Antibody that specifically binds to CLL1, method for preparing the same, and use thereof

Abstract

An antibody that specifically binds to CLL1, a method for preparing the same, and its use. Specifically, the present invention provides an anti-CLL1 nanobody or a binding fragment thereof. The antibody has good specificity. Furthermore, the antibody, and CLL1-targeted immune effector cells prepared using the antibody, show good therapeutic efficacy in treating or alleviating diseases with CLL-1 positive expression.

Description

[Technical Field]

[0001] This invention relates to the field of engineered immunotherapy, and more particularly to antibodies that specifically bind to CLL1, methods for preparing the same, and the use of the same. [Background technology]

[0002] Acute myeloid leukemia (AML) is a disease characterized by heterogeneity in both treatment response and survival. It is primarily characterized by the arrest of differentiation of primitive progenitor cells and immature myeloid cells in the bone marrow and peripheral blood, resulting in abnormal proliferation and accumulation of myeloblasts at various differentiation stages, while functionally normal red blood cells, platelets, and white blood cells are dramatically reduced. AML is generally prevalent across all age groups, but is particularly common in the elderly (over 60 years of age), and is the most common type of acute leukemia in adults. AML is primary, refractory, relapsing, and therapeutically fatal, making it a deadly disease. Commonly used treatments include chemotherapy and hematopoietic stem cell transplantation.

[0003] Chimeric antigen receptor T cells (CAR T cells) are a novel immunotherapy that targets specific antigens on the surface of tumor cells. They are currently being applied to the development of cell therapies for tumors.

[0004] Camel-derived antibodies (or single-domain antibodies) offer advantages over conventional antibodies in terms of their stable properties, good water solubility, low immunogenicity, stronger affinity, smaller molecular weight, and stronger tissue penetration. Single-domain antibodies can be used as diagnostic tools and also for CAR-T therapy and targeted drug delivery.

[0005] Therefore, in this field, there is an urgent need to develop novel CAR-T receptors with stronger affinity, higher specificity, and / or lower immunogenicity for the treatment of acute myeloid leukemia. [Overview of the Initiative]

[0006] The object of the present invention is to provide an antibody that has high affinity and high biological activity and can specifically recognize the CLL1 antigen, as well as its use.

[0007] In a first aspect of the present invention, a nanobody that specifically binds to CLL1 is provided. The complementarity-determining regions (CDRs) of the VHH chain of the nanobody include the following CDR1, CDR2, and CDR3: (a) CDR1: Its sequence is as shown in sequence numbers 14, 11, or 20, (b) CDR2: Its sequence is as shown in sequence numbers 12, 15, 17, or 19, (c)CDR3: Its sequence is as shown in sequence numbers 13, 16, 18, 21, or 22.

[0008] In another preferred embodiment, the complementarity-determining region (CDR) of the VHH chain of the nanobody is as follows: (Y1) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 19, and CDR3 as shown in Sequence ID 16, (Y2) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 12, and CDR3 as shown in Sequence ID 22, (Y3) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 15, and CDR3 as shown in Sequence ID 16, (Y4) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 17, and CDR3 as shown in Sequence ID 18, (Y5) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 19, and CDR3 as shown in Sequence ID 16, (Y6) CDR1 as shown in Sequence ID 11, CDR2 as shown in Sequence ID 12, and CDR3 as shown in Sequence ID 13, (Y7) Selected from the group consisting of CDR1 as shown in Sequence ID No. 20, CDR2 as shown in Sequence ID No. 12, and CDR3 as shown in Sequence ID No. 21.

[0009] In another preferred embodiment, CDR1, CDR2, and CDR3 are separated by the framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

[0010] In another preferred embodiment, the nanobody specifically binding to CLL1 includes a humanized antibody, a camel-derived antibody, and a chimeric antibody.

[0011] In another preferred embodiment, the amino acid sequence of the nanobody specifically binding to CLL1 is as shown by any one of SEQ ID NOs: 1 to 10.

[0012] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is selected from the group consisting of SEQ ID NOs: 1 to 10, or a combination thereof.

[0013] In another preferred embodiment, the CDR of the VHH chain of the nanobody contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity to any one of SEQ ID NOs: 1 to 10.

[0014] In another preferred embodiment, the amino acid sequence of the CDR of the VHH chain of the nanobody contains one or more amino acid substitutions, preferably conservative amino acid substitutions, as compared to any one of SEQ ID NOs: 1 to 10.

[0015] In another preferred embodiment, any one of the aforementioned amino acid sequences further optionally contains a derivative sequence in which at least one (for example, 1 to 3, preferably 1 to 2, more preferably 1) amino acid is added, deleted, modified, and / or substituted and retains the ability to specifically bind to CLL1.

[0016] In another preferred embodiment, CLL1 is CLL1 derived from a human or a non-human mammal.

[0017] In another preferred embodiment, CLL1 is derived from humans, mice, rats, or non-human primates (such as monkeys).

[0018] A second aspect of the present invention provides a polyvalent antibody or multiepitope-targeted antibody that specifically binds to CLL1, wherein the polyvalent antibody or multiepitope-targeted antibody comprises at least one antibody component that targets the CLL1 epitope, and the antibody component is an anti-CLL1 nanobody as described in the first aspect of the present invention.

[0019] In another preferred embodiment, the anti-CLL1 polyvalent antibody, or multiepitope-targeted antibody, comprises one or more anti-CLL1 nanobodies.

[0020] In another preferred embodiment, the anti-CLL1 antibody comprises a monomer, a dimer (bivalent antibody), a tetramer (quadrivalent antibody), and / or a multimer (multivalent antibody).

[0021] In another preferred embodiment, the anti-CLL1 antibody comprises one or more VHH chains having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 10.

[0022] In another preferred embodiment, the anti-CLL1 antibody comprises two VHH chains having the amino acid sequence shown in any one of SEQ ID NOs: 1 to 10.

[0023] In another preferred embodiment, the antibody is selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, or a combination thereof.

[0024] In a third aspect of the present invention, a recombinant protein is provided. (i) a nanobody that specifically binds to CLL1 as described in the first aspect of the present invention, or a polyvalent antibody or a multiepitope-targeted antibody that specifically binds to CLL1 as described in the second aspect of the present invention, (ii) comprising a tag sequence that promotes optional expression and / or purification.

[0025] In another preferred embodiment, the antibody is a polyvalent antibody.

[0026] In another preferred embodiment, the tag array includes an Fc tag, an HA tag, a flag tag, and a 6His tag.

[0027] In another preferred embodiment, the Fc tag includes mIgG2aFc.

[0028] In another preferred embodiment, the recombinant protein specifically binds to the CLL1 protein.

[0029] In a fourth aspect of the present invention, a chimeric antigen receptor (CAR) fusion protein is provided, wherein the chimeric antigen receptor (CAR) fusion protein is arranged from the N-terminus to the C-terminus: (i) An antigen-binding domain that specifically binds to CLL1, comprising the nanobody described in the first aspect of the present invention, (ii) Transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) Includes the activation domain.

[0030] In another preferred embodiment, the antigen-binding domain is monovalent or polyvalent.

[0031] In another preferred embodiment, the antigen-binding domain is derived from the nanobody described in the first aspect of the present invention or the recombinant protein described in the third aspect of the present invention.

[0032] In another preferred embodiment, the CAR has a structure represented by formula Ia: L-VHH-FH-TM-C-CD3ζ (Ia) During the ceremony, Each "-" independently represents a linker peptide or peptide bond. L is a signal peptide sequence, VHH is an antigen-binding domain that specifically binds to CLL1. F does not exist, or it is a Flag tag. H is the hinge region, TM is a transmembrane domain, C is a co-stimulatory signaling domain, and CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including the wild type or its variants / modified versions).

