CN120399078A

CN120399078A - Nanobodies targeting CD117 and their applications

Info

Publication number: CN120399078A
Application number: CN202510592573.6A
Authority: CN
Inventors: 朱晰; 陈红; 洛文靖; 赵小平
Original assignee: Shanghai Tianze Yuntai Biomedical Co ltd
Current assignee: Shanghai Tianze Yuntai Biomedical Co ltd
Priority date: 2025-05-08
Filing date: 2025-05-08
Publication date: 2025-08-01

Abstract

The application relates to a CD 117-targeted nano antibody and application thereof. The application also relates to antibodies or antigen binding fragments thereof that target CD117 and uses thereof.

Description

CD 117-targeting nanobody and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a CD 117-targeted nano antibody and application thereof.

Background

Hematopoietic Stem Cells (HSCs) are the basic stone cells of the blood and immune systems, with the ability to self-renew and differentiate into all blood cell lineages. Clinically, bone marrow suppression with HSCs as a core is a curative therapy for treating various blood diseases including leukemia, lymphoma, and severe genetic blood diseases such as beta-thalassemia and sickle cell disease. In gene therapy, HSCs can be used as a breakthrough in curing diseases, and the patient's own HSCs can be genetically corrected in vitro and then returned to the in vivo to reconstitute a healthy blood system, which is reflected in emerging genome editing therapies for hemoglobinopathies. However, HSC basal therapies face challenges such as limited donor, graft versus host disease risk in allografts, and complexity of HSC handling in vitro. Therefore, it would be of great interest to develop a system that could be targeted for delivery directly in a patient to modify or eliminate HSCs, which would make transplantation and gene therapy safer and easier to obtain.

CD117, also known as c-Kit, is a transmembrane receptor tyrosine kinase capable of binding to Stem Cell Factor (SCF). CD117 is critical to the biological function of Hematopoietic Stem Cells (HSC), the interaction of SCF with c-Kit can promote HSC survival, proliferation and differentiation, and CD117 is widely used as a marker defining HSC and early progenitor cells. Almost all pluripotent HSCs and progenitor cells highly express CD117. In view of its key role, CD117 has been the target in HSC-based therapies. For example, monoclonal antibodies directed against CD117 can block SCF signaling and deplete HSC, a method that is being developed as a non-genotoxic conditioning regimen for removing bone marrow from patients prior to transplantation. anti-CD 117 antibody therapies (including antibody drug conjugates, ADCs) have been shown in preclinical models to promote therapeutic efficacy of donor cell engraftment by selective removal of HSCs while avoiding chemotherapy or radiotherapy conditioning. In addition to the transplantation field, CD117 is also expressed on certain leukemic or myelodysplastic stem cells, and anti-CD 117 immunotherapy is being explored for HSCs that clear these lesions. In summary, CD117 is a key identifier and function regulator of HSCs, making them attractive targets for direct therapy delivery to the HSC region.

Existing antibody targeting strategies for Hematopoietic Stem Cells (HSCs) and their markers, while showing promise, have significant limitations. Traditional full length monoclonal antibodies (typically IgG) are relatively high in molecular weight (about 150 kDa), may have limited tissue penetration (e.g., difficult to access to the bone marrow microenvironment), and circulate for a relatively long period of time in vivo. While IgG antibodies or Antibody Drug Conjugates (ADCs) directed against HSC markers such as CD117 may be effective in targeting HSCs, their size and structure are not suitable for certain applications, such as delivering gene therapy payloads into cells. Standard antibodies are not normally internalized into cells with high efficiency unless engineered to have this capability and their Fc segment may trigger immune effector functions (complement activation, ADCC) resulting in non-selective inflammatory responses or cell loss. Furthermore, HSC specific targeting using whole IgG may not be accurate enough that markers such as CD117 are also present on other cell types (e.g., T cell subsets or progenitor cells), so systemic administration of a potent IgG or ADC may affect non-targeted cells or require careful dosing. Gene therapy vectors (e.g., lentiviral or AAV vectors) face different challenges in that they often lack the natural chemotaxis for HSCs, and in vivo HSCs are mostly in a quiescent state and in a protective microenvironment where the circulating vector is difficult to reach. To overcome these obstacles, there is a need for targeting ligands that bind both highly specific to HSC markers and have smaller size and modular properties in order to be able to attach to a variety of delivery vehicles. Bispecific or multivalent forms of antibodies have also been proposed to increase specificity by binding to multiple HSC markers, as they require simultaneous recognition of both markers, but construction of stable large antibody complexes can be complex. In general, although existing antibody-based approaches demonstrate the concept of HSC targeting, there remains a need for improved targeting agents for applications such as in vivo gene delivery, precise cell isolation, and safe conditioning.

Single domain antibodies have unique advantages in therapeutic and diagnostic applications against CD117 and other Hematopoietic Stem Cell (HSC) markers. Typical single domain antibodies structurally comprise a VHH fragment. VHH are single domain antigen binding fragments derived from camelid heavy chain antibodies, typically only about 12-15KD in size. Because of its small size and monodomain structure, VHH exhibit high antigen binding affinity and specificity under a range of conditions, while having excellent stability. With these small and robust antibodies, the invention aims to provide tools and methods that significantly improve the specificity and efficiency of HSC targeting for a variety of therapeutic, prophylactic and diagnostic purposes.

Disclosure of Invention

The present application provides an antibody or antigen binding fragment thereof, preferably a nanobody, that is capable of specifically binding to human CD117 and targeting hematopoietic stem cells (Hematopoietic stem cell, HSCs) expressing CD 117. In particular embodiments, the antibodies or antigen binding fragments thereof may be used to detect the level of CD117 in a mixed system, to detect the level of hematopoietic stem cells in a mixed system, and to treat hematopoietic stem cell-related disorders by conjugation to other drugs.

The application provides antibodies or antigen binding fragments thereof that target CD117, which may have one or more of the following properties 1) are capable of specifically binding CD117 and/or cells expressing CD117, 2) are capable of inhibiting the binding of CD117 to at least one ligand thereof, 3) are capable of inhibiting CD 117-mediated signal transduction, 4) are capable of inhibiting migration, accumulation, recruitment and/or infiltration of cells expressing CD117, 5) are capable of mediating killing of cells expressing CD117, 6) are capable of inducing endocytosis of CD117 receptors on the cell surface, thereby reducing activation of immune cells, 7) are capable of being used in the treatment of CD 117-related diseases and/or disorders, 8) are capable of being used in the detection of CD117 and/or cells expressing CD 117.

The application also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, expression vectors, host cells, pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, methods of making the antibodies or antigen-binding fragments thereof, and uses of the antibodies or antigen-binding fragments thereof of the application.

In a first aspect, the application provides an antibody or antigen-binding fragment thereof that specifically binds human CD117. The antibody or antigen binding fragment thereof specifically binds human CD117. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH). The heavy chain variable region includes heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2), and heavy chain complementarity determining region 3 (HCDR 3).

In certain embodiments, the HCDR1, HCDR2, HCDR3 is selected from any one of the following groups:

(1) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 2, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 3, the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 4, or

(2) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 6, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 7, the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 8, or

(3) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 10, the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 11, and the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 12.

In certain embodiments, the VH comprises an amino acid sequence as set forth 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 to an amino acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 9.

In certain embodiments, the antibody is selected from one or more of the group consisting of a monospecific antibody, a multispecific antibody, a human antibody, a humanized antibody, and a chimeric antibody.

In certain embodiments, the antibody is selected from one or more of the group consisting of monovalent antibodies and multivalent antibodies.

In certain embodiments, the antigen binding fragment is selected from one or more of the group consisting of VH, fab, fv, F (ab') 2 and single chain Fv (scFv).

In a preferred embodiment, the antibody or antigen-binding fragment thereof is a VH that specifically binds human CD 117.

In a preferred embodiment, the antibody or antigen-binding fragment thereof is a nanobody. More preferably, the nanobody has a molecular weight of 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.

In a preferred embodiment, the antibody or antigen binding fragment thereof is a single domain antibody.

In certain embodiments, the antibody or antigen binding fragment thereof comprises an Fc fragment.

In a second aspect, the application provides a fusion protein. Comprising an antibody or antigen-binding fragment thereof of any one of the first aspects.

In certain embodiments, the fusion protein can be a Chimeric Antigen Receptor (CAR).

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

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

In a third aspect, the application provides an isolated nucleic acid molecule encoding any of the antibodies of the first aspect or antigen binding fragments thereof or any of the fusion proteins of the second aspect.

In certain embodiments, the isolated nucleic acid molecule may be produced or synthesized by (i) in vitro amplification, e.g., by Polymerase Chain Reaction (PCR) amplification, (ii) by clonal recombination, (iii) purification, e.g., by cleavage and gel electrophoresis fractionation, or (iv) synthesized, e.g., by chemical synthesis.

In a fourth aspect, the present application provides a carrier. Comprising an isolated nucleic acid molecule of any of the third aspects.

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

In certain embodiments, the RNA vector is an mRNA vector.

In a fifth aspect, the application provides a cell. Comprising an isolated nucleic acid molecule of any of the third aspects or a vector of any of the fourth aspects.

In certain embodiments, the cell comprises a host cell.

In particular embodiments, the host cell is a eukaryotic cell, such as a cell from a plant, a fungal or yeast cell, or the like.

In specific embodiments, the cell is a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2/0 cell, a NS0 cell, or a myeloma cell. More specifically, it may be an engineered cell, such as 293T cell, that produces antibodies.

In a sixth aspect, the application provides a method of producing an antibody or antigen-binding fragment thereof of any one of the first aspects. Specifically, the method comprises:

(1) Constructing an expression vector comprising a gene sequence encoding said antibody or antigen binding fragment thereof,

(2) Transforming the expression vector into a host cell to induce expression, and

(3) Isolating the antibody or antigen binding fragment thereof from the expression product.

In a seventh aspect, the present application provides a pharmaceutical composition. Comprising any of the antibodies or antigen binding fragments thereof of the first aspect, any of the fusion proteins of the second aspect, any of the isolated nucleic acid molecules of the third aspect, any of the vectors of the fourth aspect, any of the cells of the fifth aspect, and/or optionally a pharmaceutically acceptable carrier.

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

In certain embodiments, the pharmaceutical composition is used in combination with other drugs. Preferably, the other drug is a small molecule targeted anticancer agent, other antibody drug, adoptive cell therapy and/or oncolytic virus enhancer.

In certain embodiments, the pharmaceutical composition may be a conjugate of any one of the antibodies of the first aspect or antigen binding fragments thereof with other molecules. More specifically, the other molecule may be a small molecule drug, may be an antibody targeting an antigen other than CD117, may be a Lipid Nanoparticle (LNP).

In an eighth aspect, the application provides the use of any of the antibodies or antigen binding fragments thereof of the first aspect, any of the fusion proteins of the second aspect, any of the isolated nucleic acid molecules of the third aspect, any of the vectors of the fourth aspect, any of the cells of the fifth aspect, and/or any of the pharmaceutical compositions of the seventh aspect for the preparation of a medicament for the prevention and/or treatment of a disease.

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

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

In a tenth aspect, the application provides the use of any of the antibodies or antigen binding fragments thereof of the first aspect, any of the fusion proteins of the second aspect, any of the isolated nucleic acid molecules of the third aspect, any of the vectors of the fourth aspect, any of the cells of the fifth aspect, and/or any of the pharmaceutical compositions of the seventh aspect for the prevention and/or treatment of a disease.

