CN115109157A - Antibody or antigen binding fragment thereof, preparation method and medical application thereof - Google Patents
Antibody or antigen binding fragment thereof, preparation method and medical application thereof Download PDFInfo
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- CN115109157A CN115109157A CN202210287884.8A CN202210287884A CN115109157A CN 115109157 A CN115109157 A CN 115109157A CN 202210287884 A CN202210287884 A CN 202210287884A CN 115109157 A CN115109157 A CN 115109157A
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- antigen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention relates to an antibody or an antigen binding fragment thereof, a preparation method thereof and medical application thereof, namely the antibody or the antigen binding fragment thereof for resisting a C-type lectin-like molecule 1(CLL-1) receptor, and further relates to a fully human antibody or an antigen binding fragment thereof for resisting the CLL-1 receptor, medical application thereof, a pharmaceutical composition containing the fully human antibody or the antigen binding fragment thereof for resisting the CLL-1 receptor, and medical application thereof as a medicament for treating myeloproliferative diseases, particularly acute myelogenous leukemia.
Description
Technical Field
The present invention relates to a CLL-1 antibody or an antigen-binding fragment thereof, and further, the present invention relates to a CLL-1 fully human antibody or an antigen-binding fragment thereof comprising CDR regions; the invention also relates to a pharmaceutical composition containing the CLL-1 fully human antibody or the antigen binding fragment thereof, and application thereof as a diagnostic agent and a therapeutic drug for CLL-1 related diseases.
Background
Acute Myelogenous Leukemia (AML) is a malignant tumor caused by abnormal proliferation of myeloid hematopoietic stem cells, and the current clinical treatment is mainly chemotherapy. Most AML patients are in remission after chemotherapy, but most suffer relapse or death after remission. In 2005, monoclonal antibody against CD33, developed by feverfew, was approved as the first biopharmaceutical for AML. However, in subsequent studies, Mylotary did not achieve the desired results and was released from the united states in 2010. Although immunotherapy has shown excellent therapeutic effects in many malignant tumors, AML has been studied at a slow pace due to the heterogeneity of myeloid cells and low specificity of target antigens. Currently, therapeutic drug development for AML is more focused on new targets with novel mechanisms of action.
C-type lectin-like molecule 1(CLL-1, also known as CLEC12A/MICL/DCAL2) belongs to a member of the C-type lectin family, which is functionally diverse, such as cell adhesion, signaling, and a role in inflammation and immune response. CD34 expressed in CLL-1 in most AML + CD38 - On stem cells, normal human CD34 + CD38 - The stem cells do not express. Therefore, the cell surface antigen can be used as a marker for AML diagnosis, andit has also been shown to be an ideal target for treating AML. Currently, the development strategy for CLL-1 is mainly focused on the field of bispecific antibodies and CAR-T, and many are in early development stages, and only one type of bispecific antibody named MCLA-117(CD3/CLL-1) is undergoing phase I clinical research. Therefore, there is a need to further develop CLL-1 fully human antibodies with higher activity, high affinity and high stability for therapeutic research and application of related diseases.
Phage display technology has been increasingly used for antibody discovery in recent years, and some therapeutic monoclonal antibodies developed by the technology have been used for clinical treatment, such as adalimumab for treating various autoimmune diseases such as arthritis, belimumab for treating systemic lupus erythematosus, monoclonal antibodies for treating various tumors such as non-small cell lung cancer, gastric cancer, colon cancer, head and neck cancer, such as ramucirumab, ranibizumab, resisbank and the like.
The full-human natural phage display library is a phage library constructed by reverse transcribing mRNA from human peripheral blood cells into cDNA, and the library has rich capacity, low immunogenicity and fast and high efficiency in antibody screening. The technology integrates the coding gene of the exogenous protein into a phage genome, expresses the exogenous target protein on the surface of a phage capsid protein through transcription and translation, screens a phage library by gradually reducing the concentration of antigen protein through a solid phase-liquid phase, selects out affinity specificity positive clone, then performs DNA sequencing, and utilizes sequence characteristic cloning to construct a full-length antibody, thereby shortening the early discovery time of the antibody. Phage display libraries are classified into natural libraries and immune libraries according to the source of the antigen, and into Fab fragments, ScFv, light chain, heavy chain libraries, and the like, according to the protein displayed by each display library.
Disclosure of Invention
The invention utilizes a fully human natural phage display library to screen out a specific anti-human CLL-1 Fab fragment, and then utilizes a human natural Fc framework to construct a fully human anti-human CLL-1 antibody.
The present invention provides an anti-CLL-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; wherein said heavy chain variable region comprises at least 1 HCDR1 selected from the group consisting of seq id nos: 1, 7, 13, 19 and 25; and at least 1 HCDR2 selected from the group consisting of seq id no: HCDR 2: 2, 8, 14, 20 and 26; and at least 1 HCDR2 selected from the group consisting of seq id no: HCDR 3: 3, 9, 15, 21, 27 and 33,
the light chain variable region comprises at least 1 LCDR1 selected from the group consisting of seq id nos: 4, 10, 16, 22 and 34; and at least 1 LCDR2 selected from the group consisting of seq id no:5, 11, 17, 23 and 35; and at least 1 LCDR3 selected from the group consisting of seq id no:6, 12, 18, 24, 30 and 36.
The present invention also relates to a preferred embodiment, according to the present invention, there is provided an anti-CLL-1 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises:
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively;
or, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9, respectively;
or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15, respectively;
or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21, respectively;
or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27, respectively;
or, HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 33, respectively.