[0033] In another preferred embodiment, L is a protein signal peptide selected from the group consisting of CD28, 4-1BB, GM-CSF, CD3, CD8a, or a combination thereof.

[0034] In another preferred embodiment, L is a signal peptide derived from CD8.

[0035] In another preferred embodiment, L comprises an amino acid sequence as shown in Sequence ID No. 27.

[0036] In another preferred embodiment, the amino acid sequence of VHH is as shown in any one of SEQ ID NOs: 1 to 10.

[0037] In another preferred embodiment, H includes the hinge region of CD28.

[0038] In another preferred embodiment, TM includes a transmembrane region derived from CD28.

[0039] In another preferred embodiment, the amino acid sequences of H and TM include the amino acid sequences shown in SEQ ID NO: 29.

[0040] In another preferred embodiment, C is a transmembrane region of a protein selected from the group consisting of CD28, 4-1BB, CD8a, or a combination thereof.

[0041] In another preferred embodiment, C comprises a co-stimulatory signaling molecule derived from 4-1BB.

[0042] In another preferred embodiment, C comprises an amino acid sequence as shown in SEQ ID NO: 30.

[0043] In another preferred embodiment, CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 31.

[0044] In another preferred embodiment, the CAR fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 32-41.

[0045] In a fifth aspect of the present invention, an antibody-drug conjugate is provided, the antibody-drug conjugate is, (a) a nanobody according to the first aspect of the present invention, a polyvalent antibody or multiepitope-targeted antibody according to the second aspect of the present invention, or a recombinant protein according to the third aspect of the present invention, (b) A conjugate moiety conjugated to an antibody moiety, the conjugate moiety comprising a conjugate moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0046] In another preferred embodiment, the antibody moiety is conjugated to the conjugate moiety via a chemical bond or a linker.

[0047] A sixth aspect of the present invention provides a polynucleotide encoding a protein, selected from the group consisting of a nanobody described in the first aspect of the present invention, a multivalent antibody or multiepitope-targeted antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a CAR fusion protein described in the fourth aspect of the present invention.

[0048] A seventh aspect of the present invention provides an expression vector comprising the polynucleotide described in the sixth aspect of the present invention.

[0049] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0050] In another preferred embodiment, the expression vector is a lentiviral vector.

[0051] An eighth aspect of the present invention provides a host cell that contains an expression vector as described in the seventh aspect of the present invention, or has a polynucleotide as described in the sixth aspect of the present invention incorporated into its genome, or expresses a nanobody as described in the first aspect of the present invention, a multivalent antibody or multiepitope-targeted antibody as described in the second aspect of the present invention, a recombinant protein as described in the third aspect of the present invention, or a CAR fusion protein as described in the fourth aspect of the present invention.

[0052] In another preferred embodiment, the cells are isolated cells and / or the cells are genetically engineered cells.

[0053] In another preferred embodiment, the cells are mammalian cells.

[0054] In another preferred embodiment, the cells are T cells.

[0055] In another preferred embodiment, the host cells are engineered immune cells.

[0056] In another preferred embodiment, the manipulated immune cells are as follows: (i) Chimeric antigen receptor αβ T cells (CAR-T cells), (ii) Chimeric antigen receptor γδ T cells (CAR-T cells), (iii) Chimeric antigen receptor NKT cells (CAR-NKT cells), (iv) Chimeric antigen receptor NK cells (CAR-NK cells), and (v) Selected from a group consisting of chimeric antigen receptor macrophages.

[0057] A ninth aspect of the present invention provides a method for preparing engineered immune cells, comprising the step of introducing a nucleic acid molecule described in the sixth aspect of the present invention, or a vector described in the seventh aspect of the present invention, into T cells or NK cells to obtain engineered immune cells that express a CAR fusion protein described in the fourth aspect of the present invention.

[0058] In another preferred embodiment, the method further includes the step of testing the function and efficacy of the resulting manipulated immune cells.

[0059] A tenth aspect of the present invention provides a formulation comprising a nanobody described in the first aspect of the present invention, a polyvalent antibody or multiepitope-targeted antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, a CAR fusion protein described in the fourth aspect of the present invention, a vector described in the seventh aspect of the present invention, or a host cell described in the eighth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0060] In another preferred embodiment, the host cells are engineered immune cells.

[0061] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, (i) an immune cell expressing a nanobody according to the first aspect of the present invention, a polyvalent antibody or multiepitope-targeted antibody according to the second aspect of the present invention, a recombinant protein according to the third aspect of the present invention, or a CAR fusion protein according to the fourth aspect of the present invention, and (ii) Provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0062] In another preferred embodiment, the conjugate portion of the immunoconjugate is a drug, toxin, and / or therapeutic isotope.

[0063] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating an immune system disorder or a tumor disorder.

[0064] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating a CLL1-related disease or condition.

[0065] In another preferred embodiment, the CLL1-related disease or condition is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), or a combination thereof.

[0066] A twelfth aspect of the present invention provides a kit comprising a nucleic acid molecule described in the sixth aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a vector described in the seventh aspect of the present invention.

[0067] In another preferred embodiment, the kit is used to prepare immune cells expressing the receptor CAR fusion protein described in the fourth aspect of the present invention.

[0068] A thirteenth aspect of the present invention provides the use of immune cells expressing the nanobody described in the first aspect of the present invention, the polyvalent antibody or multiepitope-targeted antibody described in the second aspect of the present invention, the recombinant protein described in the third aspect of the present invention, or the CAR fusion protein described in the fourth aspect of the present invention, in the preparation of drugs for the prevention and / or treatment of CLL1-related cancer or tumor.

[0069] A fourteenth aspect of the present invention provides a method for treating a disease, comprising administering immune cells expressing a nanobody described in the first aspect of the present invention, a multivalent antibody or multiepitope-targeted antibody described in the second aspect of the present invention, a recombinant protein described in the third aspect of the present invention, or a CAR-fusion protein described in the fourth aspect of the present invention to a subject in need of treatment.

[0070] In another preferred embodiment, the disease is a tumor having CLL1-positive expression.

[0071] Within the scope of the present invention, it should be understood that the above-described technical features of the present invention and the technical features specifically described below (as in the examples) can be combined to form new or preferred technical solutions. Due to space limitations, such technical solutions are not detailed herein.

[0072] Within the scope of the present invention, it should be understood that the above-described technical features of the present invention and the technical features specifically described below (as in the examples) can be combined to form new or preferred technical solutions. Due to space limitations, such technical solutions are not detailed herein. [Brief explanation of the drawing]

[0073] [Figure 1] This shows CLL1 expression on the surface of target cells. [Figure 2] This shows the CAR positivity rate on the surface of CLL1 C-CAR T cells. [Figure 3] The cytotoxicity of CLL1 C-CAR-T against wild-type HeLa cells and CLL1-overexpressing HeLa cells is demonstrated (RTCA assay). [Figure 4] The cytotoxicity of CLL1 C-CAR-T against K562, KG1, and HL60 cells is demonstrated (luciferase assay). [Figure 5] This demonstrates the recognition of antigens on the surface of target cells by the CLL1 antibody. [Figure 6] This shows the CAR positivity rate on the surface of CLL1 F-CAR T cells. [Figure 7] This shows a schematic flowchart of flow cytometry analysis of in vitro multi-round killing by CAR-T cells. [Figure 8] This shows the results of multiple rounds of killing CLL1 C-CAR T cells and CLL1 F-CAR T cells against HL60 cells. [Figure 9]This shows the results of multiple rounds of killing CLL1 C-CAR T cells and CLL1 F-CAR T cells against KG1 cells. [Figure 10] This shows the CAR positivity rate after CAR-NK92 sorting. [Figure 11] This demonstrates the cytotoxicity of CAR-NK92 against tumor cells (luciferase assay). [Figure 12] This shows the lethality results of the CAR-N92K over multiple rounds. [Modes for carrying out the invention]

[0074] After extensive and thorough research, the inventors unexpectedly obtained, for the first time, an anti-CLL1 antibody with excellent affinity and high antitumor activity. Specifically, after extensive screening, the inventors obtained, for the first time, multiple nanobodies that specifically bind to CLL1. In this invention, recombinant antibodies, humanized antibodies, and chimeric antigen receptors targeting CLL1 were further prepared based on the nanobodies. Based on alpaca heavy chain antibodies that specifically bind to CLL1, this invention provides CAR-T cells that have higher affinity, stronger specificity to the target antigen, and lower immunogenicity compared to conventional antibodies. The CAR-T cells of this invention have excellent target cell killing activity and in vivo tumor suppressor effects. Based on these findings, the invention was completed.

