WO2022171113A1 - 人cd33抗体及其用途 - Google Patents
人cd33抗体及其用途 Download PDFInfo
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- WO2022171113A1 WO2022171113A1 PCT/CN2022/075621 CN2022075621W WO2022171113A1 WO 2022171113 A1 WO2022171113 A1 WO 2022171113A1 CN 2022075621 W CN2022075621 W CN 2022075621W WO 2022171113 A1 WO2022171113 A1 WO 2022171113A1
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- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C—CHEMISTRY; METALLURGY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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- C—CHEMISTRY; METALLURGY
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
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- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- C07K2319/00—Fusion polypeptide
Definitions
- the invention belongs to the fields of bioengineering and biomedicine, and relates to human CD33 antibody, nucleic acid encoding the same, a method for preparing the antibody, a pharmaceutical composition containing the antibody, and related uses of the pharmaceutical composition for treating tumors.
- CD33 is a cell surface antigen specifically expressed on myeloid cells including myeloid leukemia cells. It is the smallest member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family. CD33 has a molecular weight of 67kDa and is a type I transmembrane receptor protein composed of 364 amino acids.
- the N-terminus is extracellular, and the terminal amino acids form a conserved V-set immunoglobulin-like domain and a variable C2-set domain, in which V-set specifically recognizes and binds to sialic acid; cytoplasmic tail There is an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like structure at the end, which transmits inhibitory signals to the cell by binding to tyrosine phosphatase, thereby regulating cell growth. Purpose.
- the ITIM sequence in the CD33 molecule differs from other Siglecs in that the hydrophobic amino acids in front of tyrosine are replaced by leucine and threonine. The analysis of its primary structure shows that the CD33 molecule is highly conserved in various organisms.
- CD33 is expressed on early multilineage hematopoietic progenitors and myelomonocyte precursors. No expression in pluripotent hematopoietic stem cells (Andrews et al., Journal of Experimental Medicine 169, 1721-1731, 1989). Its expression is downregulated on mature granulocytes, but is retained on macrophages, monocytes and dendritic cells (Andrews et al., Blood 62, 24-132, 1983). In addition to myelomonocytic cells, Valent et al. found that CD33 is also expressed on human mast cells and blood basophils (Blood 15, 73(7): 1778-85, 1989).
- the sialic acid-binding extracellular domain of CD33 is involved in cell-to-cell adhesion.
- Intracellular immunoreceptor tyrosine-based inhibitory motifs impart inhibitory signals to cells, thereby affecting proliferation and cell survival.
- the actual signaling pathway of CD33 is poorly understood, but von Gunten et al. suggest that it is involved in the recruitment of ITIM and ITIM-like motifs and tyrosine phosphatases (Ann. N.Y. Acad. Sci. 1143:61-82, 2008).
- Brinkman-Van der Linden et al. have defined a murine CD33 homolog, but its functional comparability with human CD33 remains questioned (Mol Cell Biol., 23(12):4199-206, 2003).
- the functional role of human CD33 on normal and malignant leukocytes remains unknown.
- CD33 is a stable cell surface marker expressed on primary AML and CML cells in 70% to 100% of tested patients (Plesa et al, Cancer 112(3), 572-80, 2007; Hauswirt et al, Eur J ClinInvest. Jan 73-82, 2007; Scheinberg et al., Leukemia, Vol. 3, 440-445, 1989).
- CD33 plays an important role in malignant myeloid blasts, which represent the majority of malignant cells in the peripheral blood and bone marrow of leukemia patients, and leukemia stem cells, a relatively small number of poorly differentiated cells in the bone marrow characterized by their ability to self-renew and maintain leukemia clone level), while only partially normal hematopoietic stem cells express low levels of CD33.
- CD33 can regulate the function of leukocytes, so CD33 is an ideal target for the treatment of acute myeloid leukemia.
- Nanobodies are genetically engineered antibodies that contain only a single domain.
- Belgian scientist Hamers-Casterman C found a natural heavy chain antibody containing only heavy chain but no light chain in camel blood (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, et al. al.Naturally occurring antibodies devoid of light chains.Nature.363(6428):446–8(1993)), although the heavy chain antibody lacks the light chain compared with the ordinary antibody, it still retains the ability to bind to the antigen.
- Nanobodies are not only 1/10 of the molecular weight of ordinary antibodies, but also have more flexible chemical properties, good stability, high solubility, easy expression, high tumor tissue penetration, and easy coupling to other molecules. Therefore, nanobody technology has broad application prospects in the field of therapeutic antibodies.
- the present invention provides antibodies or antigen-binding fragments capable of binding to human CD33, nucleic acids, vectors, cells encoding the same, methods for preparing the antibodies or antigen-binding fragments, pharmaceutical compositions containing the antibodies or antigen-binding fragments, and Uses related to the treatment of tumors.
- the present invention provides an antibody or antigen-binding fragment that specifically binds to CD33, the antibody or antigen-binding fragment comprising: CDR1, CDR2 and CDR3; the CDR1, CDR2 and CDR3 have the following Any sequence combination or sequence combination with 1, 2, 3 or more amino acid insertions, deletions and/or substitutions compared to said sequence combination:
- the CDR1 can be selected from SEQ ID NOs: 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83 , 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, 155, 158 , 161, 164, 167, 170, 173, 176, 179, 182, 185, 188, 226, 227;
- the CDR2 can be selected from SEQ ID NOs: 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 , 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, 156, 159 , 162, 165, 168, 171, 174, 177, 180, 183, 186, 189, 229, 230;
- the CDR3 can be selected from SEQ ID NO: 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85 , 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, 154, 157, 160 , 163, 166, 169, 172, 175, 178, 181, 184, 187, 190, 228;
- Each CDR1, CDR2 and CDR3 is encoded according to the pass analysis method of KABAT, Chothia or IMGT;
- the substitutions are conservative amino acid substitutions.
- the CDR1, CDR2 and CDR3 respectively comprise CDR1, CDR2 and CDR3 selected from the VHH domains set forth in any one of SEQ ID NOs: 11-28 or 193-223;
- the antibody or antigen-binding fragment comprises CDR1, CDR2 and CDR3 according to the KABAT, Chothia or IMGT numbering system:
- Described CDR1 is selected from SEQ ID NO: 29, 83, 137, described CDR2 is selected from SEQ ID NO: 30, 84, 138, and described CDR3 is selected from SEQ ID NO: 31, 85, 139;
- the CDR1 is selected from SEQ ID NO: 32, 86, 140
- the CDR2 is selected from SEQ ID NO: 33, 87, 141
- the CDR3 is selected from SEQ ID NO: 34, 88, 142;
- the CDR1 is selected from SEQ ID NO: 35, 89, 143, 226, 227
- the CDR2 is selected from SEQ ID NO: 36, 90, 144
- the CDR3 is selected from SEQ ID NO: 37, 91, 145;
- the CDR1 is selected from SEQ ID NO: 38, 92, 146
- the CDR2 is selected from SEQ ID NO: 39, 93, 147
- the CDR3 is selected from SEQ ID NO: 40, 94, 148;
- the CDR1 is selected from SEQ ID NO: 41, 95, 149
- the CDR2 is selected from SEQ ID NO: 42, 96, 150, 229
- the CDR3 is selected from SEQ ID NO: 43, 97, 151, 228;
- the CDR1 is selected from SEQ ID NO: 44, 98, 152
- the CDR2 is selected from SEQ ID NO: 45, 99, 153
- the CDR3 is selected from SEQ ID NO: 46, 100, 154;
- the CDR1 is selected from SEQ ID NO: 47, 101, 155
- the CDR2 is selected from SEQ ID NO: 48, 102, 156
- the CDR3 is selected from SEQ ID NO: 49, 103, 157;
- Described CDR1 is selected from SEQ ID NO: 50, 104, 158, described CDR2 is selected from SEQ ID NO: 51, 105, 159, and described CDR3 is selected from SEQ ID NO: 52, 106, 160;
- the CDR1 is selected from SEQ ID NO: 53, 107, 161
- the CDR2 is selected from SEQ ID NO: 54, 108, 162, 230
- the CDR3 is selected from SEQ ID NO: 55, 109, 163;
- the CDR1 is selected from SEQ ID NO: 56, 110, 164
- the CDR2 is selected from SEQ ID NO: 57, 111, 165
- the CDR3 is selected from SEQ ID NO: 58, 112, 166;
- the CDR1 is selected from SEQ ID NO: 59, 113, 167
- the CDR2 is selected from SEQ ID NO: 60, 114, 168
- the CDR3 is selected from SEQ ID NO: 61, 115, 169;
- the CDR1 is selected from SEQ ID NO: 62, 116, 170
- the CDR2 is selected from SEQ ID NO: 63, 117, 171
- the CDR3 is selected from SEQ ID NO: 64, 118, 172;
- the CDR1 is selected from SEQ ID NO: 65, 119, 173
- the CDR2 is selected from SEQ ID NO: 66, 120, 174
- the CDR3 is selected from SEQ ID NO: 67, 121, 175;
- the CDR1 is selected from SEQ ID NO: 68, 122, 176
- the CDR2 is selected from SEQ ID NO: 69, 123, 177
- the CDR3 is selected from SEQ ID NO: 70, 124, 178;
- the CDR1 is selected from SEQ ID NO: 71, 125, 179
- the CDR2 is selected from SEQ ID NO: 72, 126, 180
- the CDR3 is selected from SEQ ID NO: 73, 127, 181;
- the CDR1 is selected from SEQ ID NO: 74, 128, 182, the CDR2 is selected from SEQ ID NO: 75, 129, 183, the CDR3 is selected from SEQ ID NO: 76, 130, 184;
- the CDR1 is selected from SEQ ID NO: 77, 131, 185
- the CDR2 is selected from SEQ ID NO: 78, 132, 186
- the CDR3 is selected from SEQ ID NO: 79, 133, 187;
- the CDR1 is selected from SEQ ID NO: 80, 134, 188
- the CDR2 is selected from SEQ ID NO: 81, 135, 189
- the CDR3 is selected from SEQ ID NO: 82, 136, 190; or,
- the antibody or antigen-binding fragment comprises a combination of CDR1, CDR2 and CDR3 sequences selected from SEQ ID NOs: 11-28 or 193-223; Sequences having at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity compared to CDR3.
