Nanobodies targeting CD90 and their applications

Abstract

This application relates to a CD90-targeting nanobody and its application. This application also relates to an antibody or antigen-binding fragment targeting CD90 and its application.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a nanobody targeting CD90 and its applications. Background Technology

[0002] Hematopoietic stem cells (HSCs) are the cornerstone cells of the blood and immune system, possessing the ability to self-renew and differentiate into all blood cell lineages. Clinically, myelosuppression centered on HSCs is a curative therapy used to treat a variety of blood disorders, including leukemia, lymphoma, and severe hereditary blood disorders such as β-thalassemia and sickle cell disease. In gene therapy, HSCs can serve as a breakthrough in curing diseases; a patient's own HSCs can be genetically corrected in vitro and then returned to the body to rebuild a healthy blood system, as demonstrated in emerging genome editing therapies for hemoglobinopathies. However, HSC-based therapies face numerous challenges, such as limited donors, the risk of graft-versus-host disease in allogeneic transplantation, and the complexity of in vitro HSC manipulation. Therefore, developing systems that can directly target and deliver HSCs to modify or eliminate them within the patient is of great significance, potentially making transplantation and gene therapy safer and more readily available.

[0003] CD90 (also known as Thy-1) is a 25-37 kDa, highly N-glycosylated glycoprotein anchored to the cell membrane via glycosphingomyelinositol (GPI). It is expressed on human hematopoietic stem cells (HSCs), particularly those with long-term remodeling capacity, and is typically identified as CD34. + CD90 + CD90 is detected in HSCs (Hodgkin's stem cells) and other cell types, including thymocytes, neurons, endothelial cells, fibroblasts, and mesenchymal stem cells. Functionally, CD90 is an adhesion and signaling molecule; although its exact role in HSCs is still being elucidated, it marks a group of HSCs with strong engraftment potential. Notably, CD90 has gained attention in oncology as a cancer stem cell marker and a prognostic indicator for solid tumors. For example, in hepatocellular carcinoma and glioblastoma, CD90-positive cells have shown enhanced tumor initiation capacity and are associated with poor patient prognosis. [The text then abruptly shifts to a different topic:] Targeting CD90... + Cancer stem cells have been proposed as a therapeutic strategy, and studies have shown that inhibiting CD90 signaling (e.g., via antibodies or shRNA) can reduce tumor progression. Therefore, CD90 is not only a useful HSC marker, but also a marker for cancer cells with high CD90 levels. + Potential therapeutic targets in cancers involving malignant stem cell populations.

[0004] While existing antibody-targeting strategies against hematopoietic stem cells (HSCs) and their markers have shown promise, they also have significant limitations. Traditional full-length monoclonal antibodies (typically IgG) have large molecular weights (approximately 150 kDa), which may limit their tissue penetration (e.g., difficulty entering the bone marrow microenvironment) and result in long circulation times in vivo. Although IgG antibodies or antibody-drug conjugates (ADCs) targeting HSC markers (such as CD90) may be effective in targeting HSCs, their size and structure are not suitable for certain applications, such as delivering gene therapy payloads intracellularly. Standard antibodies typically do not internalize efficiently into cells unless engineered to do so, and their Fc fragments may trigger immune effector functions (complement activation, ADCC), leading to non-selective inflammatory responses or cell loss. Furthermore, HSC-specific targeting using whole-grain IgG may not be precise enough: markers like CD90 are also present in 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 dose adjustment. Gene therapy vectors (such as lentiviruses or AAV vectors) face various challenges: they often lack natural tropism for HSCs, and HSCs in vivo are mostly quiescent and located in protective microenvironments that are difficult for circulating vectors to reach. To overcome these obstacles, targeting ligands are needed that can bind to HSC markers with high specificity while also being small in size and modular to allow them to be attached to a variety of delivery vectors. Bispecific or multivalent antibody forms have also been proposed to improve specificity by binding to multiple HSC markers, as they need to recognize two markers simultaneously; however, constructing stable, large antibody complexes can be complex. Overall, while existing antibody-based approaches demonstrate the concept of HSC targeting, improved targeting agents are still needed for applications such as in vivo gene delivery, precise cell isolation, and safe prototyping.

[0005] Single-domain antibodies offer unique advantages in therapeutic and diagnostic applications targeting CD90 and other hematopoietic stem cell (HSC) markers. A typical single-domain antibody structurally comprises a VHH fragment. VHHs are single-domain antigen-binding fragments derived from heavy-chain antibodies from camels, typically only about 12–15 kDa in size. Due to their small size and single-domain structure, VHHs exhibit high antigen-binding affinity and specificity under a range of conditions, while also possessing excellent stability. This invention aims to provide tools and methods to significantly improve the specificity and efficiency of HSC targeting for a variety of therapeutic, preventative, and diagnostic purposes using these small and robust antibodies. Summary of the Invention

[0006] This application provides an antibody or antigen-binding fragment thereof targeting CD90, preferably a nanobody, which can specifically bind to human CD90 and target hematopoietic stem cells (HSCs) expressing CD90. In specific embodiments, the antibody or antigen-binding fragment thereof can be used to detect the content of CD90 in a mixed system, to detect the content of hematopoietic stem cells in the mixed system, and can also be conjugated with other drugs to treat hematopoietic stem cell-related diseases.

[0007] The antibody or antigen-binding fragment targeting CD90 provided in this application may have one or more of the following properties: 1) specifically binding to CD90 and / or cells expressing CD90; 2) inhibiting the binding of CD90 to at least one of its ligands; 3) inhibiting CD90-mediated signal transduction; 4) inhibiting the migration, accumulation, recruitment, and / or infiltration of cells expressing CD90; 5) mediating the killing effect on cells expressing CD90; 6) inducing endocytosis of CD90 receptors on the cell surface, thereby reducing the activation of immune cells; 7) being used to treat CD90-related diseases and / or symptoms; and 8) being used for the detection of CD90 and / or cells expressing CD90.

[0008] This application also provides nucleic acid molecules encoding the antibody or its antigen-binding fragment, expression vectors, host cells, pharmaceutical compositions containing the antibody or its antigen-binding fragment, methods for preparing the antibody or its antigen-binding fragment, and uses of the antibody or its antigen-binding fragment described in this application.

[0009] In a first aspect, this application provides an antibody or antigen-binding fragment thereof that specifically binds to human CD90. The antibody or antigen-binding fragment thereof specifically binds to human CD90. The antibody or antigen-binding fragment thereof includes a heavy chain variable region (VH). The heavy chain variable region includes heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3).

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

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

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

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

[0014] In some embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, or comprises a sequence homology of 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% with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9.

[0015] In some embodiments, the antibody is selected from one or more of the group consisting of: monospecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, and chimeric antibodies.

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

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

[0018] In a preferred embodiment, the antibody or its antigen-binding fragment is a VH that specifically binds to human CD90.

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

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

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

[0022] Secondly, this application provides a fusion protein. It comprises any one of the antibodies or its antigen-binding fragments described in the first aspect.

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

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

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

[0026] Thirdly, this application provides an isolated nucleic acid molecule that encodes any of the antibodies or antigen-binding fragments thereof in the first aspect or any of the fusion proteins in the second aspect.

[0027] In some embodiments, the isolated nucleic acid molecules may be produced or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR); (ii) clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0028] Fourthly, this application provides a vector comprising any of the isolated nucleic acid molecules described in the third aspect.

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

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

[0031] Fifthly, this application provides a cell. It comprises any of the nucleic acid molecules isolated according to the third aspect or any of the vectors described in the fourth aspect.

[0032] In some implementations, the cell comprises a host cell.

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

[0034] In specific embodiments, the cells are bacterial cells (e.g., Escherichia coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. More specifically, they can be engineered cells for antibody production, such as 293T cells.

[0035] Sixthly, this application provides a method for producing any of the antibodies or antigen-binding fragments of the first aspect. Specifically, the method includes:

[0036] (1) Construct an expression vector containing a gene sequence encoding the antibody or its antigen-binding fragment.

[0037] (2) The expression vector was transformed into host cells to induce expression, and

[0038] (3) The antibody or its antigen-binding fragment is isolated from the expression product.

[0039] In a seventh aspect, this application provides a pharmaceutical composition comprising any of the antibodies or antigen-binding fragments thereof (as described in the first aspect), any of the fusion proteins (as described in the second aspect), any of the isolated nucleic acid molecules (as described in the third aspect), any of the carriers (as described in the fourth aspect), any of the cells (as described in the fifth aspect), and / or optionally a pharmaceutically acceptable carrier.

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

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

[0042] In some embodiments, the pharmaceutical composition may be a conjugate of any of the antibodies or antigen-binding fragments of the first aspect with other molecules. More specifically, the other molecules may be small molecule drugs, antibodies targeting antigens other than CD90, or lipid nanoparticles (LNPs).

[0043] Eighthly, this application provides the use of any of the antibodies or antigen-binding fragments thereof from the first aspect, any of the fusion proteins from the second aspect, any of the isolated nucleic acid molecules from the third aspect, any of the vectors from the fourth aspect, any of the cells from the fifth aspect, and / or any of the pharmaceutical compositions from the seventh aspect in the preparation of a medicament for the prevention and / or treatment of a disease.

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

[0045] Ninthly, this application provides a method for preventing and / or treating a disease. The method comprises the use of any of the antibodies or antigen-binding fragments thereof (as described in the first aspect), any fusion protein (as described in the second aspect), any isolated nucleic acid molecule (as described in the third aspect), any vector (as described in the fourth aspect), any cell (as described in the fifth aspect), and / or any pharmaceutical composition (as described in the seventh aspect).

[0046] Tenthly, this application provides the use of any antibody or antigen-binding fragment thereof (as described in the first aspect), any fusion protein (as described in the second aspect), any isolated nucleic acid molecule (as described in the third aspect), any vector (as described in the fourth aspect), any cell (as described in the fifth aspect), and / or any pharmaceutical composition (as described in the seventh aspect) for the prevention and / or treatment of diseases.

[0047] In some embodiments, the diseases described in aspects eight, nine, or ten may be hematological diseases. Specifically, the diseases may be hematopoietic stem cell-related diseases. More specifically, the hematopoietic stem cell-related diseases may be selected from one or more of the following: aplastic anemia, myelodysplastic syndrome, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myeloproliferative neoplasm, myelofibrosis, immunodeficiency diseases, thalassemia, and sickle cell disease.

[0048] Eleventhly, this application provides a reagent or kit. The reagent or kit comprises any of the antibodies or antigen-binding fragments thereof (as described in the first aspect), any fusion protein (as described in the second aspect), any isolated nucleic acid molecule (as described in the third aspect), any vector (as described in the fourth aspect), any cell (as described in the fifth aspect), and / or any pharmaceutical composition (as described in the seventh aspect).