[0075] Specifically, in this invention, alpacas are immunized using human-derived CLL1 antigen protein to obtain a high-quality immunonanobody gene library. The CLL1 protein molecule is then conjugated to an ELISA plate to display the precise spatial structure of the CLL1 protein. Next, this form of antigen is used to screen the immunonanobody gene library (alpaca heavy chain antibody phage display gene library) using phage display technology, thereby obtaining specific nanobody genes for the CLL1 protein. Subsequently, the genes are introduced into Escherichia coli (E. coli) to obtain nanobody strains that can be efficiently expressed in E. coli and exhibit high specificity.

[0076] term To facilitate understanding of this disclosure, certain terms are defined first. Where used in this application, unless expressly otherwise specified herein, the following terms shall have the meanings set forth below. Further definitions are provided throughout this application.

[0077] The term “approximately” may mean that a value or composition is within a specific tolerance range for that value or composition as determined by those skilled in the art, which depends in part on how the value or composition is measured or determined. For example, as used herein, the expression “approximately 100” includes 99 and 101 and all values ​​in between (e.g., 99.1, 99.2, 99.3, 99.4).

[0078] As used herein, the terms “comprise” or “contain” may be open-ended, semi-closed-ended, or closed-ended. In other words, the terms also include “substantially composed of” or “composed of.”

[0079] Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions in which the same residues appear. Typically, sequence identity is expressed as a percentage. Methods for measuring sequence identity of nucleotide sequences are well known to those skilled in the art.

[0080] As used herein, the terms “heavy chain variable region” and “VH” may be used interchangeably.

[0081] As used herein, the terms “variable region” and “complementarity-determining region (CDR)” can be used interchangeably.

[0082] In the present invention, the terms "antibody of the present invention", "single domain antibody of the present invention", or "protein of the present invention" or "polypeptide of the present invention" can be used interchangeably, and all refer to an antibody that specifically binds to CLL1, such as a protein or polypeptide having a heavy chain variable region (for example, the amino acid sequences set forth in SEQ ID NOs: 1 to 10). They may or may not contain a starting methionine.

[0083] CLL1 C-type lectin domain family 12 member A (CLEC12A), or C-type lectin-like molecule 1 (CLL1), also known as CD371, is a type II transmembrane protein. CLL1 is a glycoprotein receptor and a member of a large family of C-type lectin-like receptors involved in immune regulation. CLL1 is expressed on hematopoietic cells, mainly on innate immune cells including monocytes, DCs, and granulocytes, as well as on bone marrow progenitor cells.

[0084] CLL-1 is also found in acute myeloid leukemia (AML) blasts and leukemia stem cells (for example, CD34 + / CD38 - ). CLL1 can also be expressed on the surface of cells of other myeloid leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS).

[0085] Studies have shown that CLL1 is expressed on AML stem cells (CD34 + / CD38 - ) and a small fraction of hematopoietic progenitor cells (CD34 + / CD38 + , or CD34 + / CD33 + ), but not on normal hematopoietic stem cells (CD34 + / CD38 - , or CD34 + / CD33 - ).

[0086] Therefore, myeloid leukemias with high CLL1 expression can be treated using single-domain antibodies targeting CLL1 and CAR-modified immune cells (e.g., CAR-T cells), including, but not limited to, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS).

[0087] antibody As used herein, the term “antibody” refers to immunoglobulin, which is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains linked by interchain disulfide bonds.

[0088] As used herein, the terms “single-domain antibody (VHH)” and “nanobody” have the same meaning and refer to the variable region of the heavy chain of a cloned antibody. A single-domain antibody (VHH) consisting of only one heavy chain variable region is constructed, and this is the smallest antigen-binding fragment with full function. Generally, an antibody lacking a light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody's heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0089] Existing antibody numbering schemes include the following: 1. The Kabat scheme (Kabat et al., 1991) is based on the location of regions with high sequence variation between sequences of the same domain type. Antibody heavy chain (VH) and light chain (Vλ and Vκ) variable domains are numbered differently. 2. Chothia's scheme (Al-Lazikani, 1997) is identical to Kabat's scheme, but modifies it so that insertions correspond to structural loops when they are annotated around the first VH complementarity determination region (CDR). Similarly, the Enhanced Chothia scheme (Abhinandan and Martin, 2008) performs further structural correction of indel locations. 3. In contrast to these Kabat-like schemes, IMGT (Lefranc, 2003) and AHo (Honegger and Pluckthun, 2001) both define unique schemes for antibody and T cell receptor (TCR) (Vα and Vβ) variable domains. Therefore, equivalent residue positions can be easily compared between domain types. IMGT and AHo differ in the number of positions they annotate (128 and 149, respectively) and the locations where indels are thought to arise.

[0090] Immunoglobulins differ in antigenicity due to differences in the amino acid composition and sequence of the heavy chain constant region. Therefore, immunoglobulins can be classified into five classes or immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Each class of Ig can be subdivided into different subclasses according to differences in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4. The light chain can be a κ or λ chain depending on its constant region. Each of the five classes of Ig may have a κ or λ chain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

[0091] The light chain of the antibody according to the present invention may further include a light chain constant region, the light chain constant region including a human or mouse κ chain, λ chain, or a variant thereof.

[0092] In the present invention, the heavy chain of the antibody according to the present invention may further include a heavy chain constant region, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or their variants. The sequence of approximately 110 amino acids toward the N-terminus of the antibody's heavy and light chains changes significantly and constitutes a variable region (Fv region). The sequence of other amino acids toward the C-terminus is relatively stable and constitutes a constant region. The variable region includes three hypervariable regions (HVRs) and four framework regions (FRs) having relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, which are arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 toward the carboxyl terminus, respectively. The three CDRs of the light chain are designated LCDR1, LCDR2, and LCDR3. The three CDRs of the heavy chain are designated HCDR1, HCDR2, and HCDR3.

[0093] In the present invention, the term "mouse antibody" refers to an anti-CLL1 monoclonal antibody prepared in accordance with the knowledge and techniques of the art. During preparation, a CLL1 antigen is injected into a test subject, and then a hybridoma expressing an antibody having a desired sequence or functional characteristics is isolated. In preferred embodiments of the present invention, the mouse CLL1 antibody, or its antigen-binding fragment, may further comprise the light chain constant region of a mouse κ chain, a λ chain, or a variant thereof, or the heavy chain constant region of mouse IgG1, IgG2, IgG3, or a variant thereof.

[0094] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody. Chimeric antibodies can mitigate the immune response induced by mouse antibodies.

[0095] The term "humanized antibody," also known as CDR transplantation antibody, refers to an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework, i.e., a different type of human germline antibody framework sequence. Humanized antibodies can overcome heterogeneous reactions induced by chimeric antibodies containing large amounts of mouse protein components. Such framework sequences can be obtained from publicly available DNA databases containing germline antibody gene sequences or from publicly available references. To avoid a decrease in activity due to reduced immunogenicity, Human antibody variable region framework sequences can be subjected to minimal reverse or back mutations to maintain activity.