The present invention also relates to a preferred embodiment, which provides an anti-CLL-1 antibody or an antigen-binding fragment thereof according to the present invention, wherein the light chain variable region comprises:
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively;
or LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, respectively;
or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, respectively;
or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, respectively;
or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO 16, SEQ ID NO 5 and SEQ ID NO 30, respectively;
or LCDR1, LCDR2 and LCDR3 shown as SEQ ID NO 34, SEQ ID NO 35 and SEQ ID NO 36, respectively.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or an antigen-binding fragment thereof provided according to the present invention, wherein:
the heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2 and HCDR3 shown in SEQ ID NO. 3, and the light chain variable region comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6; or the like, or, alternatively,
the heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 7, HCDR2 shown in SEQ ID NO. 8 and HCDR3 shown in SEQ ID NO. 9; and the variable region of the antibody light chain comprises LCDR1 shown in SEQ ID NO. 10, LCDR2 shown in SEQ ID NO. 11 and LCDR3 shown in SEQ ID NO. 12; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 13, HCDR2 shown in SEQ ID NO. 14 and HCDR3 shown in SEQ ID NO. 15; and the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO. 16, LCDR2 shown in SEQ ID NO. 17 and LCDR3 shown in SEQ ID NO. 18; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 19, HCDR2 shown in SEQ ID NO. 20 and HCDR3 shown in SEQ ID NO. 21; and the antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO. 22, LCDR2 shown in SEQ ID NO. 23 and LCDR3 shown in SEQ ID NO. 24; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 25, HCDR2 shown in SEQ ID NO. 26 and HCDR3 shown in SEQ ID NO. 27; and the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO. 16, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 30; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 7, HCDR2 shown in SEQ ID NO. 8 and HCDR3 shown in SEQ ID NO. 33; and the antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO. 34, LCDR2 shown in SEQ ID NO. 35 and LCDR3 shown in SEQ ID NO. 36.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of those represented by seq id nos: 37, 39, 41, 43, 45 or 47 SEQ ID NO,
the present invention also relates to a preferred embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the group consisting of those represented by the following sequences, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: 38, 40, 42, 44, 46 or 48.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or an antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or an antigen-binding fragment thereof comprises:
the heavy chain variable region shown in SEQ ID NO. 37 and the light chain variable region shown in SEQ ID NO. 38; or the like, or a combination thereof,
the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO: 40; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO. 41 and the light chain variable region shown in SEQ ID NO. 42; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO. 43 and the light chain variable region shown in SEQ ID NO. 44; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO. 45 and the light chain variable region shown in SEQ ID NO. 46; or the like, or, alternatively,
the heavy chain variable region shown by SEQ ID NO. 47 and the light chain variable region shown by SEQ ID NO. 48.
The present invention also relates to a preferred embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, or IgG4, or a variant thereof;
preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1 or a variant thereof;
more preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as set forth in SEQ ID NO 28.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises a light chain constant region further comprising a light chain derived from a human antibody kappa chain, lambda chain or a variant thereof;
preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a kappa chain, a lambda chain of a human antibody;
further preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID NO. 29 or SEQ ID NO. 31.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein said anti-CLL-1 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of the heavy chains represented by the following sequences, or a heavy chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequences: 49, 51, 53, 55, 57 or 59 SEQ ID NO.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises a light chain selected from the group consisting of a light chain represented by the following sequence, or a light chain having at least 80%, 85%, 90%, 95% or 99% identity compared to the following sequence: 50, 52, 54, 56, 58 or 60.
The present invention also relates to a preferred embodiment, an anti-CLL-1 antibody or an antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody comprises:
the heavy chain shown as SEQ ID NO. 49 and the light chain shown as SEQ ID NO. 50; or
The heavy chain shown as SEQ ID NO. 51 and the light chain shown as SEQ ID NO. 52; or
A heavy chain shown as SEQ ID NO. 53 and a light chain shown as SEQ ID NO. 54; or
The heavy chain shown as SEQ ID NO. 55 and the light chain shown as SEQ ID NO. 56; or
The heavy chain shown as SEQ ID NO. 57 and the light chain shown as SEQ ID NO. 58; or
The heavy chain shown as SEQ ID NO. 59 and the light chain shown as SEQ ID NO. 60.
The present invention also relates to a preferred embodiment, the anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the present invention, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof is selected from a fully human antibody or antigen-binding fragment thereof.
The invention further provides a polynucleotide encoding the anti-CLL-1 antibody or antigen-binding fragment thereof described above.
The present invention further provides an expression vector comprising the polynucleotide described above.
The present invention further provides a host cell into which the above-described expression vector is introduced or contained.
In a preferred embodiment of the present invention, there is provided a host cell according to the present invention, wherein said host cell is selected from the group consisting of a bacterial, yeast or mammalian cell; wherein, the bacteria is preferably Escherichia coli, Pichia yeast and the mammalian cell is preferably CHO cell or HEK293 cell.
The present invention further provides a method for producing an anti-CLL-1 fully human antibody or an antigen-binding fragment thereof, comprising the steps of:
a) culturing the host cell of the invention;
b) isolating the antibody or antigen-binding fragment thereof from the culture; and the number of the first and second groups,
c) purifying the antibody or antigen-binding fragment thereof of step b).
The invention further provides a pharmaceutical composition comprising an anti-CLL-1 antibody or antigen-binding fragment thereof of the invention, and a pharmaceutically acceptable excipient, diluent or carrier.
The invention further provides a detection or diagnostic kit comprising an anti-CLL-1 antibody or antigen-binding fragment thereof of the invention, optionally further comprising one or more reagents capable of detecting the binding of the anti-CLL-1 antibody or antigen-binding fragment thereof to CLL-1.
The invention further provides a detection or diagnostic kit comprising an anti-CLL-1 antibody or antigen-binding fragment thereof of the invention and an excipient, diluent or carrier useful for detection or diagnosis.
In a preferred embodiment of the invention there is provided the use of an anti-CLL-1 antibody or antigen-binding fragment thereof of the invention, or a pharmaceutical composition of the invention, in the manufacture of a medicament for the treatment or prevention of a CLL-1 mediated disease or condition.
In a preferred embodiment of the invention, the anti-CLL-1 antibody or antigen-binding fragment thereof provided according to the invention, or the pharmaceutical composition provided by the invention, is used for the preparation of a kit for the detection, diagnosis, prognosis of a CLL-1 mediated disease or disorder.
In a preferred embodiment of the invention, there is provided a use according to the invention, wherein:
the disease or condition is a myeloproliferative disease;
acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), multiple myeloma, plasmacytoma, and myelofibrosis are preferred.
In a preferred embodiment of the invention, there is provided an anti-CLL-1 antibody or antigen-binding fragment thereof according to the invention, or a pharmaceutical composition according to the invention, or a kit according to the invention, for use in the detection, diagnosis, prognosis of CLL-1 mediated diseases; the disease is selected from: acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, multiple myeloma, plasmacytoma, and myelofibrosis.