- the antibody or antigen-binding fragment comprises a FR region in the VHH domain shown in any one of SEQ ID NOs: 11-28 or 193-223; alternatively, the antibody or antigen The binding fragment comprises at least 80, 85%, 90%, 91%, 92%, 93%, 94% compared to the FR region in the VHH domain set forth in any one of SEQ ID NOs: 11-28 or 193-223 , 95%, 96%, 97%, 98%, 99% or 100% identical sequences; or, alternatively, the antibody or antigen-binding fragment comprises any of SEQ ID NOs: 11-28 or 193-223 Occurs at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 compared to the FR regions in the VHH domains shown in one item , 3, 2 or 1 mutated sequence; the mutations may be selected from insertions, deletions and/or substitutions, preferably conservative amino acid substitutions.
- the antibody or antigen-binding fragment comprises the sequence shown in any one of SEQ ID NOs: 11-28 or 193-223; NO: have at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% compared to the sequences shown in any one of 11-28 or 193-223 , 99% or 100% identical sequences; or, alternatively, the antibody or antigen-binding fragment comprises up to 20 occurrences of, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 , 2 or 1 mutated sequence; the mutations may be selected from insertions, deletions and/or substitutions, preferably conservative amino acid substitutions.
- the dissociation constant (KD) of the antibody or antigen-binding fragment for binding to human CD33 is not greater than 100 nM; the dissociation constant (KD) for binding to monkey CD33 is not greater than 100 nM.
- the antibody or antigen-binding fragment includes or does not include an antibody heavy chain constant region; optionally, the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat , rabbit or sheep; alternatively, the antibody heavy chain constant region can be selected from IgG, IgM, IgA, IgE or IgD, and the IgG can be selected from IgG1, IgG2, IgG3 or IgG4; alternatively, the heavy chain The chain constant region may be selected from the group consisting of an Fc region, a CH3 region, a heavy chain constant region in the absence of a CH1 fragment, or an intact heavy chain constant region; preferably, the heavy chain constant region is a human Fc region, more preferably having a region such as SEQ ID NO: 191 The indicated amino acid sequence; preferably, the antibody or antigen-binding fragment is a heavy chain antibody.
- the antibody heavy chain constant region can be selected from human, alpaca, mouse, rat , rabbit or sheep; alternatively, the antibody
- the antibody or antigen-binding fragment is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or, (3) a fully human antibody or its fragment Fragment.
- the antibody or antigen-binding fragment is further coupled with a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from radioisotopes, cytotoxic agents or immunomodulatory agents, the The tracer is selected from radiographic contrast agents, paramagnetic ions, metals, fluorescent labels, chemiluminescent labels, ultrasound contrast agents and photosensitizers; more preferably, the cytotoxic agent is selected from alkaloids, methotrexate methotrexate, doxorubicin or taxanes.
- the antibody or antigen-binding fragment is further linked with another functional molecule, and the functional molecule can be selected from one or more of the following: signal peptide, protein tag, or cytokine.
- the present invention provides a multispecific antibody comprising the antibody or antigen-binding fragment of the first aspect; preferably, the multispecific antibody further comprises a specific binding An antigen other than CD33 or an antibody or antigen-binding fragment that binds to a different CD33 epitope from the antibody or antigen-binding fragment of the first aspect.
- the antigen other than CD33 can be selected from: CD3, preferably CD3 ⁇ ; CD16, preferably CD16A; CS32B; PD-1; PD-2; PD-L1; NKG2D; CD19; CD20; CD40 ;CD47;4-1BB;CD137;EGFR;EGFRvIII;TNF-alpha;MSLN;HER2;HER3;HSA;CD5;CD27;EphA2;EpCAM;MUC1;MUC16;CEA;Claudin18.2;folate receptor;Claudin6;WT1 ; NY-ESO-1; MAGE3; ASGPR1 or CDH16.
- the multispecific antibody may be a bispecific, trispecific or tetraspecific antibody, and the multispecific antibody may be bivalent, tetravalent or hexavalent.
- the present invention provides a chimeric antigen receptor (CAR) comprising at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, the The extracellular antigen binding domain comprises an antibody or antigen binding fragment optionally from the first aspect.
- CAR chimeric antigen receptor
- the present invention provides an immune effector cell, the immune effector cell expressing the chimeric antigen receptor of the third aspect, or comprising a nucleic acid fragment encoding the chimeric antigen receptor of the third aspect;
- the immune effector cells are selected from T cells, NK cells (natural killer cells), NKT cells (natural killer T cells), DNT cells (double negative T cells), monocytes, macrophages, dendritic cells cells or mast cells, the T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cells are autoimmune effector cells or allogeneic immune effector cells.
- the present invention provides an isolated nucleic acid fragment capable of encoding the antibody or antigen-binding fragment of the first aspect above, the multispecific antibody of the second aspect, or the chimeric antigen receptor of the third aspect.
- the present invention provides a vector comprising the isolated nucleic acid fragment of the fifth aspect.
- the present invention provides a host cell, the host cell comprising the vector described in the sixth aspect; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
- the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (Escherichia coli), fungi (yeast), insect cells or mammalian cells (CHO cell line or 293T cell line).
- the present invention also provides a method for preparing an antibody, an antigen-binding fragment, or a multispecific antibody, the method comprising culturing the cells of the seventh aspect, and isolating the cells under suitable conditions The expressed antibody or antigen-binding fragment, or the isolated multispecific antibody expressed by the cell.
- the present invention also provides a method for preparing immune effector cells, the method comprising introducing a nucleic acid fragment encoding the CAR of the third aspect into the immune effector cells, optionally, the method further comprising The immune effector cells are activated to express the CAR of the third aspect.
- the present invention also provides a pharmaceutical composition
- a pharmaceutical composition comprising the antibody or antigen-binding fragment optionally from the first aspect, or the multispecific optionally from the second aspect
- the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.
- the present invention also provides a method for preventing and/or treating tumors, comprising administering to a patient in need thereof an effective amount of the antibody or antigen-binding fragment optionally described in the first aspect, or any Selected from the multispecific antibody described in the second aspect, or the immune effector cell described in the fourth aspect, or the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect; or the eighth and ninth aspects
- the tumor is preferably myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and promyelocytic leukemia (PML).
- MDS myelodysplastic syndrome
- AML acute myeloid leukemia
- CML chronic myeloid leukemia
- PML promyelocytic leukemia
- the present invention provides an antibody or antigen-binding fragment optionally according to the first aspect, or a multispecific antibody optionally according to the second aspect, or an immune effector according to the fourth aspect cells, or the nucleic acid fragment described in the fifth aspect, or the vector described in the sixth aspect; or the product prepared by the methods described in the eighth and ninth aspects; or the pharmaceutical composition described in the tenth aspect in the preparation of preventive and/or or use in a medicament for the treatment of tumors; preferably myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and promyelocytic leukemia (PML).