[0049] In some embodiments, the kit is used to detect the CD90 content in a mixed system.

[0050] In some embodiments, the kit is used to detect the amount of CD90-expressing cells in the mixed system.

[0051] For example, the CD90-expressing cells are selected from one or more of the following groups: hematopoietic stem cells, mesenchymal stem cells, keratinocyte stem cells, neurons, glial cells, endothelial cells, fibroblasts, stromal cells, activated endothelial cells, tumor cells, and tumor-associated fibroblasts.

[0052] In a twelfth aspect, this application provides a method for detecting CD90. The method comprises the use of any of the antibodies or antigen-binding fragments thereof (as described in the first aspect), any of the fusion proteins (as described in the second aspect), any of the isolated nucleic acid molecules (as described in the third aspect), any of the vectors (as described in the fourth aspect), any of the cells (as described in the fifth aspect), and / or any of the pharmaceutical compositions (as described in the seventh aspect).

[0053] In some embodiments, the CD90 may be free CD90 within a mixed system.

[0054] In some implementations, the CD90 may be CD90 expressed on cells.

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

[0056] In some embodiments, the CD90 may be coupled with other molecules.

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

[0058] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

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

[0060] Figure 1 The diagram shows the binding ability of serum from mice immunized with CD90 antigen and then enhanced with CD90 antigen to CD90-expressing (CHO-CD90) cells and control cells (WT).

[0061] Figure 2 The diagram shows the binding ability of the recombinant antibody in Example 2 to CD90 positive cells (CHO-CD90 and HEL) and control cells (CHO-K1).

[0062] Figure 3 The figure shows the binding efficiency of different concentrations of candidate antibodies in Example 4 to human CD90-CHO, monkey CD90-CHO or HEL cells.

[0063] Figure 4 The results shown are from Example 4, which used the ELISA method to evaluate the binding efficiency of the candidate antibody to the mouse CD90 antigen.

[0064] Figure 5The image shown is an SDS-PAGE image of the purified antibody from Example 5. NR indicates that no reducing agent was added to the sample, and R indicates that a reducing agent was added to the sample.

[0065] Figure 6 The diagram shows the structure of the modified ligand, linker, and ligand-linker coupling formed by their coupling in Example 6.

[0066] Figure 7 The diagram shows the steps of a method for preparing targeted lipid nanoparticles (tLNPs) by coupling ligands on the surface of lipid nanoparticles (LNPs) via bioorthogonal click chemistry.

[0067] Figure 8 This study shows a comparative study of the in vitro delivery efficiency of tLNPs coupled with different VHH clones.

[0068] Figure 9 The demonstration shows the gene delivery and editing capabilities of tLNP for in vitro delivery of gene editing components. Detailed Implementation

[0069] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0070] Terminology Definition

[0071] In this application, the term "CD90," also known as "Thy-1," generally refers to a glycosylphosphatidylinositol (GPI)-anchored glycoprotein belonging to the immunoglobulin superfamily. It is expressed in various cell types, including hematopoietic stem cells, mesenchymal stem cells, nerve cells, fibroblasts, stromal cells, activated endothelial cells, and glioblastoma stem cells. CD90 plays an important role in physiological processes such as cell adhesion, proliferation, and signal transduction. In this application, unless otherwise specified, CD90 can include all its subtypes and all species. In this application, CD90 can include CD90.1 (Thy-1.1) and CD90.2 (Thy-1.2). In this application, CD90 can include any naturally occurring CD90 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. In this application, the CD90 may include "full-length", unprocessed CD90, and any form of CD90 derived from cell processing; it may also include naturally occurring variants of CD90, such as splice variants or allelic variants. For example, the complete amino acid sequence of human CD90 is accessed at Uniprot website (https: / / www.uniprot.org / ) under accession number P04216.

[0072] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component is found in nature as an "isolated" substance, it may mean that its natural environment has changed, or that the substance has been isolated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance. In this application, unless otherwise specified, the antibody or its antigen-binding fragment is also isolated.

[0073] In this application, the term "antigen-binding protein" generally refers to a protein with antigen-binding ability. For example, an antigen-binding protein may include isolated antigen-binding proteins. An antigen-binding protein may contain, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted CDR or a CDR derivative. Such frameworks include, but are not limited to, antibody-derived framework regions containing mutations introduced, for example, to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic framework regions containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen-binding proteins include, but are not limited to: Fab, Fab', Fv fragments, F(ab')2, F(ab)2, scFv, di-scFv, dAb, VHH, human antibodies, humanized antibodies, chimeric antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies, monovalent antibodies, multivalent antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies and fragments thereof.

[0074] In this application, the term "CDR," also known as "complementarity-determining region," typically refers to a region within the variable domain of an antibody whose sequence is highly variable and / or forms a structurally defining loop. Typically, the variable domain of the heavy chain (VH) of an antibody includes three CDRs (HCDR1, HCDR2, and HCDR3). Antibodies composed solely of the heavy chain can function normally and stably even in the absence of the light chain, for example, naturally occurring camel antibodies; see, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, AbM, Chothia, IMGT, and a combination of Kabat / Chothia. These coding systems are known in the art, and for example, see www.bioinf.org.uk / abs / index.html#kabatnum. For example, the amino acid sequence number of the antigen-binding protein can be in accordance with the IMGT numbering scheme (IMGT, the international ImMunoGeneTics informationsystem@imgt.cines.fr; imgt.cines.fr; Lefranc et al., 1999, Nucleic Acids Res. 27:209-212; Ruiz et al., 2000, Nucleic Acids Res. 28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefranc et al., 2003, Nucleic Acids Res. 31:307-310; Lefranc et al., 2005, DevComp Immunol 29:185-203). For example, the CDR of the antigen-binding protein can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, Department of Health and Human Services, NIH Publication No. 91-3242).

[0075] In this application, the term "variable" generally refers to the fact that certain segments of the variable region may differ significantly in sequence between antibodies. The variable region mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire variable region. It is typically concentrated in three segments within the light or heavy chain variable region, known as the hypervariable region (CDR or HVR). The more highly conserved portions of the variable region are called the frame region (FR).

[0076] In this application, the term "FR" generally refers to a more conserved portion of the antibody variable domain, which is called the frame region. Typically, the natural heavy chain variable domain contains four FR regions: H-FR1, H-FR2, H-FR3, and H-FR4.

[0077] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide that includes an antigen-binding site, whether it is produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," such as in "complete antibody," for the purposes of this application, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, VHH, dAb, and other antibody fragments that retain antigen-binding function (e.g., capable of specifically binding CD90).

[0078] In this application, the term "antigen-binding fragment" generally refers to one or more fragments having the ability to specifically bind to an antigen (e.g., CD90). In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0079] In this application, the term "single-domain antibody" generally refers to an antibody lacking a light chain. The single-domain antibody described in this application may include a heavy-chain antibody (HcAb), which includes a heavy-chain variable region and conventional heavy-chain CH2 and CH3 regions. The single-domain antibody described in this application may include the smallest binding unit of an antigen-binding protein. The single-domain antibody described in this application may include an antibody fragment consisting only of the antibody heavy-chain variable region.

[0080] In this application, the term "VHH (variable domain of heavy chain of heavy chain antibody)" generally refers to the variable region antigen-binding domain of a heavy chain antibody (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P et al., 2011, Antiviral Research 92, 389-407).

[0081] In this application, the term "nanobody" refers to an antibody with a very small molecular weight. In a preferred embodiment, the nanobody consists only of the variable region of a heavy chain antibody, and its molecular weight is typically less than 15 kDa, which is 1 / 10 of that of a conventional antibody. This structure endows nanobodies with a series of unique properties, including high affinity, high stability, good water solubility, low immunogenicity, and strong tissue penetration ability.

[0082] In this application, "single-domain antibody" and "nanobody" refer to a complete antibody capable of functioning independently, and "VHH" refers to a structural domain within a complete antibody. Furthermore, "single-domain antibody" emphasizes the constituent parts of the antibody (e.g., containing only a single heavy chain), while "nanobody" emphasizes the molecular weight of the antibody. In this application, single-domain antibodies may include, but are not limited to, nanobodies.

[0083] In this application, the term "Fc fragment" generally refers to a C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. This term includes native sequence Fc regions and variant Fc regions. The Fc region of an immunoglobulin generally contains two constant domains—a CH2 domain and a CH3 domain—and optionally includes a CH4 domain. For example, the Fc region may not contain a CH1 domain. Amino acid residue substitutions in the Fc moiety that alter antibody effector functions are known in the art (U.S. Patents 5,648,260; 5,624,821, Winter et al.). The Fc moiety of an antibody mediates several important effector functions, such as cytokine induction, ADCC, phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance of the antibody and antigen-antibody complex. Depending on the therapeutic purpose, these effector functions are desirable for therapeutic antibodies in some cases, but may be unnecessary or even detrimental in others.

[0084] In this application, the term "monoclonal antibody" generally refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibodies typically exhibit high specificity against a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (which usually contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized through hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and is not to be interpreted as requiring the production of antibodies through any particular method. For example, the monoclonal antibodies used in this application can be prepared using recombinant DNA methods.

[0085] In this application, the terms "monospecific antibody" and "multispecific antibody" generally refer to antibodies capable of specifically binding to one or more antigen sites. When multiple antigen sites are specifically bound, these multiple antigen sites may originate from the same antigen or from different antigens, preferably from different antigens.

[0086] In this application, the terms "monovalent antibody" and "multivalent antibody" refer to antibody molecules having one or more antigen-binding sites. As those skilled in the art will know, classic antibodies (e.g., IgG antibodies) are bivalent antibodies, having two antigen-binding sites. In this application, "monovalent antibody" and "multivalent antibody" may contain a constant region and / or an Fc fragment, or may not contain a constant region and an Fc fragment. When the "monovalent antibody" or "multivalent antibody" does not contain a constant region and an Fc fragment, the multivalent antibody may be formed by linking two or more antigen-binding fragments end-to-end, preferably via covalent bonding. The linking method of the multivalent antibody can be N-terminal to C-terminal, N-terminal to N-terminal, or C-terminal to C-terminal.

[0087] In this 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 from a laboratory animal such as a camel ("parental antibody"), and the constant region is derived from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in human individuals compared to parental (e.g., camel-derived) antibodies.

