Nanometer antibody targeting CD117 and application thereof

Abstract

The invention relates to a nano antibody targeting CD117 and application of the nano antibody. The invention also relates to a CD117-targeting antibody or an antigen binding fragment thereof and an application of the CD117-targeting antibody or the antigen binding fragment thereof.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically relates to a nanobody targeting CD117 and its applications. Background Art

[0002] Hematopoietic stem cells (HSCs) are the foundational cells of the blood and immune systems, possessing the ability to self-renew and differentiate into all blood cell lineages. Clinically, bone marrow ablation centered around HSCs is a curative therapy for treating a variety of blood diseases, including leukemia, lymphoma, and severe hereditary blood diseases such as β-thalassemia and sickle cell disease. In gene therapy, HSCs can serve as a breakthrough point for curing diseases. A patient's own HSCs can be genetically corrected in vitro and then returned to the body to reconstruct a healthy blood system, as demonstrated in emerging genome editing therapies for hemoglobinopathies. However, HSC-based therapies face numerous challenges, such as limited donors, the risk of graft-versus-host disease in allogeneic transplantation, and the complexity of in vitro HSC manipulation. Therefore, developing systems that can directly target and deliver to modify or eliminate HSCs in patients in vivo is of great significance, which may make transplantation and gene therapy safer and more accessible.

[0003] CD117, also known as c-Kit, is a transmembrane receptor tyrosine kinase that can bind to stem cell factor (SCF). CD117 is crucial for the biological functions of hematopoietic stem cells (HSCs): the interaction between SCF and c-Kit can promote the survival, proliferation, and differentiation of HSCs, and CD117 is widely used as a marker to define HSCs and early progenitors. Almost all multipotent HSCs and progenitors highly express CD117. Given its key role, CD117 has been a target in HSC-based therapies. For example, monoclonal antibodies against CD117 can block SCF signaling and deplete HSCs; this approach is being developed as a non-genotoxic conditioning regimen for clearing a patient's bone marrow before transplantation. Anti-CD117 antibody therapies (including antibody-drug conjugates, i.e., ADCs) have shown efficacy in preclinical models in promoting donor cell engraftment by selectively clearing HSCs, while avoiding chemotherapy or radiotherapy conditioning. Beyond the transplantation field, CD117 is also expressed on certain leukemic or myelodysplastic stem cells, and anti-CD117 immunotherapy is being explored for clearing these diseased HSCs. In summary, CD117 is a key identifier and functional regulator of HSCs, making them an attractive target for directly delivering therapies to the HSC compartment.

[0004] Existing antibody - targeting strategies against hematopoietic stem cells (HSCs) and their markers, while showing some promise, also have significant limitations. Traditional full - length monoclonal antibodies (usually IgG) have a relatively large molecular weight (about 150 kDa), which may limit their tissue - penetrating ability (e.g., difficult to enter the bone marrow microenvironment) and have a relatively long circulation time in the body. Although IgG antibodies or antibody - drug conjugates (ADCs) against HSC markers (such as CD117) may be effective in targeting HSCs, their size and structure are not suitable for some applications, such as delivering gene - therapy payloads intracellularly. Standard antibodies generally do not internalize efficiently into cells unless engineered to do so, and their Fc regions may trigger immune effector functions (complement activation, ADCC), leading to non - selective inflammatory responses or cell loss. In addition, using whole IgG for HSC - specific targeting may not be precise enough: markers like CD117 are also present on other cell types (e.g., T - cell subsets or progenitors), so systemic administration of a potent IgG or ADC may affect non - target cells or require careful dose adjustment. Gene - therapy vectors (such as lentiviral or AAV vectors) face different challenges: they generally lack a natural tropism for HSCs, and most HSCs in the body are in a quiescent state and located in protective microenvironments that are difficult for circulating vectors to reach. To overcome these obstacles, targeting ligands are needed that can bind to HSC markers with high specificity and have a small size and modular properties to be able to be conjugated to various delivery vectors. Bispecific or multivalent forms of antibodies have also been proposed to increase specificity by binding to multiple HSC markers, but constructing stable large - antibody complexes can be complex. Overall, although existing antibody - based methods have demonstrated the concept of HSC targeting, improved targeting agents are still needed for applications such as in vivo gene delivery, precise cell isolation, and safe conditioning.

[0005] Single - domain antibodies have unique advantages in therapeutic and diagnostic applications against CD117 and other hematopoietic stem cell (HSC) markers. A typical single - domain antibody structurally contains a VHH fragment. VHH is a single - domain antigen - binding fragment derived from the heavy - chain antibodies of camelids, with a typical size of only about 12 - 15 kDa. Due to its small size and single - domain structure, VHH exhibits high antigen - binding affinity and specificity under a range of conditions, along with excellent stability. With these small and robust antibodies, the invention aims to provide tools and methods to significantly improve the specificity and efficiency of HSC targeting for a variety of therapeutic, prophylactic, and diagnostic purposes. Summary of the Invention

[0006] The present application provides an antibody or antigen-binding fragment thereof targeting CD117, preferably a nanobody, which can specifically bind to human CD117 and target hematopoietic stem cells (HSCs) expressing CD117. In a specific embodiment, the antibody or antigen-binding fragment thereof can be used to detect the content of CD117 in a mixed system, can detect the content of hematopoietic stem cells in a mixed system, or can be conjugated with other drugs to treat diseases related to hematopoietic stem cells.

[0007] The antibody or antigen-binding fragment thereof targeting CD117 provided by the present application may have one or more of the following properties: 1) being able to specifically bind to CD117 and / or cells expressing CD117; 2) being able to inhibit the binding of CD117 to at least one of its ligands; 3) being able to inhibit CD117-mediated signal transduction; 4) being able to inhibit the migration, accumulation, recruitment, and / or infiltration of cells expressing CD117; 5) being able to mediate the killing effect on cells expressing CD117; 6) being able to induce the endocytosis of CD117 receptor on the cell surface, thereby reducing the activation of immune cells; 7) being able to be used for treating CD117-related diseases and / or disorders; 8) being able to be used for the detection of CD117 and / or cells expressing CD117.

[0008] The present application also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, an expression vector, a host cell, a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, a method for preparing the antibody or antigen-binding fragment thereof, and the use of the antibody or antigen-binding fragment thereof described in the present application.

[0009] In a first aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to human CD117. The antibody or antigen-binding fragment thereof specifically binds to human CD117. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH). The heavy chain variable region comprises a heavy chain complementarity determining region 1 (HCDR1), a heavy chain complementarity determining region 2 (HCDR2), and a heavy chain complementarity determining region 3 (HCDR3).

[0010] In certain embodiments, the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups:

[0011] (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:4; or

[0012] (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8; or

[0013] (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:12.

[0014] In certain embodiments, the VH comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9, or comprises about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence homology with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9.

[0015] In certain embodiments, the antibody is selected from one or more of the following groups: monospecific antibody, multispecific antibody, human antibody, humanized antibody and chimeric antibody.

[0016] In certain embodiments, the antibody is selected from one or more of the following groups: monovalent antibody and multivalent antibody.

[0017] In certain embodiments, the antigen-binding fragment is selected from one or more of the following groups: VH, Fab, Fv, F(ab’)2 and single-chain Fv (scFv).

[0018] In a preferred embodiment, the antibody or its antigen-binding fragment is a VH that specifically binds to human CD117.

[0019] In a preferred embodiment, the antibody or its antigen-binding fragment is a nanobody. More preferably, the molecular weight of the nanobody is less than 50KD, still more preferably less than 45KD, less than 40KD, less than 35KD, less than 30KD, less than 25KD, less than 15KD, less than 10KD, less than 5KD.

[0020] In a preferred embodiment, the antibody or its antigen-binding fragment is a single-domain antibody.

[0021] In certain embodiments, the antibody or its antigen-binding fragment comprises an Fc fragment.

[0022] Second aspect, the present application provides a fusion protein. It comprises any one of the antibodies or antigen-binding fragments thereof in the first aspect.

[0023] In certain embodiments, the fusion protein may be a chimeric antigen receptor (CAR).

[0024] In certain embodiments, the fusion protein is a bispecific antibody.

[0025] In certain embodiments, the fusion protein is a multispecific antibody.

[0026] Third aspect, the present application provides an isolated nucleic acid molecule encoding any one of the antibodies or antigen-binding fragments thereof in the first aspect or any one of the fusion proteins in the second aspect.

[0027] In certain embodiments, the isolated nucleic acid molecule can be produced or synthesized by the following methods: (i) amplified in vitro, for example, by polymerase chain reaction (PCR); (ii) produced by cloning and recombination; (iii) purified, for example, by digestion with enzymes and separation by gel electrophoresis; or (iv) synthesized, for example, by chemical synthesis.

[0028] Fourth aspect, the present application provides a vector. It comprises any one of the isolated nucleic acid molecules in the third aspect.

[0029] In certain embodiments, the vector comprises an expression vector. In certain embodiments, the vector comprises a DNA vector and an RNA vector.

[0030] In certain embodiments, the RNA vector is an mRNA vector.

[0031] Fifth aspect, the present application provides a cell. It comprises any one of the isolated nucleic acid molecules in the third aspect or any one of the vectors in the fourth aspect.

[0032] In certain embodiments, the cell comprises a host cell.

[0033] In specific embodiments, the host cell is a eukaryotic cell, such as a cell from a plant, a fungal or yeast cell, etc.

[0034] In specific embodiments, the cell is a bacterial cell (e.g., Escherichia coli), a yeast cell or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NS0 cells or myeloma cells. More specifically, it can be an engineered cell for producing antibodies, such as 293T cells.

[0035] Sixth aspect, the present application provides a method for producing any one of the antibodies or antigen-binding fragments thereof in the first aspect. Specifically, the method comprises:

[0036] (1) constructing an expression vector, the expression vector containing a gene sequence encoding the antibody or antigen-binding fragment thereof,

[0037] (2) transforming the expression vector into a host cell for induced expression, and

[0038] (3) isolating the antibody or antigen-binding fragment thereof from the expression product.

[0039] Seventh aspect, the present application provides a pharmaceutical composition. It comprises any one of the antibodies or antigen-binding fragments thereof in the first aspect, any one of the fusion proteins in the second aspect, any one of the isolated nucleic acid molecules in the third aspect, any one of the vectors in the fourth aspect, any one of the cells in the fifth aspect, and / or optionally a pharmaceutically acceptable carrier.

[0040] In certain embodiments, the pharmaceutical composition is used as a monotherapy.

[0041] In certain embodiments, the pharmaceutical composition is used in combination with other drugs. Preferably, the other drugs are small molecule targeted anti-cancer agents, other antibody drugs, adoptive cell therapy, and / or oncolytic virus enhancers.

[0042] In certain embodiments, the pharmaceutical composition may be a conjugate of any one of the antibodies or antigen-binding fragments thereof in the first aspect and other molecules. More specifically, the other molecules may be small molecule drugs, may be antibodies targeting antigens other than CD117, or may be lipid nanoparticles (LNP).

[0043] Eighth aspect, the present application provides the use of any one of the antibodies or antigen-binding fragments thereof in the first aspect, any one of the fusion proteins in the second aspect, any one of the isolated nucleic acid molecules in the third aspect, any one of the vectors in the fourth aspect, any one of the cells in the fifth aspect, and / or any one of the pharmaceutical compositions in the seventh aspect in the preparation of a drug for preventing and / or treating a disease.

[0044] In certain embodiments, the drug may be a bispecific antibody, may be a multispecific antibody, may be an antibody-drug conjugate (ADC), or may be an antibody-conjugated lipid nanoparticle.

[0045] Ninth aspect, the present application provides a method for preventing and / or treating a disease. The method comprises the use of any one of the antibodies or antigen-binding fragments thereof in the first aspect, any one of the fusion proteins in the second aspect, any one of the isolated nucleic acid molecules in the third aspect, any one of the vectors in the fourth aspect, any one of the cells in the fifth aspect, and / or any one of the pharmaceutical compositions in the seventh aspect.

[0046] In a tenth aspect, the present application provides the use of any antibody or antigen-binding fragment thereof according to the first aspect, any fusion protein according to the second aspect, any isolated nucleic acid molecule according to the third aspect, any vector according to the fourth aspect, any cell according to the fifth aspect, and / or any pharmaceutical composition according to the seventh aspect in the prevention and / or treatment of diseases.