- MDS myelodysplastic syndromes
- AML acute myeloid leukemia
- CML chronic myeloid leukemia
- PML promyelocytic leukemia
- the present invention provides a kit comprising an antibody or antigen-binding fragment optionally from the first aspect, or a multispecific antibody optionally from the second aspect, or a fourth
- the present invention provides an in vitro method for inhibiting the proliferation or migration of CD33-expressing cells, characterized in that a complex can be formed between the antibody or antigen-binding fragment optionally described in the first aspect and CD33 The cells are contacted with the antibody or antigen-binding fragment optionally from the first aspect under conditions.
- CD33 herein is the smallest member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family. It is a type I transmembrane receptor protein composed of 364 amino acids with a molecular weight of 67kDa.
- the N-terminus is extracellular, and the terminal amino acids form a conserved V-set immunoglobulin-like domain and a variable C2-set domain, in which V-set specifically recognizes and binds to sialic acid; cytoplasmic tail There is an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like structure at the end, which transmits inhibitory signals to the cell by binding to tyrosine phosphatase, thereby regulating cell growth. Purpose.
- the ITIM sequence in the CD33 molecule differs from other Siglecs in that the hydrophobic amino acids in front of tyrosine are replaced by leucine and threonine.
- the analysis of its primary structure shows that the CD33 molecule is highly conserved in various organisms.
- the term "CD33” includes the CD33 protein of any human and non-human animal species, and specifically includes human CD33 as well as non-human mammalian CD33.
- the term "specifically binds" herein refers to an antigen-binding molecule (eg, an antibody) that specifically binds an antigen and a substantially identical antigen, usually with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
- high affinity generally refers to having about 10-7 M or less, about 10-8 M or less, about 1 ⁇ 10-9 M or less, about 1 ⁇ 10-10 M or less, KD of 1 ⁇ 10-11 M or lower or 1 ⁇ 10-12 M or lower.
- the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.
- antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
- Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen, but is unrelated to the structure of an antibody.
- antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPin), nucleic acid aptamer or Kunitz-type domain peptide.
- antibody is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to enable specific binding to an antigen or peptide combination.
- Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
- Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, eg, biocompatible polymers.
- Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
- Antibody herein includes a typical "quad-chain antibody”, which is an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); heavy chain refers to a polypeptide chain that is The N-terminal to C-terminal direction consists of the heavy chain variable region (VH), the heavy chain constant region CH1 domain, the hinge region (HR), the heavy chain constant region CH2 domain, the heavy chain constant region CH3 domain; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction
- the polypeptide chain composed of the region (CL); the heavy chain and the heavy chain, and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure.
- immunoglobulins Due to the different amino acid composition and arrangement sequence of the constant region of immunoglobulin heavy chain, its antigenicity is also different. Accordingly, the "immunoglobulins" herein can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are ⁇ and ⁇ chains, respectively. , ⁇ chain, ⁇ chain and ⁇ chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain.
- IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2.
- Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
- Each of the five classes of Ig can have a kappa chain or a lambda chain.
- Antibody herein also includes antibodies that do not contain a light chain, such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other heavy-chain antibodies (heavy-chain antibodies, HCAbs) and sharks and other cartilaginous fish found in the new immunoglobulin antigen receptors (Ig new antigen receptor, IgNAR).
- a light chain such as those produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and alpaca ( Vicugna pacos) and other heavy-chain antibodies (heavy-chain antibodies, HCAbs) and sharks and other cartilaginous fish found in the new immunoglobulin antigen receptors (Ig new antigen receptor, IgNAR).
- heavy chain antibody herein refers to a second type of antibody known to occur naturally in camelid species that is naturally deficient in the light chain and the CH1 constant region.
- Heavy-chain antibodies consist of two heavy chains linked by covalent disulfide bonds. One end of each heavy chain in an HCAb has a variable domain. To distinguish them from the variable domains (VH) of camelid "conventional" antibody heavy chains, the variable domains of HCAbs are referred to as "VHH". VHH domains are quite different from VH domains, they are encoded by different gene segments in the camelid genome.
- VHH domain and “nanobody” and “single domain antibody” (sdAb) herein have the same meaning and can be used interchangeably, and refer to the variable region of cloning heavy chain antibodies, constructing A single-domain antibody consisting of only one heavy chain variable region, it is the smallest fully functional antigen-binding fragment.
- a heavy chain antibody that naturally lacks light chain and heavy chain constant region 1 (CH1) is obtained first, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
- an “antibody” herein can be derived from any animal, including, but not limited to, humans and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as camelid, llama , ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fishes (eg sharks).
- the term "antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind a target antigen.
- the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
- Antibody fragments can be Fab, F(ab')2, scFv, SMIP, diabodies, tribodies, affibodies, Nanobodies, aptamers or domain antibodies.
- binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab) 2 Fragment, a bivalent fragment comprising two Fab fragments connected at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of an antibody one-arm Constituent Fv fragments; (V) dAbs comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546, 1989); (vii) VH or VL (viii
- the two domains of the Fv fragment, VL and VH are encoded by separate genes, the two domains can be joined using recombinant methods by a linker that enables it to be made in which the VL and VH regions are paired to form A single protein chain of a monovalent molecule (called a single-chain Fv (scFv); see eg, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 , 1988).
- scFv single-chain Fv
- These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies.
- Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
- the term "monoclonal antibody” herein refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method by which the antibody is produced.
- multispecific herein refers to having at least two antigen-binding sites, each of which is associated with a different epitope of the same antigen or with a different epitope of a different antigen combine.
- terms such as “bispecific”, “trispecific”, “tetraspecific” etc. refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
- valency herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule.
- the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites, respectively, in an antibody/antigen binding molecule the existence of points.
- full-length antibody “intact antibody,” and “intact antibody” are used interchangeably herein to mean having a structure that is substantially similar to that of a native antibody.
- Antigen-binding fragment and “antibody fragment” are used interchangeably herein and do not possess the full structure of an intact antibody, but only include partial or partial variants of the intact antibody that have the ability to bind antigenic capacity.
- the "antigen-binding fragments” and “antibody fragments” may be single domain antibodies.
- chimeric antibody herein refers to an antibody having variable sequences of immunoglobulins derived from one source organism (eg, rat, mouse, rabbit, or alpaca) and derived from a different organism (eg, human ) of the constant regions of immunoglobulins.
- Methods for producing chimeric antibodies are known in the art. See, eg, Morrison, 1985, Science 229(4719): 1202-7; Oi et al, 1986, Bio Techniques 4: 214-221; Gillies et al, 1985 J Immunol Methods 125: 191-202; incorporated by reference above This article.
- humanized antibody refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase homology to the sequence of a human antibody.
- CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (eg, variable FR and/or constant regions) are derived from human Immunoglobulins (receptor antibodies).
- Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, and the like.
- Fully human antibody refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences.
- Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
- variable region herein refers to the region of an antibody heavy or light chain that is involved in binding an antibody to an antigen.
- "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”.
- VL is used interchangeably with "VL”, “LCVR”.
- the variable domains (VH and VL, respectively) of the heavy and light chains of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, eg, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007).
- a single VH or VL domain may be sufficient to confer antigen binding specificity.
- complementarity determining region and “CDR” are used interchangeably herein, and generally refer to the variable region of the heavy chain (VH) or the hypervariable region (HVR) of the light chain variable region (VL), which is spatially structured It can form precise complementarity with the antigenic epitope, so it is also called the complementarity determining region.
- the heavy chain variable region CDR can be abbreviated as HCDR
- the light chain variable region CDR can be abbreviated as LCDR.
- framework region or “FR region” are used interchangeably and refer to those amino acid residues other than the CDRs in the variable region of the heavy or light chain of an antibody.
- a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (see Kabat et al., Sequences of Protein so of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; incorporated herein by reference).
- CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, which constitute the heavy chain variable region (VH).
- CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, the second CDR and the first CDR of the light chain variable region (VL), respectively Three CDRs that make up the CDR combination of the light chain (or its variable region) (VLCDR combination).
- CDRs may be labeled and defined by means known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including, but not limited to, the AbRSA website (http://cao.labshare.
- CDRs herein include overlaps and subsets of amino acid residues differently defined.
- Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
- Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, eg, Chothia & Lesk (1987) J. Mol. Biol 196:901-917; Chothia et al. (1989) Nature 342:878-883).