The present invention further provides a method of treating or preventing CLL-1 mediated diseases comprising the steps of:
providing a therapeutically or prophylactically effective amount of an anti-CLL-1 antibody or antigen-binding fragment thereof of the invention to a subject; or providing a therapeutically or prophylactically effective amount of a pharmaceutical composition of the invention to a subject; wherein the CLL-1 mediated disease is selected from: acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, multiple myeloma, plasmacytoma, and myelofibrosis.
Detailed Description
Term of
In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The three letter codes and the one letter codes for amino acids used in the present invention are as described in j.biol.chem, 243, p3558 (1968).
The term "antibody" as used herein refers to an immunoglobulin, which is a tetrapeptide chain structure formed by two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The constant regions of immunoglobulin heavy chains differ in their amino acid composition and arrangement, and thus, their antigenicity. Accordingly, immunoglobulins can be classified into five classes, otherwise known as the isotype of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. The same class of igs can be divided into different subclasses according to differences in amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain, and for example, iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Light chains are classified as either kappa or lambda chains by differences in the constant regions. Each of the five classes of Ig may have either a kappa chain or a lambda chain.
In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region comprising a human or murine kappa or lambda chain or a variant thereof.
In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
The sequences of the antibody heavy and light chains, near the N-terminus, are widely varied by about 110 amino acids, being variable regions (V-regions); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable regions include 3 hypervariable regions (HVRs) and 4 Framework Regions (FRs) which are relatively sequence conserved. The 3 hypervariable regions determine the specificity of the antibody, and are also known as Complementarity Determining Regions (CDRs). Each of the light chain variable region (VL) and the heavy chain variable region (VH) is composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR 3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR 3. The CDR amino acid residues in the VL and VH regions of the antibodies or antigen-binding fragments of the invention conform in number and position to the rules of the known Kabat or Chothia or ABM definitions (http:// bio in. org. uk/abs /).
The term "antigen presenting cell" or "APC" is a cell that displays foreign antigens complexed with MHC on its surface. T cells recognize this complex using the T Cell Receptor (TCR). Examples of APCs include, but are not limited to, Dendritic Cells (DCs), Peripheral Blood Mononuclear Cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.
The term "antigen presentation" refers to the process by which APCs capture antigens and enable them to be recognized by T cells, for example as a component of an MHC-I/MHC-II conjugate.
The term "recombinant human antibody" includes human antibodies made, expressed, created or isolated by recombinant methods, involving techniques and methods well known in the art, such as:
1. antibodies isolated from a transgene of human immunoglobulin genes, a transchromosomal animal (e.g., a mouse), or a hybridoma prepared therefrom;
2. antibodies isolated from host cells transformed to express the antibodies, such as transfectomas;
3. antibodies isolated from a library of recombinant combinatorial human antibodies; and
4. antibodies produced, expressed, created or isolated by methods such as splicing of human immunoglobulin gene sequences to other DNA sequences.
Such recombinant human antibodies comprise variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as occur during antibody maturation.
The term "human antibody" or "fully human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" or "fully human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences (i.e., "humanized antibodies").
The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, an antibody fragment retains at least 10% of the parent binding activity when expressed as activity on a molar basis. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: fab, Fab ', F (ab') 2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, nat. biotechnol.23: 1126, 1136.
A "Fab fragment" consists of one light and one heavy chain of CH1 and the variable domains. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form a F (ab ')2 molecule.
An "F (ab') 2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F (ab ')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.
The "Fv region" comprises variable regions from both the heavy and light chains, but lacks the constant region.
The term "multispecific antibody" is used in its broadest sense to encompass antibodies having polyepitopic specificity. These multispecific antibodies include, but are not limited to: an antibody comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitopic specificity; an antibody having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; an antibody having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full length antibodies, antibody fragments, diabodies (diabodies), bispecific diabodies and triabodies (triabodies), antibody fragments that have been covalently or non-covalently linked together, and the like.
The term "single-chain antibody" is a single-chain recombinant protein formed by connecting a heavy chain variable region VH and a light chain variable region VL of an antibody via a linker peptide, and is the smallest antibody fragment having a complete antigen-binding site.
The term "domain antibody fragment" is an immunologically functional immunoglobulin fragment containing only heavy chain variable regions or light chain variable regions. In certain instances, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target the same or different antigens.
The term "binds to CLL-1" in the context of the present invention means capable of interacting with human CLL-1.
The term "antigen binding site" of the present invention refers to a three-dimensional spatial site recognized by an antibody or antigen binding fragment of the present invention.
The term "epitope" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes can be formed from contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after denaturing solvent treatment. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods of determining what epitope is bound by a given antibody are well known in the art and include immunoblot and immunoprecipitation detection assays, and the like. Methods of determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
The terms "specific binding", "selectively binding" and "selective binding" as used herein refer to binding of an antibody to an epitope on a predetermined antigen. Typically, when human CLL-1 is used as the analyte and an antibody is used as the ligand, as determined by Surface Plasmon Resonance (SPR) techniques in an instrument, the antibody is present at a level of about less than 10 -7 M or even smaller equilibrium dissociation constant (K) D ) Binds to a predetermined antigen and binds to the predetermined antigen with at least twice the affinity as it binds to a non-specific antigen other than the predetermined antigen or closely related antigens (e.g., BSA, etc.). The term "antigen-recognizing antibody" is used interchangeably herein with the term "specifically binding antibody".
The term "cross-reactive" refers to the ability of an antibody of the invention to bind to CLL-1 from a different species. For example, an antibody of the invention that binds to human CLL-1 can also bind to CLL-1 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or binding or functional interactions with cells that physiologically express CLL-1. Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.
The terms "inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of a ligand preferably reduces or alters the normal level or type of activity that occurs in the absence of inhibition or blocking when ligand binding occurs. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with an anti-CLL-1 antibody compared to a ligand not contacted with an anti-CLL-1 antibody.
The term "inhibit growth" (e.g., in relation to a cell) is intended to include any measurable decrease in cell growth.
The terms "induce an immune response" and "enhance an immune response" are used interchangeably and refer to stimulation (i.e., passive or adaptive) of an immune response to a particular antigen. The term "induction" with respect to induction of CDC or ADCC refers to stimulation of a specific direct cell killing mechanism.