[0047] In certain embodiments, the diseases described in the eighth, ninth, or tenth aspect may be hematological diseases. Specifically, the diseases may be diseases related to hematopoietic stem cells. More specifically, the hematopoietic stem cell-related diseases may be selected from one or more of the following: aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative neoplasms, myelofibrosis, immunodeficiency diseases, thalassemia, and sickle cell disease.

[0048] In an eleventh aspect, the present application provides a reagent or kit. The reagent or kit contains any antibody or antigen-binding fragment thereof according to the first aspect, any fusion protein according to the second aspect, any isolated nucleic acid molecule according to the third aspect, any vector according to the fourth aspect, any cell according to the fifth aspect, and / or any pharmaceutical composition according to the seventh aspect.

[0049] In certain embodiments, the kit is used to detect the content of CD117 in a mixed system.

[0050] In certain embodiments, the kit is used to detect the content of cells expressing CD117 in a mixed system.

[0051] For example, the cells expressing CD117 are selected from one or more of the following groups: hematopoietic stem cells, mesenchymal stem cells, keratinocyte stem cells, neurons, glial cells, endothelial cells, fibroblasts, stromal cells, activated endothelial cells, tumor cells, and tumor-associated fibroblasts.

[0052] In a twelfth aspect, the present application provides a method for detecting CD117. The method includes the use of any antibody or antigen-binding fragment thereof according to the first aspect, any fusion protein according to the second aspect, any isolated nucleic acid molecule according to the third aspect, any vector according to the fourth aspect, any cell according to the fifth aspect, and / or any pharmaceutical composition according to the seventh aspect.

[0053] In certain embodiments, the CD117 may be free CD117 within a mixed system.

[0054] In certain embodiments, the CD117 may be CD117 expressed on cells.

[0055] For example, the cells may be selected from one or more of the following group: hematopoietic stem cells, mesenchymal stem cells, keratinocyte stem cells, neurons, glial cells, endothelial cells, fibroblasts, stromal cells, activated endothelial cells, tumor cells, and tumor-associated fibroblasts.

[0056] In certain embodiments, the CD117 may be conjugated to other molecules.

[0057] For example, the other molecules may be selected from one or more of the following group: antibodies, small molecule ligands, targeting peptides, cell-penetrating peptides, antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), CRISPR / Cas systems, glycans, lipids, magnetic nanoparticles, photosensitive materials, and tissue engineering scaffolds.

[0058] Those skilled in the art can readily appreciate other aspects and advantages of the present application from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of the present application enables those skilled in the art to make modifications to the specific embodiments disclosed without departing from the spirit and scope of the invention involved in the present application. Accordingly, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive. Brief Description of the Drawings

[0059] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0060] Figure 1 Shows the binding ability of the serum of mice immunized with CD117 antigen and boosted immunization in Example 1 to cells expressing CD117 (CHO-CD117) and control cells (WT).

[0061] Figure 2 Shows the binding ability of the recombinant antibody in Example 2 to CD117-positive cells (CHO-CD117 and HEL) and control cells (CHO-K1).

[0062] Figure 3 Shows the binding efficiency of different concentrations of candidate antibodies to human CD117-CHO, monkey CD117-CHO, or HEL cells in Example 4.

[0063] Figure 4 Shows the results of evaluating the binding efficiency of candidate antibodies to mouse CD117 antigen using the ELISA method in Example 4.

[0064] Figure 5The figure shows the SDS-PAGE identification image of the purified antibody in Example 5. Among them, NR represents that no reducing agent is added to the sample, and R represents that a reducing agent is added to the sample.

[0065] Figure 6 The figure shows the structural schematic diagrams of the modified ligand, linker, and the ligand-linker conjugate formed by the coupling of the two in Example 6.

[0066] Figure 7 The figure shows the step schematic diagram of the method for preparing targeted lipid nanoparticles (tLNP) by conjugating ligands on the surface of lipid nanoparticles (LNP) through bioorthogonal click chemical reactions.

[0067] Figure 8 The figure shows the differences in the delivery efficiency of lipid nanoparticles with different lengths of ligands conjugated on the surface in cells expressing CD117.

[0068] Figure 9 The figure shows the comparison of the delivery efficiency of tLNP containing different hinges in cell lines.

[0069] Figure 10 The figure shows the evaluation of the gene delivery and editing capabilities of tLNP for delivering gene editing components in vitro. Detailed implementation manners

[0070] The following specific examples illustrate the implementation manners of the invention of this application. Those familiar with this technology can easily understand other advantages and effects of the invention of this application from the content disclosed in this specification.

[0071] Term definitions

[0072] In the present application, the term "CD117", also known as "c-kit", is a type III transmembrane protein tyrosine kinase receptor encoded by the proto-oncogene 4q11-q12. It is expressed in a variety of cell types, including hematopoietic stem cells, mast cells, melanocytes, spermatogonia, oocytes, and Cajal cells of the gastrointestinal tract. The ligand of CD117 is stem cell factor (also known as mast cell growth factor), and after this ligand binds to CD117, it can activate a series of intracellular signaling pathways, thereby regulating cell growth, survival, differentiation, migration, and adhesion. In the present application, unless otherwise specified, CD117 may include all its subtypes and all species. In the present application, the CD117 may include any natural CD117 from any vertebrate source, and the vertebrates include mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. In the present application, the CD117 may include "full-length", unprocessed CD117, and any form of CD117 derived from processing in cells; it may also include naturally occurring variants of CD117, such as splice variants or allelic variants. For example, the complete amino acid sequence of human CD117 has the accession number P10721 on the Uniprot website (https: / / www.uniprot.org / ).

[0073] In the present application, the term "isolated" generally refers to being obtained by artificial means from its natural state. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both situations have occurred. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal body, and the highly purified same polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance. In the present application, unless otherwise specified, the antibody or its antigen-binding fragment is also isolated.

[0074] In the present application, the term "antigen-binding protein" generally refers to a protein having antigen-binding ability. For example, an antigen-binding protein may include an isolated antigen-binding protein. An antigen-binding protein may comprise, for example, a protein framework region (FR) of antibody origin or an alternative protein framework region or an artificial framework region having grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein and fully synthetic framework regions containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen-binding proteins include, but are not limited to: Fab, Fab’, Fv fragments, F(ab’)2, F(ab)2, scFv, di-scFv, dAb, VHH, human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies, monovalent antibodies, multivalent antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies and fragments thereof.

[0075] In the present application, the term "CDR" also known as "complementary determining region" generally refers to the regions in the variable domains of antibodies, the sequences of which are highly variable and / or form structurally defined loops. Generally, three CDRs (HCDR1, HCDR2, HCDR3) are included in the variable domain of the heavy chain (VH) of an antibody. Antibodies consisting only of heavy chains can function normally and stably in the absence of light chains. For example, naturally occurring camel antibodies, see, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined by a variety of coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, taking into account Kabat / Chothia, etc. These coding systems are known in the art, see, e.g., www.bioinf.org.uk / abs / index.html#kabatnum for details. For example, the amino acid sequence numbering of the antigen-binding protein can be according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system @imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, Dev Comp Immunol 29:185-203). For example, the CDRs of the antigen-binding protein can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242).

[0076] In the present application, the term "variable" generally refers to the fact that certain segments in the variable regions may vary significantly in sequence among antibodies. The variable regions mediate antigen binding and determine the specificity of a particular antibody for its specific antigen. However, the variability is not evenly distributed throughout the variable regions. It is typically concentrated in three segments called hypervariable regions (CDR or HVR) in the variable region of the light or heavy chain. The more highly conserved portions of the variable regions are called framework regions (FR).

[0077] In the present application, the term "FR" generally refers to the more highly conserved portions of the antibody variable domain, which are called framework regions. Typically, a native heavy chain variable domain contains four FR regions, namely H-FR1, H-FR2, H-FR3, and H-FR4.

[0078] In the present application, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, encompassing any polypeptide including an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present application, the term "antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that retain antigen-binding function (e.g., capable of specifically binding CD117).

[0079] In the present application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind an antigen (e.g., CD117). In the present application, the antigen-binding fragment may include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, VHH, and / or dAb.

[0080] In the present application, the term "single-domain antibody" generally refers to an antibody lacking a light chain. The single-domain antibody described in the present application may include a heavy-chain antibody (HcAb, heavy-chain antibody), which includes a heavy-chain variable region and conventional heavy-chain CH2 and CH3 regions. The single-domain antibody described in the present application may include the smallest binding unit of an antigen-binding protein. The single-domain antibody described in the present application may include an antibody fragment consisting only of the variable region of the antibody heavy chain.

[0081] In the present application, the term "VHH (variable domain of heavy chain of heavy chain antibody, VHH)" generally refers to the variable region antigen-binding domain of a heavy chain antibody (see Nguyen V.K. et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and the review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407).

[0082] In the present application, the term "nanobody" refers to an antibody with a very small molecular weight. In a preferred embodiment, the nanobody consists only of the variable region of a heavy chain antibody, usually having a molecular weight of less than 15 KD, which is one-tenth that of a conventional antibody. This structure endows nanobodies with a series of unique properties, including high affinity, high stability, good water solubility, low immunogenicity, and strong tissue penetration ability.

[0083] In the present application, "single-domain antibody" and "nanobody" refer to complete antibodies that can independently perform functions, and "VHH" refers to a domain in a complete antibody. Further, "single-domain antibody" emphasizes the component of the antibody (e.g., containing only a single heavy chain), while "nanobody" emphasizes the molecular weight of the antibody. In the present application, single-domain antibodies can include, but are not limited to, nanobodies.

[0084] In the present application, the term "Fc fragment" generally refers to the C-terminal region of an immunoglobulin heavy chain that contains at least a part of the constant region. This term includes the native sequence Fc region and variant Fc regions. The Fc region of an immunoglobulin generally contains two constant domains - the CH2 domain and the CH3 domain, and optionally contains the CH4 domain. For example, the Fc region may not contain the CH1 domain. Amino acid residue substitutions in the Fc portion that alter the effector function of the antibody are known in the art (Winter et al. U.S. Patent Nos. 5,648,260; 5,624,821). The Fc portion of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of antibodies and antigen-antibody complexes. Depending on the therapeutic purpose, in some cases, these effector functions are desired for therapeutic antibodies, but in other cases, they may be unnecessary or even harmful.

[0085] In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules composed of a single molecule. Monoclonal antibodies are generally highly specific for a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used in the present application can be prepared by recombinant DNA methods.

[0086] In the present application, the terms "monospecific antibody" and "multispecific antibody" generally refer to antibodies that are capable of specifically binding to a single or multiple antigenic sites. When the specifically bound antigenic sites are multiple, the multiple antigenic sites can be from the same antigen or from different antigens, preferably the multiple antigenic sites are from different antigens.

[0087] In the present application, the terms "monovalent antibody" and "multivalent antibody" refer to antibody molecules having a single and multiple antigen-binding sites. As is known to those skilled in the art, classical antibodies (such as IgG antibodies) are bivalent antibodies having two antigen-binding sites. In the present application, the "monovalent antibody" and "multivalent antibody" can include a constant region and / or an Fc fragment, or can not include a constant region and an Fc fragment. When the "monovalent antibody" or "multivalent antibody" does not include a constant region and an Fc fragment, the multivalent antibody can be formed by connecting two or more antigen-binding fragments end to end, and the connection is preferably carried out by a covalent bond. The connection mode of the multivalent antibody can be N-terminal docking C-terminal, or N-terminal docking N-terminal, or C-terminal docking C-terminal.

[0088] In the present application, the term "chimeric antibody" generally refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species. Generally, the variable region is derived from an antibody of an experimental animal such as a camelid ("parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual compared to the parent (e.g., camel-derived) antibody.

[0089] In the present application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR regions of a non-human antibody (such as a camel antibody) are replaced with the corresponding amino acids derived from a human immunoglobulin. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permitted, provided that they still retain the ability of the antibody to bind a specific antigen. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains the antigen specificity similar to the original antibody. A "humanized" form of a non-human (such as camel) antibody may minimally comprise a chimeric antibody having sequences derived from a non-human immunoglobulin. In certain cases, the CDR region residues in a human immunoglobulin (recipient antibody) may be replaced with CDR region residues of a non-human species (donor antibody) having the desired properties, affinities and / or capabilities, such as a camel, alpaca, mouse, rat, rabbit or non-human primate. In certain cases, the FR region residues of a human immunoglobulin may be replaced with the corresponding non-human residues. In addition, a humanized antibody may comprise amino acid modifications not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve the performance of the antibody, such as binding affinity.