- IMGT numbering system herein generally refers to the numbering system based on The International ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.
- IMGT International ImMunoGeneTics information system
- variable domains refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, relative to the availability of the antibody
- the variable domains have more conserved amino acid sequences.
- a “heavy chain constant region” comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
- “Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a substantially similar structure to that of natural antibody constant region, while the latter includes only "full-length heavy chain constant region” part".
- a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include the CH1 domain.
- a typical "heavy chain constant region fragment" can be selected from an Fc or CH3 domain.
- light chain constant region refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding the antibody to an antigen, which light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
- Fc region is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc regions and variant Fc regions.
- a human IgG heavy chain Fc region can extend from Cys226 or Pro230 to the carboxy terminus of the heavy chain.
- antibodies produced by host cells may undergo post-translational cleavage, cleavage of one or more, particularly one or two amino acids, from the C-terminus of the heavy chain.
- an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleavage variant of the full-length heavy chain.
- This may be the case when the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to the Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present.
- the IgG Fc region comprises the IgG CH2 and IgG CH3 domains, optionally, the entire or partial hinge region, but not the CH1 domain.
- the "CH2 domain" of a human IgG Fc region generally extends from the amino acid residue at about position 231 to the amino acid residue at about position 340. In one embodiment, the carbohydrate chain is attached to the CH2 domain.
- a CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain.
- the "CH3 domain" comprises that stretch of residues in the Fc region that is C-terminal to the CH2 domain (ie, from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG).
- a CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (eg having a "knob” ("knob”, knob) introduced in one chain thereof and a correspondingly introduced “cavity” in the other chain thereof ("hole", hole) of the CH3 domain; see US Patent No. 5,821,333, expressly incorporated herein by reference).
- a variant CH3 domain eg having a "knob” ("knob”, knob) introduced in one chain thereof and a correspondingly introduced “cavity” in the other chain thereof ("hole”, hole) of the CH3 domain; see US Patent No. 5,821,333, expressly incorporated herein by reference).
- such variant CH3 domains can be used to promote heterodimerization of two non-identical antibody heavy chains.
- the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National As described in Institutes of Health, Bethesda, MD, 1991.
- amino acids generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
- amino acids within each of the following groups belong to each other's conserved amino acid residues, and substitutions of amino acid residues within a group belong to conservative amino acid substitutions:
- percent (%) sequence identity and “percent (%) identity” are used interchangeably herein and refer to alignment of sequences and introduction of gaps, if necessary, to achieve maximum percent sequence identity (eg, in order to For optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and for comparison purposes, non-homologous sequences may be ignored) followed by the amino acid (or nucleotide) residues of the candidate sequence The percent identical to the amino acid (or nucleotide) residues of the reference sequence.
- alignment can be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAIi) software.
- a reference sequence aligned for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleotide) residues of the candidate sequence to 100% sequence identity.
- the length of candidate sequences aligned for comparison purposes may be, for example, at least 30% (eg, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. .
- chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and to specifically bind an antigen, comprising at least (1) an extracellular antigen binding domain, eg, an antibody The variable heavy or light chain, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
- CARs can utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
- nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
- Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), sugar (ie deoxyribose or ribose) and a phosphate group.
- cytosine C
- G guanine
- A adenine
- T thymine
- U uracil
- nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
- the sequence of bases is generally represented as 5' to 3'.
- nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as synthetic forms of DNA or RNA. A mixed polymer of one or more of these molecules.
- Nucleic acid molecules can be linear or circular.
- nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
- nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
- nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the invention in vitro and/or in vivo, eg, in a host or patient.
- DNA eg, cDNA
- RNA eg, mRNA
- the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1).
- An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment.
- An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.
- vector herein includes nucleic acid vectors such as DNA vectors (eg, plasmids), RNA vectors, viruses or other suitable replicons (eg, viral vectors).
- DNA vectors eg, plasmids
- RNA vectors eg. RNA vectors
- viruses or other suitable replicons eg, viral vectors.
- Various vectors have been developed for the delivery of polynucleotides encoding foreign proteins into prokaryotic or eukaryotic cells.
- the expression vectors of the present invention contain polynucleotide sequences and additional sequence elements, eg, for expressing proteins and/or integrating these polynucleotide sequences into the genome of mammalian cells.
- Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids containing regulatory sequences (eg, promoter and enhancer regions) that direct gene transcription.
- kits for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct efficient transcription of genes carried on expression vectors.
- Expression vectors of the present invention may also contain polynucleotides encoding markers for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.
- host cell herein refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
- the progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.
- the steps of transforming host cells with recombinant DNA described in the present invention can be performed using conventional techniques well known to those skilled in the art.
- the obtained transformants can be cultured by conventional methods, and the polypeptides encoded by the genes of the present invention can be expressed.
- the medium used in the culture can be selected from various conventional media depending on the host cells used.
- the host cells are cultured under conditions suitable for growth of the host cells.
- pharmaceutical composition refers to a formulation that is in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain unacceptable toxicity to the subject to whom the pharmaceutical composition is administered of additional ingredients.
- subject refers to an organism receiving treatment for a particular disease or disorder (eg, cancer or infectious disease) as described herein.
- a particular disease or disorder eg, cancer or infectious disease
- subjects and patients include mammals such as humans, primates, pigs, goats, rabbits, hamsters, cats, dogs, Guinea pigs, bovid family members (such as domestic cattle, bison, buffalo, elk and yak, etc.), cattle, sheep, horses and bison, etc.
- treatment refers to surgical or therapeutic treatment for the purpose of preventing, slowing (reducing) unwanted physiological changes or pathologies, such as cell proliferative disorders (eg, cancer or infectious diseases) in the subject being treated. disease) progression.
- beneficial or desirable clinical outcomes include, but are not limited to, reduction of symptoms, reduction in disease severity, stable disease state (ie, no worsening), delayed or slowed disease progression, improvement or alleviation of disease state, and remission (whether partial remission or complete remission), whether detectable or undetectable.
- Those in need of treatment include those already suffering from the disorder or disease as well as those prone to develop the disorder or disease or for whom the disorder or disease is to be prevented.
- alleviation, alleviation, weakening, alleviation, alleviation, etc. the meanings also include elimination, disappearance, non-occurrence, etc.
- an effective amount herein refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent, to a cell, tissue, or subject, is effective to prevent or alleviate a disease condition or progression of the disease.
- Effective amount also refers to an amount of the compound sufficient to relieve symptoms, eg, treat, cure, prevent or alleviate related medical conditions, or an increased rate of treatment, cure, prevention or alleviation of such conditions.
- a therapeutically effective dose refers to that ingredient alone.
- a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, consecutively or simultaneously.
- cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Benign and malignant cancers are included in this definition.
- tumor or “neoplastic” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
- cancer and “tumor” are not mutually exclusive when referred to herein.
- Figure 1 Detection results of human CD33-V-his and CD33-C2-his protein samples on SDS-PAGE reducing gel and non-reducing gel: 1-hCD33-V-his (non-reducing); 2-hCD33-V-his (reducing) ); 3-hCD33-C2-his (non-reduced); 4-hCD33-C2-his (reduced); M-marker.
- FIG. 2A ELISA detects the binding reaction of control antibody to human CD33-his protein.
- FIG. 2B ELISA detects the binding reaction of control antibody to human CD33-V-his protein.
- FIG. 2C ELISA detects the binding reaction of control antibody to human CD33-C2-his protein.
- Figure 4 FACS screening test results of CHO-K1 cells transfected with human CD33 protein.
- Figure 5 FACS screening test results of HEK293T cells transfected with monkey CD33 protein.
- FIG. 6A ELISA detection of serum antibody titers of alpaca after human CD33 protein immunization.
- Figure 6B FACS detection of serum antibody titers of alpaca after human CD33 protein immunization.
- FIGS. 7A, 7B ELISA detects the binding reaction of VHH antibody to human CD33-his protein.
- FIG. 8A, 8B FACS detection of the binding reaction of VHH antibody to CHO-K1-human CD33.
- FIGS 8C and 8D FACS detects the binding reaction of VHH antibody to U937.
- FIGS. 9A and 9B ELISA detects the binding reaction of VHH antibody to murine CD33-His protein.
- FIG. 9C, 9D ELISA detects the binding reaction of VHH antibody to monkey CD33-His protein.
- FIGS. 10A, 10B FACS detects the binding reaction of VHH antibody to HEK293T-monkey CD33.