The term "ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, as used herein refers to the direct killing of antibody-coated target cells by Fc fragments of cells expressing Fc receptors through recognition of the antibody. The ADCC effector function of an antibody may be enhanced or reduced or eliminated by modification of the Fc portion of the IgG. The modification refers to mutation in the heavy chain constant region of the antibody.
The engineered antibodies or antigen binding fragments of the invention can be prepared and purified using conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into the GS expression vector. Recombinant immunoglobulin expression vectors can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bind to antigens of human origin. Positive clones were expanded in bioreactor serum-free medium to produce antibodies. The antibody-secreting culture medium can be purified and collected by conventional techniques. The antibody can be concentrated by filtration by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.
"administration," "administering," and "treating," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration," "administering," and "treating" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of the cells comprises contacting the reagent with the cells and contacting the reagent with a fluid, wherein the fluid is in contact with the cells. "administering", "administering" and "treating" also mean treating, for example, a cell in vitro and ex vivo by an agent, a diagnostic, a binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
By "treating" is meant administering a therapeutic agent, such as any one of the antibodies of the invention, either internally or externally to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically relevant degree. The amount of therapeutic agent effective to alleviate any particular disease symptom (also referred to as a "therapeutically effective amount") can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test commonly used by physicians or other health professional to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., methods of treatment or articles of manufacture) may be ineffective in alleviating the symptoms of the target disease in each patient, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any of the statistical tests known in the art, such as Student's t-test, chi-square test, U-test by Mann and Whitney, Kruskal-Wallis test (H-test), Jonckhere-Terpstra test, and Wilcoxon test.
The term "consisting essentially of … …" or variants thereof as used throughout the specification and claims is meant to encompass all such elements or groups of elements, and optionally other elements of similar or different nature than the elements described, which other elements do not materially alter the basic or novel characteristics of a given dosing regimen, method or composition.
The term "naturally occurring" as applied to an object in accordance with the present invention refers to the fact that the object may be found in nature. For example, a polypeptide sequence or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally occurring.
An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the method and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.
"exogenous" refers to a substance that is to be produced outside an organism, cell, or human body by background.
"endogenous" refers to a substance produced in a cell, organism, or human body by background.
"homology" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, two sequences are 60% homologous if there are 6 matches or homologies at 10 positions in the two sequences when the sequences are optimally aligned. In general, comparisons are made when aligning two sequences to obtain the greatest percentage of homology.
As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny thereof. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where different names are intended, they are clear from the context.
"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a particular sequence may, but need not, be present.
"pharmaceutical composition" means a composition containing one or more antibodies or antigen-binding fragments thereof described herein, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.
Detailed Description
The present invention will be further described with reference to the following examples, which are not intended to limit the scope of the present invention. The experimental method of the present invention, in which the specific conditions are not specified, is usually performed according to conventional conditions, such as the antibody technical laboratory manual of cold spring harbor, molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw material or the goods. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.
Example 1 screening of Fab fragments of anti-CLL-1 antibody from fully human native phage display library
Screening of anti-CLL-1 antibody Fab fragment with capacity of 2.25 × 10 10 cfu/mL, full human native phage display library screening with integrity and diversity. Firstly, carrying out negative enrichment screening on a fully human natural phage display library, co-incubating the fully human natural phage display library and Dynabeads which are sealed by 2% BSA, collecting magnetic beads by a Kingfisher magnetic bead screening system, and collecting bacteria liquid after negative screening. Then, positive screening is carried out, biotinylated CLL-1-Fc (Acrobiosystems, Cat: CLA-H5266) or His label antigen (Acrobiosystems, Cat: CLA-H5245) coated Dynabeads are sealed by 2% BSA solution and incubated with a phage library after negative screening, then, a solid-liquid phase alternate elutriation mode is adopted, CLL-1 protein concentration is gradually reduced, 3 rounds of elutriation are carried out, positive clones are screened out, DNA sequencing is carried out on the positive clones, sequence specific molecules are selected for expression and purification, and further, affinity and verification are carried out.
During the 3 rounds of panning, round 1 screening was performed with 2.5% BSA as blocking solution, in each case, group 1: 100ug/ml of CLL-1-Fc antigen on solid phase, and group 2: liquid phase 300nM biotinylated CLL-1-Fc antigen and negatively screened full human natural phage display library are incubated and screened, and combined positive clone is digested and eluted by trypsin; 2, 2.5% BSA is used as a blocking solution, a solid phase of 30ug/ml CLL-1-His antigen and a liquid phase of 200nM biotinylated CLL-1-His antigen are respectively incubated with the positive clones enriched in the group 1 and the group 2 selected in the 1 st round, and after washing, the combined positive clones are digested and eluted by trypsin; the positive clones from the previous two rounds of screening were washed and eluted by trypsinization after incubation with 10ug/ml of CLL-1-Fc antigen in solid phase using 2.5% BSA as blocking solution for round 3 screening. Plates were coated with 2ug/ml CLL-1Fc antigen, incubated overnight at 4 ℃, washed 3 times with 1 XPBST, blocked with 5% PBS-Milk at room temperature for 1h, and washed 3 times with PBST. Mixing the positive clones screened in the previous 3 rounds, diluting with 1x PBS by 4-fold gradient, incubating at room temperature for 1h, washing the plate for 3 times by PBST, adding a secondary antibody Anti-M13-HRP diluted by 1:8000 and incubating at room temperature for 1h, washing the plate for 5 times by PBST, adding 30 mu L TMB for developing at room temperature for 5-10min, then adding 30 mu L2M HCl for terminating the reaction, reading data by an enzyme labeling instrument OD450 to obtain 109 positive clones in total, and then performing DNA sequencing to obtain 37 specific clones and present gene diversity. The 37 specific clones were subjected to affinity sequencing by ELISA as described above to obtain 6 higher affinity Fab clones, Fab1, Fab2, Fab3, Fab4, Fab5 and Fab 6.
The amino acid sequence of the antibody variable region is shown in table 1:
TABLE 1 heavy and light chain variable region sequences of antibodies
Note: the CDR regions are underlined.