In certain embodiments, the disease described in the eighth, ninth or tenth aspect may be a disease of the blood system. In particular, the disease may be a hematopoietic stem cell-related disease. More specifically, the hematopoietic stem cell-related disorder may be selected from one or more of aplastic anemia, myelodysplastic syndrome, acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, myeloproliferative neoplasm, myelofibrosis, immunodeficiency disease, thalassemia, and sickle cell disease.

In an eleventh aspect, the application provides a reagent or kit. The agent or kit comprises any of the antibodies or antigen binding fragments thereof of the first aspect, any of the fusion proteins of the second aspect, any of the isolated nucleic acid molecules of the third aspect, any of the vectors of the fourth aspect, any of the cells of the fifth aspect, and/or any of the pharmaceutical compositions of the seventh aspect.

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

In certain embodiments, the kit is used to detect the content of CD 117-expressing cells in a mixed system.

For example, the CD 117-expressing cells are selected from one or more of the group consisting of 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.

In a twelfth aspect, the application provides a method of detecting CD 117. The method comprises the use of any of the antibodies or antigen binding fragments thereof of the first aspect, any of the fusion proteins of the second aspect, any of the isolated nucleic acid molecules of the third aspect, any of the vectors of the fourth aspect, any of the cells of the fifth aspect, and/or any of the pharmaceutical compositions of the seventh aspect.

In certain embodiments, the CD117 may be free CD117 within the mixed system.

In certain embodiments, the CD117 may be CD117 expressed on a cell.

For example, the cells may be selected from one or more of the group consisting of 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.

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

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

Other aspects and advantages of the present application will become readily apparent to those skilled in the art 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 present disclosure enables one skilled in the art to make modifications to the disclosed embodiments without departing from the spirit and scope of the application as claimed. Accordingly, the drawings and descriptions of the present application are to be regarded as illustrative in nature and not as restrictive.

Drawings

The specific features of the application related to the application are shown in the appended claims. A better understanding of the features and advantages of the application in accordance with the present application will be obtained by reference to the exemplary embodiments and the accompanying drawings that are described in detail below. The drawings are briefly described as follows:

FIG. 1 shows the binding capacity of serum from mice immunized with CD117 antigen and immunopotentiated in example 1 to cells expressing CD117 (CHO-CD 117) and control cells (WT).

FIG. 2 shows the binding capacity of the recombinant antibodies of example 2 to CD117 positive cells (CHO-CD 117 and HEL) and control cells (CHO-K1).

FIG. 3 shows the binding efficiency of different concentrations of candidate antibodies to human CD117-CHO, monkey CD117-CHO or HEL cells in example 4.

FIG. 4 shows the results of evaluating the binding efficiency of a candidate antibody to mouse CD117 antigen using ELISA method in example 4.

FIG. 5 shows SDS-PAGE identification of purified antibodies in example 5. Wherein NR represents that no reducing agent is added to the sample, and R represents that the reducing agent is added to the sample.

FIG. 6 shows a schematic structure of the modified ligand, linker and ligand-linker conjugate formed by coupling the modified ligand and linker of example 6.

Fig. 7 shows a schematic step diagram of a method for preparing targeted lipid nanoparticles (tLNP) by bio-orthogonal click chemistry to couple ligands on the surface of Lipid Nanoparticles (LNP).

Figure 8 shows the differences in delivery efficiency of lipid nanoparticles surface-coupled with ligands of different lengths in cells expressing CD 117.

Figure 9 shows a comparison of the delivery efficiency of tLNP comprising different hinges in a cell line.

FIG. 10 shows an evaluation tLNP of the gene delivery and editing capacity of in vitro delivery of gene editing components.

Detailed Description

Further advantages and effects of the present application will become readily apparent to those skilled in the art from the present disclosure, by describing embodiments of the present application with specific examples.

Definition of terms

In the present application, the term "CD117", also called "c-kit", is a type III transmembrane protein tyrosine kinase receptor encoded by the proto-oncogene 4q11-q 12. It is expressed in a variety of cell types including hematopoietic stem cells, mast cells, melanocytes, spermatogonia, egg cells, and Cajal cells of the gastrointestinal tract. The ligand for CD117 is a stem cell factor (also known as mast cell growth factor) that, when bound to CD117, activates a range of intracellular signaling pathways, thereby regulating cell growth, survival, differentiation, migration and adhesion. In the present application, CD117 may include all subtypes and all species thereof unless otherwise specified. In the present application, the CD117 may include any natural CD117 of any vertebrate origin, including 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 a cell, and 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 accession number P10721 at the Uniprot website (https:// www.uniprot.org /).

In the present application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude the incorporation of artificial or synthetic substances, nor the presence of other impure substances that do not affect the activity of the substance. In the present application, the antibody or antigen-binding fragment thereof is also isolated unless specifically stated.

In the present application, the term "antigen binding protein" generally refers to a protein having antigen binding ability. For example, the antigen binding protein may comprise an isolated antigen binding protein. The antigen binding proteins may comprise, for example, an antibody-derived protein Framework Region (FR) or an alternative protein framework region or artificial framework region with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, framework regions comprising antibody sources that are introduced, for example, to stabilize mutations in the three-dimensional structure of the antigen binding protein, as well as fully synthetic framework regions comprising, for example, biocompatible polymers. See, e.g., korndorfer et al, 2003,Proteins:Structure,Fun ction,andBioinformatics,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 ', 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.

In the present application, the term "CDR" is also referred to as "complementarity determining region", and generally refers to a region in an antibody variable region whose sequence is highly variable and/or forms a structurally defined loop. Typically, three CDRs (HCDR 1, HCDR2, HCDR 3) are included in the heavy chain variable region (variabledomain of HEAVY CHAIN, VH) of the antibody. Antibodies composed of heavy chains alone are also able to function normally and stably in the absence of light chains, e.g. 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, a combination of Kabat/Chothia et al. Such coding systems are known in the art and can be found, for example, in www.bioinf.org.uk/abs/index. Html # kabatnum. For example, the amino acid sequence numbering of the antigen binding proteins may be according to the IMGT numbering scheme (IMGT, the international ImMunoGeneTics information system@imgt.cines.fr; IMGT. Circuits. Fr; lefranc et al, 1999,Nucleic Acids Res.27:209-212; ruiz et al, 2000Nucleic 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,DevComp Immunol 29:185-203). For example, the CDRs of the antigen binding protein may be determined according to the Kabat numbering system (see, e.g., kabat EA & Wu TT (1971) ANN NY ACADSCI 190:190:382-391 and Kabat EAet al.,(1991)Sequences of Proteins ofImmunological Interest,FifthEdition,U.S.Department of Health and Human Services,NIH Publication No.91-3242).

In the present application, the term "variable" generally refers to the fact that certain segments of the variable region may differ greatly in sequence between antibodies. The variable region mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable region. It is typically concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light or heavy chain variable regions. The more highly conserved portions of the variable regions are called Framework Regions (FR).

In the present application, the term "FR" generally refers to the more highly conserved portion of the antibody variable domain, which is referred to as the framework region. Typically, the native heavy chain variable domain comprises four FR regions, namely H-FR1, H-FR2, H-FR3 and H-FR4.

In the present application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, and encompasses any polypeptide comprising 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 antibodies", 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., are capable of specifically binding to CD 117).

In the present application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (e.g., CD 117). In the present application, the antigen binding fragment may comprise a Fab, fab ', F (ab) 2, fv fragment, F (ab') 2, scFv, di-scFv, VHH and/or dAb.

In the present application, the term "single domain antibody" generally refers to an antibody lacking a light chain. The single domain antibodies of the present application may include heavy chain antibodies (HcAb) that include a heavy chain variable region and conventional heavy chain CH2, CH3 regions. The single domain antibodies of the application may comprise the smallest binding unit of an antigen binding protein. The single domain antibodies of the application may include antibody fragments consisting only of the antibody heavy chain variable regions.

In the present application, the term "VHH (variabledomain of HEAVY CHAIN of HEAVY CHAIN anti-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 reviewed Vanlandschoot P. Et al, 2011,Antiviral Research 92,389-407).

In the present application, the term "nanobody" refers to an antibody having a small molecular weight. In a preferred embodiment, the nanobody consists of only the variable region of the heavy chain antibody, typically having a molecular weight of less than 15KD, which is 1/10 of that of a conventional antibody. This structure confers a unique set of properties to nanobodies, including high affinity, high stability, good water solubility, low immunogenicity, and strong tissue penetration.

In the present application, "single domain antibody" and "nanobody" refer to an intact antibody that can function independently, and "VHH" refers to a domain in an intact antibody. Still further, "single domain antibodies" emphasize a component of an antibody (e.g., comprising only a single heavy chain), while "nanobodies" emphasize the molecular weight of an antibody. In the present application, single domain antibodies may include, but are not limited to, nanobodies.

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 portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally a CH4 domain. For example, the Fc region may not comprise a 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. Pat. No. 5,648,260;5,624,821). The Fc portion of antibodies mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement Dependent Cytotoxicity (CDC), and half-life/clearance of antibodies and antigen-antibody complexes. Depending on the therapeutic purpose, in some cases these effector functions are desirable for therapeutic antibodies, but in other cases may be unnecessary or even detrimental.

In the present application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules consisting of single molecules. 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, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in the present application may be prepared by recombinant DNA methods.

In the present application, the terms "monospecific antibody" and "multispecific antibody" generally refer to antibodies capable of specifically binding to a single or multiple antigenic sites. When there are a plurality of antigen sites to which specific binding is to be imparted, the plurality of antigen sites may be derived from the same antigen or may be derived from different antigens, preferably the plurality of antigen sites are derived from different antigens.

In the present application, the terms "monovalent antibody" and "multivalent antibody" refer to antibodies having single and multiple sites on the molecule that bind antigen. Classical antibodies (e.g. IgG antibodies) are bivalent antibodies, with two sites for binding antigen, as informed by the person skilled in the art. In the present application, "monovalent antibody" and "multivalent antibody" may or may not contain a constant region and/or an Fc fragment. When the "monovalent antibody" or "multivalent antibody" does not comprise a constant region and an Fc fragment, the multivalent antibody may be formed by joining two or more antigen binding fragments end to end, preferably by covalent bonding, and the multivalent antibody may be joined N-terminally to C-terminally, N-terminally to N-terminally, or C-terminally to C-terminally.

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. Typically, the variable region is derived from an antibody of an experimental animal, such as a camelid (the "parent antibody"), and the constant region is derived from a human antibody, such that the resulting chimeric antibody has a reduced likelihood of eliciting an adverse immune response in a human individual as compared to the parent (e.g., camelid-derived) antibody.

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 (e.g., a camelid antibody) are replaced with the corresponding amino acids derived from a human immunoglobulin. Small additions, deletions, insertions, substitutions or modifications of amino acids in the CDR regions may also be permissible, provided that they still retain the ability of the antibody to bind to a particular antigen. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region. "humanized antibodies" retain antigen specificity similar to the original antibody. A "humanized" form of a non-human (e.g., camelid) antibody may minimally comprise chimeric antibodies derived from sequences of non-human immunoglobulins. In some cases, 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, affinity and/or ability, such as camel, alpaca, mouse, rat, rabbit or non-human primate. In some cases, the FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, the humanized antibody may comprise amino acid modifications that are not in the recipient antibody or in the donor antibody. These modifications may be made to further improve the properties of the antibody, such as binding affinity.

