Anti-CD6 nano antibody and application thereof

Abstract

The invention relates to an anti-CD6 nano antibody and application thereof. The invention provides a CD6 binding molecule which comprises an anti-CD6 nano antibody or an antigen binding fragment thereof, and a complementary determining region CDR of the anti-CD6 nano antibody comprises CDR1, CDR2 and CDR3. The invention also provides targeted delivery drugs (such as antibody drug conjugates ADC, gene vectors) containing the CD6 binding molecules and expression cells thereof. The antibodies and cells described herein have good safety and therapeutic effects of targeting CD6.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals and immunotherapy, specifically relating to anti-CD6 nanobodies and their use in the preparation of CD6-targeted delivery drugs (such as ADCs and gene vectors), CD6-targeted chimeric antigen receptors (CARs), and in the treatment of CD6-positive diseases (such as T-cell leukemia and T-cell lymphoma). Background Technology

[0002] Cancer is one of the leading causes of death worldwide. Traditional chemotherapy and radiotherapy for cancer treatment can damage normal tissues and have limited effectiveness, while surgical resection does not cover all cancers. In recent years, targeted therapies at the cellular and molecular level have emerged. These therapies are designed to target specific points in the body. Once in the body, the drugs specifically bind to these targets, causing tumor cells to die without damaging the surrounding normal tissue cells.

[0003] CD6 is a member of the immunoglobulin superfamily, a type I transmembrane glycoprotein, mainly expressed in mature T cells, thymocytes, and some activated immune cells. Its natural ligand is CD166, and the binding of the two can regulate T cell activation, proliferation, and migration, maintaining normal immune function. Under pathological conditions, CD6 is abnormally highly expressed in T-cell malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL), peripheral T-cell lymphoma (PTCL), and adult T-cell leukemia / lymphoma (ATLL), and its expression level is closely related to tumor invasiveness and poor patient prognosis. Meanwhile, CD6 is limited to immune cells in normal tissues and is not widely expressed in solid organs, making it an ideal target for tumor targeted therapy.

[0004] In the field of targeted therapy, antibody-drug conjugates (ADCs) and chimeric antigen receptor T-cell (CAR-T) therapy are two core technologies. ADCs consist of a targeting antibody, a cytotoxic drug, and a linker. By specifically binding to tumor surface antigens, the antibody precisely delivers the cytotoxic drug to tumor cells, achieving targeted killing and effectively reducing the systemic toxicity of traditional chemotherapy. Currently, ADCs have achieved breakthroughs in the treatment of various solid tumors and hematological malignancies, such as CD22-targeted ADCs used to treat B-cell leukemia. However, research on CD6-targeted ADCs is still in its infancy—existing ADCs mostly use single-chain antibodies (scFv) or intact monoclonal antibodies, which have problems such as complex structures, poor tissue penetration, and high immunogenicity, making it difficult to meet the treatment needs of CD6-positive T-cell tumors.

[0005] CAR-T cells are created by embedding the variable single-chain region (scFv) of an antibody or a nanobody (VHH) onto a T cell surface receptor. They primarily consist of three parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain, often a single-chain antibody (scFv) or nanobody (VHH), is responsible for recognizing and binding to the target antigen. The transmembrane domain is a hinge or spacer that anchors the scFv or VHH to the cell membrane. The intracellular signaling domain contains the CD3 signaling domain. When the antigen is recognized and bound, a stimulating signal is generated and transmitted to the intracellular signaling domain, activating the T cell and enabling it to perform effector functions.

[0006] First-generation CARs lacked co-stimulatory domains, resulting in limited efficacy due to insufficient signal strength and durability. Therefore, second-generation and subsequent CAR designs incorporated one or more co-stimulatory domains to enhance and maintain T-cell activation signals. Due to their modular structure, CARs can be constructed targeting various tumor antigens, providing an easily adaptable platform for treating multiple types of cancer. For example, CAR-T cells targeting the CD19 antigen expressed on B cells were the first FDA-approved gene therapy product, achieving a complete response rate of up to 90% in patients with relapsed or refractory B-cell malignancies. However, despite the promising results of adoptive T-cell therapy in treating hematologic malignancies, it has yielded limited success in treating the vast majority of cancers—solid tumors. Solid tumors present unique challenges to CAR-T cell therapy compared to hematologic malignancies. First, the high antigenic heterogeneity in solid tumors provides them with an effective mechanism to escape CAR-T cells, as CAR-T cells typically encode specificity for a single antigen target, thus failing to recognize all cancer cells in the tumor. However, expanding the specificity of T cells to multiple antigens increases the risk of extra-target cytotoxicity. Secondly, solid tumors are typically surrounded by physical barriers, such as a collagen-rich matrix, which effectively prevents T cell infiltration. In addition to physical barriers, T cells must also contend with a highly immunosuppressive tumor microenvironment (TME) characterized by cellular, molecular, and metabolic features, ultimately leading to T cell exhaustion and dysfunction. To date, CAR-T cells are insufficient to overcome these additional barriers posed by solid tumors.

[0007] Nanobodies (VHHs), as a preferred material for targeting domains in CAR-T cell therapy, are providing a new direction for overcoming the bottlenecks of traditional CAR-T technology. Traditional CARs often use single-chain antibodies (scFvs) as their targeting domains. However, scFvs are prone to aggregation due to poor structural folding, which may trigger non-specific activation or toxicity of CAR-T cells. They also exhibit weak stability, are easily degraded in vivo, and may induce anti-CAR immune responses due to murine sequences, affecting efficacy and safety. In contrast, alpaca nanobodies, as the smallest antigen-binding unit (15kDa), have a compact structure. When used to construct CARs, they can reduce inter-chain aggregation, thus lowering the risk of off-target toxicity. Their high stability (tolerant to pH 2-12, 60-80℃) ensures that CARs maintain their targeting function continuously in the complex in vivo environment, prolonging the anti-tumor activity window of CAR-T cells. Furthermore, they share over 80% homology with human antibodies and have low immunogenicity, reducing the production of anti-CAR antibodies in patients and improving safety. More importantly, its superior tissue penetration performance helps CAR-T break through the barrier of solid tumors. Compared with scFv, it penetrates the tumor stroma more efficiently, making it easier for CAR-T to reach deep tumor cells and solving the pain point of traditional CAR-T's "difficulty in reaching" solid tumors. Currently, CAR-T based on this antibody has shown potential in research on targets such as CD19 and BCMA. For example, the nanobody CAR-T targeting BCMA showed stronger targeting binding ability and a lower incidence of cytokine release syndrome in pretreated patients.

