is displayed on the phage surface as the phage reassembles. Conventional antibody development often uses recombinant antigens for multiple biopanning and enrichment to obtain positive clones. However, recombinant antigens cannot guarantee the conformation of the natural antigen, resulting in antibodies that may not recognize cell surface antigens, thus hindering their application and transformation. Therefore, this invention utilizes a constructed stable cell line overexpressing CD117 for antibody development, ensuring that the obtained candidate antibodies specifically recognize cell surface antigens, thereby improving the sorting efficiency of hematopoietic stem cells and playing an important role in the treatment of thalassemia.

[0006] Currently, most CD117 antibodies are obtained through hybridoma technology and are mostly traditional monoclonal antibodies. Traditional antibody production is costly, and commercially available antibodies have complex compositions and unknown sequences, which hinders subsequent modification and further applications. Currently, CD117 antibodies under development can perform various biological functions, such as immune regulation, anti-CD117 immunotoxin / antibody-drug conjugates, bispecific T cell conjugation and activation antibodies, and anti-CD117 CAR-T cells. However, most of these are still early-stage studies based on murine antibodies, and immunogenicity issues may exist for future clinical applications. Summary of the Invention

[0007] To overcome the high production cost and potential immunogenicity issues of traditional antibodies in existing technologies, this invention provides an anti-human CD117 nanobody and its applications. The nanobody exhibits good binding ability and specificity to CD117, with a half-maximal effective concentration (EC50) reaching the nM level.

[0008] To address the aforementioned technical problems, one of the technical solutions provided by this invention is: a nanobody targeting human CD117, which comprises at least one VHH chain, wherein the VHH chain includes CDR1, CDR2, and CDR3.

[0009] The amino acid sequence of CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 6.

[0010] Alternatively, the amino acid sequence of CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9;

[0011] Alternatively, the amino acid sequence of CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 12.

[0012] In some embodiments, the VHH chain in the nanobody targeting human CD117 comprises FR1, FR2, FR3, and FR4.

[0013] The amino acid sequence of FR1 is shown in SEQ ID NO: 13, the amino acid sequence of FR2 is shown in SEQ ID NO: 14, the amino acid sequence of FR3 is shown in SEQ ID NO: 15, and the amino acid sequence of FR4 is shown in SEQ ID NO: 16.

[0014] Alternatively, the amino acid sequence of FR1 is shown in SEQ ID NO: 17, the amino acid sequence of FR2 is shown in SEQ ID NO: 18, the amino acid sequence of FR3 is shown in SEQ ID NO: 19, and the amino acid sequence of FR4 is shown in SEQ ID NO: 20.

[0015] Alternatively, the amino acid sequence of FR1 is shown in SEQ ID NO: 17, the amino acid sequence of FR2 is shown in SEQ ID NO: 21, the amino acid sequence of FR3 is shown in SEQ ID NO: 22, and the amino acid sequence of FR4 is shown in SEQ ID NO: 20.

[0016] In some embodiments, the VHH chain of the nanobody targeting human CD117 comprises an amino acid sequence or a variant thereof as shown in any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3;

[0017] The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence from which it originates.

[0018] In some embodiments, the nanobody targeting human CD117 further comprises the Fc region of an immunoglobulin; in this case, the nanobody is also referred to as a heavy chain antibody.

[0019] In some specific implementations, the Fc region includes at least one of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD, and IgE constant regions.

[0020] In some specific implementations, the Fc region is the Fc region of human IgG4, and its amino acid sequence is shown in SEQ ID NO: 23.

[0021] The second technical solution provided by the present invention is: an isolated nucleic acid that encodes a nanobody as described in the first technical solution.

[0022] The third technical solution provided by the present invention is: a recombinant expression vector comprising the isolated nucleic acid as described in the second technical solution.

[0023] In some implementations, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector.

[0024] The fourth technical solution provided by the present invention is: a host cell, wherein the host cell contains the nucleic acid or the recombinant expression vector described in the claims.

[0025] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0026] In some specific implementations, the eukaryotic cells are yeast cells or mammalian cells; wherein, the mammalian cells are, for example, HEK293 cells.

[0027] The fifth technical solution provided by this invention is: a method for preparing a nanobody targeting human CD117, the method comprising the following steps:

[0028] The host cells were cultured under suitable growth and fermentation conditions to obtain nanobodies against human CD117 from the culture.