[0088] In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDR region of a non-human antibody (e.g., a camel antibody) are replaced by corresponding amino acids derived from human immunoglobulins. Small additions, deletions, insertions, substitutions, or modifications of amino acids within the CDR region are also permissible, as long as they retain the antibody's ability to bind to a specific antigen. A humanized antibody may optionally contain at least a portion of the constant region of a human immunoglobulin. A "humanized antibody" retains antigen specificity similar to that of the original antibody. The "humanized" form of a non-human (e.g., camel) antibody may minimally contain a chimeric antibody with a sequence derived from a non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (receptor antibody) may be replaced with CDR region residues from a non-human species (donor antibody) (such as a camel, alpaca, mouse, rat, rabbit, or non-human primate) having the desired properties, affinity, and / or capabilities. In some cases, FR region residues in a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications not found in receptor antibodies or in donor antibodies. These modifications can be made to further improve antibody performance, such as binding affinity.

[0089] In this application, the term "fusion protein" refers to a protein that is produced by linking the gene sequences of two or more different proteins together using genetic engineering techniques, thereby expressing two or more functions within the same polypeptide chain. This protein fusion can endow the fusion protein with new functional properties or enhance its original functions. Fusion proteins have advantages such as functional diversity, enhanced stability, improved targeting, and ease of production, and can be applied in various fields such as biopharmaceuticals, vaccine development, cell therapy, and diagnostic tools. In specific embodiments, the fusion protein can be a viral membrane fusion protein, a cell fusion protein, a bispecific antibody, or an Fc fusion protein.

[0090] In this application, the term "nucleic acid molecule" generally refers to a nucleotide, deoxyribonucleotide, or ribonucleotide of any length in an isolated form, or an analogue isolated from its natural environment or synthesized artificially.

[0091] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein into itself and enable the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material elements they carry to be expressed within the host cells. For example, vectors can include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors can include retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0092] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this application. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro using the vectors described in this application. 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, COS-1 cells, and NSO cells. Cells may also include engineered cells.

[0093] In this application, the term "pharmaceutical composition" generally refers to a composition for the prevention / treatment of a disease or condition. The pharmaceutical composition may comprise the antibody or its antigen-binding fragment described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions described in this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0094] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0095] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target is determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) may be an antibody that binds to the target with greater affinity, strength, ease, and / or duration than it binds to other targets. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across proteins of different species. In some embodiments, specific binding may include, but is not required to be, exclusive binding.

[0096] In this application, the term "hematopoietic stem cell-related diseases" generally refers to a range of hematologic disorders caused by functional abnormalities, developmental defects, or malignant transformation of hematopoietic stem cells. These diseases typically involve bone marrow hematopoietic dysfunction, immune deficiencies, or malignant proliferation of blood cells. These diseases may be caused by genetic factors, environmental exposures, or unknown factors, and symptoms include anemia, bleeding, infection, and organ enlargement. Diagnosis and treatment usually require a multidisciplinary, comprehensive approach.

[0097] "Conservative substitution" of amino acids is well known in the art and generally refers to the alteration of one amino acid residue with another amino acid residue having a structurally or functionally similar side chain. For example, an exemplary list of conservative substitutions is provided in the table below.

[0098]

[0099]

[0100] In this 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.

[0101] In this application, the proteins, peptides, and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0102] In this application, the variant can be, for example, a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof capable of specifically binding CD90). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the 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 polypeptide prior to the alteration. For example, the substitution may be a conserved substitution.

[0103] In this application, the homolog can be a protein or polypeptide having at least about 80% (e.g., having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof capable of specifically binding to CD90).

[0104] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, U) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in WRPearson and DJ Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and DJ Lipman and WRPearson, “A Fast and Sensitive Search for Protein Similarity,” Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, EW Myers, and D. Lipman, “A Basic Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0105] In this application, the term "comprising" generally means "including," "summarizing," "containing," or "encompassing," and these terms are used interchangeably. In some cases, it also means "to be" or "composed of."

[0106] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Invention Details

[0108] Antibody or its antigen-binding fragment

[0109] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, 1-30, 1-20, or 1-10 amino acid substitutions, deletions, and / or insertions, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions; homologs are also included, which can 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 higher) sequence homology with the amino acid sequence of the CDR. In some embodiments, the antigen-binding protein described in this application can be defined by the KABAT coding system.

[0110] This application provides an antibody or antigen-binding fragment thereof that specifically binds to human CD90. The antibody or antigen-binding fragment includes a heavy chain variable region (VH). The heavy chain variable region includes heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3).

[0111] In some embodiments, HCDR1, HCDR2, and HCDR3 are selected from any one of the following groups:

[0112] (1) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:4; or

[0113] (2) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:6, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:8; or

[0114] (3) The HCDR1 includes the amino acid sequence shown in SEQ ID NO:10, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:11, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:12.

[0115] In some embodiments, the VH comprises an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, or comprises an amino acid sequence having about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% sequence homology with the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, and all amino acid differences are in the non-CDR region.

[0116] Preferably, the above-mentioned amino acid differences are conservative substitutions of amino acids.

[0117] In this application, the antibody or its antigen-binding fragment may contain at least one CDR from the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, and the CDR may include a CDR partitioned in any manner. Any CDR partitioned in any manner, if its sequence is identical to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:9, falls within the protection scope of the claims of this application.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof described in this application comprises an antigen-binding protein having an HCDR1 having an amino acid sequence as shown in SEQ ID NO:2, SEQ ID NO:6, or SEQ ID NO:10, an HCDR2 having an amino acid sequence as shown in SEQ ID NO:3, SEQ ID NO:7, or SEQ ID NO:11, and an HCDR3 having an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:8, or SEQ ID NO:12, and a humanized antigen-binding protein thereof. In some embodiments, the humanized antigen-binding protein may have one or more amino acid mutations in HCDR1, one or more amino acid mutations in HCDR2, and / or one or more amino acid mutations in HCDR3, while the humanized antigen-binding protein still has the ability to bind CD90.

[0119] In some embodiments, the isolated antigen-binding protein described in this application includes a variable region having an amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:5 or SEQ ID NO:9, and a humanized antigen-binding protein thereof.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof described in this application may include a single-domain antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof described in this application may include a heavy-chain antibody or antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment thereof described in this application may include a nanobody.

[0121] In some embodiments, the antibody or its antigen-binding fragment may comprise an antibody consisting only of a heavy chain. In some embodiments, the heavy chain-only antibody comprises a variable region antigen-binding domain, which is composed of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some embodiments, the heavy chain-only antibody comprises an antigen-binding domain, at least a partial hinge region, and CH2 and CH3 domains. In some embodiments, the heavy chain-only antibody comprises an antigen-binding domain, at least a partial hinge region, and a CH2 domain. In some embodiments, the heavy chain-only antibody comprises an antigen-binding domain, at least a partial hinge region, and a CH3 domain. Heavy chain-only antibodies in which the CH2 and / or CH3 domains are truncated are also included herein. In another embodiment, the heavy chain comprises an antigen-binding domain and at least one CH (CH1, CH2, CH3, or CH4) domain, but without a hinge region. The heavy chain-only antibody may be in dimer form, wherein the two heavy chains are bonded by disulfide bonds, or otherwise covalently or non-covalently linked together. Antibodies consisting solely of heavy chains may belong to the IgG subclass, but antibodies belonging to other subclasses, such as IgM, IgA, IgD, and IgE subclasses, are also included herein. In specific embodiments, heavy chain antibodies may be IgG1, IgG2, IgG3, or IgG4 subtypes, particularly the IgG1 subtype.

[0122] In some embodiments, the antibody may be selected from one or more of the following groups: monoclonal antibodies, chimeric antibodies, and humanized antibodies.

[0123] In a preferred embodiment, the antibody may be a nanobody, more preferably a single-domain antibody within a nanobody. Single-domain antibodies offer unique advantages in therapeutic and diagnostic applications targeting CD90 and other hematopoietic stem cell (HSC) markers. The most important component of a single-domain antibody is the VHH, a single-domain antigen-binding fragment derived from heavy-chain-only antibodies from camels, typically only about 12–15 kDa in size. Due to its small size and single-domain structure, VHHs exhibit high antigen-binding affinity and specificity under a range of conditions, while also possessing excellent stability. These properties enable single-domain antibodies to overcome many limitations of conventional antibodies: they can more easily penetrate tissues and cellular microenvironments, can be recombinantly produced in low-cost microbial systems, and can be easily engineered with a variety of payloads and formats via gene fusion or chemical conjugation. Importantly, VHHs are often able to recognize unique or hidden epitopes (thanks to their relatively long CDR3 loops) and tend to be efficiently internalized when binding to cell surface targets, especially if they are multivalent or linked to triggers that promote endocytosis. For example, nanobodies have been shown to reach intracellular targets when used as delivery agents, a key characteristic for gene therapy payload delivery. They inherently lack an Fc region, thus avoiding unwanted Fc-mediated immune effects; this is an advantage for imaging agents (reducing background uptake in organs rich in Fc receptors), or can be mitigated by adding Fc or other polymeric tags when effector function or extended half-life is required. Overall, the stability, high affinity, and modularity of single-domain antibodies make them ideal ligands for innovative HSC-targeted therapies.

[0124] In this application, the antibody or its antigen-binding fragment may further comprise an Fc fragment. In some embodiments, the N-terminus of the Fc fragment is directly or indirectly linked to the C-terminus of the heavy chain variable region. In some embodiments, the N-terminus of the Fc fragment and the C-terminus of the heavy chain variable region may be linked via a hinge region. In some embodiments, the Fc fragment may be derived from the Fc fragment of human IgG. In some embodiments, the antigen-binding protein of this application comprises a wild-type Fc fragment derived from human IgG. The amino acid sequence of the Fc fragment derived from human IgG is known in the art. In some embodiments, the Fc fragment may comprise an Fc fragment derived from any of the following immunoglobulins: IgG1, IgG2, IgG3, and IgG4. In some embodiments, the antigen-binding protein of this application comprises a wild-type Fc fragment derived from any of the following immunoglobulins: IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the Fc fragment derived from IgG1, IgG2, IgG3, or IgG4 is known in the art. For example, the Fc fragment may comprise an Fc fragment derived from human IgG1. For example, the Fc fragment may comprise an Fc fragment derived from human IgG3. For example, the Fc fragment may comprise an Fc fragment derived from human IgG4.

[0125] In some embodiments, the antibody or antigen-binding fragment described in this application comprises an Fc fragment variant having one or more amino acid mutations relative to the wild-type Fc fragment. The half-life of the antibody or antigen-binding fragment can be prolonged and / or the effector function of the antibody or antigen-binding fragment can be enhanced by optimizing the Fc fragment sequence.

