CN117106086A

CN117106086A - CLL1 antibody and application thereof

Info

Publication number: CN117106086A
Application number: CN202310854805.1A
Authority: CN
Inventors: 白大勇; 张超; 张其猛; 周立
Original assignee: Heyuan Kanghua Pharmaceutical Technology Beijing Co ltd
Current assignee: Heyuan Kanghua Pharmaceutical Technology Beijing Co ltd
Priority date: 2022-08-09
Filing date: 2022-08-09
Publication date: 2023-11-24
Also published as: CN115850476B; CN115850476A

Abstract

The application provides a CLL1 antibody and application thereof, wherein the CLL1 antibody or an antigen binding site thereof comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO:56 or SEQ ID NO:58, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO:57 or SEQ ID NO: 59.

Description

CLL1 antibody and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a CLL1 antibody and application thereof.

Background

Acute myeloid leukemia (Acute Myeloid Leukemia, AML) is a malignant disease of myeloid hematopoietic stem/progenitor cells. The method is mainly characterized by abnormal proliferation of primary and naive marrow system cells in marrow and peripheral blood, and clinically manifests as anemia, hemorrhage, infection, fever, viscera infiltration, metabolism abnormality and the like, and most cases are critical in illness state and dangerous in prognosis, and life can be endangered if not treated in time. AML is more likely to occur in infants than adults, and the disease accounts for 30% of infantile leukemia. The current treatment methods are: chemotherapy, supportive treatment, hematopoietic stem cell transplantation.

Chimeric antigen receptors (Chimeric Antigen Receptor, CARs) are core components of CAR cell therapeutics that can include an antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. Up to now, antigen recognition domains are derived from the single chain variable region of antibodies (Single Chain Variable Fragment, abbreviated scFv), or from receptor ligand interactions, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR); the most common source of these is scFv antibodies. CAR-T cell immunotherapy is considered one of the most promising approaches to combat tumors. The CAR-T cells are characterized in that the T cells express CAR proteins by using a genetic modification method, and the CAR proteins have the capability of recognizing complete proteins on the surface of a membrane under the condition of not depending on antigen presentation, thereby causing activation and functional effects of the T cells. Currently, CAR-T cell immunotherapy has achieved significant success in the treatment of a variety of hematological tumors, such as: CD19 CAR-T for the treatment of B cell lymphomas and BCMA CAR-T for the treatment of multiple myeloma have been marketed. However, due to the heterogeneity of AML, it is more difficult to find ideal CAR-T targets for the treatment of AML, existing targets for AML are CD33, CD123, leY, NKG2D, etc., but none of these targets have marketed CAR-T drugs.

The research shows that CLL1 (C-type Lectin-like Molecule-1) is an ideal target with the prospect of treating AML, because CLL1 is not expressed in normal hematopoietic stem cells and is highly expressed in AML primitive cells and leukemia stem cells, the search for antibodies which take CLL1 as a target and are suitable for medicines, especially antibodies which take CLL1 as a target and are suitable for CAR-T cell treatment medicines has practical significance.

Disclosure of Invention

The application provides a CLL1 antibody and application thereof, the inventor develops a plurality of antibodies targeting CLL1, takes corresponding scFv antibodies as extracellular antigen recognition domains of a CLL1CAR structure, constructs a chimeric antigen receptor expression vector, prepares a CAR-T cell targeting CLL1, verifies a plurality of indexes of the CLL1CAR and the CLL1 CAR-T cell at the cell level, and selects 3 antibodies with best performance.

A CLL1 antibody or antigen binding portion thereof, comprising a heavy chain variable region having amino acid sequences of CDR1, CDR2, CDR3 that are identical to the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the heavy chain variable region of the antibody shown in SEQ ID No. 54, and a light chain variable region having amino acid sequences of CDR1, CDR2, CDR3 that are identical to the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the light chain variable region of the antibody shown in SEQ ID No. 55;

Or, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO. 56, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO. 57;

or, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody shown in SEQ ID NO. 58, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody shown in SEQ ID NO. 59.

A CLL1 antibody or antigen binding portion thereof, comprising a heavy chain variable region, the amino acid sequences of CDR1, CDR2, CDR3 of the heavy chain variable region comprising the amino acid sequences shown as SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, respectively, and a light chain variable region, the amino acid sequences of CDR1, CDR2, CDR3 comprising the amino acid sequences shown as SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, respectively;

Or, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47;

or, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 48, SEQ ID NO. 49 and SEQ ID NO. 50, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53.

In certain embodiments, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 54 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 55;

or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 56, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 57;

alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 58 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 59.

The CLL1 antibody or antigen binding portion thereof is, in some embodiments, a CLL1 scFv antibody, CLL1Sc (Fv) ₂ Antibodies, CLL1[ Sc (Fv) ₂ ] ₂ An antibody.

In certain embodiments, the CLL1 scFv antibody or antigen binding portion thereof comprises any one of the sequences selected from the group consisting of seq id nos: an amino acid sequence-linker sequence shown as SEQ ID NO. 54-an amino acid sequence shown as SEQ ID NO. 55-an amino acid sequence-linker sequence shown as SEQ ID NO. 55-an amino acid sequence shown as SEQ ID NO. 54-an amino acid sequence-linker sequence shown as SEQ ID NO. 56-an amino acid sequence shown as SEQ ID NO. 57-an amino acid sequence-linker sequence shown as SEQ ID NO. 57-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence-linker sequence shown as SEQ ID NO. 58-an amino acid sequence shown as SEQ ID NO. 59-an amino acid sequence shown as SEQ ID NO. 58. Optionally, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id no: an amino acid sequence-linker sequence shown as SEQ ID NO. 55-an amino acid sequence shown as SEQ ID NO. 54-an amino acid sequence-linker sequence shown as SEQ ID NO. 57-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence-linker sequence shown as SEQ ID NO. 59-an amino acid sequence shown as SEQ ID NO. 58; further alternatively, the CLL1 scFv antibody comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 14.

In some embodiments, the binding sequence of any one of the CLL1 antibodies or antigen binding portions thereof is selected from one or more of the following: SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68.

The application also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the CLL1 antibody or antigen binding portion thereof described above.

In certain embodiments, the isolated nucleic acid molecule described above comprises a nucleotide sequence encoding the CLL1 antibody or antigen binding portion thereof described above:

1) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 54, optionally as SEQ ID NO. 60; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 55, optionally as set forth in SEQ ID NO. 61; or (b)

2) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 56, optionally as shown as SEQ ID NO. 62; and a nucleotide sequence encoding an amino acid sequence of a light chain variable region as set forth in SEQ ID NO. 57, optionally as set forth in SEQ ID NO. 63; or (b)

3) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as set forth in SEQ ID NO. 58, optionally as set forth in SEQ ID NO. 64; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 59, optionally as set forth in SEQ ID NO. 65.

The application also provides a vector comprising the isolated nucleic acid molecule described above.

The application also provides a cell comprising any of the CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules or vectors described above.

The application also provides a pharmaceutical composition comprising any one of the above CLL1 antibodies or antigen binding portions thereof, an isolated nucleic acid molecule, a vector or a cell, and pharmaceutically acceptable excipients.

The application also provides an antibody medicament comprising any one of the CLL1 antibodies or antigen binding portions thereof.

In certain embodiments, the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.

The application also provides an antibody drug conjugate comprising any of the CLL1 antibodies or antigen binding sites thereof described above.

The application also provides an application of the CLL1 antibody or an antigen binding portion thereof, an isolated nucleic acid molecule, a vector or a cell in preparing a medicament for treating diseases or symptoms related to the expression of CLL 1.