[0090] In the present application, the term "fusion protein" refers to a protein in which the gene sequences of two or more different proteins are linked together by genetic engineering techniques, so as to express a protein having two or more functions in the same polypeptide chain. Such protein fusion can endow the fusion protein with new functional characteristics or enhance its original functions. Fusion proteins have the advantages of diverse functions, enhanced stability, improved targeting and easy production, and can be applied to different fields such as biopharmaceuticals, vaccine development, cell therapy and diagnostic tools. In a specific embodiment, the fusion protein may be a viral membrane fusion protein, a cell fusion protein, or a bispecific antibody, an Fc fusion protein.

[0091] In the present application, the term "nucleic acid molecule" generally refers to any length of isolated form of nucleotides, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or synthesized artificially.

[0092] In the present application, the term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed. The vector can be transformed, transduced or transfected into a host cell so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors can include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors can include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector may contain a variety of elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain an origin of replication. The vector may also include components that assist its entry into cells, such as virus particles, liposomes or protein coats, but not only these substances.

[0093] In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a plasmid or vector of a subject, which includes the nucleic acid molecule described in the present application or the vector described in the present application. The cell can include the progeny of a single cell. Due to natural, accidental or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or genomically). The cell can include cells transfected in vitro with the vector described in the present application. The cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells. The cell can also include cells that have been engineered.

[0094] In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing / treating a disease or disorder. The pharmaceutical composition can contain the antibody or its antigen-binding fragment described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application and / or the cell described in the present application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition can also contain a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions described in the present application include, but are not limited to, liquid, frozen and lyophilized compositions.

[0095] In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers, which are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, counterions for salt formation such as sodium; and / or nonionic surfactants.

[0096] In the present application, the term "specific binding" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, which can determine the presence of the target in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which can be an epitope) can be an antibody that binds to the target with greater affinity, avidity, more readily, and / or for a greater duration than it binds to other targets. In certain embodiments, the antibody specifically binds to an epitope on a protein, and the epitope is conserved among proteins of different species. In certain embodiments, specific binding can include but does not require exclusive binding.

[0097] In the present application, the term "hematopoietic stem cell-related diseases" generally refers to a series of blood system diseases caused by abnormal functions, developmental defects or malignant transformation of hematopoietic stem cells. These diseases usually involve bone marrow hematopoietic dysfunction, immune function defects or malignant proliferation of blood cells. These diseases may be caused by genetic factors, environmental exposures or unknown factors, and the symptoms include anemia, bleeding, infection and organ enlargement. Diagnosis and treatment usually require a multidisciplinary approach.

[0098] "Conservative substitution" of amino acids is well known in the art and generally refers to the change of one amino acid residue to another amino acid residue having a structurally or functionally similar side chain. For example, an exemplary list of conservative substitutions is provided in the following table.

[0099]

[0100]

[0101] In the present application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

[0102] In the present application, the protein, polypeptide and / or amino acid sequences involved should also be understood to include at least the following ranges: variants or homologs having the same or similar functions as the said protein or polypeptide.

[0103] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or a fragment thereof capable of specifically binding to CD117). For example, the functional variant may comprise a protein or polypeptide having an amino acid change by substitution, deletion and / or insertion of at least 1, such as 1 - 30, 1 - 20 or 1 - 10, and further such as 1, 2, 3, 4 or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or the polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or the polypeptide before the change. For example, the substitution may be a conservative substitution.

[0104] In the present application, the homologue may be a protein or polypeptide having at least about 80% (e.g., having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or a fragment thereof capable of specifically binding to CD117).

[0105] In the present application, the term "homology" generally refers to the similarity, resemblance or relatedness between two or more sequences. The "percent sequence homology" can be calculated by comparing two sequences to be aligned in a comparison window to determine the number of positions at which the same nucleic acid bases (e.g., A, T, C, G, U) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exist in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence homology. The alignment for determining the percent sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment within the full-length sequences being compared or within the target sequence regions. The homology can also be determined by the following methods: FASTA and BLAST. The description of the FASTA algorithm can be found in "Improved Tools for Biological Sequence Comparison" by W.R. Pearson and D.J. Lipman, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and "Rapid and Sensitive Protein Similarity Searches" by D.J. Lipman and W.R. Pearson, Science, 227: 1435-1441, 1989. The description of the BLAST algorithm can be found in "Basic Local Alignment Search Tool" by S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, Journal of Molecular Biology, 215: 403-410, 1990.

[0106] In the present application, the term "comprising" generally means "including", "encompassing", "containing" or "comprehending", and these terms can be used interchangeably. In some cases, it also means "consisting of".

[0107] In the present application, the term "about" generally means varying within a range of 0.5% - 10% above or below a specified value, for example, varying within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value. DETAILED DESCRIPTION OF THE INVENTION

[0109] Antibody or its antigen-binding fragment, fusion protein

[0110] The CDR of an antibody, also known as the complementarity-determining region, is part of the variable region. The amino acid residues in this region can contact the antigen or epitope. Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia, etc. These coding systems are known in the art. For specific references, see, for example, www.bioinf.org.uk / abs / index.html#kabatnum. A person skilled in the art can determine the CDR region according to the sequence and structure of the antibody using different coding systems. Using different coding systems, the CDR regions may vary. In the present application, the CDR encompasses CDR sequences obtained according to any CDR partitioning method; it also encompasses variants thereof, where the variants include the amino acid sequence of the CDR with one or more amino acids substituted, deleted, and / or added. For example, 1 - 30, 1 - 20, or 1 - 10 amino acids, and for another example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; it also encompasses homologs thereof, where the homologs can be amino acid sequences having at least about 85% (for example, having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the CDR. In certain embodiments, the antigen-binding protein described in the present application can be defined by the Kabat coding system.

[0111] The present application provides an antibody or an antigen-binding fragment thereof that specifically binds to human CD117. The antibody or its antigen-binding fragment includes a heavy-chain variable region (VH). The heavy-chain variable region includes heavy-chain complementarity-determining region 1 (HCDR1), heavy-chain complementarity-determining region 2 (HCDR2), and heavy-chain complementarity-determining region 3 (HCDR3).

[0112] In certain embodiments, the HCDR1, HCDR2, and HCDR3 are selected from any of the following groups:

[0113] (1) The HCDR1 includes the amino acid sequence shown in SEQ ID NO:2, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:3, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:4; or

[0114] (2) The HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:6, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:7, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:8; or

[0115] (3) The HCDR1 comprises the amino acid sequence as shown in SEQ ID NO:10, the HCDR2 comprises the amino acid sequence as shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence as shown in SEQ ID NO:12.

[0116] In certain embodiments, the VH comprises the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9, or comprises an amino acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% sequence homology with the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9 and all amino acid differences are in the amino acid sequence of the non-CDR region.

[0117] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0118] In the present application, the antibody or its antigen-binding fragment may comprise at least one CDR of the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9, and the CDR may include CDRs divided in any manner. If the sequence of the CDR delimited by any one way is the same as the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9, it falls within the protection scope of the claims of the present application.

[0119] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present application includes an antigen-binding protein having an HCDR1 with an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 10, an HCDR2 with an amino acid sequence as shown in SEQ ID NO: 3, SEQ ID NO: 7, or SEQ ID NO: 11, and an HCDR3 with an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 12, as well as the humanized antigen-binding protein thereof. In certain embodiments, the HCDR1 of the humanized antigen-binding protein may have more than one amino acid mutation, the HCDR2 of the humanized antigen-binding protein may have more than one amino acid mutation, and / or the HCDR3 of the humanized antigen-binding protein may have more than one amino acid mutation, while the humanized antigen-binding protein still has the ability to bind to CD117.

[0120] In certain embodiments, the isolated antigen-binding protein described in the present application includes a variable region having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 9, as well as the humanized antigen-binding protein thereof.

[0121] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present application may include a single-domain antibody or its antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present application may include a heavy-chain antibody or its antigen-binding fragment. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present application may include a nanobody.

[0122] In certain embodiments, the antibody or antigen-binding fragment thereof may include a heavy-chain only antibody. In certain embodiments, the heavy-chain only antibody consists of a variable region antigen-binding domain composed of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In certain embodiments, the heavy-chain only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In certain embodiments, the heavy-chain only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH2 domain. In certain embodiments, the heavy-chain only antibody consists of an antigen-binding domain, at least a portion of the hinge region, and CH3 domain. Also included herein are heavy-chain only antibodies in which the CH2 and / or CH3 domains are truncated. In additional embodiments, the heavy chain consists of an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but without a hinge region. The heavy-chain only antibody may be in a dimeric form, in which the two heavy chains are bonded by disulfide bonds or are otherwise covalently or non-covalently linked to each other. The heavy-chain only antibody may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD, and IgE subclasses, are also included herein. In certain embodiments, the heavy-chain antibody may be an IgG1, IgG2, IgG3, or IgG4 subtype, particularly the IgG1 subtype.

[0123] In certain embodiments, the antibody may be selected from one or more of the following group: monoclonal antibodies, chimeric antibodies, and humanized antibodies.

[0124] In a preferred embodiment, the antibody can be a nanobody, more preferably a single-domain antibody among nanobodies. Single-domain antibodies have unique advantages in therapeutic and diagnostic applications against CD117 and other hematopoietic stem cell (HSC) markers. The main component of single-domain antibodies is VHH, which is a single-domain antigen-binding fragment derived from the heavy-chain-only antibodies of camelids, and its typical size is only about 12–15 kDa. Due to its small size and single-domain structure, VHH exhibits high antigen-binding affinity and specificity under a series of conditions, and at the same time has excellent stability. These characteristics enable single-domain antibodies to overcome many limitations of traditional antibodies: they can more easily penetrate tissues and the cellular microenvironment, can be recombinantly produced in low-cost microbial systems, and can be easily engineered with various payloads and formats through gene fusion or chemical conjugation. Importantly, VHHs are usually able to recognize unique or hidden epitopes (thanks to their longer CDR3 loops), and tend to be efficiently internalized when binding to cell surface targets, especially if they are multivalent or linked to endocytosis-promoting triggers. For example, nanobodies have been shown to be able to reach intracellular targets when used as delivery agents, which is a key property for the delivery of gene therapy payloads. They lack an Fc region themselves, thus avoiding unwanted Fc-mediated immune effects; this is an advantage for imaging agents (reducing background uptake in organs rich in Fc receptors), or when effector functions or an extended half-life are required, it can be alleviated by adding an Fc or other multimerization tags. Overall, the stability, high affinity, and modular characteristics of single-domain antibodies make them ideal ligands for innovative HSC-targeted therapies.

[0125] In the present application, the antibody or its antigen-binding fragment may further comprise an Fc fragment. In certain embodiments, the N-terminus of the Fc fragment is directly or indirectly connected to the C-terminus of the heavy chain variable region. In certain embodiments, the N-terminus of the Fc fragment and the C-terminus of the heavy chain variable region may be connected by a hinge region. In certain embodiments, the Fc fragment may be derived from the Fc fragment of human IgG. In certain embodiments, the antigen-binding protein described in the present application comprises the wild-type Fc fragment derived from human IgG. The amino acid sequence of the Fc fragment derived from human IgG is known in the art. In certain embodiments, the Fc fragment may include the Fc fragment derived from any one of the following immunoglobulins: IgG1, IgG2, IgG3, and IgG4. In certain embodiments, the antigen-binding protein described in the present application comprises the wild-type Fc fragment derived from any one of the following immunoglobulins: IgG1, IgG2, IgG3, and IgG4. The amino acid sequences of the Fc fragments derived from IgG1, IgG2, IgG3, or IgG4 are known in the art. For example, the Fc fragment may comprise the Fc fragment derived from human IgG1. For example, the Fc fragment may comprise the Fc fragment derived from human IgG3. For example, the Fc fragment may comprise the Fc fragment derived from human IgG4.

[0126] In certain embodiments, the antibody or its antigen-binding fragment described in the present application comprises an Fc fragment variant having one or more amino acid mutations relative to the wild-type Fc fragment. The half-life of the antibody or its antigen-binding fragment and / or the effector function of the antibody or its antigen-binding fragment can be extended by optimizing the Fc fragment sequence.