In the present application, the term "fusion protein" refers to a protein having two or more functions expressed in the same polypeptide chain by linking together gene sequences of two or more different proteins by genetic engineering techniques. Such protein fusion may confer new functional properties to the fusion protein or enhance its original function. The fusion protein has the advantages of multiple functions, enhanced stability, improved targeting property and simple production, and can be applied to different fields such as biopharmaceuticals, vaccine development, cell therapy and diagnostic tools. In specific embodiments, the fusion protein may be a viral membrane fusion protein, a cell fusion protein, or a bispecific antibody, an Fc fusion protein.

In the present application, the term "nucleic acid molecule" generally refers to an isolated form of a nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or synthesized synthetically.

In the present application, the term "vector" generally refers to a nucleic acid vector into which a polynucleotide encoding a protein can be inserted and the protein expressed. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. For example, vectors may include plasmids, phagemids, cosmids, artificial chromosomes such as Yeast Artificial Chromosomes (YACs), bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs), phages such as lambda or M13 phages, animal viruses and the like. Animal virus species used as vectors may include retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e.g., herpes simplex virus), poxvirus, baculovirus, papilloma virus (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these.

In the present application, the term "cell" generally refers to a single cell, cell line or cell culture that may or may not be the recipient of a subject plasmid or vector, which comprises a nucleic acid molecule according to the present application or a vector according to the present application. Cells may include progeny of a single cell. The offspring may not necessarily be identical to the original parent cell (either in the form of the total DNA complement or in the genome) due to natural, accidental or deliberate mutation. Cells may include cells transfected in vitro with the vectors of the application. The cells may be bacterial cells (e.g., E.coli), yeast cells, or other eukaryotic cells, such as COS cells, chinese Hamster Ovary (CHO) cells, CHO-K1 cells, LNCAP cells, heLa cells, HEK293 cells, CO S-1 cells, NS0 cells. Cells may also include engineered cells.

In the present application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or disorder. The pharmaceutical composition may comprise an antibody or antigen binding fragment thereof according to the application, a nucleic acid molecule according to the application, a vector according to the application and/or a cell according to the application, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions described herein include, but are not limited to, liquid, frozen and lyophilized compositions.

In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages 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, salt-forming counter ions, such as sodium, and/or nonionic surfactants.

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

In the present application, the term "hematopoietic stem cell-related diseases" generally refers to a series of diseases of the blood system caused by abnormal function, developmental defects, or malignant transformation of hematopoietic stem cells. These diseases typically involve bone marrow hematopoietic dysfunction, immunodeficiency or malignant proliferation of blood cells. These diseases may be caused by genetic factors, environmental exposure, or unknown factors, and symptoms including anemia, hemorrhage, infection, and organ enlargement, and diagnosis and treatment often require a multidisciplinary combination of methods.

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

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.

In the context of the present application, reference to a protein, polypeptide and/or amino acid sequence is also understood to include at least a range of variants or homologues having the same or similar function as the protein or polypeptide.

In the present application, the variant may be, for example, a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof capable of specifically binding CD 117). For example, the functional variant may comprise a protein or polypeptide that has been altered in amino acids by at least 1, such as 1-30, 1-20, or 1-10, and yet another such as 1,2,3,4, or 5 amino acid substitutions, deletions, and/or insertions. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to alteration (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 capacity) of the protein or the polypeptide prior to alteration. For example, the substitution may be a conservative substitution.

In the present application, the homolog 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 more) sequence homology to the amino acid sequence of the protein and/or the polypeptide (e.g., an antibody or fragment thereof capable of specifically binding CD 117).

In the present application, the homology generally refers to similarity, similarity or association between two or more sequences. The "percent sequence homology" may be calculated by comparing two sequences to be aligned in a comparison window, determining the number of positions in the two sequences where the same nucleobase (e.g., A, T, C, G, U) or the same amino acid residue (e.g., ala, pro, ser, thr, gly, val, leu, ile, phe, tyr, trp, lys, arg, his, asp, glu, asn, gln, cy s, and Met) is present to give the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce the percent sequence homology. Alignment to determine percent sequence homology can be accomplished 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. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length sequence being compared or over the region of the target sequence. The homology can also be determined by FASTA and BLAST. A description of the FASTA algorithm can be found in "improved tools for biological sequence comparison" by W.R.Pearson and D.J.Lipman, proc. Natl. Acad. Sci., 85:2444-2448,1988, and "quick sensitive protein similarity search" by D.J.lip man and W.R.Pearson, science,227:1435-1441,1989. A description of the BLAST algorithm can be found in "a basic local contrast (alignment) search tool", journal of molecular biology, 215:403-410,1990, U.S. Altschul, W.Gish, W.Miller, E.W.Myers, and D.Lipman.

In the present application, the term "comprising" generally means "including", "summarizing", "containing" or "comprising", which terms may be used interchangeably. In some cases, the meaning of "as", "consisting of.

In the present application, the term "about" generally means ranging from 0.5% to 10% above or below the specified value, e.g., ranging from 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

Antibodies or antigen binding fragments, fusion proteins

The CDRs of an antibody, also known as complementarity determining regions, are part of the variable region. The amino acid residues of this region may be contacted with an antigen or epitope. Antibody CDRs can be determined by a variety of coding systems, such as CCG, kabat, chothia, IMGT, abM, a combination of Kabat/Chothia et al. Such coding systems are known in the art and can be found, for example, in www.bioinf.org.uk/abs/index. Html # kabatnum. The CDR regions can be determined by one skilled in the art using different coding systems depending on the sequence and structure of the antibody. Using different coding systems, CDR regions may differ. In the present application, the CDRs encompass CDR sequences partitioned according to any CDR partitioning scheme, as well as variants thereof, including amino acid sequences of the CDRs that have been substituted, deleted and/or added with one or more amino acids. Such as 1-30, 1-20, or 1-10, and also such as 1,2, 3,4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and/or insertions, and homologues thereof, which may be amino acid sequences having at least about 85% (e.g., 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 more) sequence homology to the amino acid sequence of the CDR. In certain embodiments, the antigen binding proteins of the application may be defined by the KABAT coding system.

The present application provides an antibody or antigen-binding fragment thereof that specifically binds human CD 117. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH). The heavy chain variable region includes heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2), and heavy chain complementarity determining region 3 (HCDR 3).

In certain embodiments, the VH comprises an amino acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 9, or 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 to the amino acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 9 and all amino acid differences in the non-CDR regions.

Preferably, the amino acid differences are conservative substitutions of amino acids.

In the present application, the antibody or antigen-binding fragment thereof may comprise at least one CDR in the amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 9, and the CDR may comprise a CDR divided in any manner. The CDRs delimited by any one of the means fall within the scope of the claims of the present application if the sequence is identical to the amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 9.

In certain embodiments, the antibodies or antigen binding fragments thereof of the application include antigen binding proteins having an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 6 or SEQ ID NO. 10, HCDR2 having an amino acid sequence as shown in SEQ ID NO. 3, SEQ ID NO. 7 or SEQ ID NO. 11, and HCDR3 having an amino acid sequence as shown in SEQ ID NO. 4, SEQ ID NO. 8 or SEQ ID NO. 12, and humanized antigen binding proteins 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 CD 117.

In certain embodiments, the isolated antigen binding proteins of the application include a variable region having an amino acid sequence as set forth in SEQ ID NO. 1, SEQ ID NO. 5 or SEQ ID NO. 9, and humanized antigen binding proteins thereof.

In certain embodiments, an antibody or antigen-binding fragment thereof of the application may comprise a single domain antibody or antigen-binding fragment thereof. In certain embodiments, an antibody or antigen-binding fragment thereof of the application may comprise a heavy chain antibody or antigen-binding fragment thereof. In certain embodiments, an antibody or antigen-binding fragment thereof described herein may comprise a nanobody.

In certain embodiments, the antibody or antigen binding fragment thereof may comprise an antibody having only heavy chains. In certain embodiments, heavy chain-only antibodies consist of a variable region antigen binding domain consisting of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. In certain embodiments, heavy chain-only antibodies consist of an antigen binding domain, at least a portion of the hinge region, and CH2 and CH3 domains. In certain embodiments, heavy chain-only antibodies consist of an antigen binding domain, at least a portion of a hinge region, and a CH2 domain. In certain embodiments, heavy chain-only antibodies consist of an antigen binding domain, at least a portion of a hinge region, and a CH3 domain. Heavy chain-only antibodies in which CH2 and/or CH3 domains are truncated are also included herein. In further embodiments, the heavy chain consists of an antigen binding domain and at least one CH (CH 1, CH2, CH3 or CH 4) domain, but without a hinge region. Antibodies having only heavy chains may be in the form of dimers in which the two heavy chains are linked by disulfide bonds, or else are covalently or non-covalently linked to each other. Antibodies that are heavy chain only may belong to the IgG subclass, but antibodies that belong to other subclasses, such as the IgM, igA, igD and IgE subclasses, are also included herein. In particular embodiments, the heavy chain antibody may be of the IgG1, igG2, igG3 or IgG4 subtype, in particular of the IgG1 subtype.

In certain embodiments, the antibody may be selected from one or more of the group consisting of monoclonal antibodies, chimeric antibodies, and humanized antibodies.

In a preferred embodiment, the antibody may be a nanobody, more preferably a single domain antibody in a nanobody. Single domain antibodies have unique advantages in therapeutic and diagnostic applications against CD117 and other Hematopoietic Stem Cell (HSC) markers. The most prominent component of single domain antibodies is the VHH, a single domain antigen binding fragment derived from a camelid heavy chain-only antibody, which is typically only about 12-15kDa in size. Because of its small size and monodomain structure, VHH exhibit high antigen binding affinity and specificity under a range of conditions, while having excellent stability. These properties enable single domain antibodies to overcome many of the limitations of conventional antibodies in that they can penetrate tissues and cellular microenvironments more easily, can be recombinantly produced in low cost microbial systems, and can be readily engineered with a variety of payloads and formats by gene fusion or chemical coupling. Importantly, VHHs are generally capable of recognizing unique or hidden epitopes (thanks to their longer CDR3 loops) and tend to internalize efficiently upon binding to cell surface targets, especially if they are multivalent or linked to triggers that promote endocytosis. For example, nanobodies have been demonstrated to be able to reach intracellular targets when used as delivery agents, a key feature for the delivery of gene therapy payloads. They lack the Fc region themselves, thereby avoiding unwanted Fc-mediated immune effects, which is an advantage for imaging agents (reducing background uptake in Fc receptor-rich organs), or can be alleviated by the addition of Fc or other multimerization tags when effector function or prolonged half-life is desired. Overall, the stability, high affinity and modular nature of single domain antibodies make them ideal ligands for innovative HSC-targeted therapies.