[0008] Alpaca-derived nanobodies exhibit unique advantages in the CAR-T field. They can produce VHH antibodies with extremely high specificity and affinity, which can not only recognize antigenic epitopes that are difficult for traditional scFv to access (such as further improving the recognition specificity for CD6 antigen), but also have high homology between the alpaca VHH gene and the human VH gene, resulting in relatively low immunogenicity, which is conducive to subsequent humanization and effectively reduces the risk of immune rejection in human applications. Currently, targeted therapies targeting CD6 still have significant shortcomings: firstly, there is a lack of high-affinity, functional anti-CD6 nanobodies that can be used in ADCs, making precise delivery of cytotoxic drugs impossible; secondly, existing CD6-related CAR-T therapies mostly use traditional scFv as the target domain, which has safety and efficacy defects. Therefore, developing ADC and CAR-T drugs based on anti-CD6 nanobodies is of great significance for filling the gap in the treatment of CD6-positive T-cell tumors and improving the efficacy of targeted therapy, and is also a key issue that urgently needs to be addressed in this field. Summary of the Invention

[0009] The present invention provides a CD6 binding molecule comprising an anti-CD6 nanobody or an antigen-binding fragment thereof, wherein the complementarity-determining region (CDR) of the anti-CD6 nanobody comprises CDR1, CDR2 and CDR3, wherein CDR1 is the sequence shown in SEQ ID NO:1-6, CDR2 is the sequence shown in SEQ ID NO:7-12 and CDR3 is the sequence shown in SEQ ID NO:13-18.

[0010] In one or more embodiments, the heavy chain variable region sequence of the anti-CD6 nanobody is shown in SEQ ID NO:19-24.

[0011] In one or more embodiments, the CD6 binding molecule is a monovalent or multivalent nanobody or single-domain antibody, or a multispecific nanobody or single-domain antibody, comprising one, two or more anti-CD6 nanobodies or their antigen-binding fragments, and capable of specifically binding to CD6 molecules.

[0012] In one or more embodiments, the multivalent or multispecific binding molecule is linked to multiple anti-CD6 nanobodies or their antigen-binding fragments via a linker. The linker consists of 1-15 amino acids selected from G and S.

[0013] In one or more embodiments, the nanobody is derived from alpaca heavy chain antibody.

[0014] In one or more embodiments, the CD6-binding molecule described in any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.

[0015] Another aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an optional signal peptide sequence, a CD6 binding molecule as described in any embodiment herein, a hinge region, a transmembrane region, and an intracellular region.

[0016] In one or more embodiments, the intracellular region includes an intracellular co-stimulatory domain (such as 4-1BB, CD28) and / or an intracellular signaling domain (such as CD3ζ).

[0017] In one or more embodiments, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, a CD6-binding molecule as described in any of the embodiments herein, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0018] The present invention also provides a nucleic acid molecule having a sequence selected from any of the following: (1) the coding sequence of a CD6-binding molecule described in any embodiment herein, a nanobody portion comprising a targeted drug delivery molecule, or a chimeric antigen receptor; (2) the complementary sequence of (1); (3) a 5-50 bp fragment of any of (1) or (2).

[0019] In one or more embodiments, the fragment is a primer.

[0020] The present invention also provides a nucleic acid construct comprising the nucleic acid molecules described herein.

[0021] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector (such as a vector for expressing nanobodies, targeted drug delivery, or CAR), or an integration vector.

[0022] The present invention also provides a host cell selected from: (1) expressing and / or secreting the CD6-binding molecule or chimeric antigen receptor as described in any embodiment herein; (2) containing the nucleic acid molecule described herein; and / or (3) containing the nucleic acid construct described herein.

[0023] In one or more embodiments, the host cell is an immune effector cell, preferably a T cell (such as a CAR-T cell).

[0024] The present invention also provides a method for generating a CD6-binding molecule according to any embodiment herein, comprising: culturing the host cells described herein under conditions suitable for generating CD6-binding molecules (e.g., nanobodies or antigen-binding fragments thereof, monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies), and optionally purifying the CD6-binding molecule from the culture; optionally, further comprising the step of conjugating the CD6-binding molecule to a functional payload to prepare a targeted drug delivery.

[0025] The present invention also provides a targeted delivery drug comprising a CD6 binding molecule as described in any embodiment herein, and a functional payload conjugated to the binding molecule, wherein the functional payload is a therapeutic agent or a carrier; preferably, the targeted delivery drug is an antibody-drug conjugate (ADC) or a gene vector.

[0026] In one or more embodiments, when it is an antibody-drug conjugate (ADC), it further comprises a linker through which the CD6-binding molecule is conjugated to a therapeutic agent; the therapeutic agent is selected from cytotoxic drugs, chemotherapeutic drugs, toxins, or radioisotopes; the linker is a cleavable linker (such as an acid-sensitive linker or an enzyme-cleavable linker) or a non-cleavable linker.

[0027] In one or more embodiments, when it is a gene vector, the vector carries a therapeutic nucleic acid, and the CD6 binding molecule is modified on the surface of the vector; the vector is selected from viral vectors (such as lentiviral vectors, adeno-associated virus vectors), liposomes, nanoparticles, or polymer vectors; the therapeutic nucleic acid is selected from siRNA, mRNA, CRISPR-Cas9 system components, or therapeutic genes.

[0028] The present invention also provides a pharmaceutical composition comprising a CD6-binding molecule, a targeted delivery drug, a chimeric antigen receptor, a nucleic acid molecule, a nucleic acid construct or a host cell, and pharmaceutically acceptable excipients as described in any embodiment herein.

[0029] In one or more embodiments, the pharmaceutical composition is used to treat CD6 expression-related diseases or conditions, particularly CD6-positive diseases (such as T-cell acute lymphoblastic leukemia, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma).

[0030] The present invention also provides the use of the CD6 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of activated immune cells (e.g. T cells).

[0031] The present invention also provides the use of the CD6 binding molecule, chimeric antigen receptor, targeted delivery drug, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of a drug for the prevention or treatment of CD6 expression-related diseases or conditions (particularly CD6-positive diseases such as T-cell acute lymphoblastic leukemia, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma).

[0032] The present invention has the following beneficial effects: The present invention provides a novel nanobody that specifically recognizes CD6, a targeted delivery drug containing the antibody, CAR and CAR-modified cells, etc. The antibody, drug and cells have good safety and CD6-targeting therapeutic effects, providing a new treatment or improvement approach for diseases related to CD6 expression (especially CD6-positive diseases). Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is a chromatogram of the CD6 antibody internalization assay.

[0035] Figure 2 This is an in vitro specificity map of CD6 antibody. Detailed Implementation

[0036] This invention utilizes CD6 protein to immunize alpacas, obtaining a high-quality single-domain antibody gene library. Then, phage display technology is used to screen the antibody gene library, thereby obtaining CD6-specific single-domain antibody genes. These genes are then transformed into mammalian cells, resulting in antibody strains that can be efficiently expressed in mammalian cells and exhibit high specificity. High-affinity, high-specificity, and high-functional activity nanobodies are then identified using ELISA, molecular interaction analysis, and blocking assays. The antibodies or their antigen-binding fragments possess good safety and targeting properties, specifically binding to the extracellular domain of human CD6.