[0127] In the present application, the antibody or its antigen-binding fragment may further comprise an isolated antigen-binding protein that has been further functionally optimized or modified. In certain embodiments, the antigen-binding protein described in the present application may comprise an antigen-binding protein having enhanced affinity for binding to CD117. In certain embodiments, the antigen-binding protein described in the present application may comprise an antigen-binding protein having enhanced ability to inhibit the binding of CD117 to its ligand. In certain embodiments, the antigen-binding protein described in the present application may comprise an antigen-binding protein having enhanced ability to inhibit CD117-mediated signal transduction. In certain embodiments, the antigen-binding protein described in the present application may comprise an antigen-binding protein having enhanced ability to inhibit the migration, accumulation, recruitment, and / or infiltration of cells expressing CD117. In certain embodiments, the antigen-binding protein described in the present application may comprise an antigen-binding protein having enhanced ability to mediate killing of cells expressing CD117. In certain embodiments, the antigen-binding protein described in the present application may comprise an antigen-binding protein having enhanced ability to induce endocytosis of CD117 receptor on the cell surface. In certain embodiments, the enhanced ability may be relative to the antigen-binding protein before its functional optimization or modification.

[0128] The antibody or antigen-binding fragment thereof described in the present application may include a heavy chain sequence with one or more conservative sequence modifications. The so-called "conservative sequence modification" refers to amino acid modifications that do not significantly affect or change the antibody binding properties. Such conservative modifications include amino acid substitutions, insertions, and deletions. The modifications can be introduced into the antibody or antigen-binding fragment thereof described in the present application by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are the replacement of an amino acid residue with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues in the CDR regions of the antibody or antigen-binding fragment thereof described in the present application may be replaced with other amino acid residues of the same side chain group. Those skilled in the art know that some conservative sequence modifications do not abolish antigen binding. See, for example, Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0129] In the present application, the antibody or its antigen-binding fragment can specifically bind to CD117. In certain embodiments, the binding affinity of the antibody or its antigen-binding fragment for CD117 can be detected by detecting the binding ability of the antibody or its antigen-binding fragment to cells expressing CD117. For example, it can be detected by FACS. In certain embodiments, the binding of the antibody or its antigen-binding fragment to CD117 can be detected by ELISA method. For example, the antibody or its antigen-binding fragment described in the present application can bind to CD117 with an EC50 value less than or equal to about 0.10 μg / mL, less than or equal to about 0.09 μg / mL, less than or equal to about 0.08 μg / mL, less than or equal to about 0.07 μg / mL, less than or equal to about 0.06 μg / mL, less than or equal to about 0.05 μg / mL, less than or equal to about 0.04 μg / mL, less than or equal to about 0.03 μg / mL, less than or equal to about 0.02 μg / mL or less than or equal to about 0.01 μg / mL. For example, the antibody or its antigen-binding fragment described in the present application can bind to CD117 with an IC50 value less than or equal to 50 nM, less than or equal to about 40 nM, less than or equal to 30 nM, less than or equal to 20 nM, or less than or equal to 10 nM.

[0130] The CD117 antigen-binding proteins described in the present application can be assayed, identified, or characterized by various methods known in the art. For example, the antigen-binding activity of the antibody or its antigen-binding fragment of the present application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., Western blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0131] The antibody or its antigen-binding fragment provided in the present application can be used to antagonize CD117 activity. In the present application, the antibody or its antigen-binding fragment can prevent and / or treat diseases and / or disorders.

[0132] Derivatives based on antibody or its antigen-binding fragment and their applications

[0133] The present application also encompasses variants of the disclosed anti-CD117 single-domain antibodies that have been engineered to confer or enhance properties required for specific therapeutic, diagnostic, or research applications. These variants include, but are not limited to, modifications made to improve affinity, specificity, stability, solubility, reduce immunogenicity, enhance conjugation ability, or other desired functional properties.

[0134] Variants designed to improve antigen-binding affinity and specificity can be generated by methods known in the art. These methods include affinity maturation by targeted or random mutagenesis, followed by selection techniques such as phage display or yeast display. The scope of the invention includes variants with improved binding kinetics (e.g., increased association rate, decreased dissociation rate) or enhanced selectivity. Variants with enhanced stability and solubility include modifications to the framework regions. Specific amino acid substitutions may introduce stabilizing residues or disulfide bonds to increase thermal stability, resistance to protease degradation, or reduce the tendency to aggregate. In one embodiment, mutations in the framework residues increase the solubility or stability of the antibody under physiological or production conditions while maintaining target specificity and affinity.

[0135] In certain embodiments, the variant can be a variant engineered for site-specific conjugation. The variant includes introducing one or more cysteine residues at a predetermined position within the C-terminal or N-terminal region of a single-domain antibody. These cysteine residues facilitate selective and stable conjugation of therapeutic agents, toxins, imaging agents, or functional groups via a chemical linker (e.g., maleimide-thiol reaction). Additionally, the variant may contain a flexible hinge or linker sequence adjacent to the engineered cysteine residue to optimize payload accessibility and maintain antibody function. In other embodiments, the variant incorporates non-natural amino acids with bioorthogonal reactive groups (e.g., azide, alkyne, ketone-functionalized amino acids). These residues enable highly specific, orthogonal conjugation reactions ("click chemistry") to facilitate precise ligation of diagnostic agents, cytotoxic drugs, or gene editing components.

[0136] In certain embodiments, the variant can be a fusion protein. The fusion protein can comprise a genetic fusion of a VHH domain with an additional functional domain. These fusion proteins include, but are not limited to, immunoglobulin Fc fragments (forming antibody-Fc fusion proteins that extend the antibody half-life), cytokines (e.g., IL-2, IL-15), apoptosis-inducing domains (e.g., truncated toxins), imaging tags (e.g., fluorescent proteins, radiolabeled chelators), or other therapeutically relevant polypeptides. In an illustrative example, the fusion of an anti-CD117 VHH with an Fc domain results in a bivalent antibody with an extended serum half-life and enhanced affinity.

[0137] In certain embodiments, the variant can be a bispecific or multispecific antibody. These constructs may involve the genetic fusion of two or more different VHH domains via a flexible peptide linker. Examples include bispecific antibodies containing one anti-CD90 VHH domain linked to one anti-CD117 VHH domain, capable of targeting multiple epitopes on hematopoietic stem cells simultaneously to enhance specificity and therapeutic efficacy. The variant may also contain a linker sequence designed to optimize the spacing and orientation between different functional domains. Suitable linkers include flexible glycine-serine (Gly-Ser)n repeats, rigid helix-forming sequences, or cleavable linkers responsive to a specific cellular environment (e.g., protease-cleavable sequences to facilitate intracellular payload release).

[0138] Furthermore, the present application also includes humanized variants in which the camelid-derived VHH sequences have been modified to be more similar to human immunoglobulin variable domains, reducing potential immunogenicity in therapeutic applications. Such humanization may involve grafting camelid-derived complementarity-determining regions (CDRs) onto a human variable region framework, optionally followed by selective back-mutations to restore optimal binding and stability characteristics.

[0139] The present application also relates to engineered derivatives of the disclosed anti-CD117 single-domain antibodies to enhance their utility in therapeutic, diagnostic, and research applications. Antibody derivatives within the scope of the present application include fusion proteins created by genetically linking the disclosed single-domain antibody to an additional functional protein domain. In one embodiment, the VHH domain is fused to an immunoglobulin Fc domain, providing increased antibody affinity, extended serum half-life, and Fc-mediated effector functions. In another embodiment, the single-domain antibody is genetically fused to a cell-penetrating peptide or an endoplasmic reticulum escape domain, enabling improved intracellular delivery of payloads such as nucleic acids or drugs. Additionally, the VHH domain can be fused to a fluorescent protein, a luminescent reporter protein, or an enzymatic reporter protein to facilitate its use in diagnostic assays and imaging applications.

[0140] In another embodiment, the invention includes antibody-drug conjugates (ADCs) in which the single-domain antibody is chemically conjugated to a cytotoxic drug or toxin. Suitable cytotoxic drugs include auristatins, maytansines, doxorubicin, calicheamicin, and pyrrolobenzodiazepines (PBDs). The conjugation can be carried out via a cleavable or non-cleavable linker that allows for selective release of the cytotoxic payload upon internalization of the CD117-expressing target cells.

[0141] Further embodiments include radiolabeled derivatives, where the single-domain antibody is conjugated chemically to a radioisotope such as technetium-99m, zirconium-89, iodine-125, lutetium-177, or yttrium-90. These radiolabeled derivatives can be used for diagnostic imaging in positron emission tomography (PET) or single-photon emission computed tomography (SPECT), as well as for targeted radiotherapy to selectively deplete specific cell populations.

[0142] In addition, bispecific or multispecific antibody derivatives can be constructed to bind multiple target antigens simultaneously, enhancing the specificity and potency of the treatment. For example, the antibody derivative can be a bispecific antibody comprising a VHH domain targeting CD117 and a VHH domain of another clinically relevant antigen such as CD3 for T cell binding, which provides selective targeting and a potent biological effect.

[0143] In certain embodiments, the antibody derivatives are designed for targeted gene delivery and / or gene editing. These derivatives include viral vectors such as adeno-associated virus (AAV) or lentiviral particles, whose capsid or envelope proteins are genetically engineered to incorporate the VHH antibody domain, enabling specific and efficient targeting of hematopoietic stem cells (HSCs) expressing CD117 and other relevant cell types.

[0144] In certain embodiments, the antibody derivatives can be antibody derivatives for cell therapy, such as chimeric antigen receptors (CARs). These constructs integrate the VHH sequence into engineered receptors expressed on immune effector cells (T cells, NK cells, macrophages), enabling selective immune-mediated clearance of antigen-positive target cells. Similarly, bispecific T cell engagers (BiTE-like molecules) using an anti-CD117 VHH domain linked to a T cell-binding domain such as anti-CD3 are also within the scope of this application, which can precisely direct the immune response to diseased or target cells.

[0145] In certain embodiments, the antibody derivatives include single-domain antibodies conjugated to lipid-polyethylene glycol (lipid-PEG) groups. These lipid-PEG-conjugated single-domain antibodies enable a targeted non-viral delivery platform, particularly lipid nanoparticles (LNPs), liposomes, or polymer-based nanoparticles. These derivatives provide enhanced stability, targeted cell-binding ability, and efficient internalization for intracellular delivery of nucleic acid payloads such as mRNA, siRNA, or gene editing tools.

[0146] A major aspect of this application is to clone-engineer VHH or single-domain antibodies into various forms such as fusion proteins or conjugates to achieve targeted delivery or therapeutic effects. The following are several implementation categories:

[0147] 1. Antibody-drug conjugates (ADCs) and toxin fusion proteins: Each anti-CD117 VHH can be conjugated with a cytotoxic drug to create a targeted cytotoxin capable of eliminating cells expressing the target antigen. In one embodiment, the VHH is chemically conjugated to the toxin via a cysteine residue added to the VHH. For example, a cysteine is inserted at the C-terminus (after the last framework residue) of the VHH or single-domain antibody for site-specific ligation to a drug linker with a terminal maleimide. More specifically, a maytansine derivative (DM1, a microtubule toxin) of a maleimide linker is conjugated to this cysteine to obtain an anti-CD117 VHH-DM1 conjugate. In a cell killing assay, this conjugate specifically killed CD117 + cells (such as stem / progenitor cells in culture), with no effect on CD117 - cells, demonstrating targeted cytotoxicity. Similarly, the VHH can be genetically fused to a protein toxin; for example, anti-CD117 VHH fused to truncated diphtheria toxin or an apoptotic enzyme (such as granzyme) can be used to deplete cells expressing CD117. Such an ADC can be used as a conditioning agent to deplete HSCs in the bone marrow before introducing gene-corrected cells or donor cells. In a specific embodiment, the small size of the VHH may have better tissue penetration and more uniform bone marrow distribution than larger IgG ADCs, potentially achieving more effective HSC clearance at lower doses.