In the present application, the antibody or antigen-binding fragment thereof may further comprise an Fc fragment. In certain embodiments, the N-terminus of the Fc fragment is directly or indirectly linked 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 linked by a hinge region. In certain embodiments, the Fc fragment may be derived from an Fc fragment of a human IgG. In certain embodiments, the antigen binding proteins of the application comprise an Fc fragment wild-type derived from human IgG. The amino acid sequence of an Fc fragment derived from human IgG is known in the art. In certain embodiments, the Fc fragment may comprise an Fc fragment derived from any of the immunoglobulins of the group consisting of IgG1, igG2, igG3, and IgG4. In certain embodiments, the antigen binding proteins of the application comprise wild-type Fc fragments derived from any of the immunoglobulins of the group consisting of IgG1, igG2, igG3, and IgG4. The amino acid sequences of Fc fragments derived from IgG1, igG2, igG3 or IgG4 are known in the art. For example, the Fc fragment may comprise an Fc fragment derived from human IgG 1. For example, the Fc fragment may comprise an Fc fragment derived from human IgG 3. For example, the Fc fragment may comprise an Fc fragment derived from human IgG4.

In certain embodiments, an antibody or antigen-binding fragment thereof of the application comprises an Fc fragment variant having one or more amino acid mutations relative to a wild-type Fc fragment. The half-life of an antibody or antigen binding fragment thereof may be extended and/or the effector function of an antibody or antigen binding fragment thereof may be enhanced by optimization of the Fc fragment sequence.

In the present application, the antibody or antigen binding fragment thereof may further comprise an isolated antigen binding protein that has been further functionally optimized or modified. In certain embodiments, the antigen binding proteins of the application may include antigen binding proteins having enhanced affinity for binding to CD 117. In certain embodiments, the antigen binding proteins of the application may include antigen binding proteins having enhanced ability to inhibit binding of CD117 to its ligand. In certain embodiments, the antigen binding proteins of the application may include antigen binding proteins having enhanced ability to inhibit CD 117-mediated signal transduction. In certain embodiments, the antigen binding proteins of the application may include antigen binding proteins having enhanced ability to inhibit migration, accumulation, recruitment, and/or infiltration of cells expressing CD 117. In certain embodiments, the antigen binding proteins of the application may include antigen binding proteins having an enhanced ability to mediate killing of cells expressing CD 117. In certain embodiments, the antigen binding proteins of the application may include antigen binding proteins having enhanced ability to induce endocytosis of CD117 receptors on the cell surface. In certain embodiments, the enhanced ability may be relative to the antigen binding protein prior to its functional optimization or modification.

The antibodies or antigen binding fragments thereof of the application may comprise heavy chain sequences that are modified by the presence of one or more conserved sequences. By "conservative sequence modifications" is meant amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications may be introduced into the antibodies or antigen binding fragments thereof of the application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. 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), beta-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 an antibody or antigen binding fragment thereof of the application may be replaced with other amino acid residues of the same side chain set. Those skilled in the art will appreciate that some conservative sequence modifications do not result in the loss of antigen binding, see, e.g. ,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.10:341-6and Beers et al.,(2000)Clin.Can.Res.6:2835-43.

In the present application, the antibody or antigen-binding fragment thereof is capable of specifically binding to CD117. In certain embodiments, the binding affinity of the antibody or antigen-binding fragment thereof to CD117 can be detected by detecting the binding capacity of the antibody or antigen-binding fragment thereof to cells expressing CD117. For example, detection can be performed by FACS. In certain embodiments, binding of the antibody or antigen binding fragment thereof to CD117 may be detected by ELISA methods. For example, an antibody or antigen binding fragment thereof of the application may bind to CD117 at an EC50 value of 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, an antibody or antigen binding fragment thereof of the application can bind to CD117 with an IC50 value of less than or equal to 50nM, less than or equal to about 40nM, less than or equal to 30nM, less than or equal to 20nM, or less than or equal to 10nM.

The CD117 antigen binding proteins of the application may be determined, identified, or characterized by various methods known in the art. For example, the antigen binding activity of an antibody of the application or antigen binding fragment thereof can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), immunoblotting (e.g., western blot), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, and the like.

The antibodies or antigen binding fragments thereof provided herein are useful for antagonizing CD117 activity. In the present application, the antibody or antigen binding fragment thereof is capable of preventing and/or treating a disease and/or disorder.

Derivatives based on antibodies or antigen binding fragments thereof and uses thereof

The application also encompasses variants of the disclosed anti-CD 117 single domain antibodies that are engineered to confer or enhance properties required for a particular therapeutic, diagnostic or research application. Such variants include, but are not limited to, modifications for improving affinity, specificity, stability, solubility, reducing immunogenicity, enhancing coupling ability, or other desired functional properties.

Variants designed to increase antigen binding affinity and specificity can be generated by methods known in the art. These methods involve affinity maturation by targeting 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 binding rate, decreased dissociation rate) or enhanced selectivity. Variants with enhanced stability and solubility include modifications of the framework regions. Specific amino acid substitutions may introduce stabilizing residues or disulfide bonds to increase thermostability, resistance to protease degradation, or reduce aggregation propensity. In one embodiment, mutation of the framework residues increases the solubility or stability of the antibody under physiological or production conditions while maintaining target specificity and affinity.

In certain embodiments, the variants may be variants engineered for site-specific coupling. The variants include the introduction of one or more cysteine residues at predetermined positions within the C-terminal or N-terminal regions of a single domain antibody. These cysteine residues facilitate selective and stable coupling of therapeutic drugs, toxins, imaging agents, or functional groups via chemical linkers (e.g., maleimide-thiol reactions). Furthermore, variants may contain flexible hinge or linker sequences adjacent to engineered cysteine residues to optimize payload accessibility and preserve antibody function. In other embodiments, the variants incorporate unnatural amino acids with bioorthogonal reactive groups (e.g., azide, alkynyl, keto-functionalized amino acids). These residues enable highly specific, orthogonal coupling reactions ("click chemistry") that facilitate precise ligation of diagnostic agents, cytotoxic drugs, or gene editing components.

In certain embodiments, the variant may be a fusion protein. The fusion protein may comprise a genetic fusion of a VHH domain with an additional functional domain. Such fusion proteins include, but are not limited to, immunoglobulin Fc fragments (forming antibody-Fc fusion proteins that extend 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 one illustrative example, fusion of an anti-CD 117 VHH to an Fc domain results in a diabody with an extended serum half-life and enhanced affinity.

In certain embodiments, the variant may be a bispecific or multispecific antibody. These constructs may involve genetic fusion of two or more different VHH domains via flexible peptide linkers. Examples include bispecific antibodies comprising an anti-CD 90 VHH domain linked to an anti-CD 117 VHH domain, which are capable of simultaneously targeting multiple epitopes on hematopoietic stem cells to enhance specificity and therapeutic efficacy. Variants may also comprise linking sequences 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 that are responsive to a particular cellular environment (e.g., protease cleavable sequences that facilitate intracellular payload release).

In addition, the application also encompasses humanized variants wherein the camelid derived VHH sequences have been modified to more closely resemble human immunoglobulin variable domains, reducing potential immunogenicity in therapeutic applications. Such humanisation may involve grafting camelid derived Complementarity Determining Regions (CDRs) onto a human variable region framework, optionally followed by selective back-mutagenesis, to restore optimal binding and stability characteristics.

The application also relates to derivatives engineered to the disclosed anti-CD 117 single domain antibodies to enhance their use in therapeutic, diagnostic and research applications. Antibody derivatives within the scope of the application include fusion proteins created by genetically linking the disclosed single domain antibodies with additional functional protein domains. In one embodiment, the VHH domain is fused to an immunoglobulin Fc domain, providing increased antibody affinity, prolonged serum half-life, and Fc-mediated effector function. In another embodiment, the single domain antibody is genetically fused to a cell penetrating peptide or endoplasmic reticulum escape domain, thereby enabling improved intracellular delivery of payloads such as nucleic acids or drugs. Furthermore, the VHH domain may be fused to a fluorescent protein, a luminescent reporter protein or an enzymatic reporter protein, facilitating its use in diagnostic detection and imaging applications.

In another embodiment, the invention includes Antibody Drug Conjugates (ADCs) in which a single domain antibody is chemically bound to a cytotoxic drug or toxin. Suitable cytotoxic drugs include auristatins, maytansinoids, doxorubicin, carba Li Jimei, and Pyrrolobenzodiazepines (PBDs). Coupling may be via cleavable or non-cleavable linkers that allow for selective release of the cytotoxic payload upon targeted internalization of CD 117-expressing cells.

Further embodiments include radiolabeled derivatives in which a single domain antibody is chemically bound to a radioisotope (e.g., technetium-99 m, zirconium-89, iodine-125, lutetium-177, or yttrium-90). These radiolabeled derivatives are useful for diagnostic imaging of Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), as well as targeted radiation therapy for selective depletion of specific cell populations.

In addition, bispecific or multispecific antibody derivatives can be constructed to bind multiple target antigens simultaneously, increasing the specificity and efficacy of the treatment. For example, the antibody derivative may be a bispecific antibody comprising a VHH domain that targets CD117 and a VHH domain of another clinically relevant antigen (e.g. CD3 for T cell binding), the antibody derivative serving to provide selective targeting and a powerful biological effect.

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 have been genetically engineered to integrate VHH antibody domains to achieve specific and efficient targeting of Hematopoietic Stem Cells (HSCs) expressing CD117 and other relevant cell types.

In certain embodiments, the antibody derivatives may be antibody derivatives for use in cell therapy, such as Chimeric Antigen Receptors (CARs). These constructs integrate VHH sequences into engineered receptors expressed on immune effector cells (T cells, NK cells, macrophages) to achieve selective immune mediated clearance of antigen positive target cells. Likewise, it is within the scope of the application to use bispecific T cell cements (BiTE-like molecules) in which the anti-CD 117 VHH domain is linked to a T cell binding domain (e.g. anti-CD 3), which are capable of directing the immune response precisely to the diseased or target cells.

In certain embodiments, the antibody derivative comprises a single domain antibody conjugated to a lipid polyethylene glycol (lipid-PEG) group. These lipid-PEG conjugated single domain antibodies are capable of achieving targeted non-viral delivery platforms, in particular Lipid Nanoparticles (LNPs), liposomes or polymer-based nanoparticles. These derivatives provide enhanced stability, targeted cell binding capacity, and efficient internalization for intracellular delivery of nucleic acid payloads (e.g., mRNA, siRNA, or gene editing tools).

One of the main aspects of the application is the engineering of VHH or single domain antibody clones into various forms such as fusion proteins or conjugates etc. to achieve targeted delivery or therapeutic effects. The following are several implementation categories:

1. Antibody Drug Conjugates (ADCs) and toxin fusion proteins each anti-CD 117 VHH may be linked to a cytotoxic drug to create a targeted cytotoxin capable of eliminating cells expressing the antigen of interest. In one embodiment, the VHH is chemically coupled to the toxin by a cysteine residue added to the VHH. For example, a cysteine is inserted at the C-terminus (after the last framework residue) of a VHH or single domain antibody for site-specific ligation with a terminal maleimide-conjugated drug linker. More specifically, a maytansine derivative of a maleimide linker (DM 1, a microtubule toxin) was conjugated to this cysteine to give an anti-CD 117 VHH-DM1 conjugate. In cell killing experiments, this conjugate specifically killed CD117 ⁺ cells (e.g., stem/progenitor cells in culture) while having no effect on CD117 ^- cells, demonstrating targeted cytotoxicity. Likewise, VHH can be genetically fused to a protein toxin, e.g., anti-CD 117 VHH fused to a truncated diphtheria toxin or an apoptotic enzyme (e.g., granzyme), and can be used to deplete cells expressing CD 117. Such ADCs may be used as conditioning agents to deplete HSCs in bone marrow prior to introduction of gene corrected cells or donor cells. In particular embodiments, the small size of VHH may have better tissue penetration and more uniform bone marrow distribution than larger IgG ADCs, potentially enabling more efficient HSC clearance at lower doses.