[0029] The sixth technical solution provided by the present invention is: a multispecific antibody, comprising the aforementioned nanobody targeting human CD117, and an antigen-binding molecule having another antigen-binding property operably linked to the aforementioned nanobody targeting human CD117.

[0030] The seventh technical solution provided by the present invention is: a pharmaceutical composition comprising, as described in any one of the technical solutions of the present invention, a nanobody targeting human CD117, and a pharmaceutically acceptable carrier.

[0031] In some embodiments, the pharmaceutical composition further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0032] The eighth technical solution provided by the present invention is the application of the nanobody targeting human CD117 described in technical solution one, the nucleic acid described in technical solution two, the recombinant expression vector described in technical solution three, the host cell described in technical solution four, the multispecific antibody described in technical solution six, or the pharmaceutical composition described in technical solution seven in the preparation of a drug for treating thalassemia.

[0033] The ninth technical solution provided by the present invention is: a kit comprising one or more of the following: a nanobody targeting human CD117 as described in any one of the technical solutions, a nucleic acid as described in the second technical solution, a recombinant expression vector as described in the third technical solution, a host cell as described in the fourth technical solution, a multispecific antibody as described in the sixth technical solution, or a pharmaceutical composition as described in the seventh technical solution.

[0034] In some embodiments, the kit further includes (i) a means of administering the antibody or pharmaceutical composition; and / or (ii) instructions for use.

[0035] The tenth technical solution provided by the present invention is: a medicine box containing a nanobody targeting human CD117 as described in any one of the technical solutions, a multispecific antibody as described in technical solution 6, or a pharmaceutical composition as described in technical solution 7.

[0036] The eleventh technical solution provided by the present invention is: a method for immunoassay or determination of CD117, comprising mixing a sample to be tested with a nanobody targeting human CD117 as described in any one of the technical solutions, a multispecific antibody as described in technical solution 6, or a pharmaceutical composition as described in technical solution 7.

[0037] In some implementations, the detection is for non-diagnostic purposes.

[0038] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0039] The reagents and raw materials used in this invention are all commercially available.

[0040] The positive and progressive effects of this invention are as follows:

[0041] 1) The antibody type of this invention is a nanobody. A nanobody (Nb) is a novel type of small antibody derived from the variable domain VHH of a heavy chain antibody naturally lacking a light chain in camel-like animals. Due to the natural absence of a light chain, it has a small molecular weight and can be amplified and expressed in large quantities using prokaryotic and yeast systems, resulting in relatively lower production costs.

[0042] 2) The nanobody of the present invention is an alpaca anti-human CD117 nanobody. The VHH sequence has high homology with the fully human antibody sequence, low immunogenicity, and the nanobody is easy to modify, which can meet the needs of different scenarios.

[0043] 3) The nanobody of this invention can be applied to research on magnetic bead sorting of antibodies or targeted delivery of antibodies, providing more options for hematopoietic stem-based treatment options.

[0044] 4) The half-maximal effective concentration of the nanobody of the present invention can reach the nM level, and it has a good binding effect with human CD117. Attached Figure Description

[0045] Figure 1 shows the plasma titer of CD117 before and after immunization using ELISA. The lower curve represents the pre-immunization state, and the upper curve represents the post-immunization state.

[0046] Figure 2 shows the clone ELISA screening of CD117 positive clones, with the lower curve representing the control group and the upper curve representing the experimental group.

[0047] Figure 3 shows the CD117 binding activity of candidate nanobodies detected by ELISA.

[0048] Figure 4 shows the specificity of the candidate nanobody as verified by Western blotting.

[0049] Figure 5 shows the FACS validation of the candidate nanobody's specific recognition of the CD117 target on the cell surface. Detailed Implementation

[0050] The present invention will be further illustrated by way of embodiments below, but the present invention is not limited to the scope of the embodiments.

[0051] Example 1: Construction of an Immunized Alpaca and Phage Display Library

[0052] Alpaca immunization: 200 μg of human CD117 protein (ACRO) was used for immunization, with immunizations every 2 weeks. Blood was collected before each immunization for plasma titer testing, until the plasma titer reached 1:10. 5 After immunization, approximately 30 ml of peripheral blood was collected for subsequent bank construction. The results are shown in Figure 1.