- FIGS 11A, 11C ELISA detects the binding reaction of VHH antibody to human CD33-V-his protein.
- FIGS 11B, 11D ELISA detects the binding reaction of VHH antibody to human CD33-C2-his protein.
- Figures 12A, 12B Competitive ELISA assay to detect inhibition ratios among VHH antibodies.
- FIGS 13A, 13B ELISA competition of VHH antibody with Biotin-C33B904.
- FIGS 15A-15E ELISA detects the binding reaction of VHH humanized antibody to human CD33-his protein.
- Figures 16A to 16E FACS detection of the binding reaction of VHH humanized antibody to CHO-K1-human CD33.
- FIGS 17A-17E FACS detection of the binding reaction of VHH humanized antibody to U937.
- FIGS. 18A to 18E ELISA detects the binding reaction of VHH humanized antibody and mouse CD33-his protein.
- FIGS. 18F-18J ELISA detects the binding reaction of VHH humanized antibody and monkey CD33-his protein.
- FIGS. 19A to 19E FACS detection of the binding reaction of VHH humanized antibody to HEK293T-monkey CD33.
- ELISA detects the binding reaction of VHH humanized antibody and human CD33-V-his protein.
- Example 1 Control antibody preparation, CD33 protein preparation
- CD33 protein has a conserved V-set immunoglobulin-like domain and a variable C2-set domain
- SGN-33 and C33B904 are antibodies that recognize human CD33
- the antigen-binding epitope of SGN-33 is located in V-set domain
- the antigen-binding epitope of C33B904 is located in the C2-set domain.
- the heavy chain variable region and light chain variable region sequences of SGN-33 were obtained according to patent US 9453046, and the heavy chain variable region and light chain variable region sequences of C33B904 were obtained according to patent US20190382481A1.
- the VH and VL of the antibody SGN-33 that recognizes human CD33 and the human IgG1Fc are connected in sequence from the N-terminus to the C-terminus, wherein the VH and VL are connected by 3 GGGGS linkers to form the form of scFv-hFc; the human IgG1Fc will be recognized
- the VH and VL of the antibody C33B904 to CD33 form the complete antibody form of human IgG1.
- the corresponding amino acid sequence information is shown in Table 1 below, and the italic underline indicates the antibody variable region sequence.
- the expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, Item No.: 24765-1), and used FreeStyle TM 293 (Thermofisher scientific, Item No.: 12338018) at 37°C
- the cells were cultured continuously for 5 days, and the cell components were removed by centrifugation to obtain the antibody-containing culture supernatant.
- the culture supernatant was loaded onto a protein A chromatography column (Protein A packing AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borgron, catalog numbers: AA0273 and B-1620, respectively), using PBS phosphate After washing with buffer (pH 7.4), wash with 20 mM PB, 1 M NaCl (pH 7.2), and finally use pH 3.4 citrate buffer for elution, and collect the band eluted from the protein A chromatography column.
- the Fc-tagged antibody was neutralized with 1/10 volume of 1M Tris, pH 8.0, and dialyzed overnight at 4°C with PBS. The dialyzed protein was sterile filtered through a 0.22-micron filter membrane and stored in aliquots at -80°C.
- the amino acid sequence Asp 18-His 259 protein encoding the extracellular domain (ECD, extra-cellular domain) of human CD33 protein was purchased from ACROBiosystems (Cat. No.: CD3-H5226) and named as human CD33-his.
- nucleotides encoding the amino acid sequence of the his-tagged human CD33-V domain (domain) Pro19-Ser135 (SEQ ID NO: 4) and the C2 domain (domain) Pro145-Gln228 (SEQ ID NO: 5) amino acid sequence The sequences were cloned into pTT5 vector (completed by General Biosystems (Anhui) Co., Ltd.) and plasmids were prepared according to established standard molecular biology methods. The corresponding amino acid sequence information is shown in Table 2 below. For specific methods, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
- HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
- PEI Polysciences, Cat. No. 24765-1
- FreeStyleTM 293 Thermofisher scientific, Cat. No. 12338018
- the cell culture medium was collected, and the cell components were removed by centrifugation to obtain a culture supernatant containing human CD33 protein V domain and C2 domain.
- the culture supernatant was loaded on a nickel ion affinity chromatography column HisTrap TM Excel (GE Healthcare, Cat.
- the change of the ultraviolet absorption value (A280nm) was monitored with an ultraviolet (UV) detector.
- UV ultraviolet
- the nickel ion affinity chromatography column was washed with 20mM PB, 0.5M NaCl (pH7.4) until the UV absorption value returned to the baseline, and then buffer A: 20mM PB, 0.5M NaCl (pH7.4) and buffer B : 20 mM PB, 0.5 M NaCl, 500 mM imidazole for gradient elution (2%, 4%, 8%, 16%, 50%, 100%), and the His-bands eluted from the nickel ion affinity chromatography column were collected.
- the tagged human CD33 protein was dialyzed against PBS phosphate buffered saline (pH 7.4) overnight in a refrigerator at 4°C. The dialyzed protein was sterile filtered through a 0.22-micron filter membrane and stored at -80°C to obtain
- the above CD33 protein was detected by ELISA using positive control antibodies that recognize different epitopes.
- the negative control antibody hIgG1 was the antibody anti-hel-hIgG1 against Hen Egg Lysozyme chicken egg lysozyme (purchased from Baiying, article number: B117901).
- the test results are shown in Table 3-5 and Figure 2A-2C.
- SGN-33 and C33B904 can bind human CD33-his protein
- SGN-33 can bind human CD33-V-his protein
- C33B904 can bind human CD33-C2 -his protein
- the detection result is consistent with the binding epitope reported in the literature, indicating that the protein with the above-mentioned binding activity has been prepared.
- the U937 cells were expanded to the logarithmic growth phase in a T-25 cell culture flask, the medium supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS. Incubate with buffer containing human Fc Block (purchased from BD, Cat. No. 564220) for 15 minutes, use SGN-33 antibody as primary antibody, APC-labeled secondary antibody (purchased from Biolegend, Cat. No. 409306), use FACS (FACS CantoTM) , purchased from BD Company) detection and analysis results. The analysis results are shown in Table 6 and Figure 3, U937 cells can bind to SGN-33.
- the nucleotide sequence encoding the full-length amino acid sequence of human CD33 (NCBI: XP_011525834.1) was cloned into pcDNA3.1 vector and a plasmid was prepared (completed by Universal Biosystems (Anhui) Co., Ltd.).
- CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) was transfected with plasmids ( 3000 Transfection Kit, purchased from Invitrogen, Cat. No.
- Table 7 illustrates that a series of human CD33-positive CHO-K1 monoclonal cell lines have been generated.
- the abscissa is the cell fluorescence intensity, and the ordinate is the number of cells.
- the results in FIG. 4 show that CHO-K1-human CD33B8, CHO-K1-human CD33B11, and CHO-K1-human CD33C8 are cell lines that express high levels of human CD33.
- the nucleotide sequence encoding the monkey CD33 full-length amino acid sequence (NCBI: XP_014980179.1) was cloned into pcDNA3.1 vector (purchased from Thermofisher scientific) and a plasmid was prepared.
- pcDNA3.1 vector purchased from Thermofisher scientific
- NB147 is a polyclonal antibody (prepared from Example 3) prepared from serum after immunizing alpacas with human CD33 protein. The amplified clones were screened by flow cytometry with NB147 as a positive control. The polyclonal cell line with higher fluorescence intensity was further expanded and frozen in liquid nitrogen. As shown in Table 8 and Figure 5, flow cytometry analysis of HEK293T cell line showed a single positive cell peak overexpressing monkey CD33 after puromycin screening. Can be used to detect cross-activity of antibodies.
- the first and second immunizations used human CD33 (Asp18-His259)-Pol-his protein purchased from ACROBiosystems ( Item No.: CD3-H5226), the third immunization used human CD33(Asp18-His259)-Fc(Pro100-Lys330) protein purchased from ACROBiosystems (Item No.: CD3-H5257), and the fourth immunization used CHO prepared in Example 2 -K1-human CD33-B8. After the third immunization and the fourth immunization, peripheral blood was collected and serum was separated.
- RNAiso Plus reagent Takara, Cat. No.: #9108/9109
- PrimeScript TM II 1st Strand cDNA Synthesis Kit was used (Takara, Cat. No. 6210A)
- the extracted RNA was reverse transcribed into cDNA.