[0037] The present invention also provides a chimeric antigen receptor (CAR) containing the nanobody. Using a vector containing the coding sequence of the CAR to infect immune cells can yield immune effector cells with significant killing ability against CD6-overexpressing tumor cells. These immune effector cells can be used to treat or improve CD6-expression-related diseases, thus laying the foundation for the treatment of CD6-positive tumors.

[0038] Antibody

[0039] In this article, "CD6-binding molecule" refers to a protein that specifically binds to CD6, including but not limited to antibodies, heavy chain antibodies, nanobodies, or their antigen-binding fragments.

[0040] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, and Fv. In this document, "antibody" and "immunoglobulin" are used interchangeably.

[0041] Traditional "antibodies" contain a basic four-chain antibody unit, a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. Pairs of VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, 8th Edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on their constant domain amino acid sequences. Based on relatively minor differences in CH sequence and function, the γ and α types can be further subdivided into subclasses, such as those expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0042] The "heavy chain antibody" described in this article refers to antibodies derived from camelid or cartilaginous fish. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is linked to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgGFc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgGFc.

[0043] As used herein, the terms "single-domain antibody," "anti-CD6 single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," and "VHH" are used interchangeably and all refer to single-domain antibodies that specifically recognize and bind to CD6. A single-domain antibody is the variable region of a heavy chain antibody. Typically, a single-domain antibody contains three CDRs and four FRs. Preferably, the single-domain antibody of the present invention has CDR1 shown in any one of SEQ ID NO:1-7, CDR2 shown in any one of SEQ ID NO:8-13, and CDR3 shown in any one of SEQ ID NO:14-20. A single-domain antibody is the smallest functional antigen-binding fragment. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0044] In this article, "nanobody" refers to an antibody containing the VHH described herein. It can be a heavy chain antibody as described above, or a multivalent or multispecific antibody containing multiple VHHs, or a recombinant antibody obtained by recombination of VHH and antibody Fc (e.g., CH2 and CH3 or CH2, CH3 and CH4).

[0045] A binding molecule containing two or more single-domain antibodies is a multivalent single-domain antibody; a binding molecule containing two or more single-domain antibodies with different specificities is a multispecific single-domain antibody. Multivalent or multispecific single-domain antibodies are linked together by a linker. The linker typically consists of 1-15 amino acids selected from G and S.

[0046] In this article, heavy chain antibodies and antibodies (traditional four-chain antibodies) are used to distinguish different combinations of antibodies. Due to their structural similarities, the structural descriptions of antibodies below, except for those involving light chains, also apply to heavy chain antibodies.

[0047] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0048] The term "variable" refers to the wide variation in certain segments within a variable domain within an antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain (simply referred to as CDR1, CDR2, and CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity. Several variable region labeling schemes exist for antibodies, including Chothia, Kabat, IMGT, and Contact. This article uses the IMGT labeling scheme as an example.

[0049] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is based on the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0050] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments; disulfide-linked Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased by chemical modification or by incorporation into liposomes. Antigen-binding fragments can be prepared using a variety of techniques, including but not limited to hydrolyzing and digesting complete antibody proteins, and by expression in host cells containing the antigen-binding fragment.

[0051] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure. For heavy chain antibodies or nanobodies, scFv is VHH.

[0052] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

[0053] Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

[0054] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by a human-derived polynucleotide or is identical to that antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In this invention, antibodies, single-domain antibodies, heavy-chain antibodies, etc., all include humanized variants of the aforementioned antibodies.

[0055] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0056] In some embodiments, the present invention also provides nanobodies, heavy chain antibodies, antibodies, or antigen-binding fragments thereof (e.g., single-domain antibody VHH) that bind to the same epitope on human CD6 as the antigen-binding region of any anti-CD6 nanobody of the present invention, i.e., nanobodies, heavy chain antibodies, antibodies, or antigen-binding fragments thereof that can cross-compete with the antigen-binding region of any nanobody of the present invention for binding to CD6.

[0057] In this invention, the anti-CD6 single-domain antibody has CDR1 shown in SEQ ID NO:1-12, CDR2 shown in SEQ ID NO:13-24, and CDR3 shown in SEQ ID NO:25-36.

[0058] The CD6-binding molecules described herein can be monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies, comprising one, two, or more of the anti-CD6 nanobodies or single-domain antibodies described herein. Multispecificity can be against CD6 and another antigen, or against two different epitopes of CD6.

[0059] The variants of the antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention. In some embodiments, the sequences of the variants described herein may have at least 95%, 96%, 97%, 98%, or 99% homology with their source sequences. The sequence homology described herein can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

[0060] The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques. The nanobodies of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or nanobodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the present invention, followed by culturing the host cells and purifying the antibodies. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2).

[0061] CAR

[0062] This invention also provides a chimeric antigen receptor (CAR) targeting CD6. The CAR contains an optional signal peptide sequence, an antigen recognition region (i.e., the anti-CD6 binding molecule described herein), a hinge region, a transmembrane region, and an intracellular region. The intracellular region includes one or more intracellular co-stimulatory domains and / or one or more intracellular signaling domains. The terms "hinge region," "transmembrane region," and "intracellular region" as used herein can all be selected from sequences of the hinge region, transmembrane region, and intracellular region in known CAR-T technologies.

[0063] The signal peptide, optionally selected for the CAR, can be chosen as needed. Generally, a signal peptide is a peptide sequence that directs the polypeptide to a desired site within the cell. The signal peptide directs the polypeptide to the cell's secretory pathway and allows the polypeptide to integrate and anchor to the lipid bilayer; the signal peptide can also be a membrane-localizing signal peptide. Exemplary signal peptides include CD8 signal peptide, CD28 signal peptide, CD4 signal peptide, or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art. The CD8 signal peptide suitable for use in this invention can be any of the various human CD8 signal peptide sequences commonly used in CARs in the art. In some embodiments, the amino acid sequence of said human CD8 signal peptide comprises the sequence shown in SEQ ID NO:10.

[0064] The hinge region of a chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region. The hinge region is an amino acid segment that typically exists between two domains of a protein and allows for protein flexibility and relative movement between the two domains. The hinge region can be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as IgG, IgA, IgM, IgE, or IgD antibodies) can also be used in the chimeric antigen receptor described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptor described herein. Exemplarily, the hinge region of a CAR is selected from the CD8α hinge region, the IgD hinge region, the IgG1 Fc CH2CH3 hinge region, or the IgG4 Fc CH2 CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art. The CD8α hinge region suitable for use in this invention can be any of the various human CD8α hinge region sequences commonly used in CARs in the art. In some embodiments, the human CD8α hinge region comprises the sequence shown in SEQ ID NO:11.