In certain embodiments, the disease or disorder associated with expression of CLL1 is a hematological neoplasm.

In certain embodiments, the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.

The application also provides application of the CLL1 antibody or antigen binding portion thereof, an isolated nucleic acid molecule, a vector or a cell in preparing a detection reagent for diagnosing diseases or symptoms related to the expression of the CLL 1.

The application also provides a method of treating a disease or disorder associated with expression of CLL1, comprising the steps of: administering an effective amount of a medicament comprising any one of the CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells described above to a subject in need of treatment for a disease or disorder associated with expression of CLL 1.

The application also provides a medicament comprising the CLL1 antibody or antigen binding portion thereof, an isolated nucleic acid molecule, a vector or a cell as described above for treating a disease or disorder associated with expression of CLL 1.

Drawings

Fig. 1 shows a schematic diagram of the structure of various CLL1 CARs in embodiment 2 of the present application, in which from top to bottom: CD8a signal peptide, anti-CLL 1 scFv, CD8a hinge and transmembrane regions, 4-1BB, CD3 ζ.

FIG. 2 shows groups C after antigen stimulation in example 5 of the present applicationCD3 in AR-T cells ⁺ Continuous proliferation of cells.

FIGS. 3A-3C show the lytic killing effect of various CLL1CAR-T cells, positive control M26CLL 1CAR-T cells in example 6 of the application on different targets; wherein: the target cell in FIG. 3A is HL60; the target cell in FIG. 3B is K562-CLL1; in FIG. 3C the target cell is K562.

FIG. 4 shows cytokine IFN-. Gamma.release after activation of various CLL1CAR-T cells, positive control M26CLL 1CAR-T cells, UTD cells (non-CAR transduced T cells) by positive target cells in example 7 of the present application.

Detailed Description

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

The application is further described below: in the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present application, definitions and explanations of related terms are provided below.

In the present application, the term "antibody" has the meaning conventional in the art and refers to an immunoglobulin molecule composed of four polypeptide chains, which refer to two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. By analyzing the amino acid sequences of the heavy and light chains of different antibodies, it was found that the amino acid sequences of the heavy and light chains near the N-terminus varied widely, with the other portions of the amino acid sequences being relatively constant. Thus, regions of the antibody light and heavy chains that vary greatly near the N-terminal amino acid sequence are referred to as variable regions (V), regions near the C-terminal amino acid sequence are referred to as constant regions (C), V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and C regions of the heavy and light chains are abbreviated as CH and CL, respectively. The variation of a small number of amino acid residues within the variable region of an antibody is particularly intense, and the regions of variation in the amino acid residue composition and sequence are more prone to occur are called hypervariable regions (hypervariable regions, HVR); three hypervariable regions are present in each of the V regions of the L and H chains, and these regions are also called complementarity determining regions (complementarity determining region, CDRs) because they are spatially complementary to an epitope. In antibodies, there are Kabat, abM, chothia, contact, IMGT rules for CDR partitioning, which are well known to those skilled in the art, and when a website for executing these rules is applied, the VH and VL sequences are simply inputted and the corresponding rules are selected, so that CDR sequences according to the different rules can be obtained. It will be appreciated by those skilled in the art that the scope of the present application encompasses combinations of CDR sequences obtained by analysis using different rules. The 6 CDR regions of an antibody together determine the ability and specificity of the antibody to recognize the corresponding antigen. It will be appreciated by those skilled in the art that when the application defines an amino acid sequence of 6 CDR regions, the ability of an antibody to recognize and to specifically identify the corresponding antigen is contemplated.

In the present application, the term "antigen binding site" has the meaning conventional in the art and refers to a critical site on an antibody that can specifically recognize and bind an antigen, including VH and VL regions.

In the present application, the term "monoclonal antibody" has the meaning conventional in the art, and refers to an antibody which is highly homogeneous and directed against only a specific epitope, and which can be prepared by known techniques such as hybridoma technique, antibody library technique, transgenic mouse technique or single cell PCR technique.

In the present application, the term "scFv" has the meaning conventional in the art, and refers to a single chain variable region (Single Chain Variable Fragment, abbreviated as scFv) which is an antibody formed by connecting a heavy chain variable region and a light chain variable region of an antibody through a short peptide (linker).

In the present application, the term "Sc (Fv) ₂ 、[Sc(Fv) ₂ ] ₂ "and like terms not specifically explained also have the meaning conventional in the art.

In the present application, the term "chimeric antigen receptor" (Chimeric Antigen Receptor, CAR) is a core component of CAR cell therapeutics that can include an extracellular antigen recognition domain (e.g., a portion that binds a Tumor-associated antigen (Tumor-Associated Antigen, TAA)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cellular immunotherapy is considered one of the most promising means to combat tumors. The CAR-T cells are characterized in that the T cells express CAR proteins by using a genetic modification method, and the CAR proteins have the capability of recognizing complete proteins on the surface of a membrane under the condition of not depending on antigen presentation, thereby causing activation and functional effects of the T cells.

In the present application, the term "extracellular antigen recognition domain" refers to an antigen recognition domain (Antigen Recognition Domain, ARD). CAR cell therapy products (e.g. CAR-T cells) are capable of specifically recognizing and/or binding to target antigens expressed by tumor cells, relying on extracellular antigen recognition domains, which to date have been derived from single chain variable regions of antibodies (Single Chain Variable Fragment, abbreviated scFv), or from receptor ligand interactions, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR). By far the most common source is the scFv fragment of an antibody, the scFv comprising an antibody heavy chain variable region and a light chain variable region, linked by a peptide chain, such as: a linker sequence GSTSGSGKPGSGEGSTKG consisting of 18 amino acids.

In the present application, the term "specific recognition and/or binding" refers to recognition and/or binding between a CAR and a specific target, which is binding to the target with greater affinity, avidity, ease, and/or with greater duration than the CAR binds to other targets.

In the present application, the term "hinge region" refers to a junction between an extracellular antigen recognition domain and a transmembrane domain that allows the CAR to recognize an antigen by imparting a range of motion to the antigen recognition domain. The hinge regions currently in use are derived primarily from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a. In addition, typical hinge regions also contain residues that are involved in CAR dimerization, helping to enhance antigen sensitivity.

In the present application, a "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but are not directly involved in signaling, and downstream signaling can be enhanced by interactions. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70.

Within the present application, the term "intracellular domain" includes intracellular signaling regions and may also include costimulatory signaling regions.

In the present application, the term "intracellular signaling region" refers to activation of at least one normal effector function of an immune effector cell responsible for expressing a CAR. The intracellular signaling region may be derived from one or more of cd3ζ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, fcrγ, fcrβ, CD66d, DAP10, DAP12, syk.

In the present application, the term "costimulatory signaling region" exists because many immune effector cells require costimulation in addition to stimulation of antigen-specific signals to promote cell proliferation, differentiation and survival, as well as activating effector functions of the cells. In some embodiments, the CAR may further comprise one or more costimulatory signaling regions, wherein the costimulatory signaling regions may be derived from one, two, or more than three of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, myD 88.

In the present application, the term "leader peptide" refers to a short peptide preceding an extracellular antigen recognition domain (e.g., scFv sequence) that functions to direct the export of an intracellular synthesized recombinant protein to the outside of the cell. Typical leader peptides are the human CD 8. Alpha. Signal peptide, or the human GM-CSF receptor. Alpha. Signal peptide.