- Upstream primer CTTGGTGGTCCTGGCTGC (SEQ ID NO: 6)
- Downstream primer GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO: 7)
- Upstream primer (SEQ ID NO: 8):
- the target single-domain antibody nucleic acid fragment was recovered and cloned into the phage display vector pcomb3XSS (from Sichuan Apak Biotechnology Co., Ltd.) using the restriction endonuclease SfiI (NEB, catalog number: R0123S).
- the product was then electro-transformed into E. coli electro-competent cells TG1 (from Sichuan Apak Biotechnology Co., Ltd.), and a single-domain antibody phage display library against CD33 was constructed and the library was tested.
- the size of the library volume was calculated to be 3.4 ⁇ 10 9 .
- To detect the insertion rate of the library 48 clones were randomly selected for colony PCR. The results showed that the insertion rate reached 100%.
- CD33 binding-positive phages were infected with blank E. coli and plated. Then 96 single colonies were selected for expansion and culture. The plate was coated with CD33-his protein overnight at 4°C, the phage culture supernatant was added, and the plate was incubated at 37°C for 1 hour. After washing, TMB color developing solution was added to develop color, and the optical density was measured at a wavelength of 450 nm. CD33-his positive clones were picked for sequencing. The MOE software was used to analyze the sequencing results, and the phylogenetic tree was constructed according to the amino acid sequence of the VHH-encoded protein. After removing the sequences with close distances on the phylogenetic tree according to the sequence similarity, 18 clones were obtained by screening. or IMGT software analysis, the corresponding sequence information is shown in Table 10 below. Production identification of single domain antibodies was then performed.
- the VHH variable region sequence was recombined by Taizhou Baiying Biotechnology Co., Ltd. into the expression vector BI3.4 containing the signal peptide and human IgG1Fc (human IgG1Fc sequence such as SEQ ID NO: 191, hinge region sequence such as SEQ ID NO: 192) -huIgG1, and prepare plasmids according to established standard molecular biology methods. For specific methods, see Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press).
- the expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, item number: 24765-1) and used FreeStyleTM 293 (Thermofisher scientific, item number: 12338018) at 37°C. After continuous culture for 5 days, the cell components were removed by centrifugation, and the culture supernatant containing VHH single domain antibody was obtained.
- the culture supernatant was loaded onto a protein A chromatography column (Protein A packing AT Protein A Diamond and chromatography column BXK16/26 were purchased from Borglon, catalog numbers: AA0273 and B-1620, respectively), using PBS phosphate After washing with buffer (pH7.4), wash with 20mM PB, 1M NaCl, pH 7.2, and finally use citric acid buffer pH3.4 for elution, and collect the Fc-containing Fc eluted from the protein A column.
- the labeled antibody was neutralized with 1/10 volume of 1M Tris, pH 8.0, and dialyzed with PBS at 4°C overnight. The dialyzed protein was sterile filtered at 0.22 ⁇ m and stored in aliquots at -80°C.
- Hinge region sequence (SEQ ID NO: 192): EPKSADKTHTCPPCP
- human CD33-his protein purchased from ACROBiosystems, Cat. No.: CD3-H5226
- PBS PBS+2% (w/w) BSA
- ELISA plate reader Multimode Plate Reader, EnSight, purchased from Perkin Elmer
- the ELISA results of VHH-Fc and hCD33-his are shown in Figure 7A, 7B and Table 11, Table 12, Table 11, Table 12 shows that the purified antibodies can bind to human CD33 protein at the ELISA level.
- the IgG control is hIgG1
- the data in the table is the OD450nm value.
- the desired cells were expanded to the logarithmic growth phase in T-75 cell culture flasks.
- adherent cells CHO-K1 the medium was aspirated, washed twice with PBS buffer, and then the cells were trypsinized. After terminating the digestion Cells were washed twice with PBS buffer; for U937 cells in suspension, the medium supernatant was discarded by direct centrifugation, the cell pellet was washed twice with PBS, and incubated with buffer containing human Fc Block (purchased from BD, Cat. No. 564220) for 15 minutes .
- VHH antibodies can bind to human CD33 protein on the surface of U937 cells and CHO-K1-hCD33-B8 cells ( Figures 8A-8D), but not to negative cells.
- VHH-Fc antibody In order to detect the species cross-activity of VHH-Fc antibody, commercial mouse CD33 (Sino Biological, Item No.: 90303-C08H) and monkey CD33 (Sino Biological, Item No.: 50712-M08H) were coated on ELISA plates, respectively, according to the examples. 5.1 method for ELISA detection.
- the ELISA results of VHH-Fc antibody and murine CD33 are shown in Figures 9A-9B and Tables 15 and 16, and the ELISA results of VHH-Fc antibody and monkey CD33 are shown in Figures 9C and 9D and Tables 17 and 18.
- the IgG control is hIgG1
- the data in the table is the OD450nm value.
- NB147 alpaca serum was serially diluted 10 times from 1:100.
- the HEK293T-monkey CD33 cell line was subjected to FACS detection and data analysis according to the method of Example 5.2. The analysis results are shown in Tables 19, 20 and Figures 10A-10B. Except for S006-NB147-60 and S006-NB147-225, the other VHH-Fc antibodies have binding activity to HEK293T-monkey CD33 cells, but not to HEK293T cells. binding activity.
- Human CD33 VHH-Fc antibody was captured using a Protein A chip (GE Helthcare; 29-127-558).
- Sample and running buffer were HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
- the flow-through cell was set to 25 °C.
- the sample block was set to 16°C. Both were pretreated with running buffer.
- the antibody to be tested was first captured with a Protein A chip, then a single concentration of CD33 antigen protein was injected to record the binding and dissociation process of the antibody and antigen protein, and finally Glycine pH1.5 (GE Helthcare; BR-1003- 54) Complete chip regeneration.
- Binding was measured by injecting different concentrations of recombinant human CD33-his in solution for 240 sec with a flow rate of 30 ⁇ L/min, starting at 200 nM, diluted 1:1 for a total of 5 concentrations.
- the dissociation phase was monitored for up to 600 seconds and triggered by switching from sample solution to running buffer.
- the binding rate (Kon), dissociation rate (Koff) and binding affinity (KD) of VHH-Fc antibody to human CD33-his protein are shown in Tables 21 and 22, wherein antibodies C33B904 and SGN-33 were used as positive controls. As shown in Tables 21 and 22, the affinity of VHH-Fc antibody to human CD33 was better than that of 1.40E-07M.
- VHH-Fc antibody and monkey CD33 (Sino Biological, Cat. No.: 50712-M08H) protein was determined according to the method of Example 7.1. As shown in Tables 23 and 24, the affinity of S006-NB146-20, S006-NB146-39 and S006-NB146-46 with monkey CD33 is better than 1.0E-09M.
- Example 8 Antibody antigen binding epitope (epitope) analysis
- the extracellular CD33 protein has a conserved V-set immunoglobulin-like domain (V-domain) and a variable C2-set domain (C2-domain), the V-domain is located at the distal membrane end, and the C2-domain is located at the At the membrane proximal end, the antigen-binding epitope of SGN-33 is located in the V-domain, and the antigen-binding epitope of C33B904 is located in the C2-domain.
- VHH antibodies were coated with human CD33-V-his (distal membrane end) and human CD33-C2-his (proximal membrane end), respectively.
- Distal and proximal classification as shown in Figures 11A-11D and Table 25, VHH antibodies can be divided into two categories:
- a competitive ELISA method was used for epitope sorting of VHH antibodies against control antibodies of known epitopes. According to the method of Example 5.1, 2 ⁇ g/mL antibody was coated on ELISA plate, and the human CD33-his protein was serially diluted from 30 ⁇ g/mL, and EC80 was calculated (Tables 26-27).
- Antibody name EC80( ⁇ g/mL) S006-NB146-1 0.08 S006-NB146-3 0.12 S006-NB146-17 0.09 S006-NB146-20 0.07 S006-NB146-39 0.06 S006-NB146-40 0.10 S006-NB146-46 0.07 S006-NB146-60 0.05 S006-NB146-132 0.06 S006-NB147-225 0.09 C33B904 0.03 SGN-33 0.08
- the ELISA plate was coated with 2 ⁇ g/mL human CD33-his protein according to the method in Example 5.1, and the Biotin-C33B904 antibody was serially diluted from 15 ⁇ g/mL to calculate EC80 (0.18 ⁇ g/ml).