Antibody CDR sequences are shown in table 2:
TABLE 2 CDR sequences of the heavy and light chain variable regions of the antibody
The results of the affinity levels of the ELISA method are shown in table 3:
TABLE 3 level of affinity of Fab clones for recombinant human CLL-1-Fc protein
| Fab cloning | EC50(ug/mL) |
| Fab1 | 0.47 |
| Fab2 | 0.26 |
| Fab4 | 0.90 |
| Fab5 | 0.82 |
| Fab6 | 0.79 |
| Human antibody isotype control | >5 |
Example 2 affinity assessment of Fab clones at the level of CLL-1 expressing cells
The affinity level of the Fab clones for CHO-K1-huCLL-1 was evaluated by flow cytometry. Human CLL-1 Gene (Gene ID:160364) is constructed in a lentiviral vector transfected CHO-K1 host cell, and a monoclonal antibody is selected to obtain an expressed human CLL-1 cell strain CHO-K1-huCLL-1. CHO-K1-huCLL-1 cells in good growth state were treated with Accutase (Sigma, cat: A6964) digest and single cell suspensions were prepared in 2% FBS (DBPS, pH7.4 dilution) solution, adjusting cell density to 1X106 cells/mL. Dividing the mixture into 96-well V-shaped bottom plates at 100 mu L/well, centrifuging at 300g and 5min at 4 ℃, discarding the supernatant, adding a phage Fab clone supernatant solution diluted by 3 times and 9 concentration gradients (5ug/ml-7.62x10-4ug/ml) into 100 mu L/well, and incubating for 1h at 4 ℃; centrifuging at 4 deg.C for 2 times at 300g for 5min, washing with 2% FBS, adding diluted goat anti-human IgG (Fab')2 and PE-labeled secondary antibody (abcam, cat:98606) at 5. mu.L/106 cells/well, and incubating at 4 deg.C for 1 h; 300g x 5min, centrifugation at 4 ℃, washing 2 times, adding 70 μ L of 2% FBS solution to resuspend the cells, and detecting the Mean Fluorescence Intensity (MFI) of the PE channel in a flow cytometer (Bio-Rad, ZE 5).
Data processing:
the concentration logarithm value of the supernatant of the phage Fab clone is taken as an X-axis coordinate, MFI is taken as a vertical coordinate, and the EC of the affinity of the antibody to be detected to the tumor cells is calculated by utilizing the formula of GraphPad PRISM 8.0log (aginst) vs 50 As shown in table 8 below:
TABLE 4 affinity level differences (EC) of Fab clones for CHO-K1-hu CLL-1 50 )
And (4) experimental conclusion:
the above data show that the Fab clones of the present invention have good affinity for CHO-K1-huCLL-1.
Example 3 construction and expression of anti-CLL-1 full-Length IgG antibodies
The anti-human CLL-1 protein specific Fab fragment and the humanized natural IgG1 immunoglobulin Fc fragment are recombined to construct a full-length antibody, the light chain is Kappa type or Lambda type, and the antibody type is fully humanized.
Alternatively, the heavy chain variable region and the light chain variable region of the Fab clone were linked to a heavy chain constant region derived from human IgG1 and a human antibody light chain constant region sequence, respectively. Illustratively, the antibody heavy chain constant region amino acid sequence is set forth in SEQ ID NO:28, and the light chain constant region amino acid sequence is shown as SEQ ID NO: shown at 31. Full-length IgG1 antibodies were constructed from 6 Fab clones (Fab1, Fab2, Fab3, Fab4, Fab5 and Fab6), corresponding to Ab1, Ab2, Ab3, Ab4, Ab5 and Ab6, respectively.
1. Plasmid construction
The light chain variable region and constant region (VL/CL) and heavy chain variable region and constant region (VH/CH 1) cDNA sequences of specific Fab sequence screened from the full-human natural phage display library are constructed on pcDNA3.3 expression plasmid and fused with Fc segment of human IgG1 to construct, and the heavy chain and light chain plasmids are expressed according to the weight ratio of 1: 2, transfecting ExpicHO cells transiently, inducing expression and obtaining the full-length IgG1 subtype antibody of full human origin.
2. Antibody expression purification
Antibody expression was performed using the ExpicHO transient expression system in the medium (Gibco, A29100-01) and the transfection kit (Gibco, A29129). The specific method comprises the following steps: one day before transfection, the ExpCHO cells are passaged, in a 25mL system, 25 mu g of the constructed plasmid and a transfection reagent are mixed and then dripped into 25mL of ExpCHO cells, and after the mixture is fully mixed, the mixture is expressed for 18-22 hours in a cell culture box at 37 ℃. Subsequently, the transfection mixture was supplemented with a feed medium and placed in a 32 ℃ cell incubator for further culture. On day 5 post-transfection, a second feed was added and the cells placed in a 32 ℃ cell incubator for further 10-12 days. Then, the cell suspension after expression was centrifuged at high speed, and the supernatant was collected, filtered through a 0.22 μm filter and purified by protein A/G affinity column chromatography. After purification, the target protein is eluted by 100mM glycinate (pH 3.0), concentrated, replaced, subpackaged, identified by SDS-PAGE, detected by SEC purity and identified by activity, and then stored in a warehouse for freezing storage.
TABLE 5 heavy and light chain sequences of antibodies
Note: the CDR regions are underlined.
TABLE 6 constant regions of antibody heavy and light chains and IgG 1Fc fragment sequences
TABLE 7 sequence numbering of antibodies and their heavy, light, variable regions
| Antibody numbering | HCVR | HC | LCVR | LC |
| Ab1 | SEQ ID NO：37 | SEQ ID NO：49 | SEQ ID NO：38 | SEQ ID NO：50 |
| Ab2 | SEQ ID NO：39 | SEQ ID NO：51 | SEQ ID NO：40 | SEQ ID NO：52 |
| Ab3 | SEQ ID NO：41 | SEQ ID NO：53 | SEQ ID NO：42 | SEQ ID NO：54 |
| Ab4 | SEQ ID NO：43 | SEQ ID NO：55 | SEQ ID NO：44 | SEQ ID NO：56 |
| Ab5 | SEQ ID NO：45 | SEQ ID NO：57 | SEQ ID NO：46 | SEQ ID NO：58 |
| Ab6 | SEQ ID NO：47 | SEQ ID NO：59 | SEQ ID NO：48 | SEQ ID NO：60 |
Example 4 affinity assessment of antibodies to CLL-1 antigen levels
The difference in affinity level of the designed antibody to the human CLL-1 antigen was evaluated using an indirect ELISA method, i.e., "antigen-antibody-HRP labeled secondary antibody". CLL-1, his tag protein (Acrobiosystems, Cat: CLA-H5245) was diluted to 2ug/mL using DPBS (pH7.4), added to a high affinity 96-well plate (Corning, Cat:3590) at 100. mu.L/well, incubated overnight at 4 ℃ the next day, the CLL-1 antigen solution was spun off, and the resulting solution was washed off at 200 ℃mu.L/well was added with 0.05% Tween 20PBS PH7.4(PBST), washed 3 times, in 200. mu.L/well 2% BSA (dissolved in PBST), blocked at 37 ℃ for 1h, washed 3 times in PBST, added in 100. mu.L/well, in 10-fold, 9 concentration gradients (20ug/ml-0.512X 10) -3 ug/ml), 0.5% BSA as negative control, blocking at 37 ℃ for 1h, and PBST washing 3 times; at 100. mu.L/well, add the mixture as 1: 10000 diluted goat anti-human IgG, Fc-HRP (abcam, cat: ab97225) secondary antibody solution, 37 ℃ blocking for 1h, PBST washing 3 times;
TMB (CST, cat:7004P6) substrate was added to 100. mu.L/well, and the reaction was carried out at room temperature for 3min until the color of the solution in the wells with the highest concentration of antibody became dark blue, stop solution (CST, cat:7002P6) was added to 50. mu.L/well, the reaction was stopped, and the absorbance (OD) was read at a wavelength of 450 nm.