[0065] The transmembrane region of a chimeric antibody receptor can form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of translocating the cellular phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. It can be selected from the transmembrane regions of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the α, β, or ζ chains of T-cell receptors. The human CD8α transmembrane region suitable for this invention can be any of the various human CD8α transmembrane region sequences commonly used in the art for CARs. In some embodiments, the amino acid sequence of the human CD8α transmembrane region comprises the sequence shown in SEQ ID NO:12.

[0066] In addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. The "co-stimulatory domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding chaperone on immune cells (such as T cells) that specifically binds to a co-stimulatory ligand, thereby enabling the immune cell to mediate a co-stimulatory response, such as, but not limited to, proliferation and survival. Suitable intracellular co-stimulatory domains can be selected as needed, including intracellular domains containing co-stimulatory signaling molecules, such as at least one of the intracellular domains derived from 4-1BB, CD2, CD7, CD27, CD28, CD6, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 4-1BBL. In some embodiments, the amino acid sequence of the 4-1BB co-stimulatory domain comprises the sequence shown in SEQ ID NO:13.

[0067] Intracellular signaling regions (or intracellular signal transduction regions) are responsible for activating at least one normal effector function of immune effector cells expressing chimeric antigen receptors. For example, the effector function of T cells can be lytic activity or helper activity, including cytokine secretion. While the entire intracellular signal transduction region can generally be used, in many cases, using the whole chain is unnecessary. Regarding the use of truncated portions of intracellular signal transduction regions, such truncated portions can be used instead of the whole chain as long as they transduce effector function signals. Therefore, intracellular signal transduction regions include any truncated form of intracellular signal transduction regions sufficient to transduce effector function signals. The intracellular signaling domain of a CAR can be selected as needed, including but not limited to intracellular signaling domains derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling region is derived from the human CD3ζ intracellular signaling region. Furthermore, the intracellular signaling region of the human CD3zeta has the amino acid sequence shown in SEQ ID NO:14.

[0068] The aforementioned portions forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, anti-CD6 nanobody, CD8 hinge region, CD28 transmembrane region, 4-1BBz co-stimulatory domain, and CD3ζ intracellular signaling domain, can be directly linked to each other or linked via adapter sequences. The adapter sequence can be a known antibody-compatible adapter sequence, such as a G and S-containing adapter sequence. Typically, the adapter contains one or more repeating motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the adapter sequence, with no inserted amino acid residues between the repeats. The adapter sequence can consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the adapter can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the adapter sequence is a polyglycine adapter sequence. The number of glycine residues in the linker sequence is not particularly limited, typically ranging from 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. Besides glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). In some embodiments, the linker sequence is (GGGGS)n-linked, where n is an integer from 1 to 5.

[0069] In an exemplary embodiment, the CAR contains, from the N-terminus to the C-terminus, a CD8 signal peptide, the anti-CD6 nanobody described herein or its antigen-binding fragment, a CD8α hinge region, a CD8α transmembrane region, a CD3ζ intracellular signaling domain, and a 4-1BB co-stimulatory domain. In a specific embodiment, an exemplary CAR having the above structure is shown in any of SEQ ID NO:15-16.

[0070] Nucleic acid

[0071] This invention also provides polynucleotides encoding the aforementioned antibodies or CARs. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0072] Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0073] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, a long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein. The CAR sequence can also be obtained as described above. Alternatively, the sequences of the various parts of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) can be obtained as described above and then ligated to obtain the full-length CAR.

[0074] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, DNA sequences encoding the proteins of this invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of this invention through chemical synthesis. The CAR portions can be sequentially cloned into a vector or integrated into a full-length CAR before cloning.

[0075] This invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotide sequences described herein can be manipulated in various ways to ensure the expression of the antibody or CAR. The nucleic acid constructs can be manipulated depending on the expression vector or requirements before insertion into a vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0076] In some embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of the polynucleotide sequences of the present invention is typically achieved by operably linking the polynucleotide sequences of the present invention to an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components for integrating the target sequence into the cellular genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as “flanking sequences” in some embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements.

[0077] Furthermore, the type of vector is not limited; for example, plasmids, phage particles, phage derivatives, animal viruses, and entrapments can be modified depending on the host cell to be introduced. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0078] To assess the expression of CAR peptides or portions thereof, the vector introduced into cells may also contain one or both of an optional marker gene or reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector.

[0079] cell

[0080] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, preferably immune effector cells. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS7, and 293 cells.

[0081] "Immune effector cells" are immune cells capable of performing immune effector functions. In some embodiments, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells mediating ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, immune effector cells are selected from at least one of: immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMCs), and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (same as T cells). In some embodiments, T cells may be CD4+ cells. + / CD8 - CD4 - / CD8 + CD4 + / CD8 + CD4 - / CD8 - Or a combination thereof. In some embodiments, T cells produce IL-2, IFN, and / or TNF when expressing a chimeric antigen receptor and binding to target cells. In some embodiments, CD8... + T cells lyse antigen-specific target cells when they express chimeric antigen receptors and bind to them.

[0082] The T cells suitable for use in this invention can be of various types and origins. For example, T cells can be derived from PBMCs of patients with B-cell malignancies. In some embodiments, after obtaining T cells, they can be first stimulated and activated with an appropriate amount (e.g., 30–80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in IL2 medium containing an appropriate amount (e.g., 30–80 IU / ml, such as 50 IU / ml) for later use.

[0083] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into cells by physical, chemical, or biological methods. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used. In some embodiments, transduced or transfected immune effector cells proliferate in vitro after the introduction of nucleic acids or vectors.

[0084] The obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0085] The peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0086] Uses and methods

[0087] By constructing a nanobody library, the inventors screened nanobodies and their variants that could bind to CD6. The binding ability of these antibodies to the antigen was verified through protein-level binding assays, affinity assays, competitive blocking experiments, and tissue cross-reactivity tests. Using these nanobodies, the inventors constructed CARs and CAR-T cells, and molecular and cellular level experiments verified that the CAR-T cells could be prepared and detected normally.

[0088] All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, which are diseases or conditions related to CD6 expression, referring to diseases directly or indirectly caused by abnormal CD6 expression, usually referring to diseases caused by CD6 overexpression.

[0089] This invention also includes a class of cell therapies comprising expressing the CAR described herein in immune cells (e.g., T cells) and administering a therapeutically effective amount of the cells to a recipient who requires them, the cells being capable of killing the recipient's tumor cells. Compared to antibody therapies, CAR-T cells can replicate in vivo, producing long-lasting durability that can lead to sustained tumor control. The anti-tumor immune response induced by CAR-T cells can be an active or passive immune response. Additionally, CAR-mediated immune responses can be part of an adoptive immunotherapy step, wherein CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.

[0090] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0091] Example 1: Construction and eukaryotic expression of recombinant human CD6 protein expression vector.

[0092] 1. Synthesis of the gene sequence of the CD6 amino acid range from position 29 to 463 and construction of the protein expression vector.