- the ELISA plate was coated with 2 ⁇ g/mL human CD33-his protein, and the VHH antibody to be detected started from 40 ⁇ g/mL, diluted seven concentrations at 1:2, added to the ELISA plate, and then added the Biotin-C33B904 antibody at EC80 concentration. Incubate for 2 h, wash with PBS for 5 times, add HRP-labeled anti-Streptavidin (purchased from Sigma, Cat.
- the key amino acids in the framework sequence are back-mutated to the corresponding amino acids of the single domain antibody to ensure the original affinity.
- point mutations are made to sites that are prone to chemical modification (eg, DD, NSS) in the antibody.
- two amino acids 77N and 78A were deleted in the FR3 sequence of the alpaca antibody S006-NB146-39, and these two newly introduced amino acids were removed in the S006-NB146-39 humanized antibody.
- Amino Acids (NA) See Tables 30-35 for specific mutation designs.
- S39G means mutating S to G at position 39, and so on.
- the numbering of amino acids is the natural sequence numbering.
- F27I means to mutate the 27th F to I, and so on, del77N means to delete the 77th N, del78A means to delete the 78th A, and the numbering of amino acids is the natural sequence number.
- VHH name framework region mutation CDR region mutation CDR area definition method S006-NB146-60-H3 V37H/G44Q/L45R/W47L/N50S/Y58 none IMGT S006-NB146-60-H4 V37H/G44Q/L45R/W47L/N50S/Y58N/I69M/L78V none IMGT S006-NB146-60-H5 V37H/G44Q/L45R/W47L/N50S/Y58N/N73D none IMGT S006-NB146-60-H6 V37H/G44Q/L45R/W47L/N50S/Y58N/N73D/D72N none IMGT S006-NB146-60-H7 V42H/G49Q/L50R/W52L/N55S/Y66N/R95K/A96P none IMGT
- V37H represents the mutation of V at position 37 to H, and so on.
- the numbering of amino acids is the natural sequence numbering.
- V36Y means to mutate V at position 36 to Y, and so on.
- the numbering of amino acids is the natural sequence numbering.
- V37Y represents the mutation of V at position 37 to Y, and so on.
- the numbering of amino acids is the natural sequence numbering.
- VHH name back mutation CDR region mutation CDR area definition method S006-NB147-225-H5 L4V/R95L/A96P/V101T/W118Q/M123Q none Kabat
- L4V means to mutate L at position 4 to V, and so on.
- the numbering of amino acids is the natural sequence numbering.
- Humanized heavy chain template IGHJ3*01 WGQGTMVTVSS (SEQ ID NO: 225).
- variable region sequence of the aforementioned VHH humanized antibody in this example was recombined into the expression vector BI3.4-huIgG1 containing the signal peptide and human IgG1Fc, and the humanized VHH-Fc antibody was prepared according to the method described in Example 4.1 .
- VHH-Fc humanized antibody The binding activity of VHH-Fc humanized antibody to human CD33 protein was detected with reference to the method described in Example 5.1.
- the ELISA results of VHH humanized antibody and hCD33-his are shown in Fig. 15A-Fig. 15E, the results show that VHH humanized antibody can bind to human CD33 protein at the ELISA level.
- VHH-Fc humanized antibody The binding of VHH-Fc humanized antibody to different CD33 expressing cells was detected with reference to the method described in Example 5.2.
- the analysis results are shown in Figures 16A-16E and 17A-17E, indicating that both VHH humanized antibodies can bind to human CD33 protein on the surface of CHO-K1-human CD33-B8 cells and U937 cells.
- VHH humanized antibodies did not bind to negative cells Jurkat and CHO-K1 null cells.
- Example 11 Species cross-binding activity detection of VHH-Fc humanized antibodies
- ELISA detection was carried out according to the method of Example 5.1.
- the ELISA results of VHH-Fc humanized antibody and murine CD33 are shown in Figures 18A-18E, and the ELISA results of VHH-Fc humanized antibody and monkey CD33 are shown in Figures 18F-18J.
- the humanized antibody of S006-NB146-132 other humanized antibodies basically did not bind to murine CD33, and all the humanized antibodies except S006-NB146-60 and S006-NB147-225 bound to monkey CD33.
- the HEK293T-monkey CD33 cell line was subjected to FACS detection and data analysis according to the method of Example 5.2.
- the analysis results are shown in FIGS. 19A to 19E , except the humanized antibodies of S006-NB146-60 and S006-NB147-225, the other humanized antibodies have binding activity to HEK293T-monkey CD33 cells.
- the binding rate (Kon), dissociation rate (Koff) and binding affinity (KD) of VHH-Fc humanized antibody and human CD33-his protein were detected as shown in Table 15. Only some human antibodies were detected. Antibody C33B904 and SGN-33 were used as positive controls. As shown in Table 42, the tested VHH-Fc humanized antibodies had better affinity to human CD33 than 1.61E-07M.
- VHH-Fc antibody and monkey CD33 (Sino Biological, Cat. No.: 50712-M08H) protein was determined according to the method of Example 7.2. As shown in Table 43, the affinity of the VHH-Fc humanized antibodies to monkey CD33 was all better than 1.0E-07M.
- VHH humanized antibodies can be divided into the following categories.
- the antibody After humanization of S006-NB146-17 molecule, the antibody can neither bind to human CD33-C2-his protein nor human CD33-V-his protein, which is consistent with the parent clone epitope and belongs to the spatial conformation; S006-NB146-60 molecule human After humanization, the antibody can bind to human CD33-V-his protein, and the binding epitope belongs to the V domain; after humanization of the S006-NB147-225 molecule, the antibody can bind to human CD33-C2-his protein, and the binding epitope belongs to the C2 domain.
- S006-NB146-39 molecule humanization only S006-NB146-39-aH10 and S006-NB146-39-aH11 can bind to human CD33-C2-his protein, and the binding epitope belongs to C2 domain; S006-NB146- After humanization of 173 molecules, except for S006-NB146-173-H2, the rest belong to the C2 domain consistent with the parent clone; after humanization of S006-NB146-132 molecules, the antibody can bind to human CD33-C2-his protein. Bits belong to the C2 domain.
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Abstract
Description
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-1 | 1.95E+05 | 4.38E-05 | 2.24E-10 |