2. Targeting lipid nanoparticles (tLNPs) the present application includes a single domain antibody loaded lipid nanoparticle delivery system for targeting. Lipid nanoparticles can encapsulate therapeutic payloads such as mRNA, siRNA or CRISPR ribonucleoproteins, but by default they are non-specifically distributed. By attaching anti-CD 117 VHH to the LNP surface, we created a targeting LNP (tLNP) that was able to target HSCs. In one example, CD 117-targeting LNPs was made using clone B13, and these LNPs exhibited similar specificities for CD117 ⁺ cells (including normal human CD34 ⁺CD117⁺ bone marrow cells in vitro). These tLNPs were further used for gene editing tests, which we used to load mRNA encoding the gene editor AaCas b and sgRNA targeting the HBG gene (β -globin gene locus). The treated cells showed high levels of gene editing (insertion/deletion formation) at the target site, with targeting LNPs, the editing efficiency increased with increasing dose, while the untargeted LNPs activity was significantly reduced. This demonstrates that the ability to deliver gene editing tools to HSCs can be greatly enhanced by linking our VHH to LNPs, an in vivo gene therapy strategy that can be used to treat diseases such as β -thalassemia (reactivation of fetal hemoglobin by HBG promoter editing). The method of creating such tLNPs will be further described in the examples, which generally involve the process of covalently attaching a VHH to a lipid (e.g., DSPE-PEG-maleimide) and self-assembling the nanoparticle.

3. Viral vector targeting and CAR construction the VHH of the application can be used for construction of targeted viral or chimeric antigen receptors. For viral vectors, one example is AAV capsid modification by inserting the coding sequence of the VHH against CD117 into the AAV capsid protein loop to generate a chimeric capsid comprising the VHH on the viral surface. This modified AAV specifically transduces CD117 ⁺ cells, effectively redirecting viral tropism to HSCs. Another modification is the use of an adaptor molecule-e.g., biotinylated anti-CD 117 VHH can bind to streptavidin-expressing lentiviruses, thereby anchoring the virus to CD117 ⁺ cells. These strategies allow gene delivery vectors (lentiviruses, AAV, adenoviruses, etc.) to achieve cell-specific transduction, a key step in achieving HSC gene therapy in vivo without transplantation. In the context of cell therapy, the VHH sequences herein can act as antigen binding domains of CARs (chimeric antigen receptors) on immune cells. For example, a CAR was constructed by fusing the CD117 VHH sequence to the CD8 hinge-transmembrane domain and the intracellular cd3ζ and 4-1BB signaling domains. T cells expressing the CAR specifically recognize and lyse CD117 expressing cells in vitro, demonstrating that VHH retains binding capacity in the CAR. Such CAR-T cells can be used to target and destroy HSCs (for conditioning or treating malignant tumors) and 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 tighter packaging or design of dual CARs (where two small domains are used side by side).

4. Multispecific and bispecific antibodies to increase binding affinity or targeting selectivity, multiple VHHs may be linked together. By genetically fusing two CD117 VHH domains and using a flexible linker, a bivalent anti-CD 117 construct was made, yielding a tandem VHH with essentially double the binding valency for CD 117. Such diabodies exhibit a slower dissociation rate from the antigen and increase the retention time of the antibody in bone marrow tissue in an in vivo model. For dual targeting, the anti-CD 117 VHH is linked to the anti-CD 45 VHH (CD 45 is a ubiquitin marker expressed on HSCs) creating a bispecific molecule that requires simultaneous binding of two markers to bind to cells, so the molecule preferentially binds HSCs (CD 117 ⁺CD45⁺) rather than T cells (typically CD45 ⁺ but CD117 ^- in the surrounding blood). This is one example of how a multispecific nanobody construct can be precisely targeted to a well-defined subpopulation of cells. All such tandem or fusion antibody constructs are within the scope of the application.

5. Fusion with functional groups VHH can be fused to functional protein domains in addition to binding targets. For example, fusion of a VHH against CD117 to an Fc domain (creating a VHH-Fc or "nanobody-Fc") confers IgG-like properties (bivalent and prolonged half-life due to FcRn circulation) to the molecule. Alternatively, fusion of nanobodies with enzymes can localize the activity of the enzyme to hscs—anti-CD 117 VHH can be linked to cytokines or growth factors (which become a targeted cytokine that preferentially stimulates HSCs). In one embodiment, we created an IL-2-VHH fusion in which the VHH of CD117 was fused to IL-2, and this chimeric molecule was able to specifically deliver IL-2 signaling to CD117 ⁺ cells that simultaneously expressed IL-2R, representing a cell-selective cytokine delivery platform. In addition, the present application contemplates a number of such combinations (nanobody fused to a signal molecule, fused to an apoptosis-inducing domain, fused to a fluorescent protein for imaging, etc.).

All coupling and engineering techniques used are adaptations of standard or known methods. The small molecule payload may be attached to the VHH by covalent attachment via NHS ester, maleimide-thiol reaction, click chemistry (e.g., azide-alkyne cycloaddition), or the like. And peptide/protein level integration is achieved by genetic fusion. The final product was characterized by SDS-PAGE, mass spectrometry and functional detection to confirm that the VHH retains binding function after modification.

It follows that there is a broad potential for use of single domain antibodies directed against CD117 on Hematopoietic Stem Cells (HSCs). These applications cover the research and clinical fields:

Targeted gene therapy and gene editing-gene transfer or gene editing to stem cells (e.g., delivery of CRISPR systems) is achieved using anti-CD 117 VHH directed gene therapy vectors or nanoparticles to HSCs. This can treat hereditary hematological diseases by editing HSCs in situ, avoiding the need for transplantation.

Cancer immunotherapy, targeting malignant stem cells or supportive microenvironment in cancer using VHH-based constructs. For example, anti-CD 117 VHH-based Antibody Drug Conjugates (ADCs) can clear leukemia stem cells from myeloid malignancies without extensive collateral damage.

Conditioning of HSC transplantation by selective depletion of VHH conjugates or inactivation of HSCs in patients prior to transplantation. anti-CD 117 nanobody-drug conjugates (or bispecific antibodies that direct immune effector cells to HSCs) can serve as minimal toxic opsonizing agents, making room for implantation of donor HSCs. In addition, VHHs can be used to deliver radioisotopes to bone marrow to locally illuminate the HSC microenvironment.

Cell isolation and in vitro procedures, coating of magnetic beads or column matrices with anti-CD 117 VHH, isolation of HSCs from bone marrow or circulating blood for research or therapeutic graft enrichment. The small size of VHH may allow for milder, more specific capture and release of cells than whole antibodies. Likewise, VHHs can target HSCs in culture (e.g., extracellular delivery of growth factors or gene editors).

Diagnostic and imaging the diagnosis of HSCs or HSCs-enriched tissues using labeled VHH (e.g., fluorescent or radiolabeled nanobody). Radiotracers conjugated to anti-CD 117 VHH can image the distribution of bone marrow stem cells or monitor engraftment after HSCs transplantation, taking advantage of the rapid blood clearance of VHHs to obtain high contrast images. In pathology laboratories, anti-CD 117 VHH reagents can be used as specific staining agents for the identification of HSCs or cancer stem cells in tissue sections.

Nucleic acid molecules, vectors and cells

In another aspect, the application provides an isolated nucleic acid molecule comprising a nucleotide sequence that can encode an isolated antigen binding protein of the application. For example, it may be produced or synthesized by (i) in vitro amplification, e.g., by Polymerase Chain Reaction (PCR) amplification, (ii) by clonal recombination, (iii) purification, e.g., by cleavage and gel electrophoresis fractionation, or (iv) synthesized, e.g., by chemical synthesis.

In another aspect, the application provides a vector which may comprise an isolated nucleic acid molecule of the application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in an appropriate host cell and under appropriate conditions. In addition, the vector may also contain expression control elements that allow for proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers and other control elements which regulate gene transcription or mRNA translation, and the like. The vector may be expressed by transforming, transducing or transfecting a host cell such that the genetic element carried thereby is expressed within the host cell. The vector may include, for example, a plasmid, cosmid, virus, phage, or other vector commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may include components that assist it in entering the cell, such as viral particles, liposomes, or protein shells, but not exclusively.

In another aspect, the application provides a cell, which may comprise an isolated nucleic acid molecule according to the application or a vector according to the application. In certain embodiments, each or each host cell may comprise one or more nucleic acid molecules or vectors of the application. In certain embodiments, each or each host cell may comprise a plurality (e.g., 2 or more) or a plurality (e.g., 2 or more) of the nucleic acid molecules or vectors of the application. For example, the vectors of the application may be introduced into such host cells, e.g., eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cell may be a bacterial cell (e.g., E.coli), a yeast cell, or other eukaryotic cell, such as a COS cell, a Chinese Hamster Ovary (CHO) cell, a CHO-K1 cell, a LNCAP cell, a HeLa cell, a 293T cell, a COS-1 cell, a SP2/0 cell, a NS0 cell, or a myeloma cell. The vectors of the application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamine transfection, and the like.

Method for producing antibodies

In another aspect, the application provides methods of making the antibodies or antigen-binding fragments thereof. The method may comprise culturing the host cell of the application under conditions such that the antibody or antigen-binding fragment thereof is expressed. For example, such methods are known to those of ordinary skill in the art by using an appropriate medium, an appropriate temperature, an appropriate incubation time, and the like.

In certain embodiments, the method may be a molecular biological method. For example, the method comprises preparing one or more nucleotide sequences encoding any one of the antibodies or antigen binding fragments thereof described above, constructing the one or more encoding nucleotide sequences into one or more expression vectors, and expressing the expression vectors in appropriate cells.

It will be appreciated by those skilled in the art that, given the amino acid sequence determination of proteins, different coding nucleotide sequences may be used, or the coding nucleotide sequences may be optimized, due to the codon degeneracy. Methods for performing codon optimization of coding sequences are known to those skilled in the art and include adjusting codons to host bias codons for the type of host, reducing GC content and/or reducing GC-rich regions, increasing mRNA stability, and thus increasing expression efficiency of the nucleotide of interest in a particular host.

Suitable producer cells are known to the person skilled in the art. In some embodiments, mammalian cells, such as 293T, CHO or a derived cell line thereof, are used as production cells. In some embodiments, microbial cells, such as bacterial cells or fungal cells, are used as production cells, such as e.coli or yeast. In some embodiments, insect cells are used as production cells, such as Sf9. The nucleotide sequences encoding the trispecific antibodies of the invention may be codon optimized for a particular producer cell line.

The antibody or antigen-binding fragment thereof produced is preferably purified. The purification may be carried out by a method conventional in the antibody production field, and the method may include steps of filtration, chromatography, and the like. The filtration step may be selected from one or more of depth filtration, ultrafiltration, diafiltration, nanofiltration. The chromatography step may be selected from one or more of affinity chromatography, cationic chromatography, anionic chromatography, size exclusion chromatography, hydrophobic chromatography, hydroxyapatite chromatography.