Data processing:
the concentration logarithm value of the antibody to be detected is taken as an X-axis coordinate, the OD450 absorbance value is taken as a vertical coordinate, the affinity of the antibody to the antigen is calculated by utilizing the GraphPad PRISM 8.0log (aginst) vs. s.response-visual slope (four parameters) formula, and the result is shown in Table 8:
TABLE 8 affinity of antibodies to human CLL-1 antigen
And (4) experimental conclusion:
the above data show that the antibodies of the invention have good affinity for human CLL-1 antigen.
Example 5 affinity assessment of antibodies to CLL-1 expressing cells
Differences in the affinity level of the designed antibodies to HEK293-huCLL-1 were assessed using flow cytometry.
Human CLL-1 Gene (Gene ID:160364) is constructed in a lentivirus vector to transfect HEK293 host cells, and a monoclonal is selected to obtain an expressed human CLL-1 cell strain HEK 293-huCLL-1. Accutase (Sigma, cat: A6964) digestion solution is used for treating tumor cells with good growth state, 2% FBS (DBPS, pH7.4 dilution) solution is used for preparing single cell suspension, and the cell density is adjusted to be 1x106 cells/mL. Are equally divided into 96-hole V-shaped bottom plates at 100 mu L/hole so as to300g, 5min, 4 ℃ centrifugation, discard the supernatant, add 10 fold, 10 concentration gradients (40ug/ml-0.02X 10) to 100. mu.L/well -3 ug/ml) of the diluted antibody solution to be tested, incubating at 4 ℃ for 1 h;
after centrifugation at 300g for 5min at 4 ℃ and 2% FBS washing for 2 times, a solution of mouse anti-human IgG Fc diluted at 5. mu.L/106 cells, PE-labeled secondary antibody (Biolegend, cat:409304), was added at 100. mu.L/well and incubated at 4 ℃ for 1 h; 300g 5min, 4 ℃ centrifugation, washing 2 times, adding 70 u L2% FBS solution heavy suspension cells, in the flow cytometry (Bio-Rad, ZE5) to detect PE channel average fluorescence intensity (MFI: mean fluorescence intensity).
Data processing:
the affinity of the antibody was calculated using the formula GraphPad PRISM 8.0log (aginst) vs. response-usable slope (four parameters) with the concentration logarithm of the antibody as the X-axis coordinate and MFI as the ordinate, and the results are shown in Table 9:
TABLE 9 affinity levels (EC) of antibodies for HEK293-hu CLL-1 50 )
| Antibodies | Maximum MFI | EC 50 (ng/ml) |
| Ab1 | 4275.06 | 29.47 |
| Ab2 | 5844.53 | 40.20 |
| Ab4 | 4940.16 | 56.26 |
| Ab5 | 5744.07 | 41.38 |
And (4) experimental conclusion:
the above data show that the anti-human CLL-1 antibody of the present invention has good affinity for HEK 293-huCLL-1.
Example 6 evaluation of the endocytic Activity of antibodies
The endocytosis activity of the anti-human CLL-1 antibody on CLL-1 antigen-expressing tumor cell lines HL-60 and U937 and an over-expression cell line HEK293-hu CLL-1 is evaluated by flow cytometry. Treating expression cell strain HEK293-hu CLL-1 with good growth state with Accutase (Sigma, cat: A6964) digestive juice, culturing cells with HL-60 and U937 suspension for 300g x 5min, centrifuging at room temperature, discarding supernatant, resuspending with 2% FBS (DBPS, pH7.4 dilution) solution, preparing single cell suspension, adjusting cell density to 1x10 7 cells/mL. Evenly distributing 100 mu L/hole to a 96-hole V-shaped bottom plate, adding an antibody solution to be detected with the final concentration of 20 mu g/mL, uniformly mixing, and incubating for 1h at 4 ℃;
300g x 5min, centrifuging at 4 deg.C, adding 5. mu.L/10 at 100. mu.L/well 6 Mouse anti-human IgG Fc and PE-labeled secondary antibody (Biolegend, cat:409304) which are diluted in cell proportion are incubated for 1h at 4 ℃;
300g is multiplied by 5min, centrifugation is carried out at 4 ℃, washing is carried out for 2 times, 1mL of RPMI-1640(giboco) complete culture medium pre-warmed at 37 ℃ is used for resuspending cell precipitation, and the cell precipitation is divided into four equal parts which are respectively set as a 0min group, a blank group, a 30min group and a 120min group; taking out 0min and blank groups, placing on ice, placing the rest in 37 deg.C incubator, endocytosis for 30min and 120min respectively, taking out corresponding groups at corresponding time points, placing on ice for precooling for 5min, centrifuging all treatment groups, discarding supernatant (4 deg.C, 1500rpm × 5min), washing once with FACS buffer solution, discarding supernatant; all treatment groups except the 0min group were added with 250. mu.L strip buffer, incubated at room temperature for 8min, centrifuged to discard the supernatant (4 ℃, 1500 rpm. times.5 min), washed twice with FACS buffer, and the supernatant was removed, 80. mu.L of 2% FBS-resuspended cells were added to each group of samples, and the fluorescence signals of the samples to be tested were detected in a flow cytometer (Bio-Rad, ZE 5).