[0126] In this application, the antibody or its antigen-binding fragment may further comprise a separated antigen-binding protein after further functional optimization or modification. In some embodiments, the antigen-binding protein of this application may include an antigen-binding protein with enhanced affinity for binding to CD90. In some embodiments, the antigen-binding protein of this application may include an antigen-binding protein with enhanced ability to inhibit the binding of CD90 to its ligand. In some embodiments, the antigen-binding protein of this application may include an antigen-binding protein with enhanced ability to inhibit CD90-mediated signal transduction. In some embodiments, the antigen-binding protein of this application may include an antigen-binding protein with enhanced ability to inhibit the migration, accumulation, recruitment, and / or invasion of CD90-expressing cells. In some embodiments, the antigen-binding protein of this application may include an antigen-binding protein with enhanced ability to mediate the killing effect on CD90-expressing cells. In some embodiments, the antigen-binding protein of this application may include an antigen-binding protein with enhanced ability to induce CD90 receptor endocytosis on the cell surface. In some embodiments, the enhanced ability may be relative to the antigen-binding protein before functional optimization or modification.

[0127] The antibodies or antigen-binding fragments described in this application may include heavy chain sequences with one or more conserved sequence modifications. "Conserved sequence modifications" refer to amino acid modifications that do not significantly affect or alter the antibody-binding properties. Such conserved modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies or antigen-binding fragments described in this application using standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conserved amino acid substitutions involve replacing amino acid residues with amino acid residues having similar side chains. Groups of amino acid residues with similar side chains are known in the art. These amino acid residue groups include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues in the CDR region of the antibody or its antigen-binding fragment described in this application may be replaced with other amino acid residues from the same side chain group. Those skilled in the art will know that some conserved sequence modifications do not cause the antigen to lose its binding ability. For example, see Brummell et al., (1993) Biochem 32:1180-8; de Wildt et al., (1997) Prot. Eng. 10:835-41; Komissarov et al., (1997) J. Biol. Chem. 272:26864-26870; Hall et al., (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al., (1998) Int. Immunol. 10:341-6 and Beers et al., (2000) Clin. Can. Res. 6:2835-43.

[0128] In this application, the antibody or its antigen-binding fragment is specifically capable of binding to CD90. In some embodiments, the binding affinity of the antibody or its antigen-binding fragment to CD90 can be detected by measuring the binding ability of the antibody or its antigen-binding fragment to cells expressing CD90. For example, this can be detected by FACS. In some embodiments, the binding of the antibody or its antigen-binding fragment to CD90 can be detected by an ELISA method. For example, the antibody or its antigen-binding fragment of this application can bind to CD90 at EC50 values ​​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, the antibody or its antigen-binding fragment described in this application can bind to CD90 with an IC50 value of less than or equal to 50 nM, less than or equal to about 40 nM, less than or equal to 30 nM, less than or equal to 20 nM, or less than or equal to 10 nM.

[0129] The CD90 antigen-binding protein described in this application can be determined, identified, or characterized by various methods known in the art. For example, the antigen-binding activity of the antibody or its antigen-binding fragment described in this application can be tested by known methods such as enzyme-linked immunosorbent assay (ELISA), Western blotting (e.g., protein blotting), flow cytometry (e.g., FACS), immunohistochemistry, immunofluorescence, etc.

[0130] The antibody or antigen-binding fragment thereof provided in this application can be used to antagonize CD90 activity. In this application, the antibody or antigen-binding fragment thereof is capable of preventing and / or treating diseases and / or conditions.

[0131] Based on antibodies or their antigen-binding fragments and their applications

[0132] This application also covers variants of the disclosed anti-CD90 single-domain antibodies that have been engineered to confer or enhance properties required for specific therapeutic, diagnostic, or research applications. These variants include, but are not limited to, modifications made to improve affinity, specificity, stability, solubility, reduce immunogenicity, enhance conjugation ability, or other desired functional properties.

[0133] Variants designed to improve antigen-binding affinity and specificity can be generated using methods known in the art. These methods include affinity maturation via targeted or random mutations, followed by selection techniques such as phage display or yeast display. The scope of this invention includes variants with improved binding kinetics (e.g., increased binding rate, reduced dissociation rate) or enhanced selectivity. Variants with enhanced stability and solubility include modifications to the framework region. Specific amino acid substitutions may introduce stabilizing residues or disulfide bonds to increase thermal stability, resistance to protease degradation, or reduce aggregation tendency. In one embodiment, mutations in framework residues increase antibody solubility or stability under physiological or manufacturing conditions while maintaining target specificity and affinity.

[0134] In some embodiments, the variant may be an engineered variant for site-specific coupling. The variant includes the introduction of one or more cysteine ​​residues at a predetermined position within the C-terminal or N-terminal region of a single-domain antibody. These cysteine ​​residues facilitate the selective and stable coupling of therapeutic agents, toxins, imaging agents, or functional groups via chemical linkers (e.g., maleimide-thiol reactions). Furthermore, the variant may contain flexible hinges or linker sequences adjacent to the engineered cysteine ​​residues to optimize payload accessibility and maintain antibody function. In other embodiments, the variant incorporates non-natural amino acids with bioorthogonal reactive groups (e.g., azide, alkynyl, keto-functionalized amino acids). These residues enable highly specific, orthogonal coupling reactions (“click chemistry”) that facilitate the precise linking of diagnostic agents, cytotoxic drugs, or gene-editing components.

[0135] In some 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. These fusion proteins include, but are not limited to, immunoglobulin Fc fragments (forming antibody-Fc fusion proteins with extended 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 treatment-related peptides. In one illustrative example, the fusion of an anti-CD90 VHH with an Fc domain produces a bivalent antibody with extended serum half-life and enhanced affinity.

[0136] In some embodiments, the variants may be bispecific or multispecific antibodies. These constructs may involve the genetic fusion of two or more distinct VHH domains via flexible peptide linkers. Examples include bispecific antibodies comprising an anti-CD90 VHH domain linked to an anti-CD117 VHH domain, capable of simultaneously targeting multiple epitopes on hematopoietic stem cells to enhance specificity and therapeutic efficacy. Variants may also include linker sequences designed to optimize the spacing and orientation between the different functional domains. Suitable linkers include flexible gly-serine (Gly-Ser)n repeat sequences, rigid helical formation sequences, or cleavable linkers responsive to specific cellular environments (e.g., protease-cleavable sequences that facilitate the release of intracellular payloads).

[0137] Furthermore, this application also includes humanized variants in which the camel-derived VHH sequence has been modified to more closely approximate the variable domain of human immunoglobulins, reducing potential immunogenicity in therapeutic applications. This humanization may involve transplanting camel-derived complementarity-determining regions (CDRs) onto the human variable domain framework, optionally followed by selective back mutation to restore optimal binding and stability characteristics.

[0138] This application also relates to derivatives of the disclosed anti-CD90 single-domain antibody engineered to enhance its use in therapeutic, diagnostic, and research applications. Antibody derivatives within the scope of this application include fusion proteins created by genetically linking the disclosed single-domain antibody 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 an endoplasmic reticulum escape domain, thereby improving intracellular delivery of payloads such as nucleic acids or drugs. Furthermore, the VHH domain can be fused to fluorescent proteins, luminescent reporter proteins, or enzyme-catalyzed reporter proteins, facilitating their use in diagnostic detection and imaging applications.

[0139] 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 olprestatins, maytansine, doxorubicin, calichiomycin, and pyrrolobenzodiazepines (PBDs). Conjugation can be performed via cleavable or non-cleavable linkers that allow selective release of the cytotoxic payload upon internalization into cells targeting CD90 expression.

[0140] Further embodiments include radiolabeled derivatives, wherein the single-domain antibody is chemically bound to a radioisotope (such as technetium-99m, zirconium-89, iodine-125, lutetium-177, or yttrium-90). These radiolabeled derivatives can be used for diagnostic imaging in positron emission tomography (PET) or single-photon emission computed tomography (SPECT), as well as for targeted radiotherapy to selectively deplete specific cell populations.

[0141] Furthermore, bispecific or multispecific antibody derivatives can be constructed to simultaneously bind multiple target antigens, thereby improving the specificity and efficacy of treatment. For example, the antibody derivative could be a bispecific antibody containing a VHH domain targeting CD90 and a VHH domain of another clinically relevant antigen (such as CD3 for T cell binding), which provides selective targeting and a potent biological effect.

[0142] In some embodiments, the antibody derivatives are designed to target gene delivery and / or gene editing. These derivatives include viral vectors such as adeno-associated virus (AAV) or lentiviral particles whose capsid or envelope proteins are genetically modified to integrate VHH antibody domains, thereby achieving specific and efficient targeting of CD90-expressing hematopoietic stem cells (HSCs) and other related cell types.

[0143] In some embodiments, the antibody derivative may be an antibody derivative for cell therapy, such as chimeric antigen receptors (CARs). These constructs integrate the VHH sequence into an engineered receptor expressed on immune effector cells (T cells, NK cells, macrophages), thereby achieving selective immune-mediated clearance of antigen-positive target cells. Similarly, bispecific T-cell binders (BiTE-like molecules) using an anti-CD90VHH domain linked to a T-cell binding domain (such as anti-CD3) are also within the scope of this application, enabling precise targeting of immune responses to lesions or target cells.

[0144] In some embodiments, the antibody derivatives comprise single-domain antibodies bound to lipid polyethylene glycol (lipid-PEG) groups. These lipid-PEG-bound single-domain antibodies enable targeted delivery to non-viral delivery platforms, particularly lipid nanoparticles (LNPs), liposomes, or polymer-based nanoparticles. These derivatives provide enhanced stability, targeted cell binding, and efficient internalization for intracellular delivery of nucleic acid payloads such as mRNA, siRNA, or gene-editing tools.

[0145] A key aspect of this application is the engineering of VHH or single-domain antibody clones into various forms, such as fusion proteins or conjugates, to achieve targeted delivery or therapeutic effects. The following are several implementation categories:

[0146] 1. Antibody-Drug Conjugates (ADCs) and Toxin Fusion Proteins: Each anti-CD90 VHH can be linked to a cytotoxic drug to create a targeted cytotoxic agent capable of eliminating cells expressing the target antigen. In one embodiment, the VHH is chemically conjugated to the toxin by adding a cysteine ​​residue to the VHH. For example, a cysteine ​​residue is inserted at the C-terminus (after the last framework residue) of the VHH or single-domain antibody to allow for site-specific linking to a drug linker terminally conjugated with a maleimide. More specifically, a maleimide linker is conjugated with this cysteine ​​residue to obtain the anti-CD90 VHH-DM1 conjugate. In cell killing assays, this conjugate specifically kills CD90 cells. + VHHs can target cells (such as cultured stem cells / progenitor cells) without affecting CD90- cells, demonstrating targeted cytotoxicity. Similarly, VHHs can be genetically fused with protein toxins; for example, an anti-CD90 VHH fused with a truncated diphtheria toxin or an apoptosis enzyme (such as granzyme) can be used to deplete CD90-expressing cells. Such ADCs can be used as opsonizers to deplete HSCs in the bone marrow before introducing gene-correcting cells or donor cells. In specific implementations, the smaller size of VHHs may result in better tissue penetration and more uniform bone marrow distribution than larger IgG ADCs, potentially achieving more effective HSC clearance at lower doses.