[0148] 2. Targeted lipid nanoparticles (tLNPs): This application includes a lipid nanoparticle delivery system loaded with single-domain antibodies for targeting. Lipid nanoparticles can encapsulate a therapeutically effective payload, such as mRNA, siRNA, or CRISPR ribonucleoprotein, but by default they are non-specifically distributed. By attaching anti-CD117 VHH to the LNP surface, we created a targeted LNP (tLNP) that is capable of targeting HSCs. In one example, CD117-targeted LNPs were made using clone B13, and these LNPs were specific for CD117 + cells, including normal human CD34 + CD117 +The (bone marrow cells) showed similar specificity. These tLNPs were further used for gene editing tests: We loaded them with mRNA encoding the gene editor AaCas12b and the sgRNA targeting the HBG gene (β-globin gene locus). The treated cells showed a high level of gene editing (insertion / deletion formation) at the target site. For the targeted LNPs, the editing efficiency increased with the increase in dose, while the activity of the untargeted LNPs was significantly reduced. This confirmed that conjugating our VHH to LNPs can greatly enhance the ability to deliver gene editing tools to HSCs, which is an in vivo gene therapy strategy that can be used to treat diseases such as β-thalassemia (reactivating fetal hemoglobin by editing the HBG promoter). The method for creating such tLNPs will be further described in the examples and generally involves covalently conjugating VHH to lipids (e.g., DSPE-PEG-maleimide) during the self-assembly of nanoparticles.

[0149] 3. Virus vector targeting and CAR construction: The VHH described in this application can be used for the construction of targeted viruses or chimeric antigen receptors. For virus vectors, one example is the modification of the AAV capsid: The coding sequence of the anti-CD117 VHH is inserted into the AAV capsid protein loop to generate a chimeric capsid containing VHH on the virus surface. This modified AAV specifically transduces CD117 + cells, effectively redirecting the virus tropism to HSCs. Another modification method is to use an adapter molecule - for example, biotinylated anti-CD117 VHH can bind to lentiviruses expressing streptavidin, thereby anchoring the virus to CD117 + cells. These strategies allow gene delivery vectors (lentiviruses, AAVs, adenoviruses, etc.) to achieve cell-specific transduction, which is a key step in achieving in vivo HSC gene therapy without transplantation. In the context of cell therapy, the VHH sequence here can serve as the antigen-binding domain of a CAR (chimeric antigen receptor) on immune cells. For example, a CAR was constructed by fusing the CD117 VHH sequence with the CD8 hinge-transmembrane domain and the intracellular CD3ζ and 4-1BB signaling domains. T cells expressing the CAR specifically recognized and lysed CD117-expressing cells in vitro, demonstrating that VHH retained its binding ability in the CAR. Such CAR-T cells can be used to target and destroy HSCs (for conditioning or treating malignancies) or even attack CD117 + cancer stem cells in solid tumors. The advantage of using VHH in CARs is that its small size may reduce immunogenicity and allow for a more compact packaging or design as a dual CAR (where two small domains are used side by side).

[0150] 4. Multispecific and bispecific antibodies: To increase binding affinity or target selectivity, multiple VHHs can be linked together. By genetically fusing two CD117 VHH domains with a flexible linker, a bivalent anti-CD117 construct was created, generating a tandem VHH with a substantially doubled binding valence for CD117. This bivalent nanobody showed a slower dissociation rate from the antigen and increased antibody retention time in bone marrow tissue in in vivo models. For dual targeting, the VHH against CD117 was linked to the VHH against CD45 (CD45 is a pan-leukocyte marker expressed on HSCs), creating a bispecific molecule that requires simultaneous binding of both markers to bind to cells, and thus this molecule preferentially binds to HSCs (CD117 + CD45 + ) rather than T cells (which are usually CD45 + in peripheral blood but CD117 - ). This is an example of how a multispecific nanobody construct can precisely target a well-defined cell subset. All these tandem or fusion antibody constructs are within the scope of this application.

[0151] 5. Fusion with functional groups: In addition to binding to the target, VHHs can also be fused with functional protein domains. For example, fusing the VHH against CD117 with the Fc domain (creating a VHH-Fc or "nanobody-Fc") confers IgG-like properties to the molecule (bivalency and an extended half-life due to FcRn recycling). Alternatively, fusing the nanobody with an enzyme can localize the enzyme's activity to HSCs - the VHH against CD117 can be linked to a cytokine or growth factor (turning it into a targeted cytokine that preferentially stimulates HSCs). In one embodiment, we created an IL-2-VHH fusion where the VHH of CD117 was fused with IL-2; this chimeric molecule was able to specifically deliver IL-2 signals to CD117 + cells that also express IL-2R, representing a cell-selective cytokine delivery platform. In addition to this, this application contemplates many such combinations (nanobody fused with signaling molecules, with apoptosis-inducing domains, with fluorescent proteins for imaging, etc.).

[0152] All coupling and engineering techniques used are standard or adaptations of known methods. Small molecule payloads can be covalently linked to VHHs through covalent linkages such as NHS esters, maleimide-thiol reactions, click chemistry (e.g., azide-alkyne cycloaddition), etc. And peptide / protein-level integration is achieved through genetic fusion. The final product is characterized by SDS-PAGE, mass spectrometry, and functional assays to confirm that the VHH retains its binding function after modification.

[0153] It can be seen that single-domain antibodies against CD117 on hematopoietic stem cells (HSCs) have broad application potential. These applications cover research and clinical fields:

[0154] · Targeted gene therapy and gene editing: Use VHHs against CD117 to guide gene therapy vectors or nanoparticles to HSCs to achieve gene introduction or gene editing of stem cells (e.g., delivery of the CRISPR system). This can treat inherited blood diseases by in situ editing of HSCs, thus avoiding the need for transplantation.

[0155] · Cancer immunotherapy: Use VHH-based constructs to target malignant stem cells or the supportive microenvironment in cancer. For example, antibody-drug conjugates (ADCs) based on anti-CD117 VHHs can eliminate leukemia stem cells in myeloid malignancies without causing extensive collateral damage.

[0156] · HSC transplantation conditioning: Use VHH conjugates to selectively deplete or inactivate a patient's HSCs before transplantation. Anti-CD117 nanobody-drug conjugates (or bispecific antibodies that direct immune effector cells to HSCs) can serve as minimal-toxicity conditioning agents to create space for the engraftment of donor HSCs. In addition, VHHs can be used to deliver radioactive isotopes to the bone marrow for local irradiation of the HSC microenvironment.

[0157] · Cell separation and in vitro manipulation: Coat magnetic beads or column matrices with VHHs against CD117 to isolate HSCs from bone marrow or circulating blood for enrichment in research or therapeutic transplantation. The small size of VHHs may allow for gentler and more specific capture and release of cells compared to intact antibodies. Similarly, VHHs can target HSCs in culture (e.g., deliver growth factors or gene editors extracellularly).

[0158] · Diagnosis and imaging: Use labeled VHHs (e.g., fluorescent or radiolabeled nanobodies) for diagnostic imaging of HSCs or tissues rich in HSCs. Radiotracers conjugated to anti-CD117 VHHs can image the distribution of bone marrow stem cells or monitor the engraftment after HSC transplantation, taking advantage of the rapid blood clearance rate of VHHs to obtain high-contrast images. In the pathology laboratory, anti-CD117 VHH reagents can serve as specific stains for identifying HSCs or cancer stem cells in tissue sections.

[0159] Nucleic acid molecule, vector and cell

[0160] On the other hand, the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence that can encode the isolated antigen-binding protein described in the present application. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR); (ii) produced by recombinant cloning; (iii) purified, such as by digestion with enzymes and fractionation by gel electrophoresis; or (iv) synthesized, such as by chemical synthesis.

[0161] On the other hand, the present application provides a vector that can contain the isolated nucleic acid molecule described in the present application. In addition, other genes can also be included in the vector, such as a marker gene that allows selection of the vector in a suitable host cell and under suitable conditions. In addition, the vector can also contain expression control elements that allow correct expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art and can include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. The vector can be expressed by transforming, transducing, or transfecting a host cell so that the genetic material elements carried by it are expressed within the host cell. The vector can include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in genetic engineering. For example, the vector is an expression vector. In addition, the vector can also include components that assist its entry into cells, such as virus particles, liposomes, or protein coats, but not limited to these substances.

[0162] On the other hand, the present application provides a cell that can contain the isolated nucleic acid molecule described in the present application or the vector described in the present application. In certain embodiments, each host cell can contain one or a kind of the nucleic acid molecule or vector described in the present application. In certain embodiments, each host cell can contain multiple (e.g., 2 or more) or multiple kinds (e.g., 2 or more kinds) of the nucleic acid molecules or vectors described in the present application. For example, the vector described in the present application can be introduced into the host cell, such as a eukaryotic cell, such as a cell from a plant, a fungal or yeast cell, etc. In certain embodiments, the cell can be a bacterial cell (e.g., Escherichia coli), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NS0 cells, or myeloma cells. The vector described in the present application can be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0163] Method for producing antibody

[0164] On the other hand, the present application provides a method for preparing the antibody or its antigen-binding fragment. The method may include culturing the host cell described in the present application under conditions that enable the expression of the antibody or its antigen-binding fragment. For example, by using an appropriate culture medium, appropriate temperature, culture time, etc., these methods are known to those of ordinary skill in the art.

[0165] In certain embodiments, the method may be a molecular biology method. For example, the method includes preparing one or more nucleotide sequences encoding any one of the foregoing antibodies or its antigen-binding fragment, constructing the one or more encoding nucleotide sequences into one or more expression vectors, and expressing the expression vector in a suitable cell for preparation.

[0166] Those skilled in the art can understand that, on the premise of determining the amino acid sequence of a protein, due to codon degeneracy, different encoding nucleotide sequences can be used, and the encoding nucleotide sequences can also be optimized. Methods for optimizing the codons of encoding sequences are known to those skilled in the art, including adjusting the codons to the preferred codons of the host according to the type of the host, reducing the GC content and / or reducing the GC-rich regions, and improving the mRNA stability, so as to improve the expression efficiency of the target nucleotide in a specific host.

[0167] Suitable production cells are known to those skilled in the art. In some embodiments, mammalian cells, such as 293T, CHO or their derived cell lines, are used as production cells. In some embodiments, microbial cells such as bacterial cells or fungal cells are used as production cells, such as Escherichia coli or yeast. In some embodiments, insect cells are used as production cells, such as Sf9. The nucleotide sequence encoding the trispecific antibody of the present invention can be codon-optimized for a specific production cell line.

[0168] Preferably, the produced antibody or its antigen-binding fragment is purified. The purification can be carried out by conventional methods in the field of antibody production, and the methods may include steps such as filtration, chromatography, etc. The filtration step can be selected from one or more of depth filtration, ultrafiltration, diafiltration, and nanofiltration. The chromatography step can be selected from one or more of affinity chromatography, cation chromatography, anion chromatography, size exclusion chromatography, hydrophobic chromatography, and hydroxyapatite chromatography.

[0169] Any method suitable for generating monoclonal antibodies can be used to generate the antibodies or antigen-binding fragments thereof described in this application. For example, animals can be immunized with linked or naturally occurring CD117 or fragments thereof. Appropriate immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes can be used. In certain embodiments, antibodies or antigen-binding fragments thereof against CD117 can be screened and enriched by extracting peripheral blood lymphocytes from immunized alpacas, cloning cell nucleic acid fragments into vectors, and using a phage surface display system.

[0170] Any suitable form of CD117 can be used as an immunogen (antigen) to generate antibodies specific to CD117 and screen the biological activities of the antibodies. For example, the immunizing antigen can be full-length CD117, including natural homodimers, or peptides containing single / multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art.

[0171] Pharmaceutical composition, therapeutic use and detection kit

[0172] On the other hand, this application also provides a pharmaceutical composition, which may comprise the antibodies or antigen-binding fragments thereof described in this application, the isolated nucleic acid molecules described in this application, the vectors described in this application, and / or the cells described in this application, and optionally a pharmaceutically acceptable carrier. [[ID=!0]] [[ID=!1]]

[0173] [[ID=!2]]In certain embodiments, the pharmaceutical composition may further comprise a suitable formulation of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the doses and concentrations used. The pharmaceutical compositions described in this application include, but are not limited to, liquid, frozen, and lyophilized compositions. [[ID=!3]] [[ID=!4]]

[0174] [[ID=!5]]In certain embodiments, the pharmaceutical composition may further contain more than one active compound, typically those having complementary activities that do not adversely affect each other. The type and effective amount of such drugs can depend, for example, on the amount and type of antagonist present in the formulation and the clinical parameters of the subject. [[ID=!6]] [[ID=!7]]

[0175] [[ID=!8]]In certain embodiments, the pharmaceutical composition may comprise the unseparated expression products described herein. Specifically, the expression product is an intermediate product produced in the aforementioned method for producing antibodies or antigen-binding fragments thereof, and is a mixed system containing the antibodies or antigen-binding fragments thereof protected by this application. In certain embodiments, the expression product can be a homogeneous stock solution. The preparation and analysis of the expression product are well known in the art. [[ID=!9]]

[0176] In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents that are compatible with drug administration, and are generally safe, non-toxic.

