CN118324909A

CN118324909A - Humanized CLL1 antibody and application thereof

Info

Publication number: CN118324909A
Application number: CN202310032665.XA
Authority: CN
Inventors: 张其猛; 张超; 白大勇; 高俊潇; 周立
Original assignee: Heyuan Kanghua Pharmaceutical Technology Beijing Co ltd
Current assignee: Heyuan Kanghua Pharmaceutical Technology Beijing Co ltd
Priority date: 2023-01-10
Filing date: 2023-01-10
Publication date: 2024-07-12

Abstract

The invention provides a humanized 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, amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region respectively comprise amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO. 1, and amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region respectively comprise amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO. 2.

Description

Humanized CLL1 antibody and application thereof

Technical Field

The application relates to the field of biological medicine, in particular to a humanized 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 Receptor (CAR) is a core component of CAR cell therapeutics, which 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.

It was found that CLL1 (C-TYPE LECTIN-like Molecule 1, C-lectin-like Molecule-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, it is practical to find antibodies targeting CLL1 suitable for drugs, in particular antibodies targeting CLL1 suitable for CAR-T cell therapeutic drugs.

Disclosure of Invention

The application provides a humanized CLL1 antibody and application thereof, and the inventor performs humanized modification on an autonomously developed mouse antibody targeting CLL1 so as to reduce the heterology of the antibody while maintaining the functionality and the specificity of the antibody. Based on the above, the inventor takes the humanized scFv antibody as an extracellular antigen recognition domain of the CLL1 CAR structure, so as to construct a chimeric antigen receptor expression vector, prepare a CLL 1-targeted CAR-T cell, and verify multiple indexes of the CLL1 CAR and the CLL1 CAR-T cell at the cellular level.

A humanized CLL1 antibody or antigen binding portion thereof comprising a heavy chain variable region whose amino acid sequences of CDR1, CDR2, CDR3 comprise the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the heavy chain variable region of the antibody shown in SEQ ID No. 1, and a light chain variable region whose amino acid sequences of CDR1, CDR2, CDR3 comprise the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the light chain variable region of the antibody shown in SEQ ID No. 2;

Or, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region of the antibody shown in SEQ ID NO 3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region of the antibody shown in SEQ ID NO 4.

A humanized CLL1 antibody or antigen binding portion thereof, comprising a heavy chain variable region and a light 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. 5, SEQ ID No. 6, SEQ ID No.7, respectively, the amino acid sequences of CDR1, CDR2, CDR3 of the light chain variable region comprising the amino acid sequences shown as SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, 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. 11, SEQ ID NO. 12 and SEQ ID NO. 13, 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. 14, SEQ ID NO. 15 and SEQ ID NO. 16.

In certain embodiments, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 19 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 17;

or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 19, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 18;

Or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 20, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 18;

or, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 23, and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 21;

alternatively, the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 24 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 22.

The humanized CLL1 antibody or antigen binding portion thereof described above is, in certain embodiments, a CLL1 scFv antibody or a CLL1Sc (Fv) ₂ antibody.

In certain embodiments, the humanized CLL1 antibody or antigen binding portion thereof described above, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id nos: an amino acid sequence-linker sequence shown as SEQ ID NO. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 17-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 20-an amino acid sequence-linker sequence shown as SEQ ID NO. 20-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 21-a linker sequence shown as SEQ ID NO. 23-an amino acid sequence shown as SEQ ID NO. 21-an amino acid sequence shown as SEQ ID NO. 22-a linker sequence shown as SEQ ID NO. 24-an amino acid sequence shown as SEQ ID NO. 22.

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. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 22-an amino acid sequence shown as SEQ ID NO. 24; further alternatively, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id nos: the amino acid sequence shown in SEQ ID NO. 26, the amino acid sequence shown in SEQ ID NO. 27 and the amino acid sequence shown in SEQ ID NO. 31.

In some embodiments, the linker sequence is selected from one or more of the following sequences: SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34.

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

In certain embodiments, the isolated nucleic acid molecule described above comprises a nucleotide sequence encoding the humanized 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. 19, optionally as shown as SEQ ID NO. 47; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 17, optionally as set forth in SEQ ID NO. 48; or (b)

2) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 19, optionally as shown as SEQ ID NO. 47; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 18, optionally as set forth in SEQ ID NO. 49; or (b)

3) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region shown as SEQ ID NO. 24, optionally as shown as SEQ ID NO. 50; and a nucleotide sequence encoding the amino acid sequence of the light chain variable region as set forth in SEQ ID NO. 22, optionally as set forth in SEQ ID NO. 51.

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

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

The application also provides a pharmaceutical composition comprising any one of the humanized 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 humanized CLL1 antibodies or an antigen binding portion 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 humanized CLL1 antibodies or antigen binding portions thereof described above.

The application also provides an application of the humanized 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 humanized CLL1 antibody or an 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 expression of CLL 1.

The application also provides a method of treating a disease or disorder associated with expression of CLL1, comprising the steps of: an effective amount of a medicament comprising any one of the humanized CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells described above is administered 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 humanized CLL1 antibody or an 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.

FIGS. 2A and 2B show the lytic killing effect of various CLL1 CAR-T cells of example 4 of the application against different target cells at different target cell patterns; wherein: the target cell in FIG. 2A is HL60-luc; the target cell in FIG. 2B is K562-CLL1-luc.

FIG. 3 shows the release of IFN-gamma factors from various CLL1 CAR-T cells activated by different target cells in example 5 of the present application, wherein the three groups from left to right are K562, K562-CLL1 and HL60 as target cells respectively; wherein: the release of the cytokine IFN-gamma factor after activation of the target cells by the positive control BG1805, 1-3LH-Q1, 1-3LH-Q2, 1-3LH-Q3, 3-67LH-Q1 and 3-67LH-Q2 cells is shown in each group in sequence from left to right.

FIG. 4 shows the proliferation of various CLL1 CAR-T cells according to example 6 of the present application after activation by positive target cells in a single round of continuous proliferation assay.

FIG. 5 shows the results of in vivo efficacy tests of mice in examples 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 is used in the broadest sense herein, and by analyzing the amino acid sequences of the heavy and light chains of different antibodies, it is found that the amino acid sequences of the heavy and light chains near the N-terminus vary greatly, and the other parts of the amino acid sequences are 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 variable regions of antibodies are particularly strongly varied by a small percentage of amino acid residues whose composition and sequence are more prone to mutation (hypervariable regions, HVR); three hypervariable regions are present in each of the V regions of the L and H chains, and are also known as complementarity determining regions (complementarity determining region, CDRs) because of their spatial structure which can form a precise complement with an epitope. 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 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/or VL regions.

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, terms such as "Sc (Fv) ₂" and the like not specifically explained also have their conventional meaning in the art.

In the present application, the term "humanized antibody" is also referred to as an antibody subjected to humanized modification, which has a conventional meaning in the art, and the humanized modification method is known, and the purpose of the humanized modification is to substantially maintain the affinity and specificity of the parent antibody, reduce the heterology thereof, greatly reduce the immune side reaction of the heterogenous antibody to the human body, and be advantageously applied to the human body. For example, in the case where the CDRs are obtained from a murine antibody, primers can be synthesized (corresponding primers can be obtained by the method described in WO 98/13388) which are used to ligate the CDRs of the murine antibody with the Framework Regions (FRs) of the human antibody. For human antibody FRs to which CDRs are attached, those are selected that allow the CDR regions to form good antigen binding sites.