In the present application, one of the key factors determining the efficacy of CAR-immune cell therapy is the selection of tumor target antigens. In the present application, the tumor target antigen "CLL1" selected has the meaning conventional in the art, which is also referred to as KLR1, CLEC12A, which is a type II transmembrane glycoprotein, a member of the large family of C-type lectin-like receptors involved in immunomodulation. It was found that CLL1 (C-type Lectin-like molecular 1) is an ideal target for the treatment of AML, since CLL1 is not expressed in normal hematopoietic stem cells, and is highly expressed in AML primitive cells and leukemia stem cells.

In the present application, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any structure/region of the chimeric antigen receptor of the present application. The linking sequences may be composed of different amino acid residues (e.g., glycine and serine) so that adjacent protein domains are free to move relative to each other. Longer linking sequences may be used when it is desired to ensure that two adjacent domains do not spatially interfere with each other.

In the present application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude substances which have been obtained from natural sources by artificial means, either by man or by synthesis, nor does it exclude the presence of other impure substances which do not affect the activity of the substance.

In the present application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, which may be isolated from the natural environment or an artificially synthesized analog thereof.

In the present application, the CAR gene transduction/transfection and target gene expression methods mainly include viral and nonviral methods. Such as: gene transfer by gamma retrovirus vector, lentiviral vector, adenovirus-associated virus vector, plasmid DNA-dependent vector, transposon-dependent gene transfer, mRNA-mediated gene transduction.

The term "vector" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein may be inserted and the protein expressed. The vector may be transformed, transduced or transfected into a host cell to allow expression of the genetic material elements carried thereby within the host cell. For example, the carrier comprises: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these. The term "transposon" refers to a discrete piece of DNA that has the ability to migrate and carry genetic information between chromosomal sites, such as: sleep beauty SB system and PB system derived from lepidopteran insects. In some embodiments, mRNA can also be transduced into T cells using electrotransformation methods.

In the present application, the term "immune effector cell" generally refers to a cell involved in an immune response, e.g., promoting an immune effector response. The immune effector cells may be selected from the group consisting of: t lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T lymphocytes into which pluripotent stem cells differentiate, NK cells into which pluripotent stem cells differentiate, and embryonic stem cells.

In the present application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may comprise the immune effector cells described herein, and may further comprise one or more pharmaceutically acceptable excipients, such as: one or more of a carrier, a protective agent, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative. In some embodiments, pharmaceutically acceptable excipients include protective agents such as: cell cryopreservation solution. In some embodiments, the pharmaceutical composition of the application is a cell suspension or cryopreserved cells thereof.

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

In the present application, the term "comprising" is generally intended to include the explicitly specified features, but not to exclude other elements.

In the present application, the term "about" generally refers to a range of fluctuation acceptable to those skilled in the art above or below the specified value, such as: varying within a range of + -0.5% -10%, for example within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

CLL1 antibodies or antigen binding sites thereof, corresponding nucleic acid molecules, corresponding vectors, corresponding cells, corresponding pharmaceutical compositions

In one aspect, the application provides a CLL1 antibody or antigen binding portion thereof, comprising a heavy chain variable region having amino acid sequences of CDR1, CDR2, CDR3 that are the same as the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the heavy chain variable region of the antibody shown in SEQ ID No. 54, and a light chain variable region having amino acid sequences of CDR1, CDR2, CDR3 that are the same as the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the light chain variable region of the antibody shown in SEQ ID No. 55;

In antibodies, there are Kabat, abM, chothia, contact, IMGT common rules for CDR partitioning, which are well known to those skilled in the art, and when a website for executing these rules is applied, the VH and VL sequences are simply inputted and the corresponding rules are selected, so that CDR sequences according to the different rules can be obtained. It will be appreciated by those skilled in the art that even for the same heavy and light chain variable regions, different CDR sequences may be obtained according to different CDR partitioning rules. It will be appreciated by those skilled in the art that the scope of the present application encompasses combinations of CDR sequences obtained by analysis using different rules.

In another aspect, the present application also provides a CLL1 antibody or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of CDR1, CDR2, CDR3 of the heavy chain variable region comprise the amino acid sequences shown as SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38, respectively, and the amino acid sequences of CDR1, CDR2, CDR3 of the light chain variable region comprise the amino acid sequences shown as SEQ ID No. 39, SEQ ID No. 40, SEQ ID No. 41, respectively;

In the present application, the KABAT rule is used for CDR partitioning.

In some embodiments, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 54 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 55;

According to the application, 3 antibodies with best performance, namely 1-3, 1-28 and 3-67 antibodies, are screened through experiments. Wherein, the 1-3scFv antibody comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 54 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 55; 1-28scFv antibody comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 56 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 57; the 3-67scFv antibody comprises a heavy chain variable region with an amino acid sequence shown as SEQ ID NO. 58 and a light chain variable region with an amino acid sequence shown as SEQ ID NO. 59.

Any of the above-described CLL1 antibodies, or antigen binding portions thereof, in some embodiments, are CLL1 scFv antibodies, CLL1 Sc (Fv) ₂ Antibodies, CLL1[ Sc (Fv) ₂ ] ₂ An antibody.

In some embodiments, any one of the CLL1 antibodies or antigen binding portions thereof described above, the CLL1 scFv antibody comprises any one selected from the group consisting of: an amino acid sequence-linker sequence shown as SEQ ID NO. 54-an amino acid sequence shown as SEQ ID NO. 55-an amino acid sequence-linker sequence shown as SEQ ID NO. 55-an amino acid sequence shown as SEQ ID NO. 54-an amino acid sequence-linker sequence shown as SEQ ID NO. 56-an amino acid sequence shown as SEQ ID NO. 57-an amino acid sequence-linker sequence shown as SEQ ID NO. 57-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence-linker sequence shown as SEQ ID NO. 58-an amino acid sequence shown as SEQ ID NO. 59-an amino acid sequence shown as SEQ ID NO. 58. Optionally, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id no: an amino acid sequence-linker sequence shown as SEQ ID NO. 55-an amino acid sequence shown as SEQ ID NO. 54-an amino acid sequence-linker sequence shown as SEQ ID NO. 57-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence-linker sequence shown as SEQ ID NO. 59-an amino acid sequence shown as SEQ ID NO. 58; further alternatively, the CLL1 scFv antibody comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 14. In the above description, "-" means an interconnection, and in the above description "-" has directionality, which means an interconnection from the N-terminal to the C-terminal of an amino acid.

In yet another aspect, the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any one of the CLL1 antibodies or antigen binding portions thereof described above.

In some embodiments, the isolated nucleic acid molecule described above comprises a nucleotide sequence encoding the CLL1 antibody or antigen binding portion thereof described above:

In yet another aspect, the application also provides a vector comprising the isolated nucleic acid molecule described above. The carrier may be optionally selected from one or more of the following: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40).

In yet another aspect, the application provides a cell comprising the CLL1 antibody or an antigen-binding portion thereof, an isolated nucleic acid molecule or a vector as described above.

In yet another aspect, the application also provides a pharmaceutical composition comprising any of the above CLL1 antibodies or antigen binding portions thereof, an isolated nucleic acid molecule, a vector or a cell, and a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable excipients include, but are not limited to: one or more of a carrier, a protective agent, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative.

Antibody drug comprising CLL1 antibody or antigen binding site thereof, and antibody drug conjugate

In one aspect, the application provides an antibody medicament comprising any one of the CLL1 antibodies or antigen binding sites thereof described above.

In some embodiments, the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.

In another aspect, the application also provides an antibody drug conjugate comprising any one of the CLL1 antibodies or antigen binding sites thereof described above.

Use of CLL1 antibodies or antigen binding sites thereof

In one aspect, the application provides the use of any one of the above CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells for the manufacture of a medicament for the treatment of a disease or disorder associated with expression of CLL 1.