[0096] The term “antibody antigen-binding fragment” (or simply “antibody fragment”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., CLL1). It has been shown that the antigen-binding function of an antibody can be performed using fragments of a full-length antibody. Examples of binding fragments included in the term “antibody antigen-binding fragment” include: (i) Fab fragment (a monovalent fragment consisting of VL, VH, CL, and CH1 domains), (ii) F(ab')2 fragment (a divalent fragment consisting of two Fab fragments connected by disulfide bonds in the hinge region), (iii) Fd fragment (consisting of VH domain and CH1 domain), (iv) Fv fragment (consisting of the VH domain and VL domain of one arm of the antibody).

[0097] Fv antibodies contain heavy chain variable regions and light chain variable regions, but do not have a constant region. Fv antibodies are the smallest antibody fragments that possess all antigen-binding sites. Generally, Fv antibodies can further contain a polypeptide linker between the VH domain and the VL domain to form the structure necessary for antigen binding.

[0098] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immunological interest. NIH Publication, No. 91-3242.

[0099] The terms "epitope" or "antigenic determinant" refer to a site on an antigen (e.g., a specific site on the CLL1 molecule) to which an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial configuration.

[0100] The terms "specific binding," "selective binding," "to bind selectively," and "to bind specifically" refer to the binding of an antibody to a specific epitope on an antigen. Typically, an antibody binds to approximately 10 -7 Less than M, for example, approximately 10 -8 Less than M, 10 -9 Less than M, or 10 10 It binds with an affinity (KD) of less than M.

[0101] The term "competitive binding" refers to an antibody that recognizes the same epitope (also called an antigenic determinant) or a portion of the same epitope on the extracellular domain of CLL1, and binds to the antigen in such a way that the monoclonal antibody of the present invention binds to it. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of CLL1 that is recognized by the monoclonal antibody of the present invention.

[0102] The term "KD" or "Kd" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibodies of the present invention have a dissociation equilibrium constant of about 10. -7 Less than M, for example, about 10 -8 M, 10 -9 M, or 10 -10It binds to CLL1 with a dissociation equilibrium constant (KD) less than M or less.

[0103] As used herein, the term “antigenic determinant” refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0104] The present invention includes not only the whole antibody, but also fragments of the antibody, or fusion proteins formed by the antibody and other sequences, the fragments, or fusion proteins having immunoactivity. Therefore, the present invention also includes antibody fragments, derivatives, and analogs.

[0105] In the present invention, the antibody includes mouse antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, contain both human and non-human portions and can be prepared using DNA recombination techniques well known in the art.

[0106] As used herein, the term “monoclonal antibody” refers to an antibody secreted by a clone derived from a single cell. Monoclonal antibodies are highly specific and target a single antigenic epitope. The cell may be a eukaryotic cell line, a prokaryotic cell line, or a phage clone cell line.

[0107] In the present invention, the antibody may be monospecific, bispecific, triplicate, or more multispecific.

[0108] In the present invention, the antibody of the present invention further comprises its conserved variants. A conserved variant refers to a polypeptide formed by replacing up to 10 amino acids, preferably up to 8, more preferably up to 5, and most preferably 3, amino acids with similar or close properties, compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably produced by performing amino acid substitutions according to Table A below.

[0109] [Table 1]

[0110] Humanized anti-CLL1 antibody The present invention provides a humanized anti-CLL1 antibody. Specifically, the present invention provides a humanized antibody having high specificity and high affinity for CLL1. The humanized antibody comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of the heavy chain variable region (VH), and the light chain comprising the amino acid sequence of the light chain variable region (VL).

[0111] Generally, there are two types of human framework regions that can be selected for antibody humanization: one derived from well-known mature antibodies, and the other from human germline sequences. Framework regions from well-known mature antibodies typically contain somatic mutation sites, which can lead to potential immunogenicity. Compared to mature antibodies, framework regions from human germline sequences theoretically have lower immunogenicity. Furthermore, framework regions have a more flexible structure and strong plasticity, making them more readily able to accept different CDR regions. There is a bias in the frequency of use of human antibody germline genes in the human body. Antibodies humanized by selecting and using germline framework regions with high frequency of use have advantages such as lower immunogenicity, higher expression levels, and structural stability.

[0112] In a preferred embodiment of the present invention, during humanization, multiple factors (including similarity and frequency of use in humans) are considered simultaneously, and after extensive experimental screening, a framework region with a preferred sequence is selected and humanization is performed. A human antibody germline framework region is selected and used for CDR transplantation, and the humanized antibody thus constructed has a more stable structure, higher expression levels, lower immunogenicity, and higher druggability.

[0113] In another preferred embodiment, the heavy chain steady region and / or light chain steady region may be a humanized heavy chain steady region or a humanized light chain steady region. More preferably, the humanized heavy chain steady region or light chain steady region is a heavy chain steady region such as human IgG1, IgG2, or a human κ or λ light chain steady region.

[0114] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence is preferably an amino acid sequence having at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% homology.

[0115] The antibody of the present invention may be a double-chain antibody or a single-chain antibody, and preferably a fully humanized antibody.

[0116] The antibody derivatives according to the present invention may be single-chain antibodies and / or antibody fragments such as Fab, Fab', (Fab')2, or other antibody derivatives known in the art, as well as any one or more of IgA, IgD, IgE, IgG, and IgM antibodies or other subtypes.

[0117] The antibody of the present invention may be a humanized antibody, or a CDR implant, and / or a modified antibody that targets CLL1.

[0118] In the present invention described above, the number of amino acids added, deleted, modified and / or substituted is preferably 40% or less of the total number of amino acids in the initial amino acid sequence, more preferably 35% or less, more preferably 1% to 33%, more preferably 5% to 30%, more preferably 10% to 25%, and more preferably 15% to 20%.

[0119] Antibody preparation The CLL1 antibody of the present invention can be produced using any method suitable for producing monoclonal antibodies. For example, animals can be immunized with conjugated or naturally occurring CLL1 protein or fragments thereof. Suitable immunization methods, including adjuvants, immunostimulants, and repeated booster immunizations, can be used, and one or more of these methods can be used.

[0120] Any suitable form of CLL1 can serve as an immunogen (antigen) for producing CLL1-specific non-human antibodies, which are then screened for biological activity. The immunogen can be used alone or in combination with one or more immunogenicity enhancers known in the art. The immunogen can be purified from natural sources or produced in genetically modified cells. The DNA encoding the immunogen can be either genomic or non-genomic (e.g., cDNA) with respect to the source. The DNA encoding the immunogen can be expressed using a suitable gene vector. Vectors include, but are not limited to, adenovirus vectors, baculovirus vectors, plasmids, and non-viral vectors.

[0121] Humanized antibodies can be selected from any type of immunoglobulin, including IgM, IgD, IgG, IgA, and IgE. Similarly, any type of light chain can be used in the compounds and methods herein. Specifically, κ chains or λ chains or their variants can be used in the compounds and methods of the present invention.

[0122] The DNA molecule sequences of the antibodies or fragments thereof of the present invention can be obtained using conventional techniques such as PCR amplification or genome library screening, among other methods. Furthermore, the light chain and heavy chain coding sequences can be fused together to form a single-chain antibody.

[0123] After obtaining the relevant sequence, it can be obtained in large quantities using recombination. This typically involves cloning the relevant sequence into a vector, then introducing the vector into cells, and then isolating the relevant sequence from host cells grown by conventional methods to obtain the relevant sequence.

[0124] Furthermore, especially when the fragment length is short, the associated sequence can be artificially synthesized. Generally, fragments with very long sequences can be obtained by first synthesizing several smaller fragments and then ligating them together. The DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells that are well known in the field.