Any method suitable for producing monoclonal antibodies may be used to produce the antibodies or antigen-binding fragments thereof of the application. For example, an animal may be immunized with linked or naturally occurring CD117 or a fragment thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used. In certain embodiments, antibodies or antigen binding fragments thereof to CD117 may be screened and enriched by phage surface display systems by extracting immune alpaca peripheral blood lymphocytes, extracting cell nucleic acid fragments to clone into vectors.

Any suitable form of CD117 may be used as an immunogen (antigen) for generating antibodies specific for CD117, and screening the antibodies for biological activity. For example, the priming immunogen may be full length CD117, including natural homodimers, or peptides containing single/multiple epitopes. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art.

Pharmaceutical composition, therapeutic use and detection kit

In another aspect, the application also provides a pharmaceutical composition, which may comprise an antibody or antigen binding fragment thereof according to the application, an isolated nucleic acid molecule according to the application, a vector according to the application and/or a cell according to the application, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) suitable formulations of adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosages and concentrations employed. Pharmaceutical compositions described herein include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, typically those active compounds having complementary activity that do not adversely affect each other. The type and effective amount of such drugs may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

In certain embodiments, the pharmaceutical composition may comprise an unseparated expression product as described herein. In particular, the expression product is an intermediate product produced by the aforementioned method of producing an antibody or antigen-binding fragment thereof, and is a mixed system comprising the antibody or antigen-binding fragment thereof protected by the present application. In certain embodiments, the expression product may be a homogeneous stock solution. The preparation and analysis of expression products may be well known in the art.

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

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, endoluminal, intra-arterial, intrathecal and/or intranasal administration or direct injection into tissue. 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 performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition may be administered without interruption. The uninterrupted (or continuous) administration may be accomplished by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient.

In certain embodiments, the pharmaceutical composition further comprises an additional agent conjugated to the antibody or antigen-binding fragment thereof of the application and targeted to cells expressing CD117 by the antibody or antigen-binding fragment thereof of the application and treating the associated disease.

In particular, the other drug may be selected from one or more of the group consisting of small molecule targeted anticancer agents, antibody drugs, adoptive cell therapies, oncolytic viruses and oncolytic virus enhancers.

More specifically, the other drug may be selected from one or more of the group consisting of 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.

In certain embodiments, the drug may be a carrier that encapsulates the contents, e.g., liposomes, adeno-associated viruses (AAV), lipid nanoparticles, vesicles, exosomes, lentiviral vectors, adenoviral vectors, metal nanoparticles, mesoporous silica nanoparticles, erythrocytes, and platelets. The content may be selected from one or more of the group consisting of RNA drugs (e.g., mRNA, miRNA, circular RNA), DNA drugs (e.g., encoding genes, CRISPR systems), protein drugs (e.g., antibodies), and small molecule drugs.

The coupling may be by physical means, such as coupling by interaction, or by chemical means, such as coupling by covalent, ionic or the like.

The present application relates to a method of treating a subject in need thereof, which may comprise administering to the subject a prophylactically effective amount or a therapeutically effective amount of an antibody or antigen-binding fragment thereof and/or a composition as described herein. As used herein, a "subject" may include any animal that exhibits symptoms of a disease, disorder, or condition treatable with the antibodies or antigen-binding fragments, compositions, and methods disclosed herein. Suitable subjects (e.g., patients) may include non-human primates and/or human patients. The non-human primate may be selected from one or more of laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals (e.g., horses or cattle), livestock, pets (e.g., cats or dogs).

The term "administering" according to the present application means introducing the antibody or antigen-binding fragment thereof according to the present application, the isolated nucleic acid molecule according to the present application, the vector according to the present application, the cell according to the present application and/or the pharmaceutical composition according to the present application into a subject, or contacting the antibody or antigen-binding fragment thereof according to the present application, the isolated nucleic acid molecule according to the present application, the vector according to the present application, the cell according to the present application and/or the pharmaceutical composition according to the present application with a cell and/or a tissue. Administration may be by injection, irrigation, inhalation, consumption, electro-osmosis, 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 application may be selected from one or more of the following: the method comprises the steps of auricular, buccal, conjunctival, percutaneous, transdental, cervical, sinus (endosinusial), intratracheal, enteral, epidural, interstitial, intra-articular, intra-arterial, intra-abdominal, intra-aural, intra-biliary, intra-bronchial, intra-bursal, intra-cavernous, intra-cerebral-cisterna, intra-corneal, intra-coronary, intra-intracranial, intradermal, intra-tray, intra-catheter, intra-duodenal, intra-dural, intra-epicardial, intra-epidermal, intra-oesophageal, intra-gastric, intra-gingival, intra-hepatic, intra-ileal, intra-focus, intra-lingual, intraluminal, intra-lymphatic, intra-mammary, intramedullary, intra-meningal, intramuscular, intra-nasal, intra-nodal, intra-ocular, intra-omental, intra-ovarian, intraperitoneal, intra-pericardial, intra-pleural the method comprises the steps of intraprostatic, intrapulmonary, ruminal, intraspinal, intrasynovial, intratendinous, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intraventricular, intravesical, intravestibular, intravenous, intravitreal, intralaryngeal, nasal, nasogastric, oral, ocular, oropharyngeal, parenteral, transdermal, periarticular, epidural, perinerve, periodontal, respiratory, retrobular, rectal, spinal, subarachnoid, subconjunctival, subcutaneous, subdermal, subgingival, sublingual, submucosal, subretinal, topical, transdermal, intracardiac, transmucosal, transplacental, transtracheal, tympanic, ureteral, urethral and vaginal. The mode of administration may be selected from one or more of infusion, lavage and direct injection.

The term "treating" as used herein refers to administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof and/or a composition thereof as described herein, such that the disease or disorder, or symptoms of the disease or disorder, in the subject is ameliorated. The improvement may be a disease or condition, or any improvement or treatment of symptoms of a disease or condition. The improvement may be an observable or measurable improvement, or may be an overall sensation of the subject's health condition. Thus, those skilled in the art recognize that treatment may improve a disease condition, but may not be a complete cure for the disease. "prophylactically effective amount" refers to an amount of virus, virus stock, or composition effective to achieve the desired prophylactic result. As used herein, "prevention" may refer to complete prevention of a disease symptom, delay in onset of a disease symptom, or reduction in severity of a subsequently occurring disease symptom. Typically, but not necessarily, the prophylactically effective amount is less than the therapeutically effective amount, as the prophylactic dose may be administered to the subject prior to or early in the disease.

The antibodies of the invention may be delivered by conventional methods, preferably by systemic administration, for example by intravenous infusion.

The antibodies of the invention may be administered in a therapeutically effective amount. "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject to treat a disease, or at least one clinical symptom of a disease or disorder, is sufficient to effect such treatment of the disease, disorder, or symptom. A "therapeutically effective amount" may 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.

In a preferred embodiment of the application, the amount of antibody administered (mg/kg) is based on the body weight of the subject. For example, a single dose of an antibody of the application may range from about 1ng/kg body weight to about 5mcg/kg body weight, preferably from 5ng/kg body weight to about 2.5mcg/kg body weight, and more preferably from 0.1mcg/kg body weight to about 1mcg/kg body weight. For example, administration may be at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1mcg/kg body weight.

One advantage of the antibodies of the invention is that they are capable of efficiently and completely activating T cells, and are less prone to apoptosis after activation, and in addition, can induce memory T cells to provide a sustained immune response, thereby allowing for administration at relatively low dosing intervals. In preferred embodiments, the interval between two administrations of the antibodies of the invention may be no less than 1 day, no less than 3 days, no less than 5 days, or even one week apart when administered by intravenous infusion. For example, the antibodies of the invention may be administered 7 times per week, 5 times per week, 3 times per week, twice per week, or once per week. The antibodies of the invention may be administered as a cycle over 2-4 weeks, e.g., 3 weeks, and for one or more cycles.

In another aspect, the application provides a kit comprising an antibody or antigen binding fragment thereof of the application, an isolated nucleic acid molecule of the application, a vector of the application, a cell of the application, and/or a pharmaceutical composition of the application.

In certain embodiments, the kit is used to detect CD117 content in a mixed system. In certain embodiments, the detection may be a qualitative or quantitative detection. In certain examples, the level of CD117 may characterize the abundance of hematopoietic stem cells in the mixed system, alone or in combination with other indicators.

Without intending to be limited by any theory, the following examples are presented in order to illustrate the fusion proteins, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application.

Examples

EXAMPLE 1 genetically engineered humanized mouse antigen immunization

Nanobodies were generated using AceMouseNB ^TM mice. AceMouseNB ^TM is a genetically engineered humanized mouse model designed to produce fully human heavy chain-only antibodies (HcAbs). The mouse is edited by targeted genes, the heavy chain variable region of the mouse is replaced by a heavy chain variable region corresponding to human, and meanwhile, the light chain expression is silenced, so that a humanized single domain antibody containing VHH can be generated. The model mice are capable of producing high affinity antigen recognition, low immunogenicity, single domain antibodies, which accelerate the discovery of single domain antibodies for therapeutic and diagnostic applications.

Mice were immunized with CD117 antigen expression plasmid following standard immunization procedures. The serum of mice immunized with CD117 antigen was analyzed by flow cytometry to evaluate whether specific antibodies were produced in the mice.

CHO-CD117 or CHO-WT cells were seeded in the same number in each well of a 96-well plate. The antigen-immunized mouse serum was diluted sequentially with dilutions (DPBS+3% FBS) in the proportions 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24300 and 1:72900. The diluted serum samples were added 50 μl each to the corresponding cell-containing wells, gently mixed, and incubated at 4 ℃ for 45 minutes. After centrifugation again, the cells were washed 2 times with wash buffer (DPBS+3% FBS) and then resuspended in 50. Mu.L of a 500ng/mL 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) and incubated at 4℃for 30 minutes. The cells were resuspended in 25 μl of 3% FBS, centrifuged again and washed twice with wash buffer, and samples were analyzed by flow cytometry to determine the median fluorescence intensity in PE for each cell population. As shown in fig. 1, mice developed a stable specific immune response following immunization with CD117 antigen. After multiple booster immunizations and the last booster immunization, these mice were suitable for isolating CD 117-specific monoclonal B cells.

Example 2 anti-CD 117 specific Single B cell isolation and cloning analysis

Spleen cells were sorted using Single-B cell technology. Clones were obtained from 4 96-well plates, followed by selection of 2 plates to obtain antibody genes. The antibody genes are cloned into AceMab expression vectors after PCR amplification and transiently transfected to prepare recombinant antibody expression supernatants for cloning verification. Each sample was analyzed by flow cytometry using CHO-CD117, CHO-K1 or HEL cells according to the methods described in example 1. As shown in FIG. 2, positive clones with MFI values greater than 10000 for CD117 positive cells and less than 5000 for control cells (CHO-K1) were selected and sequenced using the NGS technique.