In the present application, the term "chimeric antigen Receptor" (CHIMERIC ANTIGEN Receptor, CAR) is a core component of CAR cell therapeutics, which 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) cell immunotherapy is considered to be one of the most promising means of combating 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 the single chain variable region (SINGLE CHAIN Variable Fragment, abbreviated scFv) of antibodies, or from receptor ligand interactions, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR). The most common source to date is the scFv fragment of an antibody, scFv comprising an antibody heavy chain variable region and a light chain variable region, linked by a peptide chain.

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" is responsible for the activation of at least one normal effector function of an immune effector cell 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、MyD88.

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. But the choice of antigen target is not necessarily single. By selecting an appropriate target, the anti-tumor activity of the CAR-T cells can be optimized. The extracellular antigen recognition domain may thus further comprise an extracellular antigen recognition sequence that targets any one of the following targets: CD33, CD123, CD70, FLT3, BCL2, leY. The extracellular antigen recognition domains for two or more targets comprise VH and VL regions for different targets, and the different regions are also directly or indirectly linked by a linking sequence, in any of the following arrangements: target 1 VL-target 1 VH-target 2 VL-target 2VH, target 2 VL-target 2 VH-target 1 VL-target 1VH, target 1 VL-target 2 VH-target 1VH, target 2 VL-target 1 VH-target 2VH, and the above "-" represents ligation by a linker sequence.

In the present application, the term "linker 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 antibody, 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 from which pluripotent stem cells differentiate, NK cells from which pluripotent stem cells differentiate, induced pluripotent stem cells (ipscs), T cells from which induced pluripotent stem cells differentiate (ipscs-T), NK cells from which induced pluripotent stem cells differentiate (ipscs-NK), 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 disclosure, the terms "comprises," "comprising," and "includes" are generally intended to include the specifically-named features, but not 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.

Humanized 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 humanized 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 of CDR1, CDR2, CDR3, respectively, in the heavy chain variable region of the antibody shown in SEQ ID NO:1, and the amino acid sequences of CDR1, CDR2, CDR3 of the light chain variable region comprising the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the light chain variable region of the antibody shown in SEQ ID NO: 2;

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 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.

In another aspect, the present application also provides a humanized CLL1 antibody or an 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. 5, SEQ ID No. 6, SEQ ID No. 7, 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. 8, SEQ ID No. 9, SEQ ID No. 10, 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. 19 and the amino acid sequence of the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 17;

Any of the humanized CLL1 antibodies or antigen binding portions thereof described above, in some embodiments, are CLL1 scFv antibodies or CLL1 Sc (Fv) ₂ antibodies.

In some embodiments, any of the above humanized CLL1 antibodies or antigen binding portions thereof, the CLL1scFv antibody comprises any one selected from the group consisting of: an amino acid sequence-linker sequence shown as SEQ ID NO. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 17-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 20-an amino acid sequence-linker sequence shown as SEQ ID NO. 20-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 21-a linker sequence shown as SEQ ID NO. 23-an amino acid sequence shown as SEQ ID NO. 21-an amino acid sequence shown as SEQ ID NO. 22-a linker sequence shown as SEQ ID NO. 24-an amino acid sequence shown as SEQ ID NO. 22.

Optionally, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id no: an amino acid sequence shown as SEQ ID NO. 17-a linker sequence-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 18-a linker sequence-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 22-an amino acid sequence shown as SEQ ID NO. 24. 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;

Further alternatively, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id nos: the amino acid sequence shown in SEQ ID NO. 26, the amino acid sequence shown in SEQ ID NO. 27 and the amino acid sequence shown in SEQ ID NO. 31.

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

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

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 any one of the humanized CLL1 antibodies or an antigen binding portion thereof, a nucleic acid molecule or a vector described above.

In yet another aspect, the application provides a pharmaceutical composition comprising any of the above humanized CLL1 antibodies or antigen binding portions thereof, an isolated nucleic acid molecule, vector or 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 humanized CLL1 antibody or antigen binding portion thereof, and antibody drug conjugate

In one aspect, the application provides an antibody medicament comprising any one of the humanized CLL1 antibodies or antigen binding portions 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 of the humanized CLL1 antibodies or antigen binding portions thereof described above. The antibody drug conjugate, namely the anti-drug conjugate, is called ADC for short, which is formed by connecting a small molecular drug with biological activity to an antibody through a chemical chain, and the antibody is used as a carrier to target and transport the small molecular drug into a target cell.

Humanized CLL1 antibodies or antigen binding sites thereof, corresponding nucleic acid molecules, corresponding vectors, uses of corresponding cells

In one aspect, the application provides the use of any one of the above humanized 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 of the above humanized 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: an effective amount of a medicament comprising any one of the humanized CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells 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 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 non-human animal 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 humanized CLL1 antibodies or antigen binding portions thereof, isolated nucleic acid molecules, vectors or cells described above for use in treating a disease or disorder associated with expression of CLL 1.

In some embodiments, the disease or disorder associated with expression of CLL1 is acute myeloid leukemia. CLL1 chimeric antigen receptor, corresponding nucleic acid molecule, corresponding vector, corresponding immune effector cell and pharmaceutical composition

In one aspect, the application provides a chimeric antigen receptor comprising a humanized CLL1 antibody comprising a CLL 1-targeting extracellular antigen-recognition domain, a hinge region, a transmembrane region, and an intracellular domain, the CLL 1-targeting extracellular antigen-recognition domain comprising a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein: the CLL1 heavy chain variable region and the CLL1 light chain variable region are subjected to humanized modification;

The amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO. 1, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody light chain variable region shown in SEQ ID NO. 2;

Or, the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 heavy chain variable region respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody heavy chain variable region shown in SEQ ID NO. 3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the CLL1 light chain variable region respectively comprise the amino acid sequences of CDR1, CDR2 and CDR3 in the antibody light chain variable region shown in SEQ ID NO. 4.

In another aspect, the application also provides a chimeric antigen receptor comprising a humanized CLL1 antibody, comprising a CLL 1-targeting extracellular antigen-recognition domain, a hinge region, a transmembrane region, and an intracellular domain, the CLL 1-targeting extracellular antigen-recognition domain comprising a CLL1 heavy chain variable region and a CLL1 light chain variable region, wherein: the CLL1 heavy chain variable region and the CLL1 light chain variable region are subjected to humanized modification;

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. 5, SEQ ID NO. 6 and SEQ ID NO. 7, 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. 8, SEQ ID NO. 9 and SEQ ID NO. 10;

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. 11, SEQ ID NO. 12 and SEQ ID NO. 13, 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. 14, SEQ ID NO. 15 and SEQ ID NO. 16.

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

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. 19 and the CLL light chain variable region sequence comprises an amino acid sequence set forth in SEQ ID No. 17;

or, the amino acid sequence of the CLL1 heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO. 19, and the amino acid sequence of the CLL light chain variable region comprises an amino acid sequence shown as SEQ ID NO. 18;

Or, the amino acid sequence of the CLL1 heavy chain variable region comprises the amino acid sequence shown as SEQ ID NO. 20, and the amino acid sequence of the CLL light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 18.