In some embodiments, the disease or disorder associated with expression of CLL1 is a hematological neoplasm.

In some embodiments, the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.

In another aspect, the application also provides the use of any one of the above CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells for the preparation of a detection reagent for diagnosing a disease or disorder associated with expression of CLL 1.

In yet another aspect, the application provides a method of treating a disease or disorder associated with expression of CLL1, comprising the steps of: administering an effective amount of a medicament comprising any one of the CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells described above to a subject in need of treatment for a disease or disorder associated with expression of CLL 1.

In some embodiments, the administration may be performed by different means, such as oral, intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

In some embodiments, the dosage administered may be different for different indications; the dosage may also vary for patients with varying severity of the condition.

In some embodiments, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

In yet another aspect, the application provides a medicament comprising any one of the CLL1 antibodies or antigen binding portions thereof, an isolated nucleic acid molecule, a vector or a cell described above for use in treating a disease or disorder associated with expression of CLL 1.

CLL1 chimeric antigen receptor, corresponding nucleic acid molecule, corresponding vector, corresponding immune effector cell and pharmaceutical composition

In one aspect, the application provides a CLL 1-targeted chimeric antigen receptor comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, the CLL1 extracellular antigen recognition domain comprising a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein:

the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO. 54, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody light chain variable region shown in SEQ ID NO. 55;

Or, the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO. 56, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody light chain variable region shown in SEQ ID NO. 57;

or, the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO. 58, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody light chain variable region shown in SEQ ID NO. 59.

In another aspect, the application also provides a chimeric antigen receptor that targets CLL1, comprising a CLL1 extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, the CLL1 extracellular antigen recognition domain comprising a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein:

the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 36, SEQ ID NO. 37 and SEQ ID NO. 38, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 39, SEQ ID NO. 40 and SEQ ID NO. 41;

Or, the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47;

or, the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 48, SEQ ID NO. 49 and SEQ ID NO. 50, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 51, SEQ ID NO. 52 and SEQ ID NO. 53.

In some embodiments, the amino acid sequence of the CLL1 heavy chain variable region comprises an amino acid sequence set forth in SEQ ID No. 54 and the CLL light chain variable region sequence comprises an amino acid sequence set forth in SEQ ID No. 55;

or, the amino acid sequence of the CLL1 heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 56, and the amino acid sequence of the CLL light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 57;

alternatively, the amino acid sequence of the CLL1 heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 58, and the amino acid sequence of the CLL light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 59.

In some embodiments, any of the chimeric antigen receptors described above, the CLL1 extracellular antigen recognition domain comprises any one selected from the following structures: an amino acid sequence-linker sequence shown as SEQ ID NO. 54-an amino acid sequence shown as SEQ ID NO. 55-an amino acid sequence-linker sequence shown as SEQ ID NO. 55-an amino acid sequence shown as SEQ ID NO. 54-an amino acid sequence-linker sequence shown as SEQ ID NO. 56-an amino acid sequence shown as SEQ ID NO. 57-an amino acid sequence-linker sequence shown as SEQ ID NO. 57-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence-linker sequence shown as SEQ ID NO. 58-an amino acid sequence shown as SEQ ID NO. 59-an amino acid sequence shown as SEQ ID NO. 58. Optionally, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id no: an amino acid sequence-linker sequence shown as SEQ ID NO. 55-an amino acid sequence shown as SEQ ID NO. 54-an amino acid sequence-linker sequence shown as SEQ ID NO. 57-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence-linker sequence shown as SEQ ID NO. 59-an amino acid sequence shown as SEQ ID NO. 58; further alternatively, the CLL1 scFv antibody comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2 or SEQ ID NO. 14.

In some embodiments, the linking sequence is selected from one or more of the following: SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68.

In some embodiments, the hinge region is derived from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a; alternatively, the amino acid of the hinge region is derived from CD8 a; further alternatively, the amino acid sequence of the hinge region comprises the amino acid sequence shown as SEQ ID NO. 17.

In some embodiments, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70; alternatively, the amino acid sequence of the transmembrane region is derived from CD8 a; further alternatively, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown as SEQ ID NO. 18.

In some embodiments, any of the above chimeric antigen receptors, wherein the intracellular domain comprises an intracellular signaling region; optionally, a costimulatory signaling region is also included.

Any of the above chimeric antigen receptors, in some embodiments, wherein the intracellular signaling region is derived from one or more of cd3ζ, cd3γ, cd3δ, cd3ε, CD5, CD22, CD79a, CD79b, fcrγ, fcrβ, CD66d, DAP10, DAP12, syk; alternatively, the intracellular signaling region is derived from cd3ζ; further alternatively, the amino acid sequence of the intracellular signaling region comprises the amino acid sequence shown as SEQ ID NO. 20.

Any of the chimeric antigen receptors described above, in some embodiments, wherein the costimulatory signaling region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP, B7-H3, myD 88; optionally, the costimulatory signaling region is derived from CD28 or 4-1BB; further alternatively, the amino acid sequence of the costimulatory signaling region comprises the amino acid sequence depicted as SEQ ID NO. 19.

Any of the above chimeric antigen receptors, in some embodiments, further comprises a leader peptide located N-terminal to the amino acid sequence of the chimeric antigen receptor; optionally, wherein the guide peptide is derived from CD8 a; further alternatively, the amino acid sequence of the leader peptide comprises the amino acid sequence shown as SEQ ID NO. 16.

In some embodiments, any of the chimeric antigen receptors described above comprises an amino acid sequence as set forth in SEQ ID NO. 21, SEQ ID NO. 22, or SEQ ID NO. 34.

In yet another aspect, the application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding any of the chimeric antigen receptors described above.

In some embodiments, the isolated nucleic acid molecule described above, the nucleotide sequence encoding the chimeric antigen receptor comprises:

1) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of CLL1 as shown in SEQ ID No. 54, optionally as shown in SEQ ID No. 60; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as set forth in SEQ ID NO. 55, optionally as set forth in SEQ ID NO. 61; or (b)

2) A nucleotide sequence encoding the amino acid sequence of the CLL1 heavy chain variable region as set forth in SEQ ID NO. 56, optionally as set forth in SEQ ID NO. 62; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as set forth in SEQ ID NO. 57, optionally as set forth in SEQ ID NO. 63;

3) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of CLL1 as shown in SEQ ID No. 58, optionally as shown in SEQ ID No. 64; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as set forth in SEQ ID NO. 59, optionally as set forth in SEQ ID NO. 65.

In yet another aspect, the application also provides a vector comprising the isolated nucleic acid molecule described above. The carrier can be any one or more of the following: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40).

The above-described vector is, in some embodiments, an expression vector; in other embodiments, the vector is a viral vector; in other embodiments, the vector is a lentiviral vector.

In yet another aspect, the application provides an engineered immune effector cell comprising the chimeric antigen receptor, the isolated nucleic acid molecule, or the vector.

In some embodiments, the engineered immune effector cell is selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells into which pluripotent stem cells differentiate, NK cells into which pluripotent stem cells differentiate, and embryonic stem cells.

In some embodiments, the engineered immune effector cell is a T lymphocyte; alternatively, the source of the T lymphocytes is autologous T lymphocytes or allogeneic T lymphocytes.

The engineered immune effector cells described above, in some embodiments, are alpha beta T lymphocytes or gamma delta T lymphocytes of T lymphocytes.

In some embodiments, the engineered immune effector cells described above may express or express a chimeric antigen receptor of the application on the surface of the engineered immune effector cells.