[0177] In certain embodiments, the pharmaceutical composition may be administered parenterally, transdermally, intracavitary, intraarterially, intrathecally, and / or intranasally or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition may be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered continuously. The continuous (or uninterrupted) administration may be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body.

[0178] In certain embodiments, the pharmaceutical composition further comprises other drugs, which are conjugated to the antibody or antigen-binding fragment thereof of the present application and targeted to cells expressing CD117 by the antibody or antigen-binding fragment thereof of the present application to treat related diseases.

[0179] Specifically, the other drugs may be selected from one or more of the following groups: small molecule targeted anti-cancer agents, antibody drugs, adoptive cell therapies, oncolytic viruses, and oncolytic virus enhancers.

[0180] More specifically, the other drugs may be selected from one or more of the following groups: tyrosine kinase inhibitors, serine / threonine kinase inhibitors, immune checkpoint inhibitors, antibodies targeting co-stimulatory molecules, CAR-T, CAR-NK, CAR-M, TCR-T, TCR-NK, TCR-M, adenovirus, reovirus, herpes virus, poxvirus, paramyxovirus, rhabdovirus, picornavirus, influenza virus, and parvovirus.

[0181] In certain embodiments, the drug may be a carrier encapsulating the content. For example, liposomes, adeno-associated viruses (AAV), lipid nanoparticles, vesicles, exosomes, lentiviral vectors, adenoviral vectors, metal nanoparticles, mesoporous silica nanoparticles, red blood cells, and platelets. The content may be selected from one or more of the following groups: RNA drugs (e.g., mRNA, miRNA, circular RNA), DNA drugs (e.g., coding genes, CRISPR systems), protein drugs (e.g., antibodies), and small molecule drugs.

[0182] The coupling method may be coupling by physical methods, such as coupling by interaction forces; or may be coupling by chemical methods, such as coupling by covalent bonds, ionic bonds, etc.

[0183] This application relates to a method for treating a subject in need, which may include administering to the subject a prophylactically effective amount or a therapeutically effective amount of an antibody or an antigen-binding fragment thereof and / or a composition as described herein. As used herein, "subject" may include any animal exhibiting symptoms of a disease, disorder, or affliction that can be treated with the antibodies, antigen-binding fragments thereof, compositions, and methods disclosed herein. Suitable subjects (e.g., patients) may include non-human primates and / or human patients. Non-human primates may be selected from one or more of the following: laboratory animals (such as mice, rats, rabbits, or guinea pigs), farm animals (such as horses or cattle), domestic animals, pets (such as cats or dogs).

[0184] As used herein, "administering" refers to introducing into a subject, or bringing into contact with cells and / or tissues, the antibody or antigen-binding fragment thereof, the isolated nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition described herein. Administration can be carried out by injection, irrigation, inhalation, ingestion, electroporation, hemodialysis, iontophoresis, and / or other methods known in the art. The route of administration may vary with the location and nature of the disease being treated. The route of administration may include the site of administration and the mode of administration. The site of administration may be selected from one or more of the following: via ear, buccal, conjunctival, percutaneous, transdental, intracervical, endosinusial, intratracheal, enteral, epidural, interstitial, intra-articular, intra-arterial, intra-abdominal, intra-auricular, intra-biliary, intra-bronchial, intra-bursal, intra-cavernous sinus, intracerebral, intracisternal, intra-corneal, intra-coronary, intracranial, intradermal, intra-discal, intra-catheter, intra-duodenal, intradural, intra-epicardial, intra-epidermal, intra-esophageal, intra-gastric, intra-gingival, intra-hepatic, intra-ileal, intralesional, intra-lingual, intra-luminal, intra-lymphatic, intra-mammary, intramedullary, intra-meningeal, intramuscular, intranasal, intra-nodal, intra-ocular, intra-retinal, intra-ovarian, intra-peritoneal, intra-pericardial, intra-pleural, intra-prostatic, intra-pulmonary, intra-rumenal, intra-spinal, intra-synovial, intra-tendinous, intra-testicular, intratracheal, intrathecal, intrathoracic, intra-tubular, intra-tumoral, intra-tympanic, intra-uterine, intra-vascular, intra-ventricular, intra-vesical, intra-vestibular, intravenous, intravitreal, intra-laryngeal, via nose, nasogastric, via mouth, via eye, oropharyngeal, parenteral, percutaneous, perijoint, epidural, perineural, periodontal, respiratory, retro tubular, rectal, spinal, subarachnoid, subconjunctival, subcutaneous, sub-dermal, sub-gingival, sublingual, submucosal, sub-retinal, topical, percutaneous, intra-endocardial, transmucosal, transplacental, transtracheal, trans-tympanic, ureteral, urethral, and vaginal. The mode of administration may be selected from one or more of the following: perfusion, lavage, and direct injection.

[0185] As used herein, "treating / treatment" means administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof and / or a composition thereof as described herein to effect an improvement in a disease or disorder, or in the symptoms of a disease or disorder, in the subject. The improvement can be any improvement or alleviation of the disease or disorder, or of the symptoms of the disease or disorder. The improvement can be an observable or measurable improvement or a general sense of well-being of the subject. Thus, those skilled in the art will recognize that treatment can effect an improvement in a disease condition but may not result in a complete cure of the disease. A "prophylactically effective amount" means an amount of a virus, virus stock or composition that is effective to achieve the desired prophylactic result. As used herein, "prophylaxis" can mean complete prevention of disease symptoms, delay in the onset of disease symptoms, or reduction in the severity of subsequently occurring disease symptoms. Typically, but not necessarily, since prophylactic doses can be administered to a subject before or early in the course of disease, a prophylactically effective amount is less than a therapeutically effective amount.

[0186] The antibodies of the present invention can be delivered by conventional methods, preferably by systemic administration, such as by intravenous infusion.

[0187] The antibodies of the present invention can be administered in a therapeutically effective amount. A "therapeutically effective amount" means an amount of an antibody that is sufficient to effect such treatment of a disease, or of at least one clinical symptom of a disease or disorder, when administered to a subject to treat the disease, disorder or symptom. A "therapeutically effective amount" can vary with the antibody, the disease, disorder and / or symptoms of the disease or disorder, the severity of the disease, disorder and / or symptoms of the disease or disorder, the age of the subject to be treated, and / or the weight of the subject to be treated.

[0188] In a preferred embodiment of the present application, the dosage (mg / kg) of the antibody is based on the weight of the subject. For example, the single-dose of the antibody of the present invention can range from about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably from 5 ng / kg body weight to about 2.5 mcg / kg body weight, more preferably from 0.1 mcg / kg body weight to about 1 mcg / kg body weight. For example, the dosage can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mcg / kg body weight.

[0189] One advantage of the antibody of the present invention is that it can efficiently and completely activate T cells, and is not prone to apoptosis after activation. In addition, it can induce memory T cells to provide a sustained immune response, so that administration can be carried out at a relatively low dosing interval. In a preferred embodiment, when administered by intravenous infusion, the interval between two administrations of the antibody of the present invention can be not less than 1 day, not less than 3 days, not less than 5 days, or even one week apart. For example, the antibody of the present invention can be administered 7 times a week, 5 times a week, 3 times a week, twice a week or once a week. The antibody of the present invention can be administered in a cycle of 2-4 weeks, such as 3 weeks, and administered for one cycle or multiple cycles.

[0190] On the other hand, the present application provides a kit, which comprises the antibody or its antigen-binding fragment described in the present application, the isolated nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, and / or the pharmaceutical composition described in the present application.

[0191] In certain embodiments, the kit is used to detect the content of CD117 in a mixed system. In certain embodiments, the detection can be qualitative detection or quantitative detection. In certain instances, the content of CD117 can characterize the abundance of hematopoietic stem cells in the mixed system alone or together with other indicators.

[0192] Without being limited by any theory, the following examples are only for explaining the fusion protein, preparation method and uses of the present application, etc., and are not used to limit the scope of the invention of the present application.

[0193] Examples

[0194] Example 1. Immunization of genetically engineered humanized mouse antigen

[0195] Use AceMouseNB TM mice to generate nanobodies. AceMouseNB TM is a genetically engineered humanized mouse model designed to produce fully human heavy chain only antibodies (HcAbs). The mouse is generated by targeted gene editing, replacing the mouse heavy chain variable region with the corresponding human heavy chain variable region and silencing the light chain expression, so as to be able to produce human single domain antibodies containing VHH. This model mouse can produce single domain antibodies with high affinity antigen recognition and low immunogenicity, accelerating the discovery of single domain antibodies for therapeutic and diagnostic applications.

[0196] Immunize the mice with the CD117 antigen expression plasmid according to the standard immunization procedure. Analyze the sera of the mice after CD117 antigen immunization by flow cytometry to evaluate whether specific antibodies are produced in the mice.

[0197] CHO-CD117 or CHO-WT cells were seeded at the same number in each well of a 96-well plate. Antigen-immunized mouse sera were serially diluted with a diluent (DPBS + 3% FBS) at ratios of 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24300, and 1:72900. 50 μL of each diluted serum sample was added to the corresponding wells containing cells, gently mixed, and incubated at 4°C for 45 minutes. After centrifugation again, the cells were washed twice with a wash buffer (DPBS + 3% FBS), and then the cells were resuspended in 50 μL of a secondary antibody solution (goat anti-mouse IgG-PE, Invitrogen, 12-4010-82; or goat anti-human IgG Fc secondary antibody-PE, Invitrogen, 12-4998-82) at a concentration of 500 ng / mL and incubated at 4°C for 30 minutes. After centrifugation again and washing twice more with the wash buffer, the cells were resuspended in 25 μL of 3% FBS, and the samples were analyzed by flow cytometry to determine the median fluorescence intensity of PE for each cell population. As Figure 1 shown, the mice immunized with CD117 antigen generated a stable and specific immune response. After multiple booster immunizations and the last booster immunization, these mice were suitable for isolating CD117-specific monoclonal B cells.

[0198] Example 2. Isolation and Clonal Analysis of Anti-CD117-Specific Single B Cells

[0199] Using the Single-B cell technology, splenocytes were sorted. Clones were obtained from 4 96-well plates, and then 2 plates were selected to obtain antibody genes. The antibody genes were amplified by PCR and cloned into the AceMab expression vector and transiently transfected to prepare recombinant antibody expression supernatants for clone verification. According to the method described in Example 1, each sample was analyzed by flow cytometry using CHO-CD117, CHO-K1, or HEL cells. As Figure 2 shown, positive clones with an MFI value of CD117-positive cells greater than 10000 and an MFI value of control cells (CHO-K1) less than 5000 were selected and sequenced using NGS technology.

[0200] Example 3. Antibody Purification

[0201] The positive clone antibody was expressed using the AceMab antibody expression vector, and the cell culture supernatant was collected for antibody purification. Briefly, the cell culture supernatant was first filtered through a 0.22 μm membrane filter to remove all cell debris and particles. Then the supernatant was transferred to pre-equilibrated magnetic beads Pro-A and incubated at room temperature for 30 minutes to allow antibody binding. After incubation, the magnetic beads were collected with a magnet for 1 minute until the solution became clear, and then the supernatant was discarded. The magnetic beads were resuspended in 5 volumes of washing buffer and incubated for 5 - 10 minutes, and the medium supernatant was collected again with the magnetic beads for 1 minute. After the solution was cleared, the supernatant was removed and washed. This washing process was repeated twice to ensure thorough removal of non-specific binding proteins. 1 mL of elution buffer (0.05 M citric acid, pH = 3.0) was added to elute the antibody 2 - 3 times. Neutralization buffer (1 / 15 of the eluate volume) was added, and the antibody concentration was determined by ultraviolet spectrophotometry at 280 nm. To perform desalting and buffer exchange, the eluted antibody was processed through a G25 Sephadex chromatography column. Finally, standard SDS-PAGE identification was performed.