[0093] The extracellular amino acid sequence of CD6 was imported into an online codon optimization tool (http: / / www.jcat.de / #opennewwindow) to obtain the codon-optimized nucleic acid sequence. The gene sequence was then obtained through chemical synthesis, and the coding sequence for alpaca IgG1-Fc was added to the 3' end of this gene sequence. The spliced ​​product was cloned into pcDNA3.1 (Thermo) using the TaKaRa seamless cloning kit to obtain the expression vector.

[0094] 2. Expression, purification and activity identification of recombinant human CD6 protein.

[0095] Five days after transfecting 293T cells (ATCC) with the obtained expression vector, the culture supernatant was collected, and the recombinant human CD6-alFc protein was purified using AKTA explorer100 (GE). Due to glycosylation modifications and other reasons, the recombinant human CD6-alFc protein, after reducing SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, showed a size of approximately 75 kilodaltons.

[0096] Dilute CD6 antibody with appropriate buffer and add 100 μL to each well of the microplate. Incubate overnight at 4°C or 2-3 hours at 37°C to achieve coating. Discard the liquid in the wells and blot dry. Add 200 μL of blocking buffer to each well and incubate at room temperature for 1-2 hours to block non-specific sites. Discard the liquid and blot dry. Dilute the standard and sample appropriately with buffer and add 100 μL to each well. Incubate at 37°C for 1-2 hours to allow CD6 protein to bind to CD6 antibody. Discard the liquid and wash each well thoroughly with washing buffer to remove unbound material. Add 100 μL of diluted enzyme-labeled detection antibody to each well and incubate at 37°C for 1 hour. After washing each well, add 100 μL of freshly prepared TMB substrate working solution to each well and incubate at room temperature in the dark for 15-30 minutes. When the color development is appropriate, add 100 μL of stop solution to each well to terminate the reaction. Finally, use a microplate reader to detect the absorbance (OD) value of each well.

[0097] Example 2: Preparation of anti-human CD6 alpaca VHH antibody

[0098] 1. Immunize alpacas

[0099] 2 mg / m³ LCD6-alIgG1Fc fusion protein was used as the antigen and emulsified with an equal volume of Sigma-Aldrich complete adjuvant. Adult alpacas were subcutaneously immunized with 500 μg of antigen per alpaca. After the initial immunization, booster immunizations were administered every twenty days for a total of four subcutaneous immunizations. Seven days after the fourth immunization, 100 mL of whole blood was collected intravenously, and PBMCs were isolated.

[0100] 2. Serum titer detection

[0101] Before each booster immunization, 10 mL of blood was drawn intravenously, centrifuged to remove cells, and serum was retained. 50 ng / well of CD6-his protein (ACROBiosystems) was added to each well of an ELISA microplate and incubated overnight at 4°C. The plates were washed three times with PBS, and 200 μL / well of 1% BSA / PBS was added, with blocking at 37°C for 1 hour. Serially diluted alpaca serum was added, and binding was incubated at 37°C for 1 hour. The plates were washed three times with PBST, and 100 μL of 1:5000 diluted HRP-goat anti-alpaca IgG1-Fc (Jackson Immuno Research) was added, with binding incubated at 37°C for 1 hour. The plates were washed three times with PBST, and 100 μL / well of TMB chromogenic buffer was added, with incubation at 37°C for 10 minutes. 100 μL / well of ELISA stop solution was added, and the OD450 value was read using a microplate reader.

[0102] 3. Obtaining total cDNA from alpaca PBMCs

[0103] Total RNA was extracted from alpaca PBMCs using the Trizol RNA Extraction Kit. First-strand cDNA was synthesized using the SuperScript TMIV First-Strand Synthesis System kit, employing the extracted RNA as a template.

[0104] 4. VHH gene amplification

[0105] Using the cDNA as a template, the heavy chain gene was amplified by PCR using upstream primers for the variable domain of the heavy chain and downstream constant region CH2 primers (VH-F, CH2-R). In a 50 μL reaction system, 25 μL of Prime STAR MAX mastermix (Takara), 2.5 μL (25 pmol) of upstream primer, 2.5 μL (25 pmol) of downstream primer, 1.5 μL of LDMSO, 0.5 μL of cDNA, and 18 μL of ddH2O were added, respectively. The PCR reaction was performed according to the following program: pre-denaturation at 98 °C for 1 minute, followed by temperature cycling: denaturation at 98 °C for 110 seconds, annealing at 60 °C for 15 seconds, extension at 72 °C for 30 minutes, repeated 25 times, and a final extension at 72 °C for 10 minutes.

[0106] The amplified VHH-CH2 gene was recovered using a DNA gel extraction kit. Electrophoresis results are as follows: Figure 2 As shown. Using 100 ng of VHH-CH2 as a template, the VHH gene was amplified by PCR using upstream primer VH-F and downstream primer VH-R. In a 50 μL reaction system, 25 μL of RimeSTARMAX mastermix (Takara), 2.5 μL (25 pmol) of upstream primer, 2.5 μL (25 pmol) of downstream primer, 1.5 μL of LDMSO, 0.5 μL of VH-CH2 DNA, and 18 μL of ddH2O were added, respectively. The PCR reaction was performed according to the following program: pre-denaturation at 98℃ for 1 minute, followed by temperature cycling: denaturation at 98℃ for 110 seconds, annealing at 60℃ for 15 seconds, extension at 72℃ for 30 minutes, repeated 25 times, and a final extension at 72℃ for 10 minutes. The amplified VHH gene fragment was recovered using a gel extraction kit.

[0107] 5. Constructing an immune library

[0108] The VHH gene fragment and the pcomb3X-TT vector (Scripps Research, USA) were digested separately using SfiI DNA endonuclease. In a 50 μL reaction mixture, 2 μL of SfiI, 5 μL of 10x buffer, and 3 μg of DNA were added, followed by ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 50°C for 3 hours.

[0109] The digested VHH gene fragment and pComb3X vector were recovered using a DNA gel extraction kit. The digested VHH gene fragment and pComb3X vector were then cyclized using T4 ligase. In a 50 μL reaction mixture, 1 μL of T4 ligase, 5 μL of 10x buffer, 150 ng of VHH gene, and 1000 ng of pComb3X vector were added, followed by ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 4°C for 16 hours. A small amount of the product was then subjected to agarose gel electrophoresis to verify the ligation efficiency.

[0110] 10 μL of the above-mentioned ligation and cyclization product was added to self-made TG1 electroporation competent cells, and then electroporation was performed using an electroporator. 10 μL of the electroporated bacteria was taken out, diluted appropriately, and streaked onto a plate containing ampicillin to count the bacteria and determine the size of the phage antibody library. The remaining electroporated bacteria were added to 2xYT medium containing 100 μg / mL ampicillin and 2% glucose and incubated in a heated incubator. After incubation, the culture was centrifuged at 4000G for 10 minutes at 4°C, and an appropriate amount of glycerol was added to the precipitate. The culture was then stored at -80°C as an antibody culture library. Through multiple electroporations, an scFv immunoglobulin library with a capacity exceeding 9E+9 was obtained.