In yet another aspect, the application also provides a pharmaceutical composition comprising the engineered immune effector cell described above and a pharmaceutically acceptable adjuvant. Pharmaceutically acceptable excipients include, but are not limited to: one or more of a carrier, a protective agent, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutically acceptable adjuvant comprises a cell cryopreservation solution.

In some embodiments, the pharmaceutical composition is a cell suspension or cryopreserved cells thereof.

In some embodiments, the pharmaceutical composition is an intravenous injection.

Preparation method and application

In one aspect, the application also provides a method of preparing an engineered immune effector cell comprising the steps of: transduction of the vectors of the application into immune effector cells.

In some embodiments, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), pluripotent stem cells, T cells into which pluripotent stem cells differentiate, NK cells into which pluripotent stem cells differentiate, and embryonic stem cells.

In some embodiments, the T lymphocytes are αβ T lymphocytes or γδ T lymphocytes.

In another aspect, the application also provides the use of the chimeric antigen receptor, isolated nucleic acid molecule, vector or engineered immune effector cell described above for the preparation of a medicament for the treatment of a disease or disorder associated with expression of CLL 1.

In yet another aspect, the application provides a method of treating a disease or disorder associated with expression of CLL1, comprising the steps of: an effective amount of the engineered immune effector cells or pharmaceutical composition described above is administered to a subject in need of treatment for a disease or disorder associated with expression of CLL 1.

In some embodiments, the administration may be performed by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. For example, the mode of administration may be administered to the subject by intravenous injection. In some embodiments, an effective dose of the engineered immune effector cell or pharmaceutical composition may be administered to the subject in a single administration, or may be administered to the subject in separate administrations over a period of time, such as: once a week, once two weeks, once three weeks, once four weeks, once a month, once 3 months, or once 3-6 months.

In some embodiments, the method is an intravenous injection.

In some embodiments, the method is a method of administering an effective amount of the engineered immune effector cell or pharmaceutical composition to a subject in a single injection.

In some embodiments, the effective amount of the engineered immune effector cell or pharmaceutical composition is 1×10 ⁵ Up to 1X 10 ⁷ A dose of individual cells/kg. In some embodiments, the dosage administered may be different for different indications; the dosage may also vary for patients with varying severity of the condition. The dosage range of administration can be 1×10 ⁵ Individual CAR positive T cells/kg to 1×10 ⁷ Individual CAR positive T cells/kg, e.g., 1×10 ⁵ Individual CAR positive T cells/kg to 1×10 ⁶ CAR positive T cells/kg, 1X 10 ⁶ Individual CAR positive T cells/kg to 1×10 ⁷ CAR positive T cells/kg, 0.5X10 ⁶ CAR positive T cells/kg, 0.6X10 ⁶ CAR positive T cells/kg, 0.7X10 ⁶ CAR positive T cells/kg, 0.8X10 ⁶ CAR positive T cells/kg, 0.9X10 ⁶ CAR positive T cells/kg, 1.0X10 ⁶ CAR positive T cells/kg, 1.1X10 ⁶ CAR positive T cells/kg, 1.2X10 ⁶ CAR positive T cells/kg, 1.3X10 ⁶ CAR positive T cells/kg, 1.4X10 ⁶ Individual CAR positive T-finesCell/kg, 1.5X10 ⁶ CAR positive T cells/kg, 1.6X10 ⁶ CAR positive T cells/kg, 1.7X10 ⁶ CAR positive T cells/kg, 1.8X10 ⁶ CAR positive T cells/kg, 1.9X10 ⁶ CAR positive T cells/kg, 2.0X10 ⁶ Each CAR positive T cell/kg.

In yet another aspect, the application also provides a medicament comprising an engineered immune effector cell or pharmaceutical composition as described above for treating a disease or disorder associated with expression of CLL 1.

Without intending to be limited by any theory, the following examples are merely illustrative of CLL1 antibodies, chimeric antigen receptors, engineered immune effector cells, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the application. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: A Laboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press.

Example 1 CLL1 antibody development

CLL1 antibody development procedure: immunization was performed using BALB/C and C57bl/6 mice as immunized animals and human CLL1 as antigen according to the procedure in Table 1. And (3) performing FACS (flow cytometry fluorescence sorting technology) and ELISA detection on serum of each immunized mouse, combining the results of ELISA detection and FACS detection, taking the mouse with the best immune effect, enriching the plasma cells of the mouse by using CD138 antibodies, then paving the mouse on a Beacon screening chip, and analyzing and screening antibody positive B cell strains to obtain monoclonal cell 8561 strains and polyclonal cell 1511 strains, wherein the antibodies are 690 strains of IgG. According to the binding condition of antigen, positive B cell clone is exported, V region sequencing is carried out, the V region sequences of 64 monoclonal antibodies are obtained, and 14 representative sequences are selected for synthesizing scFv in CAR according to sequence similarity classification.

TABLE 1 animal immunization program

EXAMPLE 2 acquisition of CLL1 CAR-T cells

We obtained 14 candidate scFv targeting CLL1, the candidate scFv sequence numbers and their sequences are shown in table 2, respectively. We also obtained 1 CLL1 positive control antibody M26 (which was derived from Treatmen of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type section-like molecular 1,Molecular Therapy Vol.25No.9September 2017)

TABLE 2, 14 candidate scFv sequence numbers targeting CLL1 and sequences thereof

scFv sequence number	scFv sequence	scFv sequence number	scFv sequence
				1-3	SEQ ID NO:1	3-11	SEQ ID NO:8
1-28	SEQ ID NO:2	3-13	SEQ ID NO:9
				1-16	SEQ ID NO:3	3-16	SEQ ID NO:10
2-8	SEQ ID NO:4	3-35	SEQ ID NO:11
				2-25	SEQ ID NO:5	3-37	SEQ ID NO:12
2-31	SEQ ID NO:6	3-64	SEQ ID NO:13
				2-40	SEQ ID NO:7	3-67	SEQ ID NO:14
M26 (Positive control antibody)	SEQ ID NO:15

We selected to screen 14 candidate scFv on the second generation CAR structure. In each CAR structure, a CD8 alpha guide chain is adopted as a signal peptide (shown as SEQ ID NO: 16), a hinge region (shown as SEQ ID NO: 17) and a transmembrane region (shown as SEQ ID NO: 18) adopt a CD8 alpha structure, 4-1BB is adopted as an intracellular co-stimulatory signal (shown as SEQ ID NO: 19), and CD3 zeta is adopted as a T cell activating signal (shown as SEQ ID NO: 20), and the structural schematic diagram is shown in FIG. 1.

1. Construction of lentiviral vectors

Based on the sequences of the candidate scFv and the CAR structural assembly in Table 1, 14 CLL1 CAR structures and 1M 26 CLL1 CAR structures were synthesized, and the amino acid sequences thereof are shown in Table 3.

Tables 3 and 14 CLL1 CAR sequence numbers and sequences thereof

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The above 15 CLL1 CAR constructs were respectively constructed into modified empty lentiviral vectors (manufacturer: SBI company, cat# CD500-CD800, modified resistance as described in example 1 of WO 2021/121227) to obtain CAR expression vectors, and then the CAR expression vectors and three packaging plasmids were transfected together into 293T cells, and after collection and purification, functional lentiviral vectors were obtained. The three packaging plasmids were pMD2.G (from Biovector, product number Biovector 012259), pMDLg/pRRE (from Biovector, product number Biovector 012251), pRSV-Rev (from Biovector, product number Biovector 012253), respectively.

2. The corresponding 15 CLL1 specific CAR-T cells were prepared by lentiviral transduction.

Transduction experiments were performed according to conventional methods known to those skilled in the art, and the brief transduction procedure is as follows:

1) Sorting T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human apheresis cells, and T cells were then isolated from PBMC cells.