[0147] 2. Targeted Lipid Nanoparticles (tLNPs): This application includes a lipid nanoparticle delivery system loaded with single-domain antibodies for targeted delivery. Lipid nanoparticles can encapsulate therapeutic payloads such as mRNA, siRNA, or CRISPR ribonucleoproteins, but by default they are non-specifically distributed. By linking anti-CD90 VHH to the surface of an LNP, we created a targeted LNP (tLNP) capable of targeting HSCs. In one example, anti-CD90 VHH is lipid-anchored via a polyethylene glycol (PEG) linker: first, the VHH is modified with a linker containing a lipid tail. Then, the lipid-modified VHH is incorporated into an LNP formulation carrying reporter mRNA. Two optimized LNP formulations have different VHH loading ratios. Results show that these CD90-targeted LNPs are targeted by CD90. + Efficient cellular binding and uptake. When there is sufficient VHH density on the LNP (e.g., 0.125-0.5% of total lipids is VHH-lipid conjugate), almost all target cells (CD90) will readily accept it. + CHO cells and CD90 +Human cell lines (HEL) expressed the delivered reporter gene, while CD90-control cells showed minimal uptake. This demonstrates that VHH-loaded LNPs can selectively deliver nucleic acid payloads to HSC-like cells. Methods for creating such tLNPs will be further described in the examples and generally involve covalently linking VHH to lipids (e.g., DSPE-PEG-maleimide) and the self-assembly of the nanoparticles.

[0148] 3. Viral Vector Targeting and CAR Construction: The VHH described in this application can be used to construct targets for viral or chimeric antigen receptors. For viral vectors, an example is AAV capsid modification: the coding sequence of anti-CD90 VHH is inserted into the AAV capsid protein loop, generating a chimeric capsid containing VHH on the viral surface. This modified AAV specifically transduces CD90. + Cells can effectively redirect viral tropism to HSCs. Another modification approach involves using adapter molecules—for example, biotinylated anti-CD90 VHH can bind to lentiviruses expressing streptavidin, thereby anchoring the virus to CD90. + On cells. These strategies allow gene delivery vectors (lentiviruses, AAVs, adenoviruses, etc.) to achieve cell-specific transduction, a key step in realizing in vivo HSC gene therapy without transplantation. In the context of cell therapy, the VHH sequence here can serve as the antigen-binding domain of a CAR (chimeric antigen receptor) on immune cells. For example, a CAR was constructed by fusing the CD90 VHH sequence with the CD8 hinge-transmembrane domain and the intracellular CD3ζ and 4-1BB signaling domains. T cells expressing the CAR specifically recognized and lysed CD90-expressing cells in vitro, demonstrating that VHH retains its binding capacity in the CAR. Such CAR-T cells can be used to target and destroy HSCs (for opsonizing or treating malignancies) or even attack CD90 in solid tumors. + Cancer stem cells. The advantage of using VHH in CAR is that its small size may reduce immunogenicity and allows for tighter packaging or design as a dual CAR (where two small domains are used side by side).

[0149] 4. Multispecific and Bispecific Antibodies: To increase binding affinity or targeting selectivity, multiple VHHs can be linked together. A bivalent anti-CD90 construct was created by genetically fusing two CD90 VHH domains and using a flexible linker, resulting in a tandem VHH whose binding valence to CD90 is essentially doubled. This bivalent nanobody exhibited a slower dissociation rate from the antigen and increased antibody retention time in bone marrow tissue in in vivo models. For dual targeting, linking an anti-CD90 VHH with an anti-CD45 VHH (CD45 is a panleukocyte marker expressed on HSCs) creates a bispecific molecule that requires simultaneous binding of both markers to bind to cells; therefore, this molecule preferentially binds to HSCs (CD90). + CD45 + Instead of T cells (which are usually CD45 in peripheral blood) + However, CD90-). This is an example of how multispecific nanobody constructs can precisely target well-defined cell subpopulations. All of these tandem or fusion antibody constructs are within the scope of this application.

[0150] 5. Fusion with Functional Groups: In addition to binding to targets, VHHs can also be fused with functional protein domains. For example, fusing an anti-CD90 VHH with an Fc domain (creating a VHH-Fc or “nanobody-Fc”) endows the molecule with IgG-like properties (bivalent and an extended half-life due to FcRn cycling). Alternatively, fusing a nanobody with an enzyme can localize the enzyme’s activity to HSCs—linking an anti-CD90 VHH with a cytokine or growth factor (transforming it into a targeted cytokine that preferentially stimulates HSCs). In one embodiment, we created an IL-2-VHH fusion in which the CD90 VHH is fused with IL-2; this chimeric molecule can specifically deliver IL-2 signaling to CD90 co-expressing IL-2R. + The cell represents a cell-selective cytokine delivery platform. In addition, this application envisions many other combinations (fusion of nanobodies with signaling molecules, fusion with apoptosis-inducing domains, fusion with fluorescent proteins for imaging, etc.).

[0151] All coupling and engineering techniques used were adaptations of standard or known methods. Small molecule payloads were covalently linked to VHHs via NHS esters, maleimide-thiol reactions, and click chemistry (e.g., azide-alkyne cycloaddition). Peptide / protein level integration was achieved through genetic fusion. The final product was characterized by SDS-PAGE, mass spectrometry, and functional assays to confirm that VHHs retained their binding function after modification.

[0152] Therefore, single-domain antibodies targeting CD90 on hematopoietic stem cells (HSCs) have broad application potential. These applications cover both research and clinical fields:

[0153] • Targeted gene therapy and gene editing: Using anti-CD90 VHH-guided gene therapy vectors or nanoparticles to HSCs enables gene delivery or editing of stem cells (e.g., delivery via a CRISPR system). This can treat hereditary blood disorders by editing HSCs in situ, thus avoiding the need for transplantation.

[0154] Cancer immunotherapy: Utilizing VHH-based constructs to target malignant stem cells or the supportive microenvironment in cancer. For example, toxins targeting CD90 or CAR T cells can eliminate CD90 in solid tumors such as liver cancer or glioma. + Cancer stem cells.

[0155] • HSC transplant conditioning: VHH conjugates are used to selectively deplete or inactivate the patient's HSCs before transplantation. Anti-CD90 nanobody-drug conjugates (or bispecific antibodies that guide immune effector cells to HSCs) can serve as minimally toxic conditioning agents to make room for donor HSC implantation. In addition, VHHs can be used to deliver radioisotopes to the bone marrow to locally irradiate the HSC microenvironment.

[0156] • Cell isolation and in vitro manipulation: HSCs can be isolated from bone marrow or circulating blood by coating magnetic beads or column matrix with anti-CD90 VHHs for research or therapeutic transplant enrichment. The small size of VHHs may allow for more gentle and specific capture and release of cells than intact antibodies. Similarly, VHHs can target HSCs in culture (e.g., for extracellular delivery of growth factors or gene editors).

[0157] • Diagnostic and Imaging: Diagnostic imaging of HSCs or HSC-rich tissues is performed using labeled VHHs (e.g., fluorescent or radiolabeled nanobodies). Radiotracers conjugated with anti-CD90 VHHs can image the distribution of bone marrow stem cells or monitor HSC engraftment after transplantation, utilizing the rapid blood clearance of VHHs to obtain high-contrast images. In pathology laboratories, anti-CD90 VHH reagents can be used as specific staining agents to identify HSCs or cancer stem cells in tissue sections.

[0158] Nucleic acid molecules, vectors and cells

[0159] On the other hand, this application provides isolated nucleic acid molecules containing nucleotide sequences that can encode the isolated antigen-binding protein described in this application. For example, it can be produced or synthesized by: (i) in vitro amplification, for example by polymerase chain reaction (PCR); (ii) clonal recombination; (iii) purification, for example by enzyme digestion and gel electrophoresis fractionation; or (iv) synthesis, for example by chemical synthesis.

[0160] On the other hand, this application provides a vector that may contain the isolated nucleic acid molecules described in this application. Furthermore, the vector may also contain other genes, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. Additionally, the vector may contain expression control elements that allow the coding region to be correctly expressed 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 regulating gene transcription or mRNA translation. The vector can be transformed, transduced, or transfected into host cells to express the genetic material elements it carries within the host cells. The vector may include, for example, plasmids, granules, viruses, bacteriophages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. Furthermore, the vector may also include components that facilitate its entry into cells, such as viral particles, liposomes, or protein coats, but not only these substances.

[0161] On the other hand, this application provides a cell that may contain the isolated nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain one or more nucleic acid molecules or vectors described in this application. In some embodiments, each or every host cell may contain multiple (e.g., two or more) or more (e.g., two or more) nucleic acid molecules or vectors described in this application. For example, the vectors described in this application may be introduced into the host cell, such as eukaryotic cells, such as cells from plants, fungi, or yeast cells. In some embodiments, the cell may be a bacterial cell (e.g., *Escherichia coli*), a yeast cell, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, 293T cells, COS-1 cells, SP2 / 0 cells, NSO cells, or myeloma cells. The vectors described in this application may be introduced into the host cell by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0162] Methods for producing antibodies

[0163] On the other hand, this application provides a method for preparing the antibody or its antigen-binding fragment. The method may include culturing the host cells described in this application under conditions that allow the antibody or its antigen-binding fragment to be expressed. For example, this can be achieved by using appropriate culture media, appropriate temperatures, and culture times, methods known to those skilled in the art.

[0164] In some embodiments, the method may be a molecular biology approach. For example, the method includes preparing one or more nucleotide sequences encoding any of the aforementioned antibodies or their antigen-binding fragments, constructing the one or more encoding nucleotide sequences into one or more expression vectors, and expressing the expression vectors in appropriate cells.

[0165] Those skilled in the art will understand that, given a known amino acid sequence of a protein, different coding nucleotide sequences can be used due to codon degeneracy, and the coding nucleotide sequence can also be optimized. Methods for codon optimization of coding sequences are known to those skilled in the art, including adjusting codons to host-preferred codons based on the host type, reducing GC content and / or reducing GC-rich regions, and improving mRNA stability, thereby increasing the expression efficiency of target nucleotides in a specific host.