[0202] Example 4. Antibody Characterization

[0203] Antigen-binding efficiency

[0204] First, using the method in Example 1, the binding efficiency of the monoclonal antibody was evaluated by flow cytometry using human CD117-CHO, monkey CD117-CHO, or HEL cells. The initial concentration of each antibody was 10 μg / mL, and then a series of three-fold dilutions were performed to determine the antibody affinity and specificity at different concentrations. The data are as Figure 3 shown.

[0205] Subsequently, the binding efficiency of the monoclonal antibody to mouse CD117 antigen was evaluated by ELISA. Mouse CD117 antigen was diluted to 1 μg / mL with PBS as the coating solution, and 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the coating solution was discarded, and the 96-well plate was washed once with 200 μL of PBS; then 200 μL of blocking solution was added to each well and incubated at room temperature for 2 hours. After blocking, the antibody was diluted with dilution buffer to the following concentrations: 10000, 3333.3, 1111.1, 370.37, 123.46, 41.15, 13.72, and 4.57 ng / mL; 50 μL of each concentration of antibody was added to the 96-well plate and incubated at room temperature for 1 hour. After incubation, the 96-well plate was washed 3 times with 200 μL of washing buffer (PBS containing 0.05% Tween-20). 50 μL of HRP-conjugated goat anti-human IgG antibody (0.4 μg / mL) was added and incubated at room temperature for 1 hour; the solution was discarded, and the 96-well plate was washed 5 times with 200 μL of washing buffer. During detection, 50 μL of TMB substrate was added and incubated at room temperature in the dark for 3 - 5 minutes; 50 μl of 0.25 M sulfuric acid was added to each well to stop the reaction, and the absorbance was measured at 450 nm. The results (as Figure 4 ) showed that none of the candidate clones bound to mouse CD117.

[0206] Affinity

[0207] The affinity of the antibody for human CD117 recombinant protein was detected by biolayer interferometry. Briefly, the anti-human Fc probe (gator, 160003) was hydrated in buffer for at least 10 minutes before use. The antibody was diluted to 5 μg / mL in buffer and added to a 96-well plate sensor (beyotime, FCP966). Human CD117 recombinant protein antigen was serially diluted to 300, 100, 33.3, 11.1, 3.7, 1.23, and 0.41 nM in buffer; another control well without antigen was prepared as a baseline reference. The sensor with the antibody was immersed in the human CD117 recombinant protein with different concentrations, and the binding period was monitored for 240 seconds; subsequently, the sensor was transferred to fresh buffer for dissociation monitoring for more than 300 seconds. The data are shown in Table 1.

[0208] Table 1. Results of affinity detection of candidate antibodies.

[0209] ID koff (1 / s) kon (1 / Ms) KD (M) Anti-CD117 2.95E-03 1.41E+06 2.09E-09 H.2346B1 3.67E-04 1.71E+06 2.15E-10 H.2346B2 9.22E-04 4.85E+05 1.90E-09 H.2346B3 5.06E-04 8.96E+05 5.65E-10 H.2346B4 7.38E-05 1.48E+06 4.99E-11 H.2346B5 1.54E-03 2.09E+06 7.38E-10 H.2346B6 1.13E-03 1.73E+06 6.53E-10 H.2346B7 1.17E-03 1.10E+06 1.06E-09 H.2346B8 1.08E-03 2.04E+06 5.32E-10 H.2346B9 8.72E-04 6.74E+05 1.29E-09 H.2346B10 2.96E-03 2.71E+05 1.09E-08 H.2346B11 7.20E-04 1.68E+06 4.28E-10 H.2346B12 7.83E-04 3.98E+05 1.97E-09 H.2346B13 6.42E-04 2.96E+06 2.17E-10 H.2346B14 7.35E-04 6.02E+05 1.22E-09 H.2346B15 3.65E-03 3.47E+06 1.05E-09 H.2346B16 6.65E-04 6.06E+05 1.10E-09 H.2346B17 8.27E-04 6.62E+05 1.25E-09

[0210] Based on all the results of antigen binding and affinity, H.2346B1, H.2346B3, H.2346B4, H.2346B11, H.2346B13, and H.2346B17 were selected for the expression of CD117 monoclonal antibody.

[0211] Example 5. Purification and Identification of Modified VHH Antibodies

[0212] This example demonstrates that the candidate positive clone antibody was constructed in the form of anti-CD117 VHH, and the original anti-CD117 VHH sequence was engineered to introduce a histidine-tag, a hinge sequence, and a cysteine (C) residue at its C-terminus. It was cloned into the AceMab antibody expression vector according to the standard protocol. The recombinant expression vector was transfected into Expi293 cells for expression, and the purified antibody was obtained by His-tag affinity chromatography. All purified antibodies were identified by standard SDS-PAGE as Figure 5 shown.

[0213] Table 2 Figure 5 The antibody clones and concentrations corresponding to each lane

[0214] Lane Antibody clone Concentration (ug / ml) 1\5 H.2346B1 2700 2\6 H.2346B3 1500 3\7 H.2346B11 3450 4\8 H.2346B17 1800 9\12 H.2346B3 3880 10\13 H.2346B4 4600 11\14 H.2346B13 2100

[0215] Example 6. Synthesis of Ligand-Linker Conjugates

[0216] This example demonstrates a method for site-specific conjugation of a modified anti-CD117 VHH antibody to form a ligand-linker conjugate. First, the original anti-CD117 VHH sequence was engineered to introduce a histidine-tag, an optional hinge sequence, and a cysteine (C) residue at its C-terminus. The modified VHH sequence was transiently expressed in Expi293 cells and purified. The sequence modification enables the access of click chemistry groups at specific sites of VHH, thereby enhancing the conjugation potential without affecting the internal structural stability of VHH.

[0217] The connection mode of the cysteine residue with VHH includes direct connection (without hinge sequence) or connection via one of five hinge sequences with different structural features (named Hinge-1, Hinge-2, Hinge-4, Hinge-5, and Hinge-7) respectively. The design of each hinge sequence aims to reflect different structural characteristics. The complete amino acid sequences and corresponding component characteristics are provided in Table 2, including the percentage content of residues that enhance rigidity (such as P, Y, F, W, V, I, L), glycine (G) content, and the proportion of amino acids that promote the stability of the α-helix structure (such as A, L, E, M). Among them, Hinge-1 is the commonly used flexible hinge sequence (G4S)3, which has a high glycine content and shows good flexibility; Hinge-2 and Hinge-4 are classified as restricted hinge sequences, where Hinge-2 is rich in residues that enhance rigidity and has a very low glycine content, while Hinge-4 contains more than 80% of the amino acids that promote the stability of the α-helix structure; Hinge-5 and Hinge-7 have a rigid-enhancing amino acid ratio of approximately 50% and do not contain glycine, and are classified as rigid hinge sequences. Taking the cloning of H.2346B13 as an example, different hinge sequences were used for C-terminal extension, and the specific sequences are shown in the table.

[0218] Table 3 Different hinge sequences and their characteristics

[0219]

[0220]

[0221] Table 4 Amino acid sequences of anti-CD117 VHH with different C-terminal extension sequences

[0222]

[0223]

[0224] The structural schematic diagrams of the modified ligand, linker, and the ligand-linker conjugate formed by their conjugation are as Figure 6 shown. The specific conjugation steps include: First, reduce the modified and unmodified VHH using 2-mercaptoethanol (2-MEA), and then use Zeba TMThe desalting column (molecular weight cut-off 7K MWCO) was used to purify the reduced VHH; then the purified VHH was coupled with the linker DBCO-PEG4-maleimide at room temperature for 3 hours. The coupling site was the newly introduced cysteine residue, and the reaction conditions did not disrupt the internal disulfide bond structure of VHH itself. After the reaction was completed, an ultrafiltration method with a molecular weight cut-off value of 3kD was used to remove the free linker molecules, and at the same time, the buffer of the coupling product was changed to 10 mM PBS (pH 7.4). The purified ligand-linker conjugate was stored at -80 °C. The coupling product was analyzed and identified by size-exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS), and it was confirmed that the product had good purity and specifically incorporated the DBCO group.

[0225] Example 7 Preparation of Anchored Modified Lipid Nanoparticles

[0226] This example demonstrates a method for preparing anchored modified lipid nanoparticles (LNPs) using a polyethylene glycol (PEG) derivative containing a click group as an anchoring fragment. The anchoring fragment can be an amphiphilic polymer containing a click group, a PEG-lipid conjugate containing a click group, or a PEG-hydrophobic polymer conjugate containing a click group. By introducing an anchoring fragment containing a click reaction group, these anchored modified LNPs can be coupled with a ligand modified with a linker, thus efficiently forming the desired ligand-conjugated lipid nanoparticles. The click group refers to a specific group participating in click chemistry reactions.

[0227] The anchoring fragment containing a click group used in this example was DSPE-PEG-N3, which is a PEGylated lipid with an azide group at the end and can participate in click chemistry reactions. DSPE-PEG-N3 was incorporated into the lipid mixture used for LNP preparation. The ethanol phase of the lipid components included an ionizable cationic lipid, the neutral lipid DSPC, cholesterol, DSPE-PEG2000, and the lipid DSPE-PEG2000-N3 containing a click group. All lipid materials were purchased from Avanti Polar Lipids. The specific preparation method was to dissolve the above lipids in ethanol, and RNA was dissolved in 50 mM sodium citrate buffer (pH 4.0) at a concentration of 0.1 mg / mL, and the mass ratio of RNA to the ionizable cationic lipid was maintained at 1:10.

[0228] Preparation of LNP using microfluidic technology: The lipid solution (ethanol phase) was injected into the microfluidic mixer at a flow rate of 1 mL / min, while the aqueous solution of RNA was injected at a flow rate of 3 mL / min. The volume ratio of the aqueous phase to the ethanol phase was maintained at 3:1. Lipid nanoparticles were formed in the mixed solution. Subsequently, dialysis was performed to remove ethanol and replace the buffer with PBS (10 mM, pH 7.4). The dialysis step was carried out at 4 °C using a Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific Inc., molecular weight cut-off 100 KD) for two 2-hour dialyses against PBS. The dialyzed LNP was concentrated by ultra-high speed centrifugation.

[0229] Characterization of lipid nanoparticles: The particle size and polydispersity index (PDI) of LNP were measured using a Zetasizer Nano ZS instrument (BeNano, Bettersize), and measurements were carried out in PBS and Tris-HCl buffer respectively to confirm its stability in different media. The concentration of RNA in LNP was determined by ultraviolet-visible spectrophotometry, by recording the absorption spectrum and calculating based on a specific extinction coefficient, paying particular attention to the absorbance at 260 nm and calibrating with 330 nm as the baseline. At the same time, the QUANT-IT TM RIBOGREEN RNA assay kit (Shanghai Flash Crystal Molecular Biotechnology Co., Ltd.) was used to evaluate the encapsulation efficiency of RNA. The sample of ligand-conjugated lipid nanoparticles was diluted in TE buffer and placed in a polystyrene 96-well plate, incubated at 40 °C for 10 minutes, 1:200 diluted RIBOGREEN reagent was added, and the fluorescence intensity was measured using a Molecular Devices i3max microplate reader at wavelengths of approximately 488 nm and 525 nm respectively. The free percentage of RNA was determined by comparing the fluorescence intensity of the sample of intact ligand-conjugated lipid nanoparticles with the sample after adding Triton X-100 to disrupt the particles, and finally the encapsulation rate of RNA was calculated.

[0230] Example 8 Preparation of targeted lipid nanoparticles by bioorthogonal click reaction

[0231] This example demonstrates a method for preparing targeted lipid nanoparticles (tLNP) by conjugating ligands on the surface of lipid nanoparticles (LNP) through bioorthogonal click chemical reactions. The simple process steps include mixing the VHH-linker conjugate with the anchor-modified LNP in solution and subjecting it to ultrafiltration treatment to achieve ligand conjugation under mild conditions. The specific preparation process is as Figure 7 shown.

[0232] First, a certain amount of VHH-linker conjugate was added to the pre-prepared anchored modified LNP solution. The two were mixed at a specific ratio and incubated at room temperature for 3 hours to complete the conjugation reaction. After incubation, ultrafiltration was performed three times through an ultrafiltration device with a molecular weight cut-off value of 100 KD to remove unbound ligands and replace the buffer with 20 mM Tris-HCl (pH 7.4). To achieve long-term storage, 40% sucrose solution was added to adjust the final concentration of the buffer to 20 mM Tris-HCl (pH 7.4) with a sucrose concentration of 8%. Finally, the tLNP was aseptically filtered through a 0.22 μm filter membrane. The obtained LNP was aliquoted and stored at -80 °C for future use.