The application verifies that 5 humanized scFv antibodies and chimeric antigen receptors composed of the humanized antibodies can reduce the heterology of the humanized scFv antibodies while maintaining the functionality and the specificity. In particular, 3 humanized scFv antibodies and chimeric antigen receptors thereof are preferred.

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. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 17-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 20-an amino acid sequence-linker sequence shown as SEQ ID NO. 20-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 21-a linker sequence shown as SEQ ID NO. 23-an amino acid sequence shown as SEQ ID NO. 21-an amino acid sequence shown as SEQ ID NO. 22-a linker sequence shown as SEQ ID NO. 24-an amino acid sequence shown as SEQ ID NO. 22.

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. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 22-an amino acid sequence shown as SEQ ID NO. 24; further alternatively, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id nos: the amino acid sequence shown in SEQ ID NO. 26, the amino acid sequence shown in SEQ ID NO. 27 and the amino acid sequence shown in SEQ ID NO. 31. 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 some embodiments, the linking sequence is selected from one or more of the following: SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34.

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. 43.

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. 44.

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. 46.

Any of the above chimeric antigen receptors, in some embodiments, wherein the costimulatory signaling region is 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、MyD88; 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. 45.

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. 42.

In some embodiments, any of the chimeric antigen receptors described above comprises an amino acid sequence as set forth in SEQ ID NO. 36, SEQ ID NO. 37 or SEQ ID NO. 41.

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 CLL1 heavy chain variable region as set forth in SEQ ID NO. 19, optionally as set forth in SEQ ID NO. 47; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as set forth in SEQ ID No. 17, optionally as set forth in SEQ ID No. 48; 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. 19, optionally as set forth in SEQ ID NO. 47; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as set forth in SEQ ID NO. 18, optionally as set forth in SEQ ID NO. 49; or (b)

3) A nucleotide sequence encoding the amino acid sequence of the heavy chain variable region of CLL1 as shown in SEQ ID No. 24, optionally as shown in SEQ ID No. 50; and a nucleotide sequence encoding the amino acid sequence of the CLL1 light chain variable region as set forth in SEQ ID NO. 22, optionally as set forth in SEQ ID NO. 51.

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 a T lymphocyte, a natural killer cell (NK cell), a peripheral blood mononuclear cell (PBMC cell), an induced pluripotent stem cell, a T cell into which an induced pluripotent stem cell is differentiated, an NK cell into which an induced pluripotent stem cell is differentiated, and an embryonic stem cell.

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.

In some embodiments, the engineered immune effector cells described above are alpha beta T lymphocytes or gamma delta 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 cells or pharmaceutical composition is a dose of 1 x 10 ⁵ to1 x 10 ⁷ 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 administered can range from 1X 10 ⁵ CAR-positive T cells/kg to 1X 10 ⁷ CAR-positive T cells/kg, e.g., 1X 10 ⁵ CAR-positive T cells/kg to 1X 10 ⁶ CAR-positive T cells/kg, 1X 10 ⁶ CAR positive T cells/kg to 1X 10 ⁷ CAR positive T cells/kg, 0.5X 10 ⁶ CAR positive T cells/kg, 0.6X 10 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 CAR positive T cells/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 CAR positive T cells/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 presented merely to illustrate the chimeric antigen receptor, engineered immune effector cells, methods of preparation and use, and the like, 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 of ordinary skill in the art and are described in numerous publications, including Sambrook,J.,Fritsch,E.F.and Maniais,T.(1989)Molecular Cloning:ALaboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press.

Examples 1, 1-3 antibodies, humanized engineering of 3-67 antibodies

In the previous work, the inventor developed a plurality of antibodies targeting CLL1, and used the corresponding scFv antibodies as extracellular antigen recognition domains of CLL1 CAR structure, and constructed CAR expression vectors, prepared CLL 1-targeting CAR-T cells, and verified multiple indexes of CLL1 CAR and CLL1 CAR-T cells at the cellular level, and further selected 2 scFv antibodies 1-3, 3-67, which are well expressed, from which the 2 scFv antibodies are murine sequences, and designed for humanized modification in order to reduce the heterogeny thereof.

1. Humanized engineering design against murine 1-3scFv antibodies: when humanized engineering is performed on a murine 1-3scFv antibody (also referred to as 1-3LH in the present application), 3 humanized scFv sequences, 1-3LH-Q1, 1-3LH-Q2 and 1-3LH-Q3, respectively, are generated, and the light chain portion of the 3 humanized scFv sequences has 2 design modes, namely 1-3LH VL1 and 1-3LH VL2, and the heavy chain portion thereof has 2 design modes, namely 1-3LH VH1 and 1-3LH VH2.

2. Humanized engineering design against murine 3-67scFv antibodies: when humanized engineering is performed on a murine 3-67scFv antibody (also called 3-67LH in the application), 2 humanized scFv sequences are generated in total, namely 3-67LH-Q1 and 3-67LH-Q2, and the light chain part in the 2 humanized scFv sequences has 2 design modes, namely 3-67LH VL1 and 3-67LH VL2, and the heavy chain part also has 2 design modes, namely 3-67LH VH1 and 3-67LH VH2.

The structural composition of the 5 humanized scFv antibodies is shown in table 1.

TABLE 1 structural composition of 5 humanized scFv antibodies

The linker sequences used in Table 1 are shown in SEQ ID NO. 32.

The VH and VL of each of the murine 1-3scFv antibody, the 3-67scFv antibody and the humanized antibody are shown in Table 2; the amino acid sequences of murine 1-3scFv antibodies, 3-67scFv antibodies and humanized antibodies are shown in Table 3.

TABLE 2 VH and VL of murine 1-3scFv antibodies, 3-67scFv antibodies and 5 humanized antibodies

Sequence name	Amino acid sequence numbering	Sequence name	Amino acid sequence numbering
				1-3LH VL	SEQ ID NO.:2	3-67LH VL	SEQ ID NO.4:
1-3LH VL1	SEQ ID NO.:17	3-67LH VL1	SEQ ID NO.:21
				1-3LH VL2	SEQ ID NO.:18	3-67LH VL2	SEQ ID NO.22
1-3LH VH	SEQ ID NO.:1	3-67LH VH	SEQ ID NO.:3
				1-3LH VH1	SEQ ID NO.:19	3-67LH VH1	SEQ ID NO.:23
1-3LH VH2	SEQ ID NO.:20	3-67LH VH2	SEQ ID NO.:24

TABLE 3 amino acid sequences of murine 1-3scFv antibodies, 3-67scFv antibodies and 5 humanized antibodies

ScFv name	Amino acid sequence numbering	ScFv name	Amino acid sequence numbering
				1-3LH	SEQ ID NO.:25	3-67LH	SEQ ID NO.:29
1-3LH-Q1	SEQ ID NO.:26	3-67LH-Q1	SEQ ID NO.:30
				1-3LH-Q2	SEQ ID NO.:27	3-67LH-Q2	SEQ ID NO.:31
1-3LH-Q3	SEQ ID NO.:28

EXAMPLE 2 acquisition of CLL1 CAR-T cells

Selection to verify the function of 5 humanized scFv antibodies at the cellular level we selected to screen each candidate humanized scFv antibody 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: 42), a hinge region (shown as SEQ ID NO: 43) and a transmembrane region (shown as SEQ ID NO: 44) adopt a CD8 alpha structure, 4-1BB is adopted as an intracellular co-stimulatory signal (shown as SEQ ID NO: 45), CD3 zeta is adopted as a T cell activating signal (shown as SEQ ID NO: 46), a structure schematic is shown as figure 1, and the amino acid sequence of each CLL 1CAR is shown as table 4 (the name of each CLL 1CAR corresponds to its scFv, and the scFv used in 1-3LH CAR is the scFv used in 1-3LH,1-3LH-Q1CAR corresponding to 1-3LH-Q1 shown in table 3). BG1805 is used as positive control, its scFv amino acid sequence is shown in SEQ ID NO. 52 of the present application, and specific CAR amino acid sequence is shown in SEQ ID NO. 53 of the present application.