[0053] Example 2: Cell-phage screening of candidate nanobodies

[0054] 2.1 Removal of non-specific phages: Take phages (2 x 10⁻⁶) 11 Add pfu) to 1 ml MPBS (PBS containing 2% skim milk), incubate at 4°C with shaking for 1 hour; Negative cells (cells that do not express CD117, CD117-cells) 3 x 10 6 Each cell was resuspended in 5 ml of MPBS and incubated at 4°C for 1 hour by rotation; the cell suspension was centrifuged at 300 g at 4°C for 5 min, and the supernatant was discarded; phage solution was added and incubated at 4°C by rotation for 2 hours; the CD117-cell and phage mixture was centrifuged at 300 g at 4°C for 5 min, and the phage supernatant was transferred to a new EP tube.

[0055] 2.2 Specific phage enrichment: 3 x 10 cells overexpressing CD117 (CD117+ cells) were collected. 6 Resuspend each cell in 5 ml of MPBS and incubate at 4°C for 1 hour by rotation; centrifuge the cell suspension at 300 g at 4°C for 5 min and discard the supernatant; add the supernatant from step 2.1 and incubate at 4°C by rotation for 2 hours; centrifuge the CD117+ cell and phage mixture at 300 g at 4°C for 5 min; discard the supernatant, resuspend the precipitate in 1 ml of PBS, centrifuge at 300 g at 4°C for 5 min, discard the supernatant, and repeat the washing steps 5-10 times.

[0056] 2.3 Antigen-specific phage elution: Add 10 μg / mL trypsin solution to the precipitate from 2.2, resuspend the cells, and incubate at room temperature with rotation for 30 min; add 500 μL PBS, centrifuge at 300g, 4℃ for 5 min, and collect the supernatant into a new EP tube. Infect E. coli TG1 with the eluted phage for amplification and rescue, and harvest secondary phages; perform the next round of panning as in 2.1-2.3, and after 3-5 rounds of panning, enrich positive phages. Randomly select clones and perform clone ELISA screening as shown in Figure 2 to obtain positive clones that bind to CD117. Sequencing is used to obtain the sequence information of the positive antibody (see Tables 1-3).

[0057] Table 1: Complementarity-determining region (CDR) sequence of exemplary anti-human CD117 nanobody of the present invention

[0058] Table 2: Framework Region (FR) Sequence of Exemplary Anti-Human CD117 Nanobody of the Present Invention

[0059] Table 3: VHH sequence of exemplary anti-human CD117 nanobody of the present invention

[0060] Example 3: ELISA verification of the binding activity of candidate nanobodies to CD117

[0061] Coat an ELISA plate with 50 ng of CD117 recombinant antigen and incubate overnight at 4°C. The next day, remove the ELISA plate, discard the supernatant, and wash three times with PBST (0.05% Tween-20). Add 100 μl of blocking buffer to each well, incubate at room temperature for 2 h, and wash three times with PBST. Dilute the prepared positive nanobody protein 2-5 times with blocking buffer to 6-8 concentration gradients, repeating each gradient in 3 wells (50 μl per well), incubate at room temperature for 1 h, discard the supernatant, and wash three times with PBST. Add 50 μl of 3000-fold diluted Anti-HA tag secondary antibody, incubate at room temperature for 45 min, discard the supernatant, and wash three times with PBST. Add 100 μl of TMB chromogenic solution (abcam), develop for 10 min, add an equal volume of TMB stop buffer (abcam) to stop the development, read the OD450 value, and calculate the binding activity of the positive nanobody and CD117. As shown in Figure 3, the four nanoantibodies showed good binding ability to CD117, with half-maximal effective concentrations (EC50) reaching at least nM.

[0062] Example 4: Western blot verification of antigen specificity of candidate nanobodies