EXAMPLE 3 antibody purification

Positive clone antibody is expressed by AceMab antibody expression vector, and cell culture supernatant is 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. The supernatant was then transferred to pre-equilibrated magnetic beads Pro-A and incubated at room temperature for 30 minutes to allow antibody binding. After incubation, the beads were collected with a magnet for 1 min until the solution became clear and the supernatant was discarded. The beads were resuspended in 5 volumes of wash buffer for 5-10 minutes, the medium supernatant was again collected for 1 minute and the supernatant was removed and washed after the solution was cleared. This washing process was repeated twice to ensure complete removal of non-specific binding proteins. And adding 1mL of elution buffer (0.05M citric acid, pH=3.0) to elute the antibody for 2-3 times. The neutralizing buffer (1/15 of the volume of the eluent) was added and the antibody concentration was determined by UV spectrophotometry at 280 nm. For desalting and buffer exchange, the eluted antibodies were treated on a G25 Sephadex column. Finally, standard SDS-PAGE identification is carried out.

Example 4 characterization of antibodies

Antigen binding efficiency

First, using the method in example 1, the binding efficiency of monoclonal antibodies 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 followed by a series of three-fold dilutions to determine the affinity and specificity of the antibodies at the different concentrations. The data are shown in figure 3.

Subsequently, the binding efficiency of the monoclonal antibodies to the mouse CD117 antigen was evaluated by ELISA. Mouse CD117 antigen was diluted to 1. Mu.g/mL with PBS as coating solution, and 100. Mu.L of the coating solution was added to each well of a 96-well ELISA plate, and incubated at4℃overnight. The next day the coating was removed, the 96-well plate was washed 1 time with 200. Mu.L PBS, and then 200. Mu.L blocking solution was added to each well and incubated for 2 hours at room temperature. After blocking, the antibodies were diluted with dilution buffer to 10000, 3333.3, 1111.1, 370.37, 123.46, 41.15, 13.72 and 4.57ng/mL, each concentration of antibody was added to 50 μl to 96 well plates and incubated for 1 hour at room temperature. After incubation, 96-well plates were washed 3 times with 200. Mu.L of wash buffer (PBS containing 0.05% Tween-20). HRP conjugated goat anti-human IgG antibody 50. Mu.L (0.4. Mu.g/mL) was added, incubated for 1 hour at room temperature, the solution was discarded, and the 96-well plate was washed 5 times with 200. Mu.L of wash buffer. For detection, 50. Mu.L of TMB substrate was added, incubated at room temperature for 3-5 minutes in the absence of light, and 50. Mu.L of 0.25M sulfuric acid was added to each well to stop the reaction, and absorbance was measured at 450 nm. The results (as in FIG. 4) indicate that none of the candidate clones bound to mouse CD 117.

Affinity for

The affinity of the antibodies to human CD117 recombinant protein was detected using biolayer interferometry. Briefly, anti-human Fc probes (gator, 160003) were hydrated in buffer for at least 10 minutes prior to use. Antibodies were diluted to 5 μg/mL in buffer and added to 96-well plate sensors (beyotime, FCP 966). Human CD117 recombinant protein antigen was serially diluted to 300, 100, 33.3, 11.1, 3.7, 1.23 and 0.41nM in buffer, and an antigen-free control well was prepared as baseline reference. The binding period was monitored for 240 seconds by immersing the antibody-containing sensor in recombinant protein containing different concentrations of human CD117, and then the sensor was transferred to fresh buffer for dissociation monitoring for more than 300 seconds. The data are shown in Table 1.

Table 1. Results of affinity detection of candidate antibodies.

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

Based on all results of antigen binding, affinity, h.2346b1, h.2346b3, h.2346b4, h.2346b11, h.2346b13 and h.2346b17 were selected for expression of CD117 monoclonal antibodies.

EXAMPLE 5 purification and identification of modified VHH antibodies

This example demonstrates that candidate positive cloned antibodies are constructed in the form of anti-CD117VHH and the original anti-CD117VHH sequence is engineered with a histidine tag (histidine-tag), a hinge sequence and a cysteine (C) residue introduced at its C-terminus. It was cloned into AceMab antibody expression vectors according to standard protocols. The recombinant expression vector is transfected into an Expi293 cell for expression, and the purified antibody is obtained through His-tag affinity chromatography. All purified antibodies were identified by standard SDS-PAGE, as shown in FIG. 5.

Table 2 antibody clones and concentrations corresponding to lanes in FIG. 5

Lanes	Antibody cloning	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

EXAMPLE 6 Synthesis of ligand-linker conjugates

This example demonstrates the method of site-specific conjugation using engineered anti-CD117 VHH antibodies to form ligand-linker conjugates. The original anti-CD117 VHH sequence was first engineered, incorporating a histidine tag (histidine-tag), a hinge sequence (optional) and a cysteine (C) residue at its C-terminus. The engineered VHH sequences were transiently expressed in an Expi293 cell and purified. The sequence modification can enable a specific site of the VHH to be accessed into a click chemistry reaction group, so that coupling potential is improved, and meanwhile, the internal structural stability of the VHH is not affected.

The connection of the cysteine residues to the VHH comprises direct connection (without a Hinge sequence) or connection via one of five different structural features of the Hinge sequence (named as finger-1, finger-2, finger-4, finger-5 and finger-7, respectively). Each hinge sequence design is intended to exhibit a different structural characteristic. The complete amino acid sequences and corresponding compositional characteristics are provided in table 2, including the percentage of residues that enhance rigidity (e.g., P, Y, F, W, V, I, L), the glycine (glycine, G) content, and the proportion of amino acids that promote stabilization of the alpha helical structure (e.g., A, L, E, M). Wherein, range-1 is a commonly used flexible Hinge sequence (G ₄S)₃, which has a higher glycine content and shows good flexibility, and Range-2 and Range-4 are classified as limited Hinge sequences, wherein, range-2 is rich in residues for enhancing rigidity and has extremely low glycine content, and Range-4 contains more than 80% of amino acids for promoting alpha helix structure stabilization, and the ratio of rigidity enhancing amino acids of Range-5 and Range-7 is about 50% and does not contain glycine, and are classified as rigid Hinge sequences, and cloning H.2346B13 is taken as an example, and different Hinge sequences are adopted for C-terminal extension, and the specific sequences are shown in the table.

TABLE 3 different hinge (hinge) sequences and their characteristics

TABLE 4 anti-CD 117 VHH amino acid sequences with different C-terminal extension sequences

The structure of the ligand-linker conjugate formed by coupling the modified ligand and the linker is shown in FIG. 6. The specific coupling step comprises first subjecting the modified and unmodified VHH to a reduction treatment using 2-mercaptoethanol (2-mercaptoethanol, 2-MEA), then purifying the reduced VHH using a Zeba ^TM desalting column (molecular weight cut-off 7K MWCO), and then coupling the purified VHH with the linker DBCO-PEG ₄ -maleimide (DBCO-PEG 4-MALEIMIDE) for 3 hours at room temperature. The coupling site is a newly introduced cysteine residue and the reaction conditions do not disrupt the internal disulfide structure of the VHH itself. After the reaction was completed, the free linker molecules were removed by ultrafiltration with a molecular weight cut-off of 3kD, while the buffer of the coupled product was changed to 10mM PBS (pH 7.4). The purified ligand-linker conjugate was stored at-80 ℃. The coupled product is analyzed and identified by Size exclusion chromatography (Size-Exclusion Chromatography, SEC) and liquid chromatography-mass spectrometry (Liquid Chromatography-Mass Spectrometry, LC-MS), and the product is confirmed to have good purity and is specifically connected with DBCO groups.

EXAMPLE 7 preparation of Anchor-modified lipid nanoparticles

This example demonstrates a method of preparing anchored modified Lipid Nanoparticles (LNP) using polyethylene glycol (PEG) derivatives containing a click group as an anchor fragment. The anchor fragment may be an amphiphilic polymer comprising a click group, a PEG-lipid conjugate comprising a click group, or a PEG-hydrophobic polymer conjugate comprising a click group. By introducing anchoring fragments containing click reaction groups, the anchoring modified LNPs can be coupled with ligands modified by a connector, so that the required ligand coupled lipid nanoparticles can be efficiently formed. The click group refers to a specific group participating in click chemistry.

The anchoring fragment containing a click group used in this example was DSPE-PEG-N3, a pegylated lipid with an azido group at the end, capable of participating in click chemistry. DSPE-PEG-N3 was incorporated into the lipid mixture used for LNP preparation. The ethanol phase of the lipid component comprises an ionizable cationic lipid, neutral lipid DSPC, cholesterol, DSPE-PEG2000, and lipid DSPE-PEG2000-N3 comprising a click group. All lipid materials were purchased from Avanti Polar Lipids. The specific preparation method comprises dissolving the above lipid in ethanol, dissolving RNA in 50mM sodium citrate buffer (pH 4.0) at concentration of 0.1mg/mL, and maintaining the mass ratio of RNA to ionizable cationic lipid at 1:10.

LNP is prepared by utilizing a microfluidic technology, a lipid solution (ethanol phase) is injected into a microfluidic mixer at a flow rate of 1mL/min, and meanwhile, an aqueous phase solution of RNA is injected at a flow rate of 3mL/min, and the volume ratio of the aqueous phase to the ethanol phase is kept at 3:1. Lipid nanoparticles are formed in the mixed solution. Subsequently, dialysis was performed to remove ethanol and buffer was replaced with PBS (10 mM, pH 7.4). Dialysis procedure PBS was dialyzed twice, each for 2 hours, against Slide-a-Lyzer dialysis cartridges (Thermo FISHER SCIENTIFIC inc., molecular weight cut-off 100 KD) at 4 ℃. The dialyzed LNP was concentrated by ultra-high speed centrifugation.

Characterization of lipid nanoparticles particle size and polydispersity index (PDI) of LNP were measured using Zetasizer Nano ZS instrument (BeNano, bettersize) in PBS and Tris-HCl buffer, respectively, to confirm their stability in different media. The concentration of RNA in LNP was determined by UV-visible spectrophotometry, by recording the absorbance spectrum and calculating based on the specific extinction coefficient, with specific attention paid to the absorbance at 260nm and calibrated at 330nm as baseline. Meanwhile, QUANT-IT ^TM RIBOGREEN RNA assay kit (Shanghai flash molecular Biotechnology Co., ltd.) was used to evaluate the encapsulation efficiency of RNA. Samples of ligand-coupled lipid nanoparticles were diluted in TE buffer and placed in polystyrene 96-well plates, incubated at 40℃for 10 min, 1:200 dilution of RIBOGREEN reagent was added, and fluorescence intensities were measured with a Molecular Devices i max microplate reader at wavelengths of approximately 488nm and 525nm, respectively. The percent free RNA was determined by comparing the fluorescence intensity of the sample of intact ligand-coupled lipid nanoparticles to that of the sample after addition of Triton X-100 breaker particles, and finally the encapsulation efficiency of RNA was calculated.

Example 8 preparation of targeted lipid nanoparticles by bioorthogonal click reaction

This example demonstrates a method for preparing targeted lipid nanoparticles (tLNP) by bio-orthogonal click chemistry to couple ligands to the surface of Lipid Nanoparticles (LNP). The simple process step involves mixing the VHH-linker conjugate with the anchored modified LNP in solution and subjecting it to ultrafiltration to achieve ligand coupling under mild conditions. The specific preparation flow is shown in figure 7.