2) Activation of T cells

The isolated T cells were resuspended in T cell complete medium (X-VIVO 15 medium+5% FBS+300IU/ml IL-2 or X-VIVO15 medium+5% FBS+5ng/ml IL-15+10ng/ml IL-7) to a final concentration of (1-3). Times.10 ⁶ Each cell/ml, 1ul beads/1X 10 ⁶ Adding CD3/CD28 magnetic beads into cells, stimulating, mixing, and culturing in incubator at 37deg.C+5% CO ₂ The incubation time is at least 24 hours.

3) Lentivirus transduced T cells

The T cells which activated the culture were removed, resuspended in pure X-VIVO15 medium containing polybrene at a final concentration of 8. Mu.g/ml, and the cell suspension was obtained by mixing the suspension with a concentration of 2X 10 per 800ul of cell suspension ⁶ Cells) were slowly added with 200ul of lentiviral vector, mixed well, placed in an orifice plate, and cultured in an incubator at 37 ℃ +5% CO ₂ The incubation time is at least 4-6 hours.

4) Expanded culture of transduced T cells

Taking out the transduced cells, amplifying and culturing the transduced cells by using a T cell complete culture medium, and carrying out passage every other day to maintain the cell density to be (0.8-2) multiplied by 10 ⁶ Individual cells/ml for use in subsequent examples.

After T cells were infected with lentiviruses each comprising the CAR structure of table 3, the T cells obtained were each named for their CAR numbers, e.g., T cells obtained using 1-3 CARs, i.e., named 1-3CAR-T cells, and T cells obtained using 2-8 CARs, i.e., named 2-8CAR-T cells. Next, we screened 14 CLL1CAR structures at the cellular level to determine the merits of each scFv.

Example 3 detection of CLL1 CAR-T cell surface expressed CAR molecules and detection of their antigen recognition Capacity

1. Detection of CAR molecules: 14 CLL1 CAR-T cells obtained in example 2 were subjected to surface-expressed CAR protein molecules on the surface of cultured for 7 days after transduction, and were stained with PE fluorescent-labeled CLL1 antigen (manufacturer: ACRObiosystems, cat. No. CLA-PH2Q 3) and FITC fluorescent-labeled CLL1 antigen (manufacturer: ACRObiosystems, cat. No. CLA-HF247 25 ug), and the 14 CLL1 CAR-T cells, M26 CLL1 CAR-T cells and UTD cells obtained in example 2 (non-CAR-transduced T cells) were subjected to detection analysis by flow cytometry, and the detection results are shown in Table 4.

2. Detection of antigen recognition ability of CAR molecules: antigen recognition capability assay was performed on 14 CLL1 CAR-T cell surface-expressed CAR protein molecules obtained in example 2, and 14 CLL1 CAR-T cells, M26 CLL1 CAR-T cells and UTD cells (non-CAR-transduced T cells) obtained in example 2 were stained with FITC fluorescent-labeled IgG (manufacturer: abcam, cat# ab 98658), and antigen recognition capability assay was performed by flow cytometry, and the assay results are shown in Table 4.

As shown in table 4: in grey-shaded cells, CAR molecules are rejected in the screen due to poor expression and/or poor antigen recognition, wherein: 2-25, 2-40, 3-35; 3-11, 3-13, 3-16, but the ability to recognize antigen is poor, these cells were all discarded in later experiments.

Table 4 results of detection of cell surface expressed CAR molecules and results of detection of antigen recognition ability thereof

Remarks: CAR expression rates below 10% were considered either not expressed or expressed very poorly, below screening criteria; antigen binding recognition rates below 10% are considered poor in antigen recognition capacity, below screening criteria.

Example 4 specific detection of CLL1 CAR

Specific assays for CLL1 CARs were performed at the protein level and at the cellular level, respectively.

1) Specific detection of protein levels

The 8 CLL1 CAR-T cells (1-3, 1-28, 1-16, 2-8, 2-31, 3-37, 3-64 and 3-67CAR-T cells) obtained in example 3 were screened for protein levels using fluorescence-labeled CD19 protein (available from ACRObiosystems, cat. CD9-HP2H 3), CS1 protein (available from ACRObiosystems, cat. SL7-HP2H 3), BCMA protein (available from ACRObiosystems, cat. BCA-HF 254), GPC3 protein (available from ACRObiosystems, cat. GP3-HF2H 1), EGFRvIII protein (available from ACRObiosystems, cat. EGI-HP2E 3) and M26-T cells were tested for specificity as follows: 0.5X10 of each CAR-T cell was taken ⁶ Washing cells once with PBS, centrifuging for 5min at 200g, discarding the supernatant, re-suspending the cells into 200ul PBS, adding 0.1ug of the fluorescent-labeled antigen protein, incubating for 20min at 4 ℃ in the absence of light, washing the cells once with PBS, centrifuging for 5min at 200g, discarding the supernatant, re-suspending the cells into 200ul PBS, and detecting the binding capacity by flow cytometry; the results show that: each CAR-T cell could not bind to CD19 protein, CS1 protein, BCMA protein, GPC3 protein, EGFRvIII protein, and showed good specificity, and the detection results are shown in Table 5.

2) Specificity at the cellular level

The cell level of 8 CLL1 CAR-T cells (1-3, 1-28, 1-16, 2-8, 2-31, 3-37, 3-64 and 3-67CAR-T cells) and M26 CAR-T cells obtained by screening example 3 was specifically examined with cell lines of 11 different tissue sources, K562 (human chronic myelogenous leukemia cells), K562-CLL1 (K562 cells exogenously expressing CLL1 protein), HL60 (human promyelocytic leukemia cells), THP1 (human monocytic leukemia cells), OVCAR3 (human ovarian adenocarcinoma cells), RAJI (human lymphoma cells), mm.1s (human multiple myeloma cells), NALM6 (human acute lymphoblastic leukemia cells), SK-MEL1 (human skin melanoma cells), a549 (human lung cancer cells), HUH7 (human hepatoma cells), as follows: the K562-CLL1, HL60, THP1 and NALM6 cell lines are CLL1 positive target cells, and the rest cell lines are negative target cells. The method of detecting T cell activation in this study was the CD107a assay. The expression of CD107a on the membrane is believed to activate cytotoxic stranguria Markers for the bar cells (cd8+ T cells and NK cells) that are activated by the administration of specific antigens upon stimulation of cytotoxic lymphocytes express CD107a and thus can be used as a means to detect CAR-T cell specificity. The method comprises the following specific steps: tumor cells and transfected and untransfected T cells were counted and the tumor cells were adjusted to 2X 10 with X-VIVO medium ⁶ Per ml, T cells were adjusted to 1X 10 ⁶ /ml. For the killing plate, 20ul of CD107a antibody was added per well, then 100ul of T cells and 100ul of tumor cells were added per well, and centrifugation was performed at 400r/min for 3 minutes after the addition. 37 ℃ 5% CO ₂ Incubators were incubated for 60 minutes. After 60 minutes, 20ul of golgi stop working solution (3 ml of X-VIVO medium plus 2ul of golgi stop (BD golgi stop) ^TM Protein Transport Inhibitor, CAT:554724 For 2.5 hours). Equal volumes of CD3 and CD8 antibodies were mixed, and 10ul of the mixture was added to each well after mixing, and incubated in an incubator for 30 minutes. After 30 minutes, the mixture was centrifuged at 1500r/min for 5 minutes, the supernatant was removed, and 250ul FACS buffer (PBS+0.5% BSA) was added and mixed. Centrifuging at 1500r/min for 5 min, and removing supernatant. 200ul of FACS buffer (PBS+0.5% BSA) was added to each well, the mixture was transferred to a labeled flow tube, and 200-300ul of FACS buffer (PBS+0.5% BSA) was added thereto for machine detection. Analysis of CD3+ &Fluorescence signal of CD107a in the cd8+ positive cell population. The specific detection results are shown in Table 6.