[0111] 6. Screening for CD6 antibodies

[0112] (1) Recombinant human CD6 protein coupled to streptavidin magnetic beads

[0113] Using a biotinylation kit (EasyBio) following the kit instructions, the avi-tag of recombinant human CD6 protein was biotinylated to obtain biotinylated CD6 protein. 10 μg of the biotinylated recombinant protein was added to 100 μL of streptavidin magnetic beads (DynaBeads280) that had been washed three times with PBS. The mixture was placed on a rotary shaker at 18 rpm and coupled at room temperature for 30 minutes, followed by three washes with PBS.

[0114] (2) Blocking the phage library and magnetic beads.

[0115] Add 0.5 mL of 1% BSA / PBS to 0.5 mL of phage library, place on a rotary shaker at 18 rpm, and incubate at room temperature for 1 hour. These phages are designated Input1. Simultaneously, take 100 μL of uncoupled protein DynaBeads280, wash three times with PBS, add 1 mL of 1% BSA / PBS, and incubate under the same conditions for 1 hour. Separately, add 1 mL of 1% BSA / PBS to the CD6-coupled magnetic beads and incubate under the same conditions for 1 hour.

[0116] (3) Negative selection.

[0117] To remove antibodies that interact with the magnetic beads, negative panning is necessary. A BSA-blocked phage library and unconjugated magnetic beads are mixed and incubated under the conditions described above for 1 hour. After incubation, the phage-magnetic bead mixture is placed on a magnetic rack. Once the beads have adhered to the walls, the supernatant is transferred to a new EP tube.

[0118] (4) Positive screening.

[0119] The blocked magnetic beads coupled with CD6 protein were added to the negatively screened phage supernatant for positive screening, and incubated at room temperature for 1 hour under the conditions described above. After incubation, the magnetic beads were washed with 1 mL of PBST (0.1% Tween-20 in PBS), repeating the wash 10 times. After washing, 1 mL of 100 mM glycine (pH 2.0) was added, and the tube was placed on a rotary shaker at 18 rpm for elution for 10 minutes. After elution, the EP tube was placed on a magnetic rack, and after the magnetic beads adhered to the wall, the eluent was transferred to a new EP tube. 0.2 mL of 1 M Tris-HCl solution (pH 8.0) was added to the eluent for neutralization. The neutralized eluent was added to 30 mL of TG1 bacterial culture with an OD600 of approximately 0.6 and allowed to stand for 30 minutes for infection. Then, 20 times the number of M13KO7 bacteriophages were added and allowed to stand for another 30 minutes for infection. Finally, 100 mL of 2YT medium and ampicillin and kanamycin at a final concentration of 100 μg / mL were added, and the mixture was incubated overnight at 30°C and 220 rpm. The next day, bacteriophages were harvested using the same method described above for harvesting a bacteriophage library. The bacteriophage obtained at this point was Input2.

[0120] (5) Repeat positive screening.

[0121] The above selection method was repeated twice, that is, Input2 was subjected to another round of negative and positive selection to obtain Input3. The difference is that after the eluent obtained from Input3 was infected with TG1, M13KO7 was not added. Instead, 10 μL of bacterial culture was serially diluted, and 100 μL of each of the three dilutions (10³, 10⁴, and 10⁵) was spread on 2 YT / amp plates and incubated overnight at 30°C; the remaining bacterial culture was incubated overnight at 30°C and 220 rpm.

[0122] (6) ELISA screening for positive antibodies.

[0123] Randomly select TG1 monoclonal antibodies from the above plates and transfer them to deep-well plates containing 600 μL of 2 YT / amp. Cover the deep-well plates with a breathable membrane and incubate at 37°C and 220 rpm for 3 hours. Then, remove the breathable membrane and add IPTG to a final concentration of 1 mM. Incubate overnight at 30°C and 220 rpm. For ELISA plates, coat each well with 100 ng of recombinant human CD6 protein. The next day, centrifuge the deep-well plates at 4000 rpm for 10 minutes, remove the culture medium from the wells, retaining the bacterial pellet. Add 100 μL of LTES solution (20% sucrose, 0.1 mM EDTA, 50 mM Tris-HCl, pH 8.0) to each well, shake to resuspend the bacteria, incubate on ice for 30 minutes, add 200 μL of ultrapure water, shake to mix, and centrifuge at 4000 rpm for 10 minutes. The supernatant in the deep-well plates at this point is the periplasmic extract containing the antibody. Wash the ELISA plate three times with a plate washer, then add 200 μL of 1% BSA / PBS and block at 37°C for 1 hour. Remove the blocking solution from the ELISA plate, add 100 μL of the above periplasmic extract, incubate at 37°C for 1 hour, wash three times with a plate washer, add HRP-conjugated-GoatantiHA (horseradish peroxidase-labeled goat antiHA antibody) solution, incubate at 37°C for 1 hour, wash three times with a plate washer, add 100 μL of TMB chromogenic buffer, incubate at 37°C for 10 minutes, and stop the reaction with 100 μL of 1M hydrochloric acid. Read the OD450 value using a microplate reader. Perform Sanger sequencing on clones with OD450 values ​​three times higher than the background value to obtain the antibody gene sequence.

[0124] (7) Verify positive clones.

[0125] Based on the sequencing results, clones with significant amino acid sequence differences in antibody CDR3 were selected, re-inoculated, and induced overnight. The ELISA method described above was then used to verify again whether the selected clones could bind to CD6. Finally, antibody sequences 4A6-VHH, 4A7-VHH, 4A10-VHH, 4B2-VHH, 4B10-1-VHH, and 4D12-VHH were obtained. The amino acid sequences of the heavy chain variable region of the six nanobodies are shown in SEQ ID NO: 19-24, and the nucleotide sequences are shown in SEQ ID NO: 25-30.

[0126] Example 3: In vitro functional testing

[0127] 1. Detection of CD6 monoclonal antibody internalization function

[0128] The core of the experiment is based on the characteristics of pH-sensitive fluorescently labeled anti-human Fc secondary antibodies and the antigen-antibody binding internalization mechanism. CD6-VHH-Fc can specifically bind to CD6 molecules on the surface of T cells, and after binding, it will be internalized into the acidic endosomes of the cell along with the CD6 molecule. However, the pH-sensitive fluorescently labeled anti-human Fc secondary antibody used can only bind to the Fc fragment of CD6-VHH-Fc and emit fluorescence under neutral conditions. When CD6-VHH-Fc is internalized into the acidic endosomes, the environmental pH value decreases, causing the secondary antibody to be unable to bind to the Fc fragment, and the fluorescence signal disappears. The intensity of the cell fluorescence signal is detected by flow cytometry. If the fluorescence signal weakens or disappears, it indicates that CD6-VHH-Fc has been internalized. The negative control NT group has no antibody treatment and no fluorescence signal, which can be used as a baseline. The control trastuzumab group does not bind to CD6 on T cells because the anti-HER2 antibody does not bind to CD6, and no internalization occurs. The fluorescence generated by the secondary antibody binding is retained on the cell surface, thus eliminating non-specific internalization interference.