[0166] Suitable production cells are known to those skilled in the art. In some embodiments, mammalian cells, such as 293T, CHO, or derived cell lines thereof, are used as production cells. In some embodiments, microbial cells, such as bacterial or fungal cells, such as *Escherichia coli* or yeast, are used as production cells. In some embodiments, insect cells, such as Sf9, are used as production cells. The nucleotide sequence encoding the trispecific antibody of the present invention can be codon-optimized for a specific production cell line.

[0167] Preferably, the generated antibody or its antigen-binding fragment is purified. The purification can be performed using conventional methods in the antibody production field, which may include steps such as filtration and chromatography. The filtration step may be selected from one or more of deep filtration, ultrafiltration, percolation, and nanofiltration. The chromatography step may be selected from one or more of affinity chromatography, cation chromatography, anion chromatography, size exclusion chromatography, hydrophobic chromatography, and hydroxyapatite chromatography.

[0168] Any method suitable for producing monoclonal antibodies can be used to produce the antibodies or antigen-binding fragments thereof described in this application. For example, animals can be immunized with linked or naturally occurring CD90 or fragments thereof. Suitable immunization methods can be used, including adjuvants, immunostimulants, repeated booster immunizations, or one or more pathways. In some embodiments, antibodies against CD90 or antigen-binding fragments thereof can be extracted from immunized alpaca peripheral blood lymphocytes, cloned into a vector, and screened and enriched using a phage surface display system.

[0169] Any suitable form of CD90 can serve as an immunogen (antigen) for generating CD90-specific antibodies and screening for the biological activity of said antibodies. For example, the stimulating immunogen can be full-length CD90, including natural homodimers or peptides containing one or more epitopes. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art.

[0170] Pharmaceutical compositions, therapeutic uses and diagnostic kits

[0171] On the other hand, this application also provides pharmaceutical compositions that may comprise the antibody or its antigen-binding fragment described in this application, the isolated nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable carrier.

[0172] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions described in this application include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0173] In some embodiments, the pharmaceutical composition may also contain more than one active compound, typically those with complementary activities that do not adversely affect each other. The type and effective amount of such a drug may depend, for example, on the amount and type of antagonist present in the formulation, and on the clinical parameters of the subject.

[0174] In some embodiments, the pharmaceutical composition may comprise the unseparated expression product described herein. Specifically, the expression product is an intermediate product generated in the aforementioned methods for producing antibodies or antigen-binding fragments thereof, and is a mixed system comprising the antibody or antigen-binding fragment protected herein. In some embodiments, the expression product may be a homogeneous stock solution. The preparation and analysis of the expression product are well known in the art.

[0175] In some embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration and are generally safe and non-toxic.

[0176] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient's body.

[0177] In some embodiments, the pharmaceutical composition further comprises other drugs that are conjugated to the antibody or antigen-binding fragment thereof of this application and targeted to CD90-expressing cells to treat related diseases.

[0178] Specifically, the other drugs may be selected from one or more of the following groups: small molecule targeted anticancer agents, antibody drugs, adoptive cell therapy, oncolytic viruses and oncolytic virus enhancers.

[0179] More specifically, the other drugs may be selected from one or more of the following groups: Toll-like receptor agonists, 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, herpesvirus, poxvirus, paramyxovirus, rhabdovirus, piconemavirus, influenza virus, and parvovirus.

[0180] In some embodiments, the drug may be a carrier encapsulating its contents, such as liposomes, adeno-associated virus (AAV), lipid nanoparticles, vesicles, exosomes, lentiviral vectors, adenoviral vectors, metal nanoparticles, mesoporous silica nanoparticles, erythrocytes, and platelets. The contents may be selected from one or more of the following: RNA drugs (e.g., mRNA, miRNA, circular RNA), DNA drugs (e.g., gene-encoding, CRISPR system), protein drugs (e.g., antibodies), and small molecule drugs.

[0181] The coupling method can be physical coupling, such as coupling through interaction forces; or it can be chemical coupling, such as coupling through covalent bonds, ionic bonds, etc.

[0182] This application relates to a method of treating a subject in need, which may include administering to the subject a prophylacticly effective amount or a therapeutically effective amount of an antibody or antigen-binding fragment thereof and / or composition as described herein. As used herein, "subject" may include any animal exhibiting symptoms of a disease, condition, or disorder that can be treated with the antibodies or antigen-binding fragments thereof, compositions, and methods disclosed herein. Suitable subjects (e.g., patients) may include non-human primates and / or human patients. Non-human primates may be selected from one or more of the following: laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals (e.g., horses or cattle), livestock, and pets (e.g., cats or dogs).

[0183] The term "application" as used in this application refers to introducing the antibody or its antigen-binding fragment, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition described in this application into a subject, or contacting the antibody or its antigen-binding fragment, the isolated nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition with cells and / or tissues. Application can be performed by injection, flushing, inhalation, consumption, electroosmosis, hemodialysis, iontophoresis, and / or other methods known in the art. The route of application may vary depending on the location and nature of the disease being treated. The route of application may include the site of application and the manner of administration. The application site may be selected from one or more of the following: transauricular, transbuccal, transconjunctival, transcutaneous, transdental, intracervical, intraendosinusial, intratracheal, transintestinal, epidural, interstitial, intra-articular, intra-articular, intra-abdominal, intraauricular, intrabiliary, intrabronchial, intramucosal, intracavitary, intracerebral, intracisional, intracorneal, intracoronary artery, intracranial, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepithelial, intraepithelial, intraepithelial, intraepithelial, intraepithelial, intradermal, intraepithelial, intraepithelial, intraesophageal, intragastric, intragingival, intrahepatic, intraileum, intralesional, intralingual. Intraluminal, intralymphatic, intramammary, intramedullary, intradural, intramuscular, intranasal, intraconjunctival, intraocular, intraretinal, intraovarian, intraperitoneal, intrapericardial, intrapleural, intraprostatic, intrapulmonary, intrarumen, intravertebral, intrasynovial, intratendonal, intratesticular, intratracheal, intrasheath, intrathoracic, intracanal, intratumoral, tympanic cavity, intrauterine, intravascular, intraventricular, intrabladder, vestibule, intravenous, vitreous body, larynx, via the nose, nasogastric, via the mouth, via the eye, oropharynx, extra-gastrointestinal, percutaneous, periarticular, epidural, perineurial, periodontal, respiratory, retro-tubular, rectum, spine, subarachnoid, subconjunctival, subcutaneous, subdermal, subgingival, sublingual, submucosal, subretinal, local, percutaneous, intraendocardial, via mucosa, via placenta, via trachea, via tympanic membrane, ureter, urethra, and vagina. The method of application may be selected from one or more of the following: perfusion, irrigation and direct injection.

[0184] The term "treatment" as used herein refers to administering to a subject a therapeutically effective amount of an antibody or its antigen-binding fragment and / or a combination thereof, as described herein, to improve the subject's disease or ailment, or the symptoms thereof. The improvement may be any improvement or cure of the disease or ailment, or the symptoms thereof. The improvement may be an observable or measurable improvement, or a general feeling of well-being in the subject. Therefore, those skilled in the art will recognize that treatment may improve the condition, but may not be a complete cure. "Prophylactic effective amount" refers to the amount of virus, viral stock, or composition that effectively achieves the desired preventative outcome. As used herein, "prevention" may refer to complete prevention of disease symptoms, delay of the onset of disease symptoms, or reduction of the severity of subsequent disease symptoms. Typically, but not necessarily, the preventative effective amount is less than the therapeutic effective amount because the preventative dose may be administered to the subject before or early in the course of illness.

[0185] The antibodies of the present invention can be delivered by conventional methods, preferably by systemic administration, such as by intravenous infusion.

[0186] The antibodies of the present invention can be administered in a therapeutically effective amount. A "therapeutically effective amount" refers to the amount of antibody sufficient to make such treatment effective against a disease, condition, or symptom when administered to a subject to treat a disease, or at least one clinical symptom of a disease or condition. The "therapeuticly effective amount" can vary depending on the antibody, the disease, condition, and / or the symptoms of the disease or condition, the severity of the disease, condition, and / or the symptoms of the disease or condition, the age of the subject to be treated, and / or the weight of the subject to be treated.

[0187] In a preferred embodiment of this application, the dosage of the antibody (mg / kg) is based on the subject's body weight. For example, the single-dose dose of the antibody of the present invention can range from about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably from 5 ng / kg body weight to about 2.5 mcg / kg body weight, and more preferably from 0.1 mcg / kg body weight to about 1 mcg / kg body weight. For example, it can be administered at doses of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mcg / kg body weight.

[0188] One advantage of the antibody of the present invention is its ability to efficiently and completely activate T cells without easily inducing post-activation apoptosis. Furthermore, it can induce memory T cells to provide a sustained immune response, thereby allowing for administration at relatively short intervals. In a preferred embodiment, when administered via intravenous infusion, the interval between two doses of the antibody of the present invention can be no less than 1 day, no less than 3 days, no less than 5 days, or even one week. For example, the antibody of the present invention can be administered 7 times per week, 5 times per week, 3 times per week, twice per week, or once per week. The antibody of the present invention can be administered in cycles of 2-4 weeks, such as 3 weeks, and one or more cycles can be administered.

[0189] On the other hand, this application provides a kit comprising the antibody or antigen-binding fragment thereof described in this application, the isolated nucleic acid molecule described in this application, the vector described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application.

[0190] In some embodiments, the kit is used to detect the CD90 content in the mixed system. In some embodiments, the detection can be qualitative or quantitative. In some instances, the CD90 content can characterize the abundance of hematopoietic stem cells in the mixed system, alone or in conjunction with other indicators.

[0191] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0192] Example

[0193] Example 1. Immunization with antigens from genetically engineered humanized mice

[0194] Using AceMouseNB TM Mouse generates nanobodies. (AceMouseNB) TM This is a genetically engineered humanized mouse model designed to generate fully human heavy-chain-only antibodies (HcAbs). The mouse is created through targeted gene editing, replacing the mouse's heavy-chain variable region with the corresponding human heavy-chain variable region while silencing light-chain expression, thereby enabling the production of human-derived single-domain antibodies containing VHH. This mouse model produces single-domain antibodies with high affinity antigen recognition and low immunogenicity, accelerating the discovery of single-domain antibodies for therapeutic and diagnostic applications.

[0195] Mice were immunized with a CD90 antigen expression plasmid according to a standard immunization procedure. Flow cytometry was used to analyze the serum of mice immunized with the CD90 antigen to evaluate whether specific antibodies were produced in the mice.