TABLE 5 results of protein level detection of CAR-T cell specificity

Remarks: "-" indicates that the CAR-T cell does not bind to the protein and "+" indicates that the CAR-T cell binds to the protein.

TABLE 6 detection of CAR-T cell specificity at the cellular level

Remarks: "-" indicates that no CD107a signal was detected, and "+" indicates that a CD107a signal was detected.

As shown in table 6: 2-31CAR was activated by negative target cells, exhibiting CD107a signaling, and therefore did not meet the screening criteria at the cellular level, and was therefore also excluded in subsequent experiments.

EXAMPLE 5 sustained proliferation of CLL1 CAR-T cells

Antigen stimulation can activate CAR-T cells to proliferate the CAR-T cells, and continuous activation of the T cells can lead to cell exhaustion, so that proliferation capacity and effector function of the exhausted T cells are reduced, and the continuous proliferation of the CLL1 CAR-T cells is determined by detecting the proliferation condition of CD3+ cells (namely the proliferation condition of the T cells) after a plurality of antigen stimulation experiments.

Prior to antigen stimulation (with HL60 cells as antigen), the CAR positive ratios of the 7 CLL1 CAR-T cells (i.e., 1-3, 1-16, 1-28, 2-8, 3-37, 3-64, 3-67), M26 CAR-T cells obtained by the screens of example 3 and example 4 were adjusted to a level consistent with the set of CAR-T cells with the lowest CAR positive ratio using UTD, and in the antigen stimulation experiments, each set of CAR-T cells was co-cultured with positive target cells HL60 in 24 well plates at an effective target ratio of 1:2, 2ml of X-VIVO15 medium per well, and 3 wells were repeated per set of cells. CD3 was detected and analyzed by flow cytometry using a fluorescent-labeled CD3 antibody (manufacturer: bioLegend, cat# 300312), showing the proliferation of T cells (CD 3 is a marker distinguishing whether or not it is T cells), calculating the cell number of CD3 positive cells according to the conversion of the volume multiple, then taking out a certain amount of CAR-T cells from each group according to the calculation result, adding the corresponding positive target cells according to the effective target ratio of 1:2 for new round of stimulation, repeating the 3-4 rounds of stimulation every 2 days. The worst proliferation of 2-8 and 3-37CAR was eliminated by repeated experiments. After the rest 5 CLL1 CAR-T cells (namely 1-3, 1-16, 1-28, 3-64 and 3-67) and M26 CAR-T cells are subjected to multi-round stimulation and proliferation statistics by the method, the 1-3 and 1-28 have stronger continuous proliferation capability, which is obviously higher than that of positive control M26 CAR-T cells. 1-16 and 3-67 are also good in sustained proliferation capacity, 3-64 is relatively poor in sustained proliferation capacity, and the results are shown in FIG. 2. In the present application, the cells after multiple rounds of stimulation are almost all car+ cells, so the car+ expression rate is no longer an important indicator.

Example 6 cell killing experiments with CLL1 CAR-T cells

Cell killing experiments: 5 CLL1 CAR-T cells (namely, 1-3, 1-16, 1-28, 3-64, 3-67) obtained through screening in example 3, example 4 and example 5 and M26 CAR-T cells are respectively co-cultured with target cells in an X-VIVO15 culture medium for 16-24 hours according to the condition of different target ratios (1:1, 3:1 or 9:1) of effector cells to target cells, and then the killing proportion of the CAR-T cells to the target cells is detected by detecting the luciferase activity stably expressed in the target cells, wherein the target cells respectively adopt HL60 cells, K562-CLL1 cells and K562 cells: HL60 cells are positive target cells that endogenously express CLL1, K562-CLL1 are positive target cells that exogenously express CLL1, and K562 is a negative target cell that does not express CLL 1. The cell killing results are shown in fig. 3A-3C: the killing effect of each CAR-T in the HL60 tumor cell line is very remarkable; the killing effect of 1-16CAR-T cells in a K562-CLL1 tumor cell line is poor, and the rest CAR-T effects are very remarkable; in the K562 cell line, as expected, none of the groups had killing effect. Depending on the cell killing experiment, 1-16 may be further excluded.

Example 7 cytokine release assay by CLL1 CAR-T cells

Cytokine release assay: the concentration of IFN-gamma in the cell supernatant was measured by ELISA method after co-culturing 5 CLL1CAR-T cells (i.e., 1-3, 1-16, 1-28, 3-64, 3-67), M26 CAR-T cells and UTD cells obtained by the screening in example 3, example 4, example 5 with target cells in X-VIVO15 medium under the condition of an effective target ratio of 1:1 for 24 hours, respectively; the target cells are HL60 cells, K562-CLL1 cells and K562 cells respectively, wherein: HL60 cells are positive target cells that endogenously express CLL1, K562-CLL1 are positive target cells that exogenously express CLL1, and K562 is a negative target cell that does not express CLL 1. The cytokine release assay results are shown in figure 4: the release amount of the 1-3 and 1-28CAR-T cytokines is significantly higher than that of the positive control M26 CAR-T, and the release amount of the 3-67CAR-T cytokines is also higher than that of the positive control M26 and 3-64.

In conclusion, in vitro pharmacodynamic detection shows that the CAR taking 1-3scFv and 1-28scFv as extracellular antigen recognition domains has high expression rate on the surface of T cells, strong antigen recognition capability, good specificity at protein level and cell level, good sustained proliferation, strong in vitro cell killing capability and high cytokine release level; in particular, both its sustained proliferation and cytokine release levels were significantly better than the positive control M26 scFv. The CAR taking 3-67scFv as extracellular antigen recognition structural domain has strong antigen recognition capability, good specificity at protein level and cell level, good sustained proliferation, strong in vitro cell killing capability and high cytokine release level.

Sequence description

SEQ ID NO. 1:1-3scFv amino acid sequences;

SEQ ID NO. 2:1-28scFv amino acid sequence;

SEQ ID NO. 3:1-16scFv amino acid sequences;

SEQ ID NO. 4:2-8scFv amino acid sequence;

SEQ ID NO. 5:2-25scFv amino acid sequence;

SEQ ID NO. 6:2-31scFv amino acid sequence;

SEQ ID NO. 7:2-40scFv amino acid sequence;

SEQ ID NO. 8:3-11scFv amino acid sequence;

SEQ ID NO. 9:3-13scFv amino acid sequence;

SEQ ID NO. 10:3-16scFv amino acid sequences;

SEQ ID NO. 11:3-35scFv amino acid sequence;

SEQ ID NO. 12:3-37scFv amino acid sequence;

SEQ ID NO. 13:3-64scFv amino acid sequence;

SEQ ID NO. 14:3-67scFv amino acid sequence;

SEQ ID NO. 15: m26 scFv amino acid sequence;

SEQ ID NO. 16: the CD8 a guide chain as the amino acid sequence of the signal peptide;

SEQ ID NO. 17: amino acid sequence of CD8 a hinge region;

SEQ ID NO. 18: amino acid sequence of CD8 a transmembrane region;

SEQ ID NO. 19: amino acid sequence of 4-1BB intracellular co-stimulatory signal;

SEQ ID NO. 20: an amino acid sequence of a CD3 ζt cell activation signal;