Data processing:
the efficiency of antibody endocytosis was calculated according to this formula: the percent (%) of antibody internalization was (treatment group MIF-blank MFI)/(0min MFI-blank MFI) × 100%. The detection of the endocytosis rate of the antibody by the method is shown in the following table 10:
TABLE 10 endocytosis efficiency (%)
And (4) experimental conclusion:
the above data show that the anti-CLL-1 antibody of the present invention has good endocytosis at the cellular level.
Example 7 in vitro ADCC Effect assessment
This experiment was aimed at evaluating the in vitro ADCC effect of candidate antibody molecules. Assessed using the time-homogeneous resolved fluorescence, DELFIA EuTDA cytotoxicity assay kit (Perkinelmer, cat log: AD 0116). Firstly, a target cell is marked by a BATDA reagent, the BATDA can freely permeate into the cell, a TDA which cannot enter and exit a cell membrane is formed under the catalysis of acetyl esterase, the marked target cell and an effector cell are incubated together, the marked target cell is cracked by the effector cell to release the TDA under the mediated ADCC effect of an Fc end of an antibody, the TDA is combined with Eu3+ in a detection solution to form a chelate, a fluorescence signal is detected through a TRF channel with EX/EM of 340/615, and the ADCC effect is evaluated according to the strength of the fluorescence signal.
Target cells U937(ATCC, Cat #: TCHU159) and effector cells NK-92MI (NanJing Cobioner, Cat #: CBP61113) were centrifuged at 1000rpm/min for 5min and washed 2 times to adjust the cell density to 1x 106/mL and 2x 106/mL, respectively, after which 5uL of BATDA solution was added per mL of cell suspension and incubated at 37 ℃ for 10min, followed by centrifugation at 1000rpm/min for 5min and washing 3 times, the cells were resuspended in 96-Corning (Cat #3799) well plates containing 2Mm probenecid medium (RPMI-160(gibco, Cat #:22400105) + 10% FBS (gibco, Cat #:10099141)) and counted to adjust the cell density to 2x 105/mL, and the cell suspension was seeded into 96-Corning (Cat #3799, 50 uL/well plates, to which 100 uL/well of the diluted antibody to be tested was added. Effector cells NK-92MI, previously adjusted for cell density, were also added to the plate for co-incubation at an effective target ratio of 10:1, and the plate was placed in a cell culture incubator at 37 ℃ for 2-4 hours. Then, the plate was centrifuged at 800rpm/min for 5min, 20. mu.l of the supernatant was transferred to a new 96-well plate (Corning, Cat #:3917), 200. mu.l of leutopdium solution was added to each well, and the plate was incubated at room temperature for 30min with shaking at a low speed and then read.
And (3) calculating ADCC (ADCC) lysis killing efficiency according to the following calculation formula: specific cleavage efficiency ═ experimental group signal-natural death signal)/(maximal killer signal-natural death signal) x 100
Note:
experimental group signals: labeling target cells + effector cells + antibodies; natural death signal: labeled target cells only
Maximum release signal: labeling the target cell complete lysate; background signal: culture medium
The ADCC lytic killing efficiency of the detection antibody is shown in table 11 below:
TABLE 11 in vitro ADCC lytic killing efficiency (% average) of anti-CLL-1 antibody
| Conc.(nM) | Ab1 |
| 100 | 28.29 |
| 10 | 20.43 |
| 1 | 18.64 |
| 0.1 | 11.14 |
The experimental result shows that the candidate molecule shows good ADCC (ADCC) lysis killing effect on tumor cells in vitro and shows good dose-effect relationship.
Claims (22)
1. An anti-CLL-1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein,
the heavy chain variable region comprises at least 1 HCDR1 selected from the group consisting of seq id nos: 1, 7, 13, 19 and 25; and at least 1 HCDR2 selected from the group consisting of the sequences shown below: 2, 8, 14, 20 and 26; and at least 1 HCDR3 selected from the group consisting of the sequences shown below: 3, 9, 15, 21, 27 and 33,
the light chain variable region comprises at least 1 LCDR1 selected from the group consisting of seq id nos: 4, 10, 16, 22 and 34; and at least 1 LCDR2 selected from the group consisting of seq id no:5, 11, 17, 23 and 35; and at least 1 LCDR3 selected from the group consisting of seq id no:6, 12, 18, 24, 30 and 36.
2. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody heavy chain variable region comprises:
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3, respectively; or the like, or, alternatively,
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9, respectively; or the like, or, alternatively,
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15, respectively; or the like, or, alternatively,
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21, respectively; or the like, or a combination thereof,
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27, respectively; or the like, or, alternatively,
HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 33, respectively.
3. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 1, wherein the antibody light chain variable region comprises:
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 4, SEQ ID NO 5 and SEQ ID NO 6, respectively; or the like, or, alternatively,
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 12, respectively; or the like, or a combination thereof,
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 18, respectively; or the like, or, alternatively,
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 22, SEQ ID NO 23 and SEQ ID NO 24, respectively; or the like, or a combination thereof,
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 16, SEQ ID NO 5 and SEQ ID NO 30, respectively; or the like, or, alternatively,
LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO 34, SEQ ID NO 35 and SEQ ID NO 36, respectively.
4. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein:
the heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 1, HCDR2 shown in SEQ ID NO. 2 and HCDR3 shown in SEQ ID NO. 3, and the light chain variable region comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6; or the like, or, alternatively,
the heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 7, HCDR2 shown in SEQ ID NO. 8 and HCDR3 shown in SEQ ID NO. 9; and the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO. 10, LCDR2 shown in SEQ ID NO. 11 and LCDR3 shown in SEQ ID NO. 12; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 13, HCDR2 shown in SEQ ID NO. 14 and HCDR3 shown in SEQ ID NO. 15; and the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO. 16, LCDR2 shown in SEQ ID NO. 17 and LCDR3 shown in SEQ ID NO. 18; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 19, HCDR2 shown in SEQ ID NO. 20 and HCDR3 shown in SEQ ID NO. 21; and the antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO. 22, LCDR2 shown in SEQ ID NO. 23 and LCDR3 shown in SEQ ID NO. 24; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 25, HCDR2 shown in SEQ ID NO. 26 and HCDR3 shown in SEQ ID NO. 27; and the antibody light chain variable region comprises LCDR1 shown in SEQ ID NO. 16, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 30; or
The heavy chain variable region comprises HCDR1 shown in SEQ ID NO. 7, HCDR2 shown in SEQ ID NO. 8 and HCDR3 shown in SEQ ID NO. 33; and the antibody light chain variable region comprises: LCDR1 shown in SEQ ID NO. 34, LCDR2 shown in SEQ ID NO. 35 and LCDR3 shown in SEQ ID NO. 36.
5. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 4, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of those set forth in SEQ ID NO:37, 39, 41, 43, 45 or 47 SEQ ID NO;
and/or is selected from the group consisting of the light chain variable region set forth in seq id no, or a light chain variable region having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to seq id no:38, 40, 42, 44, 46 or 48.
6. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 5, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises:
the heavy chain variable region shown in SEQ ID NO. 37 and the light chain variable region shown in SEQ ID NO. 38; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO:39 and the light chain variable region shown in SEQ ID NO: 40; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO. 41 and the light chain variable region shown in SEQ ID NO. 42; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO. 43 and the light chain variable region shown in SEQ ID NO. 44; or the like, or, alternatively,
the heavy chain variable region shown in SEQ ID NO. 45 and the light chain variable region shown in SEQ ID NO. 46; or the like, or, alternatively,
the heavy chain variable region shown by SEQ ID NO. 47 and the light chain variable region shown by SEQ ID NO. 48.
7. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 6, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1, IgG2, IgG3, or IgG4, or a variant thereof;
preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region derived from human IgG1 or a variant thereof;
more preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as set forth in SEQ ID NO 28,
optionally, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody kappa chain, lambda chain, or a variant thereof;
preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a kappa chain, a lambda chain of a human antibody;
further preferably, the anti-CLL-1 antibody or antigen-binding fragment thereof further comprises a light chain constant region as set forth in SEQ ID NO. 29 or SEQ ID NO. 31.
8. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 6, wherein the anti-CLL-1 antibody or antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of those shown in seq id nos, or those having at least 80%, 85%, 90%, 95%, or 99% identity compared to seq id nos: 49, 51, 53, 55, 57 or 59 SEQ ID NO,
and/or, is selected from a light chain represented by the following sequences, or a light chain having at least 80%, 85%, 90%, 95%, or 99% identity compared to the following sequences: 50, 52, 54, 56, 58 or 60 SEQ ID NO.
9. The anti-CLL-1 antibody or antigen-binding fragment thereof of claim 8, wherein the anti-CLL-1 antibody comprises:
the heavy chain shown as SEQ ID NO. 49 and the light chain shown as SEQ ID NO. 50; or
The heavy chain shown as SEQ ID NO. 51 and the light chain shown as SEQ ID NO. 52; or
The heavy chain shown as SEQ ID NO. 53 and the light chain shown as SEQ ID NO. 54; or
The heavy chain shown as SEQ ID NO. 55 and the light chain shown as SEQ ID NO. 56; or
A heavy chain shown as SEQ ID NO. 57 and a light chain shown as SEQ ID NO. 58; or
The heavy chain shown as SEQ ID NO. 59 and the light chain shown as SEQ ID NO. 60.
10. The anti-CLL-1 antibody or antigen-binding fragment thereof of any one of claims 1-9, which is selected from a fully human antibody or antigen-binding fragment thereof.
11. A polynucleotide encoding the anti-CLL-1 antibody or antigen-binding fragment thereof of any one of claims 1-10.
12. An expression vector comprising the polynucleotide of claim 11.
13. A host cell into which the expression vector of claim 12 is introduced or which contains the expression vector.
14. The host cell of claim 13, wherein said host cell is selected from the group consisting of a bacterial, yeast, or mammalian cell; preferably E.coli, Pichia, CHO cells or HEK293 cells.
15. A method of producing an anti-CLL-1 antibody or antigen-binding fragment thereof, comprising the steps of:
a) culturing the host cell of claim 13 or 14;
b) isolating the antibody or antigen-binding fragment thereof from the culture; and the number of the first and second groups,
c) purifying the antibody or antigen-binding fragment thereof of step b).
16. A pharmaceutical composition comprising an anti-CLL-1 antibody or antigen-binding fragment thereof according to any one of claims 1-10, and a pharmaceutically acceptable excipient, diluent or carrier.
17. A detection or diagnostic kit comprising an anti-CLL-1 antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, optionally further comprising one or more reagents capable of detecting the binding of the anti-CLL-1 antibody or antigen-binding fragment thereof to CLL-1.
18. Use of an anti-CLL-1 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim 16 for the manufacture of a medicament for the treatment or prevention of a CLL-1 mediated disease or disorder.
19. Use of an anti-CLL-1 antibody or antigen-binding fragment thereof according to any one of claims 1-10 or a pharmaceutical composition according to claim 16 for the preparation of a kit for the detection, diagnosis, prognosis of a CLL-1 mediated disease or disorder.
20. The use of claim 18 or 19, wherein:
the disease or condition is a myeloproliferative disease;
acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia (CMML), myelodysplastic syndrome (MDS), multiple myeloma, plasmacytoma, and myelofibrosis are preferred.
21. anti-CLL-1 antibody or antigen-binding fragment thereof according to any one of claims 1-10, or a pharmaceutical composition according to claim 16, or a kit according to claim 17 for use in the detection, diagnosis, prognosis of CLL-1 mediated diseases; the disease is selected from acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, multiple myeloma, plasmacytoma, and myelofibrosis.
22. A method of treating or preventing a CLL-1 mediated disease, comprising the steps of:
providing a therapeutically or prophylactically effective amount of an anti-CLL-1 antibody or antigen-binding fragment thereof according to any one of claims 1-10 to a subject; or providing a therapeutically or prophylactically effective amount of the pharmaceutical composition of claim 16 to a subject; wherein the CLL-1 mediated disease is selected from the group consisting of acute myelogenous leukemia, chronic myelomonocytic leukemia, myelodysplastic syndrome, multiple myeloma, plasmacytoma, and myelofibrosis.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110304235 | 2021-03-22 | ||
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