TABLE 4 various CLL1 CAR sequences

CAR name	Sequence numbering	CAR name	Sequence numbering
				1-3LH CAR	SEQ ID NO:35	3-67LH CAR	SEQ ID NO:39
1-3LH-Q1 CAR	SEQ ID NO:36	3-67LH-Q1 CAR	SEQ ID NO:40
				1-3LH-Q2 CAR	SEQ ID NO:37	3-67LH-Q2 CAR	SEQ ID NO:41
1-3LH-Q3 CAR	SEQ ID NO:38	BG1805 CAR	SEQ ID NO:53

The specific method for obtaining the CLL1CAR-T cells is as follows:

1. Construction of lentiviral vectors

Based on the sequences of the various CLL1 CARs in table 4, 5 CLL1 CAR structures constructed from candidate humanized scFv antibodies, 2 CLL1 CAR structures constructed from murine scFv antibodies, and 1 positive control BG1805 CAR were each synthesized.

The nucleotide sequences encoding the 8 CLL1 CAR structures are respectively constructed into empty lentiviral vectors (manufacturer: SBI company, product number: CD500-CD800, conventional resistance modification is carried out as described in example 1 of WO 2021/121227) to obtain CAR expression vectors, then the CAR expression vectors and three packaging plasmids are transfected into 293T cells together, and the functional lentiviral vectors are obtained after collection and purification. The three packaging plasmids were pMD2.G (available from Biovector, product No. Biovector 012259), pMDLg/pRRE (available from Biovector, product No. Biovector 012251), pRSV-Rev (available from Biovector, product No. Biovector 012253), respectively.

2. The corresponding 8 CLL1CAR-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 complete lymphocyte 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 cells/ml, stimulated with CD3/CD28 beads at 1ul beads/1X 10 ⁶ cells, mixed and incubated in an incubator at 37℃+5% CO ₂ for at least 24 hours.

3) Lentivirus transduced T cells

Taking out T cells for activating culture, re-suspending with pure X-VIVO15 culture medium containing polybrene with final concentration of 8 μg/ml, mixing to obtain cell suspension, slowly adding 200ul of slow virus vector per 800ul of cell suspension (containing 2×10 ⁶ cells), mixing, placing into a pore plate, and culturing in an incubator under 37+5% CO ₂ for at least 4-6 hr.

4) Expanded culture of transduced T cells

The transduced cells were removed and cultured in complete lymphocyte culture medium, and passaged every other day to maintain the cell density at (0.8-2). Times.10 ⁶ cells/ml for use in the subsequent examples.

After T cells are infected with lentiviruses each comprising a nucleotide sequence encoding the CAR structure of table 4, the T cells obtained are each named for their CAR sequence number, e.g., T cells obtained using a 1-3LH CAR, i.e., named 1-3LH CAR-T cells, and T cells obtained using a 1-3LH-Q1 CAR, i.e., named 1-3LH-Q1 CAR-T cells. Next, we screened each CLL1 CAR structure at the cellular level to determine the merits of each humanized CLL1 scFv.

Example 3 detection of expression of each CLL1 CAR on T cell surface

Detection of CAR molecules: detection of 8 CLL1 CAR-T cell surface expressed CAR protein molecules obtained in example 2 after 7 days of incubation, we stained 5 CLL1 CAR-T cells constructed from candidate humanized scFv antibodies, 2 CLL1 CAR-T cells constructed from murine scFv antibodies, 1 CLL1 CAR-T cell constructed from positive control scFv antibodies and UTD cells (non-CAR transduced T cells) obtained in example 2 with FITC fluorescence-labeled CLL1 antigen (manufacturer: ACRObiosystems, cat No. CLA-HF247 ug) and analyzed by flow cytometry for positive ratio detection of CAR molecules, the detection results are shown in table 5: the surfaces of the 5 CLL1 CAR-T cells constructed according to the candidate humanized scFv antibodies all have corresponding CAR expression, and the CAR expression rate of the surfaces of the 3 humanized scFvs corresponding to the murine 1-3scFv (namely, 1-3LH-Q1, 1-3LH-Q2 and 1-3 LH-Q3) and the 2 humanized scFvs corresponding to the murine 3-67scFv (namely, 3-67LH-Q1 and 3-67 LH-Q2) is obviously improved compared with the CAR expression rate of the surfaces of the CAR-T cells constructed according to the murine scFv, and is equivalent to or slightly higher than the CAR expression rate of the surfaces of the CAR-T cells constructed according to the murine scFv.

Table 5, results of detection of T cell surface expressed CAR molecules

Example 4 killing experiments on individual CLL1CAR-T cells

Cell killing experiments: 8 CLL1 CAR-T cells and UTD cells (non-CAR-transduced T cells) obtained in example 2 were cultured for 6 days after transduction, and each cell was subjected to a test for the killing ratio of CAR-T cells to target cells by detecting the luciferase activity stably expressed in the target cells (the target cells were infected with a lentivirus having a nucleotide sequence encoding luciferase) under conditions in which the target cells were subjected to different target ratios (3:1 or 9:1, 1X 10 ⁴ per well) from the target cells (the weaker the fluorescent signal indicates that the tumor cells remained, the stronger the killing) respectively, in X-VIVO15 medium for 12 hours, and then the resulting cells were identified to obtain a cell line stably expressing luciferase, genBank: MK484106.1 for the luciferase gene; the luciferase was used to provide fluorescent signals, and the same cell line was used to provide a weaker fluorescent signal indicating that the tumor cells remained less and the killing ratio of CAR-T cells was stronger for the target cells, and 3 replicates per well. The target cells are HL60-luc cell, K562-CLL1-luc cell and K562-luc cell, wherein: HL60-luc cells are positive target cells stably expressing luciferase activity and endogenously expressing CLL1, K562-CLL1-luc are positive target cells stably expressing luciferase activity and exogenously expressing CLL1, and K562-luc are negative target cells stably expressing luciferase activity and not expressing CLL 1. In a negative target cell K562-luc system, each effector cell has no obvious killing on the negative target cell; in a positive target cell system, the cell killing results are shown in FIGS. 2A-2B. FIG. 2A shows the killing effect of each effector cell in a positive target cell HL60-luc system with an effective target ratio of 9:1, except UTD cells, the killing difference of each effector cell on target cells is not obvious, and can reach more than 80 percent; the effective target ratio is 3: in the 1, the killing effect of each group (namely 1-3LH-Q1, 1-3LH-Q2 and 1-3 LH-Q3) subjected to humanization in the 1-3LH series is obviously better than that of 1-3LH CAR-T cells, the killing effect of each group (namely 3-67LH-Q1 and 3-67 LH-Q2) subjected to humanization in the 3-67LH series is also obviously better than that of 3-67LH CAR-T cells, and the positive control BG1805 has the following effective target ratio of 3: and 1, the killing effect is better. FIG. 2B shows the killing effect of each effector cell in the positive target cell K562-CLL1-luc system with an effective target ratio of 3: at 1 and 9:1, each effector cell had significant killing of the target cell pair, but the difference in killing of each effector cell to the target cell was not significant.