First, a certain amount of VHH-linker conjugate was added to a pre-prepared anchor modified LNP solution, the two were mixed in a specific ratio, and incubated for 3 hours at room temperature to complete the coupling reaction. After the incubation was completed, three ultrafiltration was performed by an ultrafiltration device with a molecular weight cut-off of 100KD to remove unbound ligand and the buffer was replaced with 20mM Tris-HCl (pH 7.4). To achieve long-term storage, a 40% sucrose solution was added to adjust the final buffer concentration to 20mM Tris-HCl (pH 7.4), sucrose concentration to 8%. Finally, tLNP was subjected to aseptic filtration through a 0.22 μm filter, and the resulting LNP was packaged and stored at-80℃until use.

Example 9 in vitro evaluation study of cell lines

This example demonstrates the evaluation of delivery efficiency and off-target effects of targeted lipid nanoparticles (tLNP) by comparison of a cell line expressing a particular receptor with a cell line not expressing the receptor. The control study was performed with HEL cell line (a human erythroleukemia cell line) and CHO engineered cells overexpressing the CD117 receptor as target cells, and non-engineered CHO cells as non-target cells. HEL cells were cultured in RPMI-1640 medium containing L-glutamine (ThermoFisher), 10% fetal bovine serum, 1% penicillin-streptomycin. CHO cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded into 24-well plates at a density of 20 kilocells per well/250 μl, followed by the addition of a specific amount of tLNP encapsulating GFP or tdmamio encoding nucleic acid per well, after 6 hours of incubation, cell pellet was collected by centrifugation at 300g for 7 minutes and resuspended in 100 μl PBS for flow cytometry analysis. The flow data were processed by CytExpert software to determine the percentage of GFP or tdtomo positive cells in different cell lines using CytoFLEX flow cytometer (Beckman Coulter).

EXAMPLE 10 LNP comparative study of surface-coupled ligands of different molecular weights

This example evaluates the targeted delivery efficiency of the presently disclosed tLNP preparation method with surface coupling of ligands of different molecular weights. We selected two antibodies targeting CD117 (c-kit) for comparison, CD117 being a receptor highly expressed on Hematopoietic Stem Cells (HSCs), suitable for targeted delivery of HSCs. The two antibodies were VHH (about 16 kDa) and scFv (about 30 kDa), respectively. Ligand-linker conjugates were prepared as described in example 6, using engineered anti-CD117 VHH and anti-CD117 scFv, and specific amino acid sequences are shown in Table 5. Both the VHH and scFv sequences were engineered at their C-terminus, incorporating a histidine tag, a non (G4S) n-type hinge sequence and a cysteine residue.

TABLE 5 original and engineered antibody sequences

Subsequently, anchored modified lipid nanoparticles encapsulating mRNA encoding GFP protein were prepared according to the method of example 7, and CD 117-targeting tLNP was formulated according to the procedure of example 8. According to the previous experience of optimizing the formulation, two formulation ratios, formulation C and formulation E, were chosen, wherein the total content of PEG lipids was 1% and 1.5%, respectively, and the ratio of anchoring lipid to PEG lipid was 50%. The ionizable lipid is Compound 062. In the ligand coupling step, the amount of ligand used ranges from 0.125% to 0.5%. The information of each formula and the physicochemical characterization parameters is shown in Table 6.

TABLE 6 different LNP formulations and physicochemical characterization parameter information thereof

TLNP prepared as described above was used to transfect HEL cells (human erythroleukemia cells) and hCD117 overexpressing CHO cells (hCD 117 CHO) following the procedure of example 9. The transfection results are shown in FIG. 8. Both VHH and scFv conjugates significantly enhanced GFP gene expression levels in target cells (CD 117 expressing cells). It was observed that VHH performance was consistently better than scFv over the range of amounts of different antibody-linker conjugates. For example, in HEL cells, the average fluorescence intensity (MFI) obtained for the background-subtracted optimal VHH-conjugated LNP formulation is 10 times that obtained for the scFv-conjugated LNP optimal formulation. Furthermore, it is notable that for scFv, the efficiency of delivery to target cells tended to increase gradually as the ligand amount increased from 0.125% to 0.5%, whereas VHH already exhibited the best delivery performance at the lower ligand amount of 0.125%. The above results indicate that lower molecular weight antibody forms (especially VHH) are expected to achieve better targeted delivery using the preparation techniques disclosed herein.

Example 11 evaluation of the Effect of hinge sequences on tLNP delivery efficiency targeting CD117

To investigate the effect of hinge sequences on the efficiency of targeted delivery, we used a single domain antibody (VHH, clone h.2346b 13) against hematopoietic stem cell marker CD 117. All constructs share the same VHH framework and introduce a cysteine residue at the C-terminus to achieve site-specific coupling. The manner of attachment of the cysteine to the VHH includes direct attachment (without a Hinge sequence) or attachment via one of five different structural features of the Hinge sequence (named, respectively, hinge-1, hinge-2, hinge-4, hinge-5 and Hinge-7), each Hinge sequence being designed to exhibit a different structural feature, as shown in example 6. All VHH constructs were transiently expressed and purified (specific amino acid sequences are detailed in table 3). Site-specific coupling to the DBCO-PEG ₄ -maleimide linker was achieved by thiol-maleimide chemistry following the procedure described in example 6, following reduction of the VHH C-terminal cysteine. Subsequently, anchored modified lipid nanoparticles encapsulated with mRNA encoding GFP protein were prepared according to the method described in example 7, followed by preparation of CD 117-targeting tLNP according to the procedure of example 8, using formulation C (total PEG lipid content 1%, anchored lipid to PEG lipid ratio 50%). The composition and physicochemical properties of the final LNP are detailed in table 7.

TABLE 7 different LNP formulations and physicochemical characterization parameter information thereof

We assessed the efficiency of delivery of the CD 117-targeting LNP described above using CHO cells expressed from human CD117 as a positive target cell population and unmodified wild-type CHO cells as a negative control. In vitro transfection experiments were performed as described in example 9, and the results are shown in FIG. 9. All VHH-conjugated tLNP showed significantly higher Mean Fluorescence Intensity (MFI) in CD 117-positive CHO cells than wild-type CHO cells, confirming their specific delivery capacity. Notably, the G4S hinge sequence containing construct performed significantly better than the hinge sequence free construct, indicating that even flexible hinge sequences can increase the accessibility of the C-terminal cysteine site, facilitating the coupling reaction. Furthermore, the structural nature of the different Hinge sequences significantly affects the efficiency of delivery, as constructs containing constrained or rigid Hinge sequences (Hinge-2, hinge-4, hinge-5, hinge-7) continue to be more efficient at transfection in target cells than constructs without a Hinge or flexible G4S Hinge. Among them, constructs containing rigid Hinge sequences (Hinge-5 and Hinge-7) achieved the highest level of delivery, slightly superior to constructs containing constrained Hinge sequences. This result suggests that the steric constraints provided by the rigid hinge may be more conducive to optimizing the coupled VHH at the LNP surface display site, thereby more efficiently interacting with the receptor.

Example 12 in vitro Gene editing of HSC cells Using CRISPR/Cas12b loaded targeting LNPs

In this example, the inventors evaluated tLNP for gene delivery and editing capabilities using the CD 117-targeting tLNP disclosed herein. These tLNP are designed to deliver gene editing components that encapsulate the mRNA encoding CRISPR-AaCas12bMax and sgRNA targeting the HBG1/2 promoter. Editing of the HBG1/2 promoter is expected to induce insertional mutations, reactivating fetal globin expression, which could be a new approach to the treatment of thalassemia and sickle cell disease.

AaCas12bMax is a highly active variant of alicyclobacillus acidophilus-derived Cas12b (AaCas b) useful as a gene editing enzyme. The amino acid sequence of AaCas, 12 and bMax is shown in Table 8. mRNA encoding AaCas12bMax was synthesized by In Vitro Transcription (IVT) by inserting the coding sequence of AaCas12bMax into a plasmid to form a DNA template comprising the target protein sequence, the 5' and 3' UTR sequences and the T7 promoter upstream of the 5' UTR, and the PCR product of the above plasmid containing the poly A tail was used for in vitro transcription. T7 RNA polymerase recognizes the T7 promoter of the DNA template and initiates in vitro mRNA transcription, and the 5' cap structure of in vitro transcribed mRNA is added during in vitro transcription and n 1-methyl-pseudouracil is used instead of uracil. All in vitro transcription reactions were performed at 37 ℃ for 2 hours. DNase I was removed from the DNA template at 37 ℃ for 30min, followed by column purification to obtain full-length in vitro transcribed mRNA.

Sgrnas were designed to target the LRF binding motif of the 200bp region upstream of the HBG1/HBG2 gene promoter. The sgrnas were optimized by chemical modification and the targeting sequence was extended to 23nt. The optimized sgrnas were synthesized by Jin Wei mins and dissolved to 100 μm with water.

To achieve precise gene editing in HSC cells, CD 117-targeted LNPs encapsulating CRISPR-AaCas, bMax mRNA and sgrnas were prepared. First, ligand-linker conjugates were prepared according to example 6 using modified anti-CD 117 VHH antibodies. An anchored modified LNP was then prepared as described in example 7. Finally, the encapsulation CRISPR MRNA and the sgrnas tLNPs were prepared as described in example 8, with a weight ratio of AaCas12bMax mRNA to sgrnas of 1:1 being maintained. The final LNP concentration was determined based on the total RNA concentration in the formulation. The detailed composition and physicochemical properties of the CD 117-targeted LNP are shown in table 9.

HEL cells were exposed to LNP-922/923 (1-4. Mu.g/mL) at 4E+05/mL for 24 hours. HEL cells were then cultured for additional 48h and genomic DNA was extracted. Libraries were constructed by amplifying the region surrounding the target sgRNA binding site and sequenced by NGS. NGS results were analyzed using Cas-Analyzer (www.rgenome.net/Cas-Analyzer). The data in figure 10 shows that both LNP-922 and LNP-923 exhibit high editing efficiency and are dose dependent, indicating their great potential for editing in vivo.

TABLE 8 AaCas12bMax and sgRNA sequences

TABLE 9 VHH antibodies and formulations for different HSC targeting LNPs

TABLE 10 CDR sequences of different clones

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 human CD117, comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HCDR 1), heavy chain complementarity determining region 2 (HCDR 2), and heavy chain complementarity determining region 3 (HCDR 3), wherein the HCDR1, HCDR2, HCDR3 are selected from any one of the group consisting of:

(1) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 2, and the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 2

An amino acid sequence shown as ID NO. 3, wherein the HCDR3 comprises an amino acid sequence shown as SEQ ID NO. 4;

(2) The HCDR1 comprises an amino acid sequence shown as SEQ ID NO. 6, and the HCDR2 comprises an amino acid sequence shown as SEQ ID NO. 6

An amino acid sequence shown as ID No. 7, the HCDR3 comprises an amino acid sequence shown as SEQ ID No. 8, or

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the VH comprises an amino acid sequence as set forth 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 an amino acid sequence set forth in SEQ ID No. 1, SEQ ID No. 5 or SEQ ID No. 9.

3. The antibody or antigen-binding fragment thereof of claim 1, which is a VH that specifically binds human CD 117.

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

5. The antibody or antigen-binding fragment thereof of claim 1, comprising an Fc fragment.

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

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

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

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

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

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

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

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