[0125] The term "nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. When a nucleic acid is functionally related to another nucleic acid sequence, the nucleic acid is "operably ligated." For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably ligated to the coding sequence.

[0126] The term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is ligated. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated.

[0127] The present invention further relates to vectors comprising the above-mentioned suitable DNA sequence and suitable promoter or regulatory sequence. These vectors can be used to transform suitable host cells, enabling the host cells to express proteins.

[0128] The term "host cell" refers to the cell into which the expression vector has been introduced. The host cell may be a prokaryotic cell such as a bacterial cell, a lower eukaryotic cell such as a yeast cell, or a higher eukaryotic cell, such as a plant or animal cell (e.g., a mammalian cell).

[0129] The step of transforming host cells with recombinant DNA according to the present invention can be carried out using techniques well known in the art. The resulting transformants express the polypeptide encoded by the gene of the present invention by culturing them according to conventional methods. Culturing is carried out in a normal culture medium under appropriate conditions, depending on the host cells used.

[0130] Generally, host cells obtained from transformation are cultured under conditions suitable for the expression of the antibody of the present invention. Then, a conventional immunoglobulin purification process is carried out by conventional isolation and purification methods well known to those skilled in the art, such as protein A-Sepharose chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography.

[0131] The resulting monoclonal antibodies can be identified by conventional methods. For example, the binding specificity of monoclonal antibodies can be determined using immunoprecipitation or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0132] Antibody preparations Antibodies exhibit different stabilities in different formulation buffers, as evidenced by changes such as charge heterogeneity, antibody molecule degradation, and polymerization. These changes in quality characteristics are related to the physical and chemical properties of the antibody itself. Therefore, in antibody drug development, it is necessary to screen formulation buffers suitable for different antibodies according to their physical and chemical properties. Currently, commonly used antibody buffer systems include phosphate buffer, citrate buffer, and histidine buffer. Simultaneously, to maintain antibody stability, different concentrations of salt ions or excipients such as sorbitol, trehalose, and sucrose, as well as appropriate amounts of surfactants such as Tween, may be added depending on the antibody's characteristics.

[0133] Pharmaceutical composition The present invention further provides compositions. In preferred embodiments, the composition is a pharmaceutical composition comprising the aforementioned antibody or its active fragment, fusion protein or ADC, or corresponding CAR-T cell, and a pharmaceutically acceptable carrier. Generally, these substances may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is typically about 5 to 8, and preferably about 6 to 8, but the pH value may vary depending on the properties of the formulated substance and the disease being treated. The formulated pharmaceutical composition may be administered by conventional means including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

[0134] The antibodies of the present invention can be expressed intracellularly by nucleotide sequence for use in cell therapies such as chimeric antigen receptor T cell (CAR-T) immunotherapy.

[0135] The pharmaceutical composition of the present invention can be used directly to bind to the CLL1 protein molecule and, therefore, can be used for the prevention and treatment of CLL1-related diseases. Furthermore, other therapeutic agents can be used simultaneously.

[0136] The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001 wt% to 99 wt%, preferably 0.01 wt% to 90 wt%, more preferably 0.1 wt% to 80 wt%) of the monoclonal antibody (or its conjugate) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be adapted to the mode of administration. The pharmaceutical composition of the present invention can be formulated, for example, in the form of an injectable preparation prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injectable preparations and liquid preparations are preferably manufactured under sterile conditions. The dose of the active ingredient is a therapeutically effective amount, such as about 1 μg / kg body weight to about 5 mg / kg body weight per day. Furthermore, the polypeptide of the present invention may also be used in combination with other therapeutic agents.

[0137] When using a pharmaceutical composition, administer a safe and effective amount of the pharmaceutical composition to the mammal. A safe and effective amount is typically at least about 10 mg / kg body weight and, in most cases, does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 mg / kg body weight to about 20 mg / kg body weight. Of course, factors such as the route of administration and the patient's health condition should also be considered for a specific dose, and these are all within the scope of the skill of an experienced physician.

[0138] Detection applications and kits The antibodies of the present invention can be used for detection purposes, such as detecting samples, in order to provide diagnostic information.

[0139] In the present invention, the sample (collected material) can be a cell, tissue sample, biopsy specimen, etc. The term "biopsy" as used in the present invention should include all types of biopsies well known to those skilled in the art. Therefore, the biopsy used in the present invention may include, for example, a tissue sample prepared by endoscopy, organ puncture, or needle biopsy.

[0140] The samples used in this invention include fixed or preserved cell or tissue samples.

[0141] The present invention further provides a kit containing the antibody (or a fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, a user manual, a buffer, and the like. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

[0142] The main advantages of this invention are as follows: (a) The single-domain antibody (VHH domain) against CLL1 of the present invention has high specificity and affinity. (b) The CLL1 CAR T cells of the present invention exhibit highly specific in vitro cytotoxicity against CLL1-positive target cells. (c) The CLL1 CAR T cells of the present invention may effectively inhibit the proliferation of CLL1-positive tumors in vivo and exhibit long-term antitumor effects. (e) The antibody of the present invention may be used to specifically detect the CLL1 protein.

[0143] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used solely to illustrate the present invention and not to limit its scope. Experimental methods shown in the following examples, without specific conditions, typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts refer to weight percentages and parts by weight.

[0144] Example 1. Screening of alpaca antibodies Single-domain antibodies (i.e., alpaca antibodies, or VHH antibodies) were obtained by screening a phage antibody library. The process for establishing the alpaca antibody library is briefly described below. The antigen was mixed with an adjuvant, emulsified, and used to immunize alpacas three times consecutively by subcutaneous injection. After three immunizations, blood samples were collected and PBMCs were isolated. The total RNA of the PBMCs was extracted and reverse transcribed into cDNA. VHH was amplified by nested PCR, and the VHH fragment was inserted into phagemids. The phagemids were amplified and recovered to obtain the antibody library.

[0145] CLL1 antibody screening was performed using protein panning. The process is described as follows: CLL1 antigen was coated overnight. The following day, after blocking, the antibody library was added and incubated for 1 hour. After incubation, washing was performed 8-10 times. The bound phages were then eluted, amplified, and the amplified phages were separated and purified for the next round of panning.

[0146] After enrichment was observed during screening, the enriched phages were used to infect host bacteria, plated onto resistance plates, and ≥96 monoclones / libraries were randomly selected for culture. The supernatant of the overnight cultures was collected, and positive monoclones were identified by phage ELISA experiments. Multiple monoclones were selected for sequencing according to the OD value of the phage ELISA-positive antigen well (Pro) and the difference with the control well (PVA) (Pro / PVA). Furthermore, clones with identical sequences were combined after removing sequences that showed abnormalities during sequencing.

[0147] result Ten single-domain antibodies bound to CLL1 were ultimately obtained, according to the affinity order of the positive monoclones and the frequency of appearance of the enriched antibodies. The sequence numbers corresponding to the amino acid sequences and their CDRs are listed in Tables 1, 2, and 3 below.

[0148] [Table 2-1]

[0149] [Table 2-2]

[0150] [Table 3]

[0151] [Table 4] Note: These antibodies may have the same CDR. When using the same numbering for the same sequence, the deduplication-free numbering is as follows: among them, antibodies C6, C7, and C8 have three identical CDRs, and antibodies C9 and C10 have three identical CDRs.

[0152] Example 2. Cell culture and construction CLL1-expressing cell lines HL60-LucG cells, KG1-LucG cells, and CLL1-nonexpressing K562-LucG cells were all cultured using RPMI 1640 culture medium. 293T, wild-type, and CLL1-expressing Hela cells (Hela-WT and Hela-CLL1) were cultured using DMEM culture medium. Both culture media were supplemented with 10% (v / v) fetal bovine serum, 100 U / mL penicillin, streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. Cells were cultured at saturated humidity, 37°C, and 5% CO2.