[0063] Culture 293T cells, CD117+ cells, and CD34+ cells to the logarithmic growth phase, and wash once with physiological saline or serum-free culture medium. Add 100-200 μL of lysis buffer to each well to ensure complete cell lysis. Centrifuge at 10000-14000g for 3-5 minutes and collect the supernatant. Take 20 μL of the supernatant, add 5 μL of 5x native loading solution, and run SDS-PAGE gel at 100V for 1.5 hours. After gel running, perform transfer. Take out the PVDF membrane, activate it with methanol, and then soak it in transfer equilibration buffer for about 1 minute before transfer. Remove the clamp and place the membrane on the positive electrode in the following order: dry sponge sheet + electrophoresis gel + PVDF membrane + dry sponge sheet. Place the clamp and place the membrane in the transfer apparatus for transfer, which takes about 16 minutes. Wash the membrane 5 times with PBST for 5 minutes each time, add 5% skim milk powder, and block overnight at 4°C. The membrane was washed 5 times with PBST, 5 min each time. Primary antibody was prepared with 5% skim milk at a concentration of 2.5 μg / ml, 10 ml per antibody, and incubated at room temperature with shaking for 3.5 h. The membrane was then washed 5 times with PBST, 5 min each time. Anti-HA tag secondary antibody diluted 1:2000 was added, and the membrane was incubated at room temperature with shaking for 1.5 h. The membrane was then washed 5 times with PBST, 5 min each time, and developed with ECL chromogenic buffer. As shown in Figure 4, all four candidate nanobodies exhibited good specificity, as the lysate after treatment with CD117+ cells showed single bands.

[0064] Example 5: Determination of the affinity constant between nanobodies and antigens using the SPR method.

[0065] The CD117 recombinant antigen was conjugated to a CM5 chip. The antibody protein was diluted with HBS-P buffer, and the affinity of the candidate antibodies was detected using the SPR method. For each concentration of antibody sample, the injection time was 120 s, the dissociation time was 300 s, and the flow rate was 30 μl / min. Regeneration was performed with 10 mM pH 2.0 Glycine, and the cycle was repeated until all concentration gradients were injected. After the program ran, the built-in analysis program of the Biacore T200 (GE) instrument was used for fitting analysis to obtain the antibody affinity constants to the antigen (Table 4). The results showed that all three candidate nanobodies had high affinity for the CD117 antigen, reaching the sub-nanomo level.

[0066] Table 4 Affinity constants of CD117 nanobodies and bivalent antibodies

[0067] Example 6: Predicting Nanobody Binding Epitopes Using SPR Method

[0068] To further confirm the CD117 epitopes recognized by these three candidate antibodies, a competitive SPR assay was performed. The CD117 antigen was conjugated to a CM5 chip, and the candidate antibodies were prepared at 200 nM using HBS-P buffer. Each antibody was first subjected to antigen saturation testing, and the saturation time and response value were recorded. Then, competitive SPR detection was performed, and the relationship between the epitopes recognized by the two antibodies was determined by observing the change trends of the response values ​​and real-time curves. If the two antibodies recognize different antigen epitopes, the response values ​​of both antibodies are consistent with the response values ​​of a single antibody saturated. If the recognized epitopes are competing / same, then after antibody 1 saturates the epitope, antibody 2 cannot bind to the antigen, causing the response value of antibody 2 to decrease compared to a single antibody loading. The degree of decrease was used to determine the competition. The test results showed that the three CD117 nanobodies recognize at least two types of CD117 epitopes, with CD117-23 and CD117-66 representing one type of epitope and antibody CD117-93 representing another.

[0069] Table 5 Response values ​​when antibody is saturated alone

[0070] Table 6 Response values ​​during antibody competitive binding

[0071] Example 7: Flow Cytometry Validation of Candidate Antibody Binding Activity

[0072] To verify whether the candidate antibody could bind to the CD117 target on the cell surface, Junkat cells cultured to the logarithmic growth phase and CD117+-overexpressing Junkat cells were used. Cells were digested with trypsin, washed twice with 0.02% BSA / DPBS, and incubated at 500g for 5 min / time. Cells were then collected. Cells were resuspended in 1% BSA / DPBS and blocked at 4℃ for 30 min. Cells were washed three times with 0.02% BSA / DPBS, and incubated at 500g for 5 min / time. Cells were resuspended in 1% BSA / DPBS and the density was adjusted to 5 x 10⁶ cells / ml. 100 μL of cells (5 x 10⁵ cells) were taken, and 1 μg of antibody protein was added. The cells were incubated at 4℃ for 1 h. Cells were washed three times with 0.02% BSA / DPBS, and incubated at 500g for 5 min / time. Fluorescently labeled secondary antibody (Invitrogen) was added according to the manufacturer's instructions, and the cells were incubated at 4℃ for 20-30 min. Cells were washed three times with 0.02% BSA / DPBS, and incubated at 500g for 5 min / time. Cells were resuspended in 200 μL of DPBS, passed through a sieve, and analyzed by flow cytometry. Figure 5 shows that all three candidate antibodies exhibited good cell-binding activity.