Example 5 cytokine release assay for each CLL1 CAR-T cell

Cytokine release assay: 8 CLL1 CAR-T cells and UTD cells (non-CAR-transduced T cells) obtained in example 26 days after transduction were co-cultured with target cells in an effective target ratio of 1:1 in X-VIVO15 medium for 24 hours, respectively, and then the concentration of IFN-gamma in the cell supernatant was measured by ELISA method. The target cells are HL60 cells, K562-CLL1 cells and K562 cells respectively. The results are shown in FIG. 3: the release amount of IFN-gamma is obviously improved in each group (namely, 1-3LH-Q1, 1-3LH-Q2 and 1-3 LH-Q3) which is subjected to humanization in the 1-3LH series compared with the group (namely, 1-3 LH-3) which is not subjected to humanization, and the release amount of IFN-gamma is obviously improved in each group (namely, 3-67LH-Q1 and 3-67 LH-Q2) which is subjected to humanization in the 3-67LH series compared with the group (namely, 3-67LH-Q1 and 3-67 LH-Q2) which is not subjected to humanization. The release amount of IFN-gamma of the positive control BG1805 is also higher.

Example 6 sustained proliferation assay

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 proportion of each group of CAR-T cells (1-3 LH, 1-3LH-Q1, 1-3LH-Q2, 1-3LH-Q3, 3-67LH-Q1, 3-67LH-Q2, BG 1805) was adjusted to a 50% level using UTD, and in a continuous proliferation experiment, each group of CAR-T cells was co-cultured with positive target cells HL60 in 24 well plates at an effective target ratio of 2:1, 2ml X-VIVO15 medium per well, 3 wells were repeated. CD3 staining with fluorescence labeled CD3 antibody (manufacturer: bioLegend, cat# 300312), detection analysis by flow cytometry shows proliferation of T cells (CD 3 is a marker for distinguishing whether T cells are present or not), calculating cell number of CD3 positive cells according to conversion of volume multiple, taking out a certain amount of CAR-T cells according to each group according to calculation result, adding corresponding positive target cells according to effective target ratio of 2:1 for new round of stimulation, repeating the multi-round stimulation every 2 days, and proliferation results of any single round in the multi-round stimulation are shown in FIG. 4: the 1-3LH series tested at D9, D12 and D15, the 1-3LH group proliferated better than its humanized CAR+ T cells of each group, tested at D20, the 1-3LH group proliferated less than its humanized 1-3LH-Q1 and 1-3LH-Q2 groups; the 3-67 series was tested at D9, the proliferation of the 3-67LH group was better than that of the humanized group 3-67LH-Q1, but not as good as that of the humanized group 3-67LH-Q2, and the proliferation of the 3-67LH group was not as good as that of the humanized groups when tested at D12, D15 and D20. 1-3LH-Q1, 1-3LH-Q2, 3-67LH-Q1, 3-67LH-Q2 can be rapidly proliferated at the 20 th day of the experiment, the sustained proliferation capacity of 1-3LH-Q3 is slightly weaker than that of 1-3LH-Q1 and 1-3LH-Q2, 1-3LH-Q1, 1-3LH-Q2, 3-67LH-Q1 and 3-67LH-Q2 are verified in the subsequent experiment, and proliferation of 1-3LH-Q1, 1-3LH-Q2, 3-67LH-Q1 and 3-67LH-Q2 at the 20 th day of the experiment is superior to that of positive control 1805 group. 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 7 specificity of CAR-T cells

To further examine the specificity of CAR-T cells (1-3 LH-Q1, 1-3LH-Q2, 3-67LH-Q1, 3-67 LH-Q2) selected in the in vitro assay, we assessed the specificity of the humanized CLL1scFv antibodies by designing assays at the protein and cell levels and used BG1805 as a positive control scFv antibody.

1. Specific detection of protein levels

We performed specific detection of protein levels on 4 CLL1CAR-T cells that relatively perform better in vitro assays, positive control BG1805CAR-T cells, using fluorescence labelled CD19 protein (purchased from ACRO biosystems, cat# CD9-HP2H 3), CS1 protein (purchased from ACRO biosystems, cat# SL7-HP2H 3), BCMA protein (purchased from ACRO biosystems, cat# BCA-HF 254), GPC3 protein (purchased from ACRO biosystems, cat# GP3-HF2H 1), EGFR vIII protein (purchased from ACRO biosystems, cat# EGI-HP2E 3), CD33 protein (purchased from ACRO biosystems, cat# CD3-HF 224) 6 antigen proteins according to the following method: 5X 10 ⁵ of each CAR-T cell is taken, the cells are washed once with PBS, centrifuged for 5min at 200g, the supernatant is discarded, the cells are resuspended in 200ul PBS, 0.1ug of the fluorescent-labeled antigen protein is added, the cells are incubated at 4 ℃ in the absence of light for 20min, washed once with PBS, centrifuged for 5min at 200g, the supernatant is discarded, the cells are resuspended in 200ul PBS, and the binding of the CAR-T cells to each fluorescent-labeled protein is detected by flow cytometry. The specific detection results are shown in table 6: none of the CLL 1-expressing CAR-T cells bound to CD19 protein, CS1 protein, BCMA protein, GPC3 protein, egfrvlll and CD33 protein, i.e. CLL1CAR showed very good specificity at the protein level.

TABLE 6 detection of CAR-T cell specificity results at protein level

Remarks: "-" indicates detection of CAR-T cells not binding to the protein, and "+" indicates detection of CAR-T cells binding to the protein.

2. Specific detection of cellular levels

The expression of CD107a on the membrane is considered as a marker for activating cytotoxic lymphocytes (cd8+ T cells and NK cells) which are activated by immunity when given a specific antigen stimulus, and express CD107a, and thus can be used as a means for detecting CAR-T cell specificity.