[0153] HeLa cells expressing CLL1 were stable, transformed cell lines obtained by introducing the CLL1 antigen using a lentiviral vector, and were able to specifically express the CLL1 protein molecule. HL60-LucG cells, KG1-LucG cells, and K562-LucG cells were stable, transformed cell lines obtained by infecting cells with a lentivirus expressing firefly luciferase-GFP (T2A ligated), followed by screening.

[0154] The antigen expression on the surface of the target cells used is shown in Figure 1. Hela-CLL1 represents CLL1-overexpressing cells, Hela-WT and K562 represent CLL1-negative cells, and KG1 and HL60 represent CLL1-positive cells. The amount of CLL1 protein on the surface of HL60 cells is higher than the amount of CLL1 protein on the surface of KG1 cells.

[0155] Example 3. Preparation of CLL1 C-CAR T cells The CLL1 CAR gene consists of a CD8 signal peptide, a VHH or scFv that recognizes CLL1, a CD8 hinge, a transmembrane region, a 4-1BB signal region, and a CD3z signal region.

[0156] The CLL1 CAR gene was placed under the EF1α (EF-1α) promoter to form a CLL1 CAR expression vector. The CLL1 CAR expression vectors were numbered C1, C2, C3..., C9, and C10 respectively, according to differences in their VHH sequences. CLL1 CAR expression vectors constructed using other CLL1-recognizing scFvs were named using their respective scFv clone numbers, such as M26.

[0157] The amino acid sequence of the CLL1 C-CAR of the present invention is as shown in SEQ ID NOs: 32-41, and the amino acid sequence of the M26 CAR based on scFv is as shown in SEQ ID NO: 42.

[0158] 2.5 × 10 6 Each 293T cell was seeded in DMEM culture medium containing 10% FBS in a 150 cm² dish, and the cells were cultured overnight at 37°C and 5% CO2 under saturated humidity prior to transfection.

[0159] The following day, the helper plasmid and CLL1 CAR expression vector were added to a centrifuge tube containing 13.8 mL of Opti MEM culture medium, followed by the addition of 80 μg of PEI to obtain a mixture. The mixture was allowed to stand at room temperature for 20 minutes, after which 12 mL of Opti MEM culture medium was added to obtain the transfection culture medium. For transfection, after removing the culture medium, 293T cells were incubated with the transfection culture medium for 4-6 hours, and then the transfection culture medium was replaced with 20 mL of DMEM culture medium containing 2% FBS. After 72 hours, the culture medium was collected and centrifuged at 3000 g at 4°C for 15 minutes, and the supernatant was further centrifuged at 27000 g at 4°C for 2 hours. The pellet was collected and resuspended in 400 μL of pre-cooled X-VIVO culture medium to obtain the CLL1 CAR lentivirus suspension, which was maintained overnight at 4°C. The following day, the virus suspension was aliquoted for further use.

[0160] All T cells were cultured in X-VIVO culture medium supplemented with 1% human albumin, 4% serum substitute, and 300 IU / mL human interleukin-2 (hIL-2). Pan T cells and CD3 / CD28 Dynabeads in a 1:1 ratio (CD3 / CD28 Dynabeads:T cells) were incubated together with 300 IU / mL IL-2 to activate the cells. After 2 days, the Dynabeads were removed using a magnetic column, and then the T cells were injected with CLL1 CAR virus at a rate of 1 × 10⁶ 6 Transfected cells were cultured at 37°C at a cell density of cells / mL. Transfected cells were further cultured for proliferation. Three days after infection, before cryopreservation, samples were collected, cell counts were measured, and the percentage of CLL1 CAR-positive cells was determined using the CLL1 antigen, i.e., the CAR-positive rate of T cells was determined. Half of the culture medium was changed every 2-3 days, and CLL1 C-CAR T cells were collected on day 8 post-infection.

[0161] Figure 2 shows the CAR positivity rate of CLL1-C-CAR-T cells determined using the CLL1 antigen. The CAR positivity rate was ≥50% in all groups.

[0162] Example 4. In vitro euthanasia of CLL1 C-CAR T cells In this example, in vitro toxication experiments were performed on the CLL1 C-CAR T cells obtained above.

[0163] 4.1 RTCA assay test The cytotoxicity of CAR-T cells against target cells, namely a HeLa cell line overexpressing CLL1, was tested using an RTCA assay.

[0164] The results are shown in Figure 3. In two effector-target ratios, the NT control group did not kill Hela-CLL1 cells, while CLL1 C-CAR T cells showed a specific killing effect against CLL1. In two effector-target ratios, the killing effects of each group of CLL1 C-CAR T cells against CLL1-overexpressing Hela cell lines were similar.

[0165] 4.2 Luciferase assay The killing capacity of luciferase-labeled tumor target cells was determined. After introducing the luciferase gene into target cells, cloning, and screening, stable transformed cell lines, HL60-LucG and KG1-LucG, were obtained. During the experiment, luciferin substrate was added, and luciferase reacted with luciferin, producing fluorescence. Luciferase activity could be determined by measuring the fluorescence intensity, and the killing effect of each type of CAR-T cell could be obtained by measuring cell viability.

[0166] The results are shown in Figure 4. At the same E:T ratio, NT cells did not exhibit killing function, CLL1C-CAR T cells had a dose-dependent killing effect against HL60-LucG cells and KG1-LucG cells, and CLL1C-CAR T cells did not have killing ability against K562-LucG cells.

[0167] In summary, after co-culturing CAR-T cells with target cells (CLL1-overexpressing HeLa cells, and CLL1-positive tumor cells, HL60, and KG1 cells), the target cells could be lysed by CLL1-targeting CAR-T cells, and no nonspecific cytotoxicity was observed against non-target cells (wild-type HeLa cells and CLL1-negative K562 cells).

[0168] Example 5. Recognition of antigens on the surface of target cells by CLL1 antibody. The CLL1 antibodies used were synthesized and purified (mouse IgG2a Fc fragments were added to the C-terminus of VHH or scFv). These antibodies were serially diluted and used to titrate the CLL1 antigen on the surface of HL60 cells, and their ability to recognize the antigen on the surface of target cells was analyzed.

[0169] The results are shown in Figure 5. Compared to the scFv antibody M26, the single-domain antibodies C1, C3, C5, C7, and C9 of the present invention were able to recognize CLL1 on the surface of HL60 cells with a higher positive rate, and they had a higher average fluorescence intensity than M26 with the same amount of antibody used.

[0170] Example 6. Preparation of CLL1 F-CAR T cells Peripheral blood was collected from healthy donors, and PBMCs were isolated by centrifugation at 500-600g for 20-30 minutes using a density gradient centrifuge. T cells were sorted and enriched using magnetic beads (CD3 / CD28 Dynabeads) that bind to CD28 and CD3 antibodies. T cells bound to CD3 / CD28 Dynabeads were further incubated with 300 IU / mL of IL-2 to activate the cells. Simultaneously, the cells were injected with CLL1 CAR virus at a dose of 0.1-10 × 10⁻¹⁴. 6 Cells were transfected overnight at a cell density of cells / mL at 37°C. The following day, the cells were washed with physiological saline buffer and then frozen directly for storage. FAST CAR T cells could be obtained without further proliferation. During this process, the cells were activated, and these cells were also called CLL1 F-CAR T cells.

[0171] The CAR positivity rate of CLL1 F-CAR T cells needed to be titrated after resuscitation. CLL1 F-CAR T cells were resuscitated and then titrated at 37°C in the presence of 300 IU / mL of IL2 at a rate of 1 × 10⁶ 6 Cells were cultured at a cell density of cells / mL. On day 3 after resuscitation, 1 × 10⁶ cells were cultured. 5 Individual cell samples were collected, and the percentage of CLL1 CAR-positive cells was determined using the CLL1 antigen.