[0073] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples. Any changes or modifications to these embodiments without departing from the principles and essence of the present invention are within the scope of protection of the present invention.

Claims

1. A nanobody targeting human CD117, characterized in that, The antibody contains at least one VHH chain, and the VHH chain contains CDR1, CDR2, and CDR3. The amino acid sequence of CDR1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

6. Alternatively, the amino acid sequence of CDR1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR2 is shown in SEQ ID NO: 8, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9; Alternatively, the amino acid sequence of CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

12.

2. [Amended according to Rule 26, 27.12.2024] The nanobody targeting human CD117 according to claim 1 is characterized in that, The VHH chain includes FR1, FR2, FR3, and FR4. The amino acid sequence of FR1 is shown in SEQ ID NO:13, the amino acid sequence of FR2 is shown in SEQ ID NO:14, the amino acid sequence of FR3 is shown in SEQ ID NO:15, and the amino acid sequence of FR4 is shown in SEQ ID NO:

16. Alternatively, the amino acid sequence of FR1 is shown in SEQ ID NO:17, the amino acid sequence of FR2 is shown in SEQ ID NO:18, the amino acid sequence of FR3 is shown in SEQ ID NO:19, and the amino acid sequence of FR4 is shown in SEQ ID NO:

20. Alternatively, the amino acid sequence of FR1 is shown in SEQ ID NO:17, the amino acid sequence of FR2 is shown in SEQ ID NO:21, the amino acid sequence of FR3 is shown in SEQ ID NO:22, and the amino acid sequence of FR4 is shown in SEQ ID NO:

20.

3. The nanobody targeting human CD117 according to claim 1 or 2, characterized in that, The VHH chain comprises an amino acid sequence or a variant thereof as shown in any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3; The variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the sequence from which it originates.

4. The nanobody targeting human CD117 according to claim 3, characterized in that, It also includes the Fc region of immunoglobulins; the Fc region includes at least one of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE constant regions; Preferably, the Fc region is the Fc region of human IgG4, and its amino acid sequence is shown in SEQ ID NO:

23.

5. An isolated nucleic acid, characterized in that, The nucleic acid encodes the nanobody targeting human CD117 as described in any one of claims 1 to 4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 5; Preferably, the recombinant expression vector is a plasmid, granule, bacteriophage, or viral vector; the backbone of the plasmid is, for example, pcDNA3.

4.

7. A host cell, characterized in that, The host cell comprises the nucleic acid of claim 5 or the recombinant expression vector of claim 6; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; More preferably, the eukaryotic cell is a yeast cell or a mammalian cell; wherein the mammalian cell is, for example, a HEK293 cell.

8. A method for preparing a nanobody targeting human CD117, characterized in that, The method includes the following steps: The host cells are cultured under conditions suitable for host cell growth and fermentation as described in claim 7 to obtain nanobodies against human CD117 from the culture.

9. A multispecific antibody, characterized in that, It includes the nanobody targeting human CD117 as described in any one of claims 1 to 4, and an antigen-binding molecule having another antigen-binding property operatively linked to the nanobody targeting human CD117.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the nanobody targeting human CD117 as described in any one of claims 1 to 4, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises one or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

11. The use of the nanobody targeting human CD117 according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the host cell according to claim 7, the multispecific antibody according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating thalassemia.

12. A reagent kit, characterized in that, The kit comprises one or more of the following: the nanobody targeting human CD117 according to any one of claims 1 to 4, the nucleic acid according to claim 5, the recombinant expression vector according to claim 6, the host cell according to claim 7, the multispecific antibody according to claim 9, or the pharmaceutical composition according to claim 10; Preferably, the kit further includes (i) a means for administering the antibody or pharmaceutical composition; and / or (ii) instructions for use.

13. A medicine box, characterized in that, The kit contains a nanobody targeting human CD117 as described in any one of claims 1 to 4, a multispecific antibody as described in claim 9, or a pharmaceutical composition as described in claim 10.

14. A method for immunoassay or determination of CD117, characterized in that, The method includes mixing the sample to be tested with the nanobody targeting human CD117 as described in any one of claims 1 to 4, the multispecific antibody as described in claim 9, or the pharmaceutical composition as described in claim 10; Preferably, the detection is for non-diagnostic purposes.

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