[0129] Human T cells were first seeded at 1×10^6 cells / well in 24-well plates and pre-cultured in RPMI-1640 medium containing 10% fetal bovine serum for 1 hour at 37°C and 5% CO2. Then, each experimental group was supplemented with 6 CD6 nanoantibody preparations at a final concentration of 10 μg / mL, the NT group was supplemented with an equal volume of medium, and the trastuzumab group was supplemented with trastuzumab antibody at a final concentration of 10 μg / mL. All groups were incubated under the same conditions for 18 hours. After incubation, cells were washed three times with pre-chilled PBS at 4°C, centrifuged at 1000 rpm for 5 minutes each time to collect cells and remove free surface antibodies. Next, a 1:200 dilution of pH-sensitive fluorescently labeled anti-human Fc secondary antibody was added, and the cells were incubated at 4°C in the dark for 30 minutes with shaking every 10 minutes. Finally, cells were washed twice with pre-chilled PBS, centrifuged, and resuspended in 0.5 mL of pre-chilled PBS. The fluorescence threshold was set for the NT group, and flow cytometry was used to detect and collect data for each group. The results are shown below. Figure 1 As shown.

[0130] 2. In vitro specificity analysis of CD6 monoclonal antibodies

[0131] This experiment utilizes the core immunological principle of antigen-antibody specific binding, combined with flow cytometry fluorescence detection technology to verify the specificity of CD6 nanobodies. CD6 nanobodies can only bind to CD6 molecules on the cell surface. In the experiment, the nanobodies are first co-incubated with the cells to be tested. If the cells express CD6, the nanobodies will bind to it. Then, fluorescently labeled goat anti-human IgG secondary antibody is added. The secondary antibody binds to the nanobodies bound to the cell surface, causing the cells to carry a fluorescent signal. Flow cytometry detects the fluorescence signal to determine the binding status. The isotype control group uses an unrelated antibody of the same isotype to eliminate interference from non-specific binding.

[0132] The experiment consisted of 8 groups (6 experimental groups, 1 positive control group, and 1 isotype control group). First, K562 cells (non-CD6 expressing, used in the experimental and isotype control groups) and human peripheral blood T cells (CD6 expressing, used in the positive control group) were adjusted to 1×10^6 cells / mL with PBS. For the experimental groups, 1 mL of K562 cell suspension was added with the corresponding CD6 nanobody (final concentration 10 μg / mL), incubated in the dark for 30 min, centrifuged and washed twice, then incubated with fluorescent secondary antibody (1:200 dilution) for another 20 min and washed. The positive control group used T cells with any CD6 nanobody, and the isotype control group used K562 cells with an antibody unrelated to the same subtype. All procedures were the same as for the experimental groups. Finally, flow cytometry was used to detect the fluorescence signal data of each sample. The results are shown below. Figure 2 As shown.

[0133] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0134] The sequence of this article is as follows:

[0135] 1. Amino acid sequence of 4A6-CDR-H1

[0136] GFTFSSAF

[0137] 2. Amino acid sequence of 4A7-CDR-H1

[0138] GFTFSSAF

[0139] 3. Amino acid sequence of 4A10-CDR-H1

[0140] EFIFSDVF

[0141] 4. Amino acid sequence of 4B2-CDR-H1

[0142] GYIFSVFG

[0143] 5. Amino acid sequence of 4B10-1-CDR-H1

[0144] GYIFSVFG

[0145] 6. Amino acid sequence of 4D12-CDR-H1

[0146] GFIFSVFG

[0147] 7. Amino acid sequence of 4A6-CDR-H2

[0148] IDDIGGAT

[0149] 8. Amino acid sequence of 4A7-CDR-H2

[0150] IDDIGGAT

[0151] 9. Amino acid sequence of 4A10-CDR-H2

[0152] IDNSGRST

[0153] 10. Amino acid sequence of 4B2-CDR-H2

[0154] ISSRGSV

[0155] 11. The amino acid sequence of 4B10-1-CDR-H2

[0156] ISSRGNQ

[0157] 12. Amino acid sequence of 4D12-CDR-H2

[0158] ISSTGDTT

[0159] 13. Amino acid sequence of 4A6-CDR-H3

[0160] AIGASLRSNH

[0161] 14. The amino acid sequence of 4A7-CDR-H3

[0162] AIGATMRSNH

[0163] 15. Amino acid sequence of 4A10-CDR-H3

[0164] AIGLYGSNS

[0165] 16. The amino acid sequence of 4B2-CDR-H3

[0166] KRPLSDY

[0167] 17. The amino acid sequence of 4B10-1-CDR-H3

[0168] KRPLSNY

[0169] 18. Amino acid sequence of 4D12-CDR-H3

[0170] KKGGSTY

[0171] 19. The amino acid sequence of 4A6-VHH

[0172] QVQLVESGGGLVQPGGSLRLSCTASGFTFSSAFMSWVRQAPGKGLEWVSGIDDIGGATNYAASVKGRFTISRDNAKNTLYLQMNSLQPEDTAMYYCAIGASLRSNHRGQGTQVTVSS

[0173] 20. Amino acid sequence of 4A7-VHH

[0174] EVQVVESGGGLVQPGGSLRLSCTASGFTFSSAFMSWVRQAPGKGLEWVSGIDDIGGATNYAASVKGRFTISRDNAKNTLYLQMNSLHPEDTAMYYCAIGATMRSNHRGQGTLVTVSS

[0175] 21. The amino acid sequence of 4A10-VHH

[0176] QLQLVESGGGLVQPGGSLRLSCAASEFIFSDVFMSWVRQAPGKGLEWVSGIDNSGRSTNYLDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAIGLYGSNSRGQGTQVTVSS

[0177] 22. Amino acid sequence of 4B2-VHH

[0178] EVQLVESGGGLVQPGGSLRLSCAASGYIFSVFGMAWYRQAPGKQRELVADISSRGSVHYADFVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCKRPLSDYWGQGTLVTVS

[0179] 23. The amino acid sequence of 4B10-1-VHH

[0180] EVQVVESGGGLVQPGGSLRLSCVASGYIFSVFGMAWFRQAPGKQRELVADISSRGNQYYADFVKGRFTISRDNTKNAVYLQMNSLKPEDTAVYYCKRPLSNYWGQGTQVTVSS

[0181] 24. Amino acid sequence of 4D12-VHH

[0182] QVQLVESGGGLVQPGGSLRLSCAASGFIFSVFGMNWYRQAPGKERELVAYISSTGDTTIYADSVKGRFTISRENARNTVYLQMSSLKPEDTAVYYCKKGGSTYWGQGTQVTVSS