[0172] The results are shown in Figure 6. CLL1 CAR expression could be detected on the surface of the generated CLL1F-CAR T cells, with a positive rate of approximately 30%.

[0173] Example 7. In vitro multi-round killing of CLL1 C-CAR T cells and CLL1 F-CAR T cells On day 1, CLL1F-CAR T was resuscitated and 1 × 10⁶ times with 300 IU / mL of IL-2 at 37°C. 6 The cells were cultured and incubated at a cell density of cells / mL. On day 2, CLL1 C-CAR T cells were resuscitated and incubated with 300 IU / mL of IL2 at 37°C for 1 × 10⁶ cells. 6 The cells were cultured and incubated at a cell density of cells / mL. On day 3, 1 × 10⁶ cells were cultured. 5 Individual cell samples were collected, and the percentage of CLL1 CAR-positive cells was determined using the CLL1 antigen.

[0174] According to the CAR-positive rate of cells as shown in Figure 6, 2 × 10 5 We collected CAR-positive CLL1 C-CAR T cells and CLL1 F-CAR T cells, and each was analyzed using HL60-LucG (6 × 10⁻¹⁶). 5 (Individual cells, E:T=1:3), and KG1-LucG(4×10) 5 Cells were co-cultured with E:T=1:2 cells, and multiple rounds of toxicology analysis were performed.

[0175] On the third day of each round, a portion of the co-cultured cells was isolated for analysis by flow cytometry. The analysis process is shown in Figure 7, and the specific method is as follows: The 7AAD-negative viable cell population was circled in the major cell population. Then, T cells (GFP-negative) and target cells (GFP-positive) were distinguished by GFP signaling, and the CAR positivity rate was analyzed in the T cell population using the CLL1 antigen marker. Based on the flow cytometry and cell counting results, the same number of CLL1 CAR-T cells were removed and tumor cells were replenished to achieve a certain effector-to-target ratio (E:T for HL60-LucG was 1:3, and E:T for KG1-LucG was 1:2), and the killing experiment for the next round was carried out.

[0176] The results of multiple rounds of halting against HL60 target cells are shown in Figure 8. Figure 8A shows the change in CAR positivity of CLL1 C-CAR T cells and CLL1 F-CAR T cells during multiple rounds of halting against HL60. Figure 8B shows the number of surviving target cells (HL60) at the end of each round for CLL1 C-CAR T cells and CLL1 F-CAR T cells, and Figure 8C shows the total proliferation time of CAR T cells during multiple rounds of halting against HL60 by CLL1 C-CAR T cells and CLL1 F-CAR T cells. From Figure 8B, it can be seen that the halting ability of CLL1 F-CAR T cells against HL60 cells was higher than that of CLL1 C-CAR T cells. At the end of the fourth round, all CLL1 C-CAR T cells were no longer able to inhibit HL60 cells, and the CAR positivity rate was significantly reduced (Figure 8A). At the end of the fourth round, among the CLL1 F-CAR T groups, C9 showed a stronger inhibitory effect against HL60 than M26, and C9 F-CAR T maintained a better CAR positivity rate (Figure 8A). From Figure 8C, it can be seen that the proliferation of CLL1 F-CAR T was better than that of CLL1 C-CAR T during multiple rounds of killing of HL60, and the comparison between CLL1 F-CAR T groups showed that the proliferation time of C9 was longer than that of M26.

[0177] The results of multiple rounds of killing KG1 target cells are shown in Figure 9. Figure 9A shows the changes in CAR positivity rates of CLL1 C-CAR T cells and CLL1 F-CAR T cells during multiple rounds of killing KG1. Figure 9B shows the number of surviving target cells (KG1) at the end of each round for CLL1 C-CAR T cells and CLL1 F-CAR T cells, and Figure 9C shows the total proliferation time of CAR T cells during multiple rounds of killing KG1 by CLL1 C-CAR T cells and CLL1 F-CAR T cells. At the end of the third round, neither CLL1 C-CAR T cell group was able to adequately inhibit KG1 cells, while CLL1 F-CAR T cells were still able to adequately inhibit KG1 cells, and their CAR positivity rates were higher than those of CLL1 C-CAR T cells (Figure 9A). As can be seen in Figure 9C, during multiple rounds of killing against KG1, CLL1 F-CAR T cell proliferation was better than CLL1 C-CAR T cell proliferation, and comparisons between CLL1 F-CAR T cells showed that the proliferation time of C9 was longer than that of M26.

[0178] Example 8. NK92 cells and culture method The complete culture medium for NK92 cells was Alpha MEM supplemented with 12.5% ​​FBS, 12.5% ​​horse serum, 1% Pen / strep, 1% sodium pyruvate, 1% L-glutamine, 100U IL2.HL60, Molm13, KG-1, and THP-1. The complete culture medium for K562 cells was RPMI1640 supplemented with 10% FBS, 1% Pen / strep, 1% sodium pyruvate, and 1% L-glutamine. Cells were cultured at saturated humidity, 37°C, and 5% CO2.

[0179] Example 9. Packaging and transfection of CAR-NK92 lentivirus 293T cells were revived, and 150cm 2The cells were seeded in a culture dish and cultured in a CO2 incubator for 72 hours. After two subculturings, the cells were transfected at 150 cm². 2 Cells were seeded in a culture dish. A four-plasmid system consisting of a lentivirus expression vector, helper plasmids gag / pol, Rev, and VSV-G was mixed with PEI transfection reagent and then added to a fixed amount of serum-free DMEM. The mixture was thoroughly mixed and allowed to stand for 15 minutes. The resulting mixture was added to a 150 cm² culture dish seeded with 293T cells. The cells were gently and uniformly mixed and cultured in a 5% CO2 cell incubator at 37°C for 6 hours. After 6 hours, the culture medium was replaced with fresh medium, and the culture was continued. At 48 hours and 72 hours, the lentivirus culture supernatant was collected for infection. The collected supernatant was transferred to a centrifuge tube and centrifuged at 4000 rpm for 10 minutes with acceleration 9 and deceleration 9 to remove cell debris. All of the centrifuged LVV supernatant was transferred to a 0.45 μm filter for clarification by filtration, and the filtrate was transferred to a new centrifuge tube. The clarified lentivirus supernatant was added to ultracentrifuge tubes and equilibrated on a balance. The equilibrated ultracentrifuge tubes were placed in a suspension cup, the suspension cup was placed in the corresponding position on the rotor, and then placed together in an ultracentrifuge for centrifugation. The centrifugation temperature was 4°C, the rotation speed was 100,000 g, the centrifugation time was 90 minutes, and the acceleration and deceleration were set to maximum. After ultracentrifugation was complete, the supernatant was discarded, 0.5 mL of culture medium was added to each tube, and the mixture was resuspended at 2°C to 8°C for 2 hours. The collected viruses were added to 1e6 NK92, mixed in a 24-well plate, and incubated in a CO2 incubator for 72 hours.

[0180] Example 10. CAR-NK flow cytometry detection and sorting The cell suspension was removed, washed three times with DPBS, centrifuged at 300g for 5 minutes, the supernatant was discarded, and an antibody mixture (anti-FLAG-APC, 1:100 in DPBS) was added. The mixture was thoroughly mixed, stained at 4°C for 30 minutes, washed three times with DPBS, centrifuged at 300g for 5 minutes, resuspended in DPBS, and then used for flow cytometry detection and sorting. For sorting, PE-positive cells were selected and collected in 5 mL flow cytometry tubes. After sorting, the cells were centrifuged at 300g for 5 minutes, and the supernatant was discarded. The cells were resuspended in preheated culture medium and placed in a cell culture incubator at 37°C and 5% CO2.