We used 8 different tissue-derived cell lines HL60 (human promyelocytic leukemia cell), ovcar (human ovarian adenocarcinoma cell), RAJI (human lymphoma cell), mm.1s (human multiple myeloma cell), SK-MEL1 (human skin melanoma cell), a549 (human lung cancer cell), HUH7 (human hepatoma cell) and 786-0 (human renal carcinoma cell line) to co-culture with 5 CLL1 CAR-T cells and BG1805 CAR-T cells as follows, and evaluated the specificity of CLL1 CAR at the cellular level. 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 ⁶/ml with X-VIVO medium and T cells were adjusted to 1X 10 ⁶/ml. For the killing plate, 20ul of fluorescence labelled CD107a antibody was added per well, followed by 100ul of T cells and 100ul of tumor cells per well, and centrifugation was performed at 400r/min for 3 min after addition. The cells were incubated at 37℃for 60 minutes in a 5% CO ₂ incubator. After 60 minutes, 20ul golgi stop working solution (3 ml X-VIVO medium plus 2ul golgi stop (BD GolgiStop ^TM Protein Transport Inhibitor, CAT: 554724)) was added to each well and incubated 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. The fluorescence signal of CD107a in the cd3+ and cd8+ positive cell populations was analyzed. Specific detection results are shown in table 7, 4 CLL1 CAR-T cells to be detected and positive control BG1805 CAR-T cells can only recognize CLL1 positive expression cell line HL60 and are activated by the CLL1 positive expression cell line HL60 to generate CD107a, but are not activated by other 7 target cells to generate CD107a, and the 5 CLL1 CARs show good specificity.

TABLE 7 results of cell level detection of CAR-T cell specificity

Remarks: in a row of CLL1 expression, "-" indicates that CLL1 is not expressed on the tumor cell surface and "+" indicates that CLL1 is expressed on the tumor cell surface. In the remaining rows, "-" indicates that no CD107a signal was detected, and "+" indicates that a CD107a signal was detected.

Example 8 in vivo efficacy test

Based on in vitro pharmacodynamic studies, we developed in vivo pharmacodynamic assays of humanized CLL1 CAR-T in order to further screen and determine the function of humanized antibodies. An animal model of HL60-GFP-Luc acute myelogenous leukemia was established using NPG mice, CLL1 CAR-T cells were infused back through the tail vein and tumor-clearing effects in animals were monitored and assessed. The method comprises the following specific steps: the HL60-GFP-Luc cells (the HL60 cells are infected by slow viruses with nucleotide sequences for encoding GFP protein and luciferase, and then the obtained cells are identified to obtain a cell line for stably expressing the GFP protein and the luciferase, the luciferase is used for providing fluorescent signals, the weaker the fluorescent signals indicate that the less tumor cells remain, the stronger the killing is, the GenBank of the luciferase gene is MK484106.1, the GFP protein is used for screening the stable cell line, the GFP protein is a known sequence, the sequence is shown as SEQ ID NO. 54), the GFP protein is inoculated into tail veins of female NPG mice at 1X 10 ⁶/0.2 mL/volume, the cells are randomly grouped according to the tumor imaging signal intensity on the 5 th day after the cell inoculation, 7 groups (grouping information is shown in Table 8) of the samples are returned, and the feedback doses of the samples are calculated by CAR+ cells.

Table 8, in vivo efficacy experiment group of CAR-T cells

Note that: each CAR-T cell group was counted as car+ cells, 5×10 ⁶ car+ cells/0.2 mL/mouse, and each group was dosed at 200 μl/mouse.

The test article was administered once through the tail vein, day0 to Day14 after the CAR-T feedback, and in vivo imaging was performed 2 times per week, after which the tumor imaging signal value of the mice was recorded instead once per week. The results are shown in FIG. 5: compared with a negative control Mock-T group without transduced T cells and a negative control PBS group, each CLL 1CAR-T group has the capacity of eliminating tumor cells in vivo, and can obviously inhibit tumor growth at test doses; wherein: the 3 test groups 1-3LH-Q1CAR-T cell group, 1-3LH-Q2 CAR-T cell group and 3-67LH-Q2 CAR-T cell group perform better, and similar or better performance is achieved compared with the positive control BG1805 CAR-T cell group.

Sequence description

1 Murine antibody 1-3LH VH, i.e., 1-3LH VH;

EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKAKLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYWYDEGHAMDSWGQGTSVTVSS

2 VL of murine antibody 1-3LH, namely 1-3LH VL;

DIVMTQSLKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK

3 murine antibody 3-67LH VH, namely 3-67LH VH;

QVQLQQSGAELVKPGASVKLSCKASDYIFTSYYLYWVKQRPGQGLEWIGEINPSNGGTNFNAKFKSKATLTVDKSSNTAYMQLRSLTSEDSAVYYCTRVMWFASYAMDYWGQGTSVTVSS

VL of the murine antibody 3-67LH of SEQ ID NO. 4, namely 3-67LH VL;

DIQLTQTTASLSASLGDRVTINCKTSQDIRNNLNWYQQKPDGTIKLLIYSTSKLHSGVPSRFSGSGSGTDYSLTITNLDQEDIATYFCQQGNTLPLTFGSGTKLEIK

the VH CDR1 of the 5 murine antibody 1-3 LH;

SYVMH

the VH CDR2 of the murine antibody 1-3LH of SEQ ID NO. 6;

YINPYNDGTKYNEKFKG

7 VH CDR3 of murine antibody 1-3 LH;

GYYWYDEGHAMDS

VL CDR1 of 8 murine antibody 1-3 LH;

KASQDVSTAVA

VL CDR2 of the murine antibody 1-3LH of SEQ ID NO. 9;

SASYRYT

VL CDR3 of the murine antibody 1-3LH of SEQ ID NO. 10;

QQHYSTPLT

11 murine antibody 3-67LH VH CDR1;

SYYLY

VH CDR2 of the murine antibody 3-67LH of SEQ ID No. 12;

EINPSNGGTNFNAKFKS

13 VH CDR3 of murine antibody 3-67 LH;

VMWFASYAMDY

VL CDR1 of a murine antibody 3-67LH of SEQ ID NO. 14;

KTSQDIRNNLN

VL CDR2 of a murine antibody 3-67LH of SEQ ID NO. 15;

STSKLHS

VL CDR3 of 16 murine antibody 3-67 LH;

QQGNTLPLT

One of the humanized design modes of VL of the murine antibody 1-3LH of SEQ ID NO. 17 1-3LH VL1;DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVP SRFSGSGSGTDFTFTISSLQPEDFAVYYCQQHYSTPLTFGGGTKLEIK

Two humanized design modes of VL of 18 murine antibody 1-3LH 1-3LH VL2;DIQMTQSLSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVP DRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIK

One of the humanized design modes of VH of the murine antibody 1-3LH of SEQ ID NO. 19 1-3LH VH1;QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSS

Two 1-3LH VH 2 of humanized design modes of VH of 20 murine antibody 1-3 LH;

QVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVRQKPGQGLEWIGYINPYNDGTKYNEKFKGRAKLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSS

3-67LH VL1, one of the humanized design modes of VL of the 21 murine antibody 3-67 LH;

DIQMTQSPSSLSASVGDRVTITCKTSQDIRNNLNWYQQKPGKTIKLLIYSTSKLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPLTFGGGTKLEIK

22 murine antibody 3-67LH, two 3-67LH VL2 of the humanized design of VL of LH;

DIQLTQSPSSLSASVGDRVTINCKTSQDIRNNLNWYQQKPGKTIKLLIYSTSKLHSGVPSRFSGSGSGTDYTLTISSLDPEDFATYFCQQGNTLPLTFGGGTKLEIK

3-67LH VH1, one of the humanized design modes of the VH of the 23 murine antibody 3-67 LH;