[0183] 25. Nucleotide sequence of 4A6-VHH

[0184] CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCAGTAGCGCTTTTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTGATGACATTGGAGGCGCCACAAACTATGCAGCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGCAACCTGAGGATACGGCCATGTATTACTGTGCCATCGGAGCATCGTTGCGGTCGAATCACCGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0185] 26. Nucleotide sequence of 4A7-VHH

[0186] GAGGTGCAGGTCGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGTTCTCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCAGTAGCGCTTTTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTGATGATATTGGAGGCGCCACAAACTATGCAGCCTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGCACCCTGAGGATACGGCCATGTATTACTGTGCCATCGGAGCGACGATGCGGTCGAATCACCGGGGCCAGGGGACCCTGGTCACTGTCTCCTCA

[0187] 27. Nucleotide sequence of 4A10-VHH

[0188] CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAATTCATCTTCAGTGATGTCTTTATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTGATAACAGTGGGCGTAGCACAAACTATTTAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAACTTGAAACCTGAAGACACGGCCGTGTATTACTGTGCAATCGGGCTGTACGGTAGTAATTCGAGGGGTCAGGGAACCCAGGTCACCGTCTCCTCG

[0189] 28. Nucleotide sequence of 4B2-VHH

[0190] GAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGATCTCTGAGACTCTCCTGTGCAGCCTCTGGATACATCTTCAGTGTCTTTGGCATGGCCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTCGCAGATATCAGTAGTCGTGGCAGTGTACACTATGCGGACTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAAACGACCCCTGAGTGATTACTGGGGCCAGGGGACCCTGGTCACCGTCTCCT

[0191] 29. Nucleotide sequence of 4B10-1-VHH

[0192] GAGGTGCAGGTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGATCTCTGAGACTCTCCTGTGTAGCCTCTGGATACATCTTCAGTGTCTTTGGCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGCAGCGCGAGTTGGTTGCAGATATTAGTAGTCGTGGTAATCAGTACTATGCAGACTTCGTGAAGGGCCGATTCACCATCTCCAGAGACAACACCAAGAACGCGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAAACGACCCCTGAGTAATTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0193] 30. Nucleotide sequence of 4D12-VHH

[0194] CAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCATTTTCAGTGTCTTTGGCATGAACTGGTACCGGCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCGCATATATTTCTAGTACTGGCGATACGACAATCTATGCAGACTCCGTGAAGGGCCGATTCACCATTTCCAGAGAGAATGCCAGGAATACGGTGTATCTGCAAATGAGCAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTAAAAAGGGCGGATCGACCTACTGGGGCCAGGGAACCCAGGTCACCGTCTCCTCA

Claims

1. A CD6-targeting nanobody-binding molecule comprising an anti-CD6 nanobody or an antigen-binding fragment thereof, wherein the complementarity-determining region (CDR) of the anti-CD6 nanobody or the antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, wherein: The amino acid sequence of CDR1 is shown in SEQ ID NO: 1-6; The amino acid sequence of CDR2 is shown in SEQ ID NO:7-12; The amino acid sequence of CDR3 is shown in SEQ ID NO:13-18; The binding molecule is a monovalent or multivalent nanobody (containing one or more of the anti-CD6 nanobody or its antigen-binding fragment), which can specifically bind to CD6 molecules.

2. The CD6-targeting nanobody-binding molecule as described in claim 1, characterized in that, It also has one or more of the following features: (1) The amino acid sequence of the heavy chain variable region of the anti-CD6 nanobody is shown in SEQ ID NO:19-24. (2) The nucleotide sequence encoding the anti-CD6 nanobody is shown in SEQ ID NO:25-30. (3) The nanobody is derived from alpaca heavy chain antibody.

3. A targeted drug delivery method comprising the CD6-targeting nanobody-binding molecule of claim 1 or 2, and a functional payload conjugated to the binding molecule, wherein the functional payload is a therapeutic agent or a carrier; preferably, the targeted drug delivery method is an antibody-drug conjugate (ADC) or a gene carrier.

4. The targeted drug delivery method as described in claim 3, characterized in that, When it is an antibody-drug conjugate (ADC), it also includes a linker, through which the CD6-targeting nanobody-binding molecule is conjugated to a therapeutic agent; the therapeutic agent is selected from cytotoxic drugs, chemotherapeutic drugs, toxins or radioisotopes; the linker is a cleavable linker (such as an acid-sensitive linker or an enzyme-cleavable linker) or a non-cleavable linker.

5. The targeted drug delivery method as described in claim 3, characterized in that, When it is a gene vector, the vector carries therapeutic nucleic acid, and the CD6-targeting nanobody binding molecule is modified on the surface of the vector; the vector is selected from viral vectors (such as lentiviral vectors, adeno-associated virus vectors), liposomes, nanoparticles or polymer vectors; the therapeutic nucleic acid is selected from siRNA, mRNA, CRISPR-Cas9 system components or therapeutic genes.

6. A chimeric antigen receptor (CAR) comprising the CD6-targeting nanobody-binding molecule of claim 1 or 2, a hinge region, a transmembrane region, and an intracellular region; preferably, from the N-terminus to the C-terminus, it comprises: a signal peptide, the CD6-targeting nanobody-binding molecule, the hinge region, the transmembrane region, an intracellular co-stimulatory domain (such as 4-1BB, CD28), and an intracellular signaling domain (such as CD3ζ).

7. A nucleic acid molecule comprising a nucleotide sequence encoding a CD6-targeting nanobody-binding molecule as described in claim 1 or 2, a nanobody portion of a targeted drug delivery molecule as described in any one of claims 3-5, or a chimeric antigen receptor as described in claim 6, or a complementary sequence thereof.

8. A nucleic acid construct comprising the nucleic acid molecule of claim 7; wherein the nucleic acid construct is a cloning vector, an expression vector (such as a vector for expressing nanobodies, targeted drug delivery or CAR), or an integration vector.

9. A preparation method, comprising: (1) Cultivate host cells containing the nucleic acid construct of claim 8, express and purify the CD6-targeting nanobody-binding molecule of claim 1 or 2 and the chimeric antigen receptor of claim 6; (2) Optionally, the nanobody binding molecule is coupled to a functional payload to prepare a targeted delivery drug according to any one of claims 3-5.

10. A pharmaceutical composition comprising a targeted delivery drug according to any one of claims 3-5, a chimeric antigen receptor according to claim 6, a host cell expressing the CAR (such as CAR-T cells), a nucleic acid molecule according to claim 7 or a nucleic acid construct according to claim 8, and pharmaceutically acceptable excipients; for the treatment of CD6-positive diseases (such as T-cell acute lymphoblastic leukemia, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma).

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