[0233] Example 9 In vitro evaluation study of cell lines

[0234] This example demonstrated a comparative study of cell lines expressing specific receptors and cell lines not expressing the receptor to evaluate the delivery efficiency and off-target effects of targeted lipid nanoparticles (tLNP). The HEL cell line (a human erythroleukemia cell line) and CHO engineered cells overexpressing the CD117 receptor were used as target cells, and unmodified CHO cells were used as non-target cells for the comparative study. HEL cells were cultured in RPMI-1640 medium containing L-glutamine (ThermoFisher), 10% fetal bovine serum, and 1% penicillin-streptomycin. CHO cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The cells were seeded into 24-well plates at a density of 200,000 cells / 250 μL per well. Subsequently, a specific amount of tLNP encapsulating nucleic acids encoding GFP or tdTomato was added to each well. After incubation for 6 hours, the cell pellet was collected by centrifugation at 300 g for 7 minutes, and the cells were resuspended in 100 μL PBS for flow cytometry analysis. Analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter), and flow cytometry data were processed using CytExpert software to determine the percentage of GFP or tdTomato positive cells in different cell lines.

[0235] Example 10 Comparative study of LNPs conjugated with ligands of different molecular weights on the surface

[0236] This example evaluated the targeting delivery efficiency of the tLNP preparation method disclosed in the present invention when surface-coupled with ligands of different molecular weights. Two antibodies targeting CD117 (c-kit) were selected for comparison. CD117 is a receptor highly expressed on hematopoietic stem cells (HSCs) and is suitable for the targeting delivery of HSCs. These two antibodies are VHH (about 16 kDa) and scFv (about 30 kDa) respectively. Ligand-linker conjugates were prepared according to the method described in Example 6, using the modified anti-CD117 VHH and anti-CD117 scFv, and the specific amino acid sequences are shown in Table 5. Both the VHH and scFv sequences were modified at their C-terminals, introducing a histidine tag, a non-(G4S)n type hinge sequence, and a cysteine residue.

[0237] Table 5 Original and modified antibody sequences

[0238]

[0239]

[0240] Subsequently, anchored-modified lipid nanoparticles encapsulating mRNA encoding GFP protein were prepared according to the method of Example 7, and then tLNPs targeting CD117 were formulated according to the steps of Example 8. Based on previous optimization formula experience, two formula ratios were selected: Formula C and Formula E, where the total content of PEG lipids was 1% and 1.5% respectively, and the ratio of anchored lipid to PEG lipid was 50% in both cases. The ionizable lipid used was Compound 062. In the ligand conjugation step, the ligand usage range was 0.125% to 0.5%. The information on each formula and its physicochemical characterization parameters is shown in Table 6.

[0241]

[0242] Table 6 Information on different LNP formulations and their physicochemical characterization parameters

[0243]

[0244]

[0245] According to the steps of Example 9, the tLNPs prepared above were used to transfect HEL cells (human erythroleukemia cells) and hCD117-overexpressing CHO cells (hCD117 CHO). The transfection results are as Figure 8As shown. Both the VHH and scFv conjugates significantly enhanced the GFP gene expression level in target cells (cells expressing CD117). It was observed that within the dosage range of different antibody-linker conjugates, VHH always performed better than scFv. For example, in HEL cells, the mean fluorescence intensity (MFI) obtained from the optimal VHH-conjugated LNP formulation after background subtraction reached 10 times that of the MFI obtained from the optimal scFv-conjugated LNP formulation. In addition, it is worth noting that for scFv, when the ligand amount increased from 0.125% to 0.5%, the delivery efficiency to target cells showed a gradually increasing trend; while VHH exhibited the best delivery performance at a relatively low ligand amount of 0.125%. The above research results indicate that using the preparation technology disclosed in the present invention, antibody forms with lower molecular weights (especially VHH) are expected to achieve better targeted delivery effects.

[0246] Example 11 Evaluation of the effect of hinge sequence on the delivery efficiency of tLNP targeting CD117

[0247] To deeply investigate the effect of hinge sequence on the delivery efficiency, we used a single-domain antibody (VHH, clone H.2346B13) targeting the hematopoietic stem cell marker CD117. All constructs shared the same VHH framework structure, and a cysteine residue was introduced at the C-terminus to enable site-specific conjugation. The cysteine was conjugated to VHH either directly (without a hinge sequence) or via one of five different hinge sequences with distinct structural features (named Hinge-1, Hinge-2, Hinge-4, Hinge-5, and Hinge-7), and the design of each hinge sequence was intended to reflect different structural characteristics, as shown in Example 6. All VHH constructs were transiently expressed and purified (the specific amino acid sequences are shown in Table 3). After reducing the cysteine at the C-terminus of VHH as described in Example 6, site-specific conjugation with the DBCO-PEG4-maleimide linker was achieved through a thiol-maleimide chemical reaction. Subsequently, anchored modified lipid nanoparticles encapsulating mRNA encoding GFP protein were prepared according to the method described in Example 7, and then tLNP targeting CD117 was prepared according to the procedure of Example 8, using Formulation C (total PEG lipid content was 1%, and the ratio of anchored lipid to PEG lipid was 50%). The composition and physicochemical properties of the final LNP are shown in Table 7.

[0248] Table 7 Information on different LNP formulations and their physicochemical characterization parameters

[0249]

[0250] We used human CD117-expressing CHO cells as the positive target cell population and unmodified wild-type CHO cells as the negative control to evaluate the delivery efficiency of the above-mentioned CD117-targeting LNPs. The in vitro transfection experiment was carried out according to the method described in Example 9, and the results are as Figure 9 shown. The mean fluorescence intensity (MFI) of all tLNPs conjugated with VHHs in CD117-positive CHO cells was significantly higher than that in wild-type CHO cells, confirming their specific delivery ability. Notably, the construct containing the G4S hinge sequence performed significantly better than the construct without the hinge sequence, indicating that even a flexible hinge sequence can improve the accessibility of the C-terminal cysteine site, facilitating the conjugation reaction. In addition, the structural properties of different hinge sequences significantly affected the delivery efficiency: constructs containing restricted or rigid hinge sequences (Hinge-2, Hinge-4, Hinge-5, Hinge-7) consistently had higher transfection efficiency in target cells than constructs without a hinge or with a flexible G4S hinge. Among them, constructs containing rigid hinge sequences (Hinge-5 and Hinge-7) achieved the highest delivery level, slightly better than constructs containing restricted hinge sequences. This result suggests that the spatial constraint provided by the rigid hinge may be more helpful in optimizing the display site of the conjugated VHH on the LNP surface, thus interacting more effectively with the receptor.

[0251] Example 12. In vitro gene editing of HSC cells using targeted LNPs loaded with CRISPR / Cas12b

[0252] In this example, the inventors evaluated the gene delivery and editing capabilities of the CD117-targeting tLNPs disclosed in this application. These tLNPs were designed to deliver gene editing components, which encapsulated the mRNA encoding CRISPR-AaCas12bMax and sgRNA targeting the HBG1 / 2 promoter. Editing the HBG1 / 2 promoter is expected to induce insertion mutations and reactivate fetal globin expression, which may be a new method for treating thalassemia and sickle cell disease.

[0253] AaCas12bMax is a highly active variant of Alicyclobacillus acidoterrestris-derived Cas12b (AaCas12b) and can be used as a gene editing enzyme. The amino acid sequence of AaCas12bMax is shown in Table 8. The mRNA encoding AaCas12bMax was synthesized by in vitro transcription (IVT) as follows: the coding sequence of AaCas12bMax was inserted into a plasmid to form a DNA template, which contained the target protein sequence, 5' and 3' UTR sequences, and a T7 promoter upstream of the 5' UTR; the PCR product of the above plasmid containing a poly A tail was used for in vitro transcription. T7 RNA polymerase recognized the T7 promoter of the DNA template and initiated in vitro mRNA transcription; the 5' cap structure of the in vitro transcribed mRNA was added during in vitro transcription, and n1-methyl-pseudouridine was used instead of uracil. All in vitro transcription reactions were carried out at 37 °C for 2 hours. DNase I removed the DNA template at 37 °C for 30 minutes, followed by column purification to obtain full-length in vitro transcribed mRNA.

[0254] The sgRNA was designed to target the LRF binding motif in the region 200 bp upstream of the HBG1 / HBG2 gene promoter. The sgRNA was optimized by chemical modification and the targeting sequence was extended to 23 nt. The optimized sgRNA was synthesized by GenScript and dissolved in water to 100 μM.

[0255] To achieve precise gene editing in HSC cells, CD117-targeted LNPs encapsulating CRISPR-AaCas12bMax mRNA and sgRNA were prepared. First, a ligand-linker conjugate was prepared using a modified anti-CD117 VHH antibody according to Example 6. Subsequently, the anchored modified LNPs were prepared as described in Example 7. Finally, tLNPs encapsulating CRISPR mRNA and sgRNA were prepared as described in Example 8, where the weight ratio of AaCas12bMax mRNA to sgRNA was maintained at 1:1. The final LNP concentration was determined according to the total RNA concentration in the formulation. The detailed composition and physicochemical properties of the CD117-targeted LNPs are shown in Table 9.

[0256] HEL cells were exposed to LNP-922 / 923 (1 - 4 μg / mL) at a concentration of 4E+05 / mL for 24 hours. Subsequently, the HEL cells were cultured for another 48 h and genomic DNA was extracted. A library was constructed by amplifying the region around the target sgRNA binding site and sequenced by NGS. The NGS results were analyzed using Cas-Analyzer (www.rgenome.net / cas-analyzer). Figure 10 Data showed that both LNP-922 and LNP-923 exhibited high editing efficiency and were dose-dependent, indicating their great potential for in vivo editing.

[0257] Table 8. AaCas12bMax and sgRNA sequences

[0258]

[0259]

[0260]

[0261]

[0262] Table 9. VHH antibodies and formulations used for different HSC-targeting LNPs

[0263]

[0264] Table 10 CDR sequences of different clones

[0265] Clone name CDR1 (Kabat 31–35) CDR2 (Kabat 50–65) CDR3 (Kabat 95–102) H.2346B4 VVSGFTFGSYA SSISGIDNGTYYADSVKG AKDQEYSYDPYFFDY H.2346B11 AASGFAFSDAW GRIKGKTDGGTTDYAAPVK THHSGYDFPDTFDI H.2346B13 AASGFTFSDAS GRIKSQPDGGTIDYAAPVK CSLDPTSTGEDFFDI

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human CD117, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH), and the heavy chain variable region comprises heavy chain complementarity determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2), and heavy chain complementarity determining region 3 (HCDR3), and wherein the HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups: (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:4; (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8; or (3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

12.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, or comprises an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:

9.

3. The antibody or antigen-binding fragment thereof according to claim 1, which is a VH that specifically binds to human CD117.

4. The antibody or antigen-binding fragment thereof according to claim 1, which is a single-domain antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1, which comprises an Fc fragment.

6. A fusion protein comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5.

7. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1-5 or the fusion protein according to claim 6.

8. A vector comprising the isolated nucleic acid molecule according to claim 7.

9. A cell comprising the isolated nucleic acid molecule according to claim 7 or the vector according to claim 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the fusion protein according to claim 6, the isolated nucleic acid molecule according to claim 7, the vector according to claim 8, and / or the cell according to claim 9, and optionally a pharmaceutically acceptable carrier.

11. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the fusion protein according to claim 6, the isolated nucleic acid molecule according to claim 7, the vector according to claim 8, the cell according to claim 9, and / or the pharmaceutical composition according to claim 10 in the preparation of a medicament for preventing and / or treating a disease.

12. A reagent or kit for detecting CD117 in a sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the fusion protein according to claim 6, the isolated nucleic acid molecule according to claim 7, the vector according to claim 8, the cell according to claim 9, and / or the pharmaceutical composition according to claim 10.

13. A method for detecting CD117 in a sample, the method comprising using the antibody or antigen-binding fragment thereof according to any one of claims 1-5, the fusion protein according to claim 6, the isolated nucleic acid molecule according to claim 7, the vector according to claim 8, the cell according to claim 9, and / or the pharmaceutical composition according to claim 10.

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