QVQLVQSGAEVKKPGASVKVSCKASDYIFTSYYLYWVRQAPGQGLEWMGEINPSNGGTNFNAKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARVMWFASYAMDYWGQGTTVTVSS

24 murine antibody 3-67LH, two 3-67LH VH2 in the humanized design of VH;

QVQLVQSGAEVKKPGASVKVSCKASDYIFTSYYLYWVRQAPGQGLEWIGEINPSNGGTNFNAKFKSRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRVMWFASYAMDYWGQGTTVTVSS

SEQ ID NO:25 1-3LH scFv；

DIVMTQSLKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKAKLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYWYDEGHAMDSWGQGTSVTVSS

SEQ ID NO:26 1-3LH-Q1 scFv；

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFAVYYCQQHYSTPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSS

SEQ ID NO:27 1-3 LH-Q2 scFv；

DIQMTQSLSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSS

SEQ ID NO:28 1-3 LH-Q3 scFv；

DIQMTQSLSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVRQKPGQGLEWIGYINPYNDGTKYNEKFKGRAKLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSS

SEQ ID NO:29 3-67 LH scFv；

DIQLTQTTASLSASLGDRVTINCKTSQDIRNNLNWYQQKPDGTIKLLIYSTSKLHSGVPSRFSGSGSGTDYSLTITNLDQEDIATYFCQQGNTLPLTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASDYIFTSYYLYWVKQRPGQGLEWIGEINPSNGGTNFNAKFKSKATLTVDKSSNTAYMQLRSLTSEDSAVYYCTRVMWFASYAMDYWGQGTSVTVSS

SEQ ID NO:30 3-67 LH-Q1 scFv；

DIQMTQSPSSLSASVGDRVTITCKTSQDIRNNLNWYQQKPGKTIKLLIYSTSKLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASDYIFTSYYLYWVRQAPGQGLEWMGEINPSNGGTNFNAKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARVMWFASYAMDYWGQGTTVTVSS

SEQ ID NO:31 3-67 LH-Q2 scFv;DIQLTQSPSSLSASVGDRVTINCKTSQDIRNNLNWYQQKPGKTIKLLIYSTSKLHSGVPSRFSGSGSGTDYTLTISSLDPEDFATYFCQQGNTLPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASDYIFTSYYLYWVRQAPGQGLEWIGEINPSNGGTNFNAKFKSRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRVMWFASYAMDYWGQGTTVTVSS

SEQ ID NO. 32 linker sequence 1;

GGGGSGGGGSGGGGS

33 linker sequence 2;

GSTSGSGKPGSGEGSTKG

34 linker sequence 3;

GGGGS

SEQ ID NO:35 1-3LH CAR；

MALPVTALLLPLALLLHAARP

DIVMTQSLKFMSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGGGGSGGGGSGGGGSEVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKAKLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYWYDEGHAMDSWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:36 1-3LH-Q1 CAR；

MALPVTALLLPLALLLHAARP

DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDFAVYYCQQHYSTPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:37 1-3 LH-Q2 CAR；

MALPVTALLLPLALLLHAARP

DIQMTQSLSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYINPYNDGTKYNEKFKGRVTLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:38 1-3 LH-Q3 CAR；

MALPVTALLLPLALLLHAARP

DIQMTQSLSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKSPKLLIYSASYRYTGVPDRFSGSGSGTDFTFTISSVQPEDFAVYYCQQHYSTPLTFGAGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVRQKPGQGLEWIGYINPYNDGTKYNEKFKGRAKLTSDTSTSTAYMELSSLRSEDTAVYYCARGYYWYDEGHAMDSWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:39 3-67 LH CAR；

MALPVTALLLPLALLLHAARPDIQLTQTTASLSASLGDRVTINCKTSQDIRNNLNWYQQKPDGTIKLLIYSTSKLHSGVPSRFSGSGSGTDYSLTITNLDQEDIATYFCQQGNTLPLTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVKPGASVKLSCKASDYIFTSYYLYWVKQRPGQGLEWIGEINPSNGGTNFNAKFKSKATLTVDKSSNTAYMQLRSLTSEDSAVYYCTRVMWFASYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:40 3-67LH-Q1 CAR；

MALPVTALLLPLALLLHAARP

DIQMTQSPSSLSASVGDRVTITCKTSQDIRNNLNWYQQKPGKTIKLLIYSTSKLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGNTLPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASDYIFTSYYLYWVRQAPGQGLEWMGEINPSNGGTNFNAKFKSRVTLTRDTSTSTAYMELSSLRSEDTAVYYCARVMWFASYAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:41 3-67LH-Q2 CAR；

MALPVTALLLPLALLLHAARP

DIQLTQSPSSLSASVGDRVTINCKTSQDIRNNLNWYQQKPGKTIKLLIYSTSKLHSGVPSRFSGSGSGTDYTLTISSLDPEDFATYFCQQGNTLPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASDYIFTSYYLYWVRQAPGQGLEWIGEINPSNGGTNFNAKFKSRVTLTVDTSTSTAYMELSSLRSEDTAVYYCTRVMWFASYAMDYWGQGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

a 42CD8 alpha guide chain of SEQ ID NO;

MALPVTALLLPLALLLHAARP

the 43CD8 alpha hinge region;

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

A 44CD 8. Alpha. Transmembrane region of SEQ ID NO;

IYIWAPLAGTCGVLLLSLVITLYC

SEQ ID NO. 45-1 BB intracellular co-stimulatory signal;

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

46CD3 zeta activation signal;

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

A nucleotide sequence of SEQ ID NO. 47 encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO. 19;

CAGGTGCAGCTGGTGCAGAGCGGTGCCGAGGTGAAAAAGCCAGGCGCTTCTGTGAAGGTGTCTTGTAAAGCCTCTGGCTATACATTTACCTCCTACGTGATGCACTGGGTCAGACAGGCCCCTGGCCAGGGCCTGGAATGGATGGGATATATCAACCCCTACAATGATGGCACCAAGTACAACGAGAAGTTCAAGGGCAGAGTGACACTGACAAGCGACACCAGCACCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCTGTGTACTACTGCGCCAGAGGCTACTACTGGTACGACGAGGGCCACGCCATGGACAGCTGGGGCCAGGGCACACTGGTCACAGTGTCCAGC

a nucleotide sequence of SEQ ID NO. 48 encoding an amino acid sequence of a light chain variable region as shown in SEQ ID NO. 17;

GATATCCAAATGACCCAGAGCCCATCTTCTCTGTCTGCTTCTGTGGGCGACAGAGTCACCATTACATGCAAGGCTTCTCAGGACGTGTCCACAGCTGTCGCCTGGTATCAGCAGAAGCCCGGCAAGTCCCCTAAGCTGCTGATCTACAGCGCCAGCTACCGGTACACAGGCGTGCCCAGCAGGTTCAGCGGATCTGGATCCGGCACCGATTTTACCTTCACCATCAGCAGCCTGCAGCCTGAGGACTTCGCCGTGTACTATTGTCAGCAACACTACAGCACACCTCTGACCTTCGGCGGCGGCACAAAGCTGGAAATCAAG

a nucleotide sequence of SEQ ID NO. 49 encoding the amino acid sequence of the light chain variable region as shown in SEQ ID NO. 18;