SEQ ID NO. 21:1-3CAR amino acid sequence;

SEQ ID NO. 22:1-28CAR amino acid sequence;

SEQ ID NO. 23:1-16CAR amino acid sequence;

SEQ ID NO. 24:2-8CAR amino acid sequence;

SEQ ID NO. 25:2-25CAR amino acid sequence;

SEQ ID NO. 26:2-31CAR amino acid sequence;

SEQ ID NO. 27:2-40CAR amino acid sequence;

SEQ ID NO. 28:3-11CAR amino acid sequence;

SEQ ID NO. 29:3-13CAR amino acid sequence;

SEQ ID NO. 30:3-16CAR amino acid sequence;

SEQ ID NO. 31:3-35CAR amino acid sequence;

SEQ ID NO. 32:3-37CAR amino acid sequence;

SEQ ID NO. 33:3-64CAR amino acid sequence;

SEQ ID NO. 34:3-67CAR amino acid sequence;

SEQ ID NO. 35: m26 CAR amino acid sequence;

SEQ ID NO:36:1-3scFv VH CDR1 amino acid sequence;

37:1-3scFv VH CDR2 amino acid sequence;

38:1-3scFv VH CDR3 amino acid sequence;

39:1-3scFv VL CDR1 amino acid sequence;

SEQ ID NO:40:1-3scFv VL CDR2 amino acid sequence;

the amino acid sequence of the VL CDR3 of the scFv with the sequence of SEQ ID NO. 41:1-3;

the amino acid sequence of the VH CDR1 of the SEQ ID NO. 42:1-28 scFv;

43:1-28scFv VH CDR2 amino acid sequence;

SEQ ID NO. 44:1-28scFv VH CDR3 amino acid sequence;

SEQ ID NO. 45:1-28scFv VL CDR1 amino acid sequence;

the amino acid sequence of SEQ ID NO 46:1-28scFv VL CDR2;

the amino acid sequence of SEQ ID NO. 47:1-28scFv VL CDR3;

48:3-67scFv VH CDR1 amino acid sequence;

49:3-67scFv VH CDR2 amino acid sequence;

SEQ ID NO. 50:3-67scFv VH CDR3 amino acid sequence;

the amino acid sequence of SEQ ID NO. 51:3-67scFv VL CDR1;

52:3-67scFv VL CDR2 amino acid sequence;

53:3-67scFv VL CDR3 amino acid sequence;

SEQ ID NO. 54:1-3scFv VH amino acid sequence;

SEQ ID NO. 55:1-3scFv VL amino acid sequence;

SEQ ID NO. 56:1-28scFv VH amino acid sequence;

SEQ ID NO. 57:1-28scFv VL amino acid sequence;

SEQ ID NO. 58:3-67scFv VH amino acid sequence;

SEQ ID NO 59:3-67scFv VL amino acid sequence;

SEQ ID NO. 60: a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 54;

SEQ ID NO. 61: a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 55;

SEQ ID NO. 62: a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 56;

SEQ ID NO. 63: a nucleotide sequence encoding an amino acid sequence of a light chain variable region as set forth in SEQ ID NO. 57;

SEQ ID NO. 64: a nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 58;

SEQ ID NO. 65: a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 59;

SEQ ID NO. 66: a linking sequence;

SEQ ID NO. 67: a linking sequence;

SEQ ID NO. 68: a junction sequence.

Claims

1. A CLL1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO. 56, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are respectively identical with the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO. 57;

2. A CLL1 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 42, SEQ ID NO. 43 and SEQ ID NO. 44, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region respectively comprise the amino acid sequences shown as SEQ ID NO. 45, SEQ ID NO. 46 and SEQ ID NO. 47;

3. The CLL1 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein,

the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 56, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 57;

4. The CLL1 antibody or antigen-binding fragment thereof of claim 3, wherein,

The CLL1 antibody or antigen binding fragment thereof is a CLL1 scFv antibody, CLL1Sc (Fv) ₂ Antibodies, CLL1[ Sc (Fv) ₂ ] ₂ An antibody.

5. The CLL1 antibody or antigen binding fragment thereof of claim 4, wherein,

the CLL1 scFv antibody comprises any one selected from the group consisting of: an amino acid sequence shown as SEQ ID NO. 56-a linker sequence-an amino acid sequence shown as SEQ ID NO. 57-a linker sequence-an amino acid sequence shown as SEQ ID NO. 56-an amino acid sequence shown as SEQ ID NO. 58-an amino acid sequence shown as SEQ ID NO. 59-a linker sequence-an amino acid sequence shown as SEQ ID NO. 58.

6. The CLL1 antibody or antigen-binding fragment thereof of claim 5, wherein,

the CLL1 scFv antibody comprises any one selected from the group consisting of: the amino acid sequence shown as SEQ ID NO. 57-the linker sequence-the amino acid sequence shown as SEQ ID NO. 56-the amino acid sequence shown as SEQ ID NO. 59-the linker sequence-the amino acid sequence shown as SEQ ID NO. 58.

7. The CLL1 antibody or antigen-binding fragment thereof of claim 6, wherein the CLL1 scFv antibody comprises the amino acid sequence set forth in SEQ ID No. 2 or SEQ ID No. 14.

8. The CLL1 antibody or antigen-binding fragment thereof of claim 5, wherein,

the linking sequence is selected from one or more of the following sequences: SEQ ID NO 66, SEQ ID NO 67 and SEQ ID NO 68.

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8.

10. The nucleic acid molecule of claim 9, wherein the nucleotide sequence encoding the CLL1 antibody or antigen-binding fragment thereof comprises:

1) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 56 and a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown as SEQ ID NO. 57; or (b)

2) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 58 and a nucleotide sequence encoding the amino acid sequence of the light chain variable region shown as SEQ ID NO. 59.

11. The nucleic acid molecule of claim 10, wherein,

the nucleotide sequence of the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 56 is shown as SEQ ID NO. 62; the nucleotide sequence of the amino acid sequence of the light chain variable region shown as SEQ ID NO. 57 is shown as SEQ ID NO. 63; or (b)

The nucleotide sequence of the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 58 is shown as SEQ ID NO. 64; the nucleotide sequence of the amino acid sequence of the light chain variable region shown as SEQ ID NO. 59 is shown as SEQ ID NO. 65.

12. A vector comprising the isolated nucleic acid molecule of any one of claims 9-11.

13. A cell comprising the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8, the isolated nucleic acid molecule of any one of claims 9-11, or the vector of claim 8.

14. A pharmaceutical composition comprising the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8, the isolated nucleic acid molecule of any one of claims 9-11, the vector of claim 12 or the cell of claim 13, and a pharmaceutically acceptable adjuvant.

15. An antibody medicament comprising the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8.

16. The antibody drug of claim 15, wherein the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.

17. An antibody drug conjugate comprising the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8.

18. Use of the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8, the isolated nucleic acid molecule of any one of claims 9-11, the vector of claim 12 or the cell of claim 13 in the manufacture of a medicament for treating a disease or disorder associated with expression of CLL 1.

19. The use according to claim 18, wherein the disease or disorder associated with expression of CLL1 is a hematological neoplasm.

20. The use according to claim 19, wherein the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.

21. Use of the CLL1 antibody or antigen-binding fragment thereof of any one of claims 1-8, the isolated nucleic acid molecule of any one of claims 9-11, the vector of claim 12 or the cell of claim 13 in the preparation of a test agent for diagnosing a disease or disorder associated with expression of CLL 1.

22. The use according to claim 21, wherein the disease or disorder associated with expression of CLL1 is a hematological neoplasm.

23. The use according to claim 22, wherein the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.