[0196] CHO-CD90 or CHO-WT cells were seeded in equal numbers into each well of a 96-well plate. Antigen-immunized mouse serum was diluted sequentially with dilution buffer (DPBS + 3% FBS) at ratios of 1:100, 1:300, 1:900, 1:2700, 1:8100, 1:24300, and 1:72900. 50 μL of each diluted serum sample was added to the corresponding well containing cells, gently mixed, and incubated at 4°C for 45 minutes. After centrifugation, the cells were washed twice with washing buffer (DPBS + 3% FBS), and then resuspended in 50 μL of a 500 ng / 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°C for 30 minutes. Centrifuge again and wash twice with washing buffer. Resuspend the cells in 25 μL of 3% FBS and analyze the samples by flow cytometry to determine the median fluorescence intensity of PE for each cell population. Figure 1 As shown, mice immunized with the CD90 antigen produced a stable and specific immune response. After multiple booster immunizations and a final booster immunization, these mice were suitable for isolating CD90-specific monoclonal B cells.

[0197] Example 2. Isolation and Clonal Analysis of Anti-CD90 Specific Single B Cells

[0198] Spleen cells were sorted using Single-B cell technology. Clones were obtained from four 96-well plates, and antibody genes were subsequently obtained from two plates. The antibody genes were amplified by PCR, cloned into the AceMab expression vector, and transiently transfected to prepare recombinant antibody expression supernatant for cloning verification. Each sample was analyzed by flow cytometry using CHO-CD90, CHO-K1, or HEL cells, according to the method described in Example 1. Figure 2 As shown, positive clones with an MFI value greater than 10,000 for CD90 positive cells and an MFI value less than 5,000 for control cells (CHO-K1) were selected and sequenced using NGS technology.

[0199] Example 3. Antibody purification

[0200] Positive clone antibodies were expressed using the AceMab antibody expression vector, and the cell culture supernatant was collected for antibody purification. In short, 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 Pro-A magnetic beads and incubated at room temperature for 30 minutes to allow antibody binding. After incubation, the magnetic beads were collected with a magnet for 1 minute until the solution became clear, at which point the supernatant was discarded. The magnetic beads were resuspended in 5 times their volume of wash buffer and incubated for 5–10 minutes. The supernatant was collected again for 1 minute until the solution was clear, then the supernatant was discarded and the cells were washed. This washing process was repeated twice to ensure complete removal of non-specific binding proteins. The antibody was eluted 2–3 times with 1 mL of elution buffer (0.05 M citric acid, pH 3.0). Neutralization buffer (1 / 15 of the elution volume) was added, and the antibody concentration was determined by UV spectrophotometry at 280 nm. For desalting and buffer exchange, the eluted antibody was processed using a G25 Sephadex column. Finally, standard SDS-PAGE analysis was performed.

[0201] Example 4. Antibody Characterization

[0202] antigen binding efficiency

[0203] First, using the method described in Example 1, the binding efficiency of monoclonal antibodies was assessed by flow cytometry using human CD90-CHO, monkey CD90-CHO, or HEL cells. Each antibody was initially concentrated at 10 μg / mL, followed by a series of three-fold dilutions to determine antibody affinity and specificity at different concentrations. Data are as follows: Figure 3 As shown.

[0204] Subsequently, the binding efficiency of monoclonal antibodies to mouse CD90 antigen was assessed using ELISA. Mouse CD90 antigen was diluted to 1 μg / mL with PBS as the coating buffer; 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the 96-well plate was washed once with 200 μL PBS; then, 200 μL of blocking buffer was added to each well and incubated at room temperature for 2 hours. After blocking, the antibody was diluted with dilution buffer to the following concentrations: 10000, 3333.3, 1111.1, 370.37, 123.46, 41.15, 13.72, and 4.57 ng / mL; 50 μL of each concentration was added to a 96-well plate and incubated at room temperature for 1 hour. After incubation, the 96-well plate was washed three times with 200 μL of washing buffer (PBS containing 0.05% Tween-20). Add 50 μL (0.4 μg / mL) of HRP-conjugated goat anti-human IgG antibody and incubate at room temperature for 1 hour; discard the solution and wash the 96-well plate 5 times with 200 μL of washing buffer. For detection, add 50 μL of TMB substrate and incubate at room temperature in the dark for 3–5 minutes; add 50 μL of 0.25 M sulfuric acid to each well to stop the reaction, and measure the absorbance at 450 nm. Results (e.g.) Figure 4 This indicates that none of the candidate clones bound to mouse CD90.

[0205] Affinity

[0206] The affinity of the antibody for recombinant human CD90 protein was detected using a biolayer interferometry method. In short, the anti-human Fc probe (Gator, 160003) was hydrated in buffer for at least 10 minutes before use. The antibody was diluted to 5 μg / mL in buffer and added to a 96-well plate sensor (Beyotime, FCP966). The recombinant human CD90 protein antigen was sequentially diluted in buffer to 300, 100, 33.3, 11.1, 3.7, 1.23, and 0.41 nM; a control well without antigen was also prepared as a baseline reference. The antibody-containing sensor was immersed in different concentrations of recombinant human CD90 protein, and the binding period was monitored for 240 seconds; subsequently, the sensor was transferred to fresh buffer for dissociation monitoring for more than 300 seconds. The data are shown in Table 1.

[0207] Based on all results of antigen binding and affinity, H.2346A1, H.2346A5, and H.2346A10 were selected for the expression of CD90 monoclonal antibodies.

[0208] Table 1. Results of affinity assays for candidate antibodies

[0209]

[0210]

[0211] Example 5. Purification and identification of modified VHH antibody

[0212] This example demonstrates the construction of a candidate positive clone antibody in the form of anti-CD90 VHH, and the engineering of the original anti-CD90VHH sequence by introducing a histidine tag, a hinge sequence, and a cysteine ​​(C) residue at its C-terminus. It was cloned into the AceMab antibody expression vector according to standard protocols. The recombinant expression vector was transfected into Expi293 cells for expression, and the purified antibody was obtained by His-tag affinity chromatography. All purified antibodies were identified by standard SDS-PAGE, such as... Figure 5 As shown.

[0213] Example 6. Synthesis of ligand-linker conjugates

[0214] This embodiment demonstrates a method for site-specific conjugation of a modified anti-CD90 VHH antibody to form a ligand-linker conjugate. First, the original anti-CD90 VHH sequence was engineered by introducing a histidine tag, an optional hinge sequence, and a cysteine ​​(C) residue at its C-terminus. The modified VHH sequence was transiently expressed in Expi293 cells and then purified. This sequence modification allows for the insertion of click chemical reactive groups at specific sites on the VHH, thereby enhancing the conjugation potential without affecting the internal structural stability of the VHH. Taking the hinge sequence SPSTPPTPSPSTPP (SEQ ID NO:13) as an example, the specific sequence of the modified VHH is shown in Table 2.

[0215] Table 2. Modified anti-CD90 VHH sequences

[0216]

[0217]

[0218] The structural diagram of the modified ligand, linker, and the ligand-linker coupling formed by their coupling is shown below. Figure 6 As shown. The specific coupling steps include: first, reducing VHH with 2-mercaptoethanol (2-MEA), followed by using Zeba... TMThe reduced VHH was purified using a desalting column (7kM MWCO cutoff). The purified VHH was then coupled with the linker DBCO-PEG4-Maleimide at room temperature for 3 hours. The coupling site was a newly introduced cysteine ​​residue, and the reaction conditions did not disrupt the internal disulfide bond structure of VHH. After the reaction, free linker molecules were removed by ultrafiltration with a molecular weight cutoff of 3kD, and the buffer solution for the coupling product was replaced with 10mM PBS (pH 7.4). The purified ligand-linker coupling product was stored at -80°C. The coupling product was analyzed and identified by size-exclusion chromatography (SEC) and liquid chromatography-mass spectrometry (LC-MS), confirming good purity and the specific inclusion of the DBCO group.

[0219] Example 7: Preparation of Anchored Modified Lipid Nanoparticles

[0220] This embodiment demonstrates a method for preparing anchored modified lipid nanoparticles (LNPs) using polyethylene glycol (PEG) derivatives containing click groups as anchoring fragments. The anchoring fragment can be an amphiphilic polymer containing a click group, a PEG-lipid conjugate containing a click group, or a PEG-hydrophobic polymer conjugate containing a click group. By introducing anchoring fragments containing click reactive groups, these anchored modified LNPs can be coupled to linker-modified ligands, thereby efficiently forming the desired ligand-coupled lipid nanoparticles. The click group refers to a specific group that participates in the click chemical reaction.

[0221] The anchoring fragment containing the click group used in this embodiment is DSPE-PEG-N3, a polyethylene glycol-modified lipid with terminal azide groups that can participate in click chemistry reactions. DSPE-PEG-N3 was incorporated into the lipid mixture used for LNP preparation. The ethanol phase of the lipid components included an ionizable cationic lipid, the neutral lipid DSPC, cholesterol, DSPE-PEG2000, and the click-group-containing lipid DSPE-PEG2000-N3. All lipid materials were purchased from Avanti PolarLipids. Specifically, the lipids were dissolved in ethanol, and RNA was dissolved at a concentration of 0.1 mg / mL in 50 mM sodium citrate buffer (pH 4.0), maintaining a mass ratio of RNA to ionizable cationic lipid of 1:10.

[0222] LNPs were prepared using microfluidic technology. A lipid solution (ethanol phase) was injected into the microfluidic mixer at a flow rate of 1 mL / min, while an aqueous RNA solution was injected simultaneously at a flow rate of 3 mL / min, maintaining a 3:1 volume ratio of aqueous to ethanol. Lipid nanoparticles formed in the mixed solution. Dialysis was then performed to remove the ethanol and replace the buffer with PBS (10 mM, pH 7.4). The dialysis step was performed twice at 4°C using a Slide-A-Lyzer dialysis kit (Thermo Fisher Scientific Inc., molecular weight cutoff 100 KD), with each dialysis lasting 2 hours. The dialyzed LNPs were concentrated by ultracentrifugation.

[0223] Characterization of lipid nanoparticles: The particle size and polydispersity index (PDI) of lipid nanoparticles (LNPs) were determined using a Zetasizer Nano ZS instrument (BeNano, Bettersize). Measurements were performed in PBS and Tris-HCl buffer to confirm their stability in different media. The concentration of RNA in LNPs was determined by UV-Vis spectrophotometry. Absorption spectra were recorded and calculated based on specific extinction coefficients, with particular attention paid to the absorbance at 260 nm and calibration using 330 nm as the baseline. QUANT-IT was also used. TM The RIBOGREEN RNA assay kit (Shanghai Flash Crystal Molecular Biotechnology Co., Ltd.) was used to assess RNA encapsulation efficiency. The ligand-coupled lipid nanoparticle sample was diluted in TE buffer and placed in a polystyrene 96-well plate. After incubation at 40°C for 10 minutes, a 1:200 dilution of RIBOGREEN reagent was added, and fluorescence intensity was measured at approximately 488 nm and 525 nm using a Molecular Devices i3max microplate reader. The percentage of free RNA was determined by comparing the fluorescence intensity of the sample with intact ligand-coupled lipid nanoparticles with that of the sample after Triton X-100 was added to disrupt the particles. The RNA encapsulation efficiency was then calculated.