[0181] Sorted CAR-NK92 cells were stained with anti-FLAG-APC antibody, and CAR expression was detected by flow cytometry.

[0182] The results of flow cytometry detection are shown in Figure 10. The CAR positivity rate of sorted NK92 C9 cells reached over 98%.

[0183] Example 11. CAR-NK lethal experiment The killing capacity was determined using luciferase-labeled tumor target cells. The luciferase gene was introduced into target cells to obtain stable, transformed cell lines expressing the luciferase gene: Molm13, HL60, THP-1, and KG1. During the experiment, a luciferin substrate was added, and luciferase reacted with luciferin, producing fluorescence. Luciferase activity could be determined by measuring the fluorescence intensity, and the killing effect of effector cells could be obtained by measuring cell viability.

[0184] As shown in Figure 11, NK92 C9 exhibited more pronounced cytotoxicity against all target cells compared to NK92, suggesting CAR-mediated cytotoxicity.

[0185] Example 12. Multiple rounds of CAR-NK lethal experiment The day before the experiment, the fluorescent target cells THP1 were resuspended and counted. The cell density was 8 × 10⁻⁶. 4 The solution was adjusted to 1 / mL, and cells were plated in a 96-well plate at 100 μl per well. On the day of the experiment, the corresponding number of NK92 and CAR-NK92 cells were added to the wells. The plate was placed in Cellcyte, and the experiment was started. Imaging was performed every 4 hours in bright-field and fluorescence channels. One round was performed every 2 days. After one round of killing was completed, a portion of the supernatant was removed, and the cells were transferred to a new plate seeded with target cells. The plate was placed in Cellcyte, and a new round of killing was started. The killing index was calculated as the decrease in the number of fluorescent cells after adjusting for the moment of zero crossover and fluctuations in normal cell proliferation.

[0186] The results of multiple rounds of killing experiments are shown in Figure 12. NK92 C9 cells showed stronger killing ability than NK92 cells, suggesting that CAR-mediated killing was continuously effective.

[0187] [Table 5-1]

[0188] [Table 5-2]

[0189] [Table 5-3]

[0190] [Table 5-4]

[0191] [Table 5-5]

[0192] [Table 5-6]

[0193] [Table 5-7]

[0194] [Table 5-8]

[0195] [Table 5-9]

[0196] [Table 5-10]

[0197] [Table 5-11]

[0198] [Table 5-12]

[0199] All documents referenced herein are incorporated by reference as each document is incorporated by reference individually. Furthermore, those skilled in the art, having read the above teachings of the invention, should understand that various changes or modifications can be made to the invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A nanobody that specifically binds to CLL1, wherein the complementarity determination region (CDR) of the VHH chain of the nanobody includes the following CDR1, CDR2, and CDR3: (a) CDR1: Its sequence is as shown in sequence number 14, 11, or 20, (b) CDR2: Its sequence is as shown in sequence numbers 12, 15, 17, or 19, (c) CDR3: a nanobody whose sequence is as shown in sequence numbers 13, 16, 18, 21, or 22.

2. The complementarity determination region (CDR) of the VHH chain of the nanobody is as follows: (Y1) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 19, and CDR3 as shown in Sequence ID 16, (Y2) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 12, and CDR3 as shown in Sequence ID 22, (Y3) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 15, and CDR3 as shown in Sequence ID 16, (Y4) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 17, and CDR3 as shown in Sequence ID 18, (Y5) CDR1 as shown in Sequence ID 14, CDR2 as shown in Sequence ID 19, and CDR3 as shown in Sequence ID 16, (Y6) CDR1 as shown in Sequence ID 11, CDR2 as shown in Sequence ID 12, and CDR3 as shown in Sequence ID 13, (Y7) A nanobody according to claim 1, selected from the group consisting of CDR1 as shown in Sequence ID No. 20, CDR2 as shown in Sequence ID No. 12, and CDR3 as shown in Sequence ID No.

21.

3. The nanobody according to claim 1, wherein the amino acid sequence of the nanobody that specifically binds to CLL1 is as shown in any one of sequence numbers 1 to 10.

4. A multivalent antibody or multiepitope-targeted antibody that specifically binds to CLL1, wherein the multivalent antibody or multiepitope-targeted antibody comprises at least one antibody component that targets the CLL1 epitope, and the antibody component is an anti-CLL1 nanobody as described in claim 1.

5. Recombinant protein, wherein the recombinant protein is (i) a nanobody that specifically binds to CLL1 as described in claim 1, or a polyvalent antibody that specifically binds to CLL1 as described in claim 4, or a multiepitope-targeted antibody, (ii) Recombinant protein comprising a tag sequence that promotes optional expression and / or purification.

6. A chimeric antigen receptor (CAR) fusion protein, wherein the chimeric antigen receptor (CAR) fusion protein is arranged from the N-terminus to the C-terminus, (i) an antigen-binding domain that specifically binds to CLL1, comprising the nanobody described in claim 1, (ii) Transmembrane domain, (iii) at least one co-stimulatory domain, and (iv) A chimeric antigen receptor (CAR) fusion protein containing an activating domain.

7. The CAR has the structure shown in formula Ia: L-VHH-F-H-TM-C-CD3ζ (Ia) During the ceremony, Each "-" independently represents a linker peptide or peptide bond. L is the signal peptide sequence, VHH is an antigen-binding domain that specifically binds to CLL1, and the amino acid sequence of VHH is as shown in any one of SEQ ID NOs: 1 to 10. F does not exist, or it is a Flag tag. H is the hinge region, TM is a transmembrane domain, C is a co-stimulatory signaling domain, and The CAR fusion protein according to claim 6, wherein CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild-type or its variants / modified versions).

8. The CAR fusion protein according to claim 6, wherein the CAR fusion protein has the amino acid sequence shown in any one of SEQ ID NOs: 32 to 41.

9. An antibody-drug conjugate, wherein the antibody-drug conjugate is (a) a nanobody according to claim 1, a multivalent antibody according to claim 4, or a multiepitope-targeted antibody, or a recombinant protein according to claim 5, (b) An antibody-drug conjugate comprising a conjugate portion conjugated to the antibody portion, wherein the conjugate portion is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

10. A polynucleotide characterized by encoding a protein, selected from the group consisting of a nanobody as described in claim 1, a polyvalent antibody or multiepitope-targeted antibody as described in claim 4, a recombinant protein as described in claim 5, or a CAR fusion protein as described in claim 6.

11. An expression vector, characterized in that the expression vector comprises the polynucleotide described in claim 10.

12. A host cell, wherein the host cell contains the expression vector described in claim 11, or has a polynucleotide described in claim 10 incorporated into its genome, or expresses a nanobody described in claim 1, a multivalent antibody described in claim 4, or a multiepitope-targeted antibody, a recombinant protein described in claim 5, or a CAR fusion protein described in claim 6.

13. A pharmaceutical composition, wherein the pharmaceutical composition is (i) an immune cell expressing the nanobody described in claim 1, the multivalent antibody or multiepitope-targeted antibody described in claim 4, the recombinant protein described in claim 5, or the CAR fusion protein described in claim 6, and (ii) A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

14. Use of an immune cell expressing a nanobody according to claim 1, a multivalent antibody or multiepitope-targeted antibody according to claim 4, a recombinant protein according to claim 5, or a CAR fusion protein according to claim 6, wherein the use is for the preparation of a drug for the prevention and / or treatment of CLL1-associated tumors.

15. A method for treating a disease, characterized by administering immune cells expressing the nanobody described in claim 1, the multivalent antibody or multiepitope-targeted antibody described in claim 4, the recombinant protein described in claim 5, or the CAR fusion protein described in claim 6 to a subject in need of treatment.

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