GATATCCAGATGACACAGAGCCTCTCTAGCCTGAGCGCCTCCGTGGGCGACAGGGTGACCATTACATGCAAGGCCAGCCAGGACGTGTCCACCGCCGTGGCCTGGTATCAGCAAAAGCCCGGCAAGAGCCCTAAGCTGCTGATCTACAGCGCCAGCTACCGGTACACAGGCGTGCCTGATCGGTTCAGCGGATCTGGATCTGGCACCGACTTCACCTTCACCATCAGCTCCGTTCAACCTGAGGATTTCGCCGTGTACTACTGCCAGCAGCACTACAGCACACCTCTGACCTTTGGCGCTGGCACCAAACTGGAAATCAAG

SEQ ID NO. 50 nucleotide sequence encoding the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO. 24 ;CAGGTGCAACTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCTCTGATTACATCTTCACGAGCTACTACCTGTATTGGGTGCGGCAGGCCCCTGGACAGGGCCTGGAATGGATCGGCGAGATCAACCCCAGCAACGGCGGCACAAACTTCAACGCCAAGTTCAAGAGCAGAGTCACACTGACCGTGGACACCTCCACCAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTACCAGGGTGATGTGGTTCGCCTCTTACGCCATGGACTACTGGGGCCAGGGCACAACAGTGACCGTTTCCAGC

SEQ ID NO. 51 shows the nucleotide sequence of the amino acid sequence of the light chain variable region as shown in SEQ ID NO. 22 ;GATATTCAGCTGACCCAGAGCCCTAGCAGCCTGTCTGCCAGCGTCGGAGATAGAGTGACCATCAATTGCAAAACCAGCCAGGACATCAGAAACAACCTGAACTGGTACCAGCAAAAGCCCGGCAAGACCATCAAACTGCTGATCTACAGCACATCAAAGCTCCACAGCGGCGTGCCAAGCCGGTTCAGCGGATCCGGCTCTGGCACCGACTATACACTGACCATCAGCAGCCTGGACCCTGAGGACTTTGCTACATACTTCTGCCAGCAGGGCAATACCCTGCCTCTGACCTTTGGCGGTGGAACAAAGCTGGAAATCAAG

52 Positive control BG1805 scFv;

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSS

SEQ ID NO. 53 positive control BG1805 amino acid sequence

MALPVTALLLPLALLLHAARP

DIQMTQSPSSLSASVGDRVTITCKSSQSLLYSDNQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTGYHMHWVRQAPGQRLEWMGRINPYNGAASHNQKFKDRVTITRDTSASTAYMELSSLRSEDTAVYYCARGWDYDGGYYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Nucleotide sequence of 54 coding GFP protein

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag.

Claims

1. A humanized CLL1 antibody or antigen binding portion thereof comprising a heavy chain variable region whose amino acid sequences of CDR1, CDR2, CDR3 comprise the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the heavy chain variable region of the antibody shown in SEQ ID No. 1, and a light chain variable region whose amino acid sequences of CDR1, CDR2, CDR3 comprise the amino acid sequences of CDR1, CDR2, CDR3, respectively, in the light chain variable region of the antibody shown in SEQ ID No. 2;

2. A humanized CLL1 antibody or antigen binding portion thereof, comprising a heavy chain variable region and a light 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. 5, SEQ ID No. 6, SEQ ID No. 7, respectively, the amino acid sequences of CDR1, CDR2, CDR3 of the light chain variable region comprising the amino acid sequences shown as SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, respectively;

3. The humanized CLL1 antibody or antigen binding portion thereof of claim 1 or 2, wherein,

The amino acid sequence of the heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO. 19, and the amino acid sequence of the light chain variable region comprises an amino acid sequence shown as SEQ ID NO. 17;

4. The humanized CLL1 antibody or an antigen binding portion thereof according to claim 3, wherein,

The humanized CLL1 antibody or the antigen binding site thereof is a CLL1 scFv antibody or a CLL1Sc (Fv) ₂ antibody.

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

The CLL1 scFv antibody comprises any one selected from the group consisting of: an amino acid sequence-linker sequence shown as SEQ ID NO. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 17-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 19-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 20-an amino acid sequence-linker sequence shown as SEQ ID NO. 20-an amino acid sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 21-a linker sequence shown as SEQ ID NO. 23-an amino acid sequence shown as SEQ ID NO. 21-an amino acid sequence shown as SEQ ID NO. 22-a linker sequence shown as SEQ ID NO. 24-an amino acid sequence shown as SEQ ID NO. 22.

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. 17-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 18-an amino acid sequence shown as SEQ ID NO. 19-an amino acid sequence-linker sequence shown as SEQ ID NO. 22-an amino acid sequence shown as SEQ ID NO. 24;

Further alternatively, the CLL1 scFv antibody comprises any one selected from the group consisting of seq id nos: an amino acid sequence shown as SEQ ID NO. 26, an amino acid sequence shown as SEQ ID NO. 27, an amino acid sequence shown as SEQ ID NO. 31;

Still further alternatively, the linking sequence is selected from one or more of the following sequences: SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the humanized CLL1 antibody or an antigen binding portion thereof of any one of claims 1-5.

7. The isolated nucleic acid molecule of claim 6, wherein the nucleotide sequence encoding the humanized CLL1 antibody or antigen binding portion thereof comprises:

8. A vector comprising the isolated nucleic acid molecule of claim 6 or 7.

9. A cell comprising the humanized CLL1 antibody or antigen binding portion thereof of any one of claims 1-5, the isolated nucleic acid molecule of claim 6 or 7 or the vector of claim 8.

10. A pharmaceutical composition comprising the humanized CLL1 antibody or antigen binding portion thereof of any one of claims 1-5, the isolated nucleic acid molecule of claim 6 or 7, the vector of claim 8 or the cell of claim 9, and a pharmaceutically acceptable adjuvant.

11. An antibody drug comprising the humanized CLL1 antibody or an antigen binding portion thereof of any one of claims 1-5;

Optionally, the antibody drug is a monospecific antibody drug, a bispecific antibody drug, a trispecific antibody drug, or a tetraspecific antibody drug.

12. An antibody drug conjugate comprising the humanized CLL1 antibody or an antigen binding portion thereof of any one of claims 1-5.

13. Use of a humanized CLL1 antibody or an antigen binding portion thereof of any one of claims 1-5, an isolated nucleic acid molecule of claim 6 or 7, a vector of claim 8 or a cell of claim 9 in the manufacture of a medicament for the treatment of a disease or disorder associated with expression of CLL 1;

Alternatively, the disease or disorder associated with expression of CLL1 is a hematological neoplasm.

Further alternatively, the disease or disorder associated with expression of CLL1 is acute myeloid leukemia.

14. Use of the humanized CLL1 antibody or antigen binding portion thereof of any one of claims 1-5, the isolated nucleic acid molecule of claim 6 or 7, the vector of claim 8 or the cell of claim 9 in the preparation of a test reagent for diagnosing a disease or disorder associated with expression of CLL 1;

Alternatively, the disease or disorder associated with expression of CLL1 is a hematological neoplasm;