[0224] Example 8: Preparation of targeted lipid nanoparticles via bioorthogonal click reaction

[0225] This embodiment demonstrates a method for preparing targeted lipid nanoparticles (tLNPs) by conjugating ligands on the surface of lipid nanoparticles (LNPs) via bioorthogonal click chemistry. The simple process involves mixing a VHH-linker conjugate with anchored modified LNPs in solution and then ultrafiltration to achieve ligand conjugation under mild conditions. The specific preparation process is as follows: Figure 7 As shown.

[0226] First, a certain amount of VHH-linker conjugate was added to a pre-prepared anchored modified LNP solution. The two were mixed in a specific ratio and incubated at room temperature for 3 hours to complete the coupling reaction. After incubation, the mixture was subjected to three ultrafiltrations using an ultrafiltration device with a molecular weight cutoff of 100 KD to remove unbound ligands, and the buffer solution was replaced with 20 mM Tris-HCl (pH 7.4). For long-term storage, 40% sucrose solution was added to adjust the final buffer concentration to 20 mM Tris-HCl (pH 7.4), with a sucrose concentration of 8%. Finally, the tLNP was aseptically filtered through a 0.22 μm filter membrane, and the resulting LNP was aliquoted and stored at -80°C for later use.

[0227] Example 9: In vitro evaluation study of cell lines

[0228] This example demonstrates a comparative study of cell lines expressing a specific receptor and those not expressing the receptor to evaluate the delivery efficiency and off-target effects of targeted lipid nanoparticles (tLNPs). HEL cell lines (a human erythroleukemia cell line) and CHO engineered cells overexpressing the CD90 receptor were used as target cells, while unmodified CHO cells were used as non-target cells for comparison. HEL cells were cultured in RPMI-1640 medium containing L-glutamine (ThermoFisher), 10% fetal bovine serum, and 1% penicillin-streptomycin. CHO cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at a density of 200,000 cells / 250 μL per well in 24-well plates, followed by the addition of a specific amount of tLNPs encapsulating GFP or tdTomato nucleic acid to each well. After incubation for 6 hours, the cell pellet was collected by centrifugation at 300g for 7 minutes, and the cells were resuspended in 100 μL of PBS for flow cytometry analysis. The CytoFLEX flow cytometer (Beckman Coulter) was used for analysis, and the flow cytometry data were processed using CytExpert software to determine the percentage of GFP or tdTomato positive cells in different cell lines.

[0229] Example 10: In vitro characterization of tLNPs constructed from different anti-CD90 VHH clones in HSC cells.

[0230] This study prepared tLNPs using three different anti-CD90 VHH clones to evaluate their targeted delivery efficiency on hematopoietic stem cells (HSCs). CD90 is highly expressed on the surface of HSCs, therefore CD90-targeted LNPs have potential applications in HSC-specific delivery. All anti-CD90 VHH sequences used were engineered, with a histidine tag, hinge sequence, and cysteine ​​residue introduced at their C-terminus. The VHHs were transiently expressed in Expi293 cells and purified, followed by the preparation of VHH-linker conjugates according to the method in Example 6.

[0231] In the preparation of tLNP, two optimized formulations, namely formulation C and formulation E, were used. The preparation of anchored modified lipid nanoparticles was carried out according to the steps of Example 7, and the preparation of tLNP was carried out according to the method of Example 8, with different levels of ligand usage. The relevant characteristic parameters (including particle size, PDI, and encapsulation efficiency) of the obtained CD90-targeted LNP are detailed in Table 3.

[0232] Table 3. Different LNP formulations and their physicochemical characterization parameters

[0233]

[0234]

[0235] The targeted delivery efficiency of reporter genes was evaluated using cell line models. CHO cells were used as non-target cells, while CD90-overexpressing CHO cells and HEL cells (human erythroleukemia cells) were used as target cells in the experiments. Figure 8 As shown, under formulations C and E, almost all VHH clones induced strong expression signals in target cells at ligand conjugation ratios ranging from 0.062% to 0.5%, while maintaining low expression in non-target cells. Among them, three VHH clones exhibited the best delivery efficiency within a conjugation ratio range of 0.125% to 0.5%. This result differs from previous studies on CD5 and CD7 targeting tLNPs, which achieved optimal delivery at lower VHH conjugation ratios (0.062% to 0.25%). This difference suggests that the optimal VHH conjugation density may be influenced by target cell type and target receptor characteristics; therefore, fine-tuning the ligand density for different targets is crucial for achieving efficient targeted delivery. Among the three VHH clones tested, clone H.2346A5 exhibited the highest mean fluorescence intensity (MFI) value in both CD90-CHO and HEL cells.

[0236] Example 11. In vitro gene editing of HSC cells using targeted LNPs loaded with CRISPR / Cas12b.

[0237] In this embodiment, the inventors evaluated the gene delivery and editing capabilities of the tLNPs targeting CD90 disclosed in this application. These tLNPs are designed to deliver gene-editing components, encapsulating mRNAs encoding CRISPR-AaCas12bMax and sgRNA that target the HBG1 / 2 promoter. Editing the HBG1 / 2 promoter is expected to induce insertional mutations and reactivate fetal globin expression, potentially serving as a novel approach for treating thalassemia and sickle cell disease.

[0238] AaCas12bMax is a highly active variant of Cas12b (AaCas12b) derived from *A. acidophilus* and can be used as a gene-editing enzyme. The amino acid sequence of AaCas12bMax is shown in Table 4. The mRNA encoding AaCas12bMax is synthesized via in vitro transcription (IVT) as follows: The coding sequence of AaCas12bMax is inserted into a plasmid to form a DNA template containing the target protein sequence, 5' and 3' UTR sequences, and a T7 promoter upstream of the 5' UTR; the PCR product of the plasmid containing a poly A tail is used for in vitro transcription. T7 RNA polymerase recognizes the T7 promoter of the DNA template and initiates in vitro mRNA transcription; the 5' cap structure of the in vitro transcribed mRNA is added during in vitro transcription, and n1-methyl-pseudouracil is used instead of uracil. All in vitro transcription reactions are performed at 37°C for 2 hours. DNase I is used to remove the DNA template at 37°C for 30 minutes, followed by column purification to obtain the full-length in vitro transcribed mRNA.

[0239] The sgRNA was designed to target the LRF binding motif 200 bp upstream of the HBG1 / HBG2 gene promoter. The sgRNA was optimized through chemical modification, extending the targeting sequence to 23 nt. The optimized sgRNA was synthesized by Genewiz and dissolved in water to 100 μM.

[0240] To achieve precise gene editing in HSC cells, CD90-targeting LNPs encapsulating CRISPR-AaCas12bMax mRNA and sgRNA were prepared. First, according to Example 6, a ligand-linker conjugate was prepared using a modified anti-CD90 VHH antibody (Clone H.2346A5). Then, as described in Example 7, anchor-modified LNPs were prepared. Finally, tLNPs encapsulating CRISPR mRNA and sgRNA were prepared according to Example 8, maintaining a 1:1 weight ratio of AaCas12bMax mRNA to sgRNA. The final LNP concentration was determined based on the total RNA concentration in the formulation. Detailed composition and physicochemical properties of the CD90-targeting LNPs are shown in Table 5.

[0241] HEL cells were exposed to LNP-922 / 923 (1-4 μg / mL) at a concentration of 4E+05 / mL for 24 hours. HEL cells were then cultured for another 48 hours, and genomic DNA was extracted. A library was constructed by amplifying the region surrounding the target sgRNA binding site, and sequencing was performed using NGS. The NGS results were analyzed using Cas-Analyzer (www.rgenome.net / cas-analyzer). Figure 9 Data show that both LNP-922 and LNP-923 exhibit high editing efficiency and are dose-dependent, indicating their great potential for in vivo editing.

[0242] Table 4. AaCas12bMax and sgRNA sequences

[0243]

[0244]

[0245]

[0246] Table 5. VHH antibodies and formulations used for different HSC-targeting LNPs

[0247]

[0248] Table 6. CDR sequences of different clones

[0249] clone CDR1(31–35) CDR2(50–65) CDR3(95–102) H.2346A1 VASGFIFSTFGMH AVIWYDGSKKFYADSVKG ARDSITGLTADPW H.2346A5 AASGFTLSNYAM STISDSGDNTYYADSVKG AKERVWNDYMDVRGKG H.2346A10 AASGFSFSSLGMN SYISNSSSIIKYADSVKG ARDDFWSGDYTGPDPW

Claims

1. A single-domain antibody that specifically binds to human CD90, said single-domain antibody comprising a heavy chain variable region (VH), said heavy chain variable region comprising heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3), wherein said HCDR1, HCDR2, and HCDR3 are: The HCDR1 is the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 is the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 is the amino acid sequence shown in SEQ ID NO:

8.

2. The single-domain antibody according to claim 1, wherein the VH is an amino acid sequence as shown in SEQ ID NO: 5, or an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:

5.

3. The single-domain antibody according to claim 1, wherein it is a VH that specifically binds to human CD90.

4. An isolated nucleic acid molecule comprising encoding a single-domain antibody according to any one of claims 1-3.

5. A carrier comprising the isolated nucleic acid molecule of claim 4.

6. A cell comprising the isolated nucleic acid molecule of claim 4 or the vector of claim 5.

7. A pharmaceutical composition comprising a single-domain antibody according to any one of claims 1-3, an isolated nucleic acid molecule according to claim 4, a vector according to claim 5 and / or a cell according to claim 6, and optionally a pharmaceutically acceptable carrier.

8. A reagent or kit for detecting CD90 in a sample, comprising the single-domain antibody of any one of claims 1-3, the isolated nucleic acid molecule of claim 4, the vector of claim 5, the cell of claim 6, and / or the pharmaceutical composition of claim 7.

9. A method for detecting CD90 in a sample for non-diagnostic purposes, the method comprising using a single-domain antibody according to any one of claims 1-3, an isolated nucleic acid molecule according to claim 4, a vector according to claim 5, a cell according to claim 6, and / or a pharmaceutical composition according to claim 7.

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