S006-NB146-3 | 2.49E+03 | 6.28E-06 | 2.52E-09 |
S006-NB146-17 | 1.19E+05 | 2.18E-05 | 1.83E-10 |
S006-NB146-20 | 4.12E+05 | 3.77E-04 | 9.17E-10 |
S006-NB146-39 | 1.79E+05 | 2.95E-05 | 1.65E-10 |
S006-NB146-40 | 1.46E+03 | 1.23E-05 | 8.39E-09 |
S006-NB146-46 | 1.98E+05 | 3.39E-05 | 1.71E-10 |
S006-NB146-60 | 1.49E+05 | 1.50E-04 | 1.01E-09 |
S006-NB146-132 | 1.31E+05 | 6.96E-05 | 5.31E-10 |
S006-NB147-225 | 2.93E+04 | 5.29E-04 | 1.80E-08 |
C33B904 | 2.60E+05 | 4.08E-05 | 1.57E-10 |
SGN-33 | 7.02E+05 | 5.57E-03 | 7.93E-09 |
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-16 | 6.01E+04 | 2.24E-03 | 3.73E-08 |
S006-NB147-25 | 1.41E+05 | 1.58E-04 | 1.12E-09 |
S006-NB146-69 | 6.71E+04 | 3.26E-03 | 4.86E-08 |
S006-NB146-139 | 2.97E+05 | 4.01E-03 | 1.35E-08 |
S006-NB146-150 | 5.22E+04 | 7.33E-03 | 1.40E-07 |
S006-NB146-173 | 7.25E+04 | 1.72E-03 | 2.38E-08 |
S006-NB146-181 | 5.54E+04 | 2.87E-03 | 5.18E-08 |
S006-NB147-220 | 1.81E+05 | 4.97E-04 | 2.75E-09 |
C33B904 | 4.79E+05 | 7.44E-05 | 1.55E-10 |
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-1 | 2.49E+05 | 2.68E-04 | 1.08E-09 |
S006-NB146-3 | 1.08E+04 | 1.63E-04 | 1.51E-08 |
S006-NB146-17 | 1.13E+05 | 1.28E-04 | 1.13E-09 |
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-20 | 9.02E+05 | 4.95E-04 | 5.48E-10 |
S006-NB146-39 | 5.90E+05 | 6.98E-05 | 1.18E-10 |
S006-NB146-40 | 1.39E+03 | 2.56E-04 | 1.83E-07 |
S006-NB146-46 | 5.84E+05 | 9.36E-05 | 1.60E-10 |
S006-NB146-132 | 2.16E+05 | 3.62E-04 | 1.67E-09 |
C33B904 | 1.29E+06 | 6.32E-04 | 4.92E-10 |
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-16 | 1.40E+05 | 1.54E-02 | 1.10E-07 |
S006-NB147-25 | 4.06E+05 | 2.34E-03 | 5.76E-09 |
S006-NB146-69 | 1.67E+05 | 1.19E-03 | 7.15E-09 |
S006-NB146-139 | 9.25E+05 | 2.78E-02 | 3.01E-08 |
S006-NB146-150 | 1.54E+05 | 6.40E-03 | 4.16E-08 |
S006-NB146-173 | 1.55E+05 | 4.24E-03 | 2.74E-08 |
S006-NB146-181 | 7.28E+04 | 1.98E-03 | 2.72E-08 |
S006-NB147-220 | 6.08E+05 | 1.48E-03 | 2.43E-09 |
C33B904 | 1.01E+06 | 4.97E-04 | 4.94E-10 |
抗体名称 | EC80(μg/mL) |
S006-NB146-1 | 0.08 |
S006-NB146-3 | 0.12 |
S006-NB146-17 | 0.09 |
S006-NB146-20 | 0.07 |
S006-NB146-39 | 0.06 |
S006-NB146-40 | 0.10 |
S006-NB146-46 | 0.07 |
S006-NB146-60 | 0.05 |
S006-NB146-132 | 0.06 |
S006-NB147-225 | 0.09 |
C33B904 | 0.03 |
SGN-33 | 0.08 |
抗体名称 | EC80(μg/mL) |
S006-NB146-16 | 0.09 |
S006-NB147-25 | 0.03 |
抗体名称 | EC80(μg/mL) |
S006-NB146-69 | 1.50 |
S006-NB146-139 | 0.02 |
S006-NB146-150 | 0.37 |
S006-NB146-173 | 0.15 |
S006-NB146-181 | 0.25 |
S006-NB147-220 | 0.02 |
C33B904 | 0.04 |
SGN-33 | 0.03 |
VHH名称 | 框架区突变 | CDR区突变 | CDR区定义方式 |
S006-NB146-60-H3 | V37H/G44Q/L45R/W47L/N50S/Y58 | 无 | IMGT |
S006-NB146-60-H4 | V37H/G44Q/L45R/W47L/N50S/Y58N/I69M/L78V | 无 | IMGT |
S006-NB146-60-H5 | V37H/G44Q/L45R/W47L/N50S/Y58N/N73D | 无 | IMGT |
S006-NB146-60-H6 | V37H/G44Q/L45R/W47L/N50S/Y58N/N73D/D72N | 无 | IMGT |
S006-NB146-60-H7 | V42H/G49Q/L50R/W52L/N55S/Y66N/R95K/A96P | 无 | IMGT |
VHH名称 | 回复突变 | CDR区突变 | CDR区定义方式 |
S006-NB147-225-H5 | L4V/R95L/A96P/V101T/W118Q/M123Q | 无 | Kabat |
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-17 | 1.18E+05 | 2.34E-05 | 1.98E-10 |
S006-NB146-17-H1 | 2.34E+04 | 3.76E-03 | 1.61E-07 |
S006-NB146-17-H2 | 4.87E+04 | 1.03E-04 | 2.11E-09 |
S006-NB146-17-H3 | 7.30E+04 | 9.69E-05 | 1.33E-09 |
S006-NB146-17-H4 | 7.83E+04 | 6.68E-05 | 8.53E-10 |
S006-NB146-17-H5 | 7.73E+04 | 7.11E-05 | 9.20E-10 |
S006-NB146-17-H6 | 7.49E+04 | 4.47E-05 | 5.98E-10 |
S006-NB146-17-H7 | 7.16E+04 | 7.60E-05 | 1.06E-09 |
S006-NB146-17-H8 | 7.17E+04 | 7.69E-05 | 1.07E-09 |
S006-NB146-17-H9 | 7.32E+04 | 8.86E-05 | 1.21E-09 |
S006-NB146-60 | 1.25E+05 | 1.47E-04 | 1.18E-09 |
S006-NB146-60-H3 | 1.86E+04 | 5.07E-04 | 2.72E-08 |
S006-NB146-60-H4 | 4.32E+04 | 1.51E-04 | 3.49E-09 |
S006-NB146-60-H5 | 1.48E+04 | 8.17E-04 | 5.53E-08 |
S006-NB146-60-H6 | 1.17E+04 | 8.15E-04 | 6.98E-08 |
S006-NB146-60-H7 | 1.85E+04 | 4.25E-04 | 2.29E-08 |
S006-NB147-225 | 8.19E+04 | 2.33E-04 | 2.84E-09 |
S006-NB147-225-H5 | 3.59E+04 | 4.98E-04 | 1.39E-08 |
S006-NB146-132 | 1.31E+05 | 6.96E-05 | 5.31E-10 |
S006-NB146-132-aH1 | 3.17E+05 | 1.42E-03 | 4.50E-09 |
S006-NB146-132-aH2 | 5.37E+05 | 2.80E-04 | 5.21E-10 |
S006-NB146-132-aH3 | 4.77E+05 | 2.94E-04 | 6.17E-10 |
S006-NB146-132-aH5 | 6.88E+05 | 2.34E-04 | 3.41E-10 |
S006-NB146-39-aH8 | 2.04E+05 | 9.00E-04 | 4.42E-09 |
S006-NB146-39-aH9 | 2.55E+05 | 1.34E-03 | 5.25E-09 |
S006-NB146-39-aH10 | 1.99E+05 | 7.85E-05 | 3.95E-10 |
S006-NB146-39-aH11 | 2.80E+05 | 1.51E-04 | 5.38E-10 |
S006-NB146-39-aH12 | 2.08E+05 | 1.50E-03 | 7.21E-09 |
S006-NB146-39-aH13 | 2.31E+05 | 1.95E-03 | 8.43E-09 |
S006-NB146-39-aH15 | 1.58E+05 | 9.10E-04 | 5.77E-09 |
S006-NB146-173 | 1.85E+05 | 1.79E-03 | 9.68E-09 |
S006-NB146-173-H5 | 9.99E+04 | 6.11E-03 | 6.12E-08 |
S006-NB146-173-H6 | 2.14E+05 | 1.63E-03 | 7.60E-09 |
C33B904 | 8.28E+05 | 6.54E-05 | 7.90E-11 |
SGN-33 | 7.02E+05 | 5.57E-03 | 7.93E-09 |
抗体名称 | Kon(1/Ms) | Koff(1/s) | KD(M) |
S006-NB146-39-aH8 | 3.71E+05 | 1.77E-02 | 4.78E-08 |
S006-NB146-39-aH9 | 4.34E+05 | 1.85E-02 | 4.26E-08 |
S006-NB146-39-aH10 | 3.65E+05 | 8.29E-04 | 2.27E-09 |
S006-NB146-39-aH11 | 5.34E+05 | 1.45E-03 | 2.72E-09 |
S006-NB146-39-aH12 | 3.77E+05 | 2.65E-02 | 7.02E-08 |
S006-NB146-39-aH13 | 3.83E+05 | 2.35E-02 | 6.14E-08 |
S006-NB146-39-aH15 | 3.08E+05 | 1.71E-02 | 5.57E-08 |
S006-NB146-173-H5 | 1.52E+05 | 1.44E-02 | 9.51E-08 |
S006-NB146-173-H6 | 3.23E+05 | 5.20E-03 | 1.61E-08 |
C33B904 | 1.11E+06 | 6.26E-04 | 5.65E-10 |
Claims (27)
- 特异性结合CD33的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含:CDR1、CDR2和CDR3;所述CDR1、CDR2和CDR3具有选自以下的任意序列组合或者与所述序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合:(1)所述CDR1可选自SEQ ID NO:53、107、161、29、32、35、38、41、44、47、50、56、59、62、65、68、71、74、77、80、83、86、89、92、95、98、101、104、110、113、116、119、122、125、128、131、134、137、140、143、146、149、152、155、158、164、167、170、173、176、179、182、185、188、226、227;(2)所述CDR2可选自SEQ ID NO:230、54、108、162、30、33、36、39、42、45、48、51、57、60、63、66、69、72、75、78、81、84、87、90、93、96、99、102、105、111、114、117、120、123、126、129、132、135、138、141、144、147、150、153、156、159、165、168、171、174、177、180、183、186、189、229;(3)所述CDR3可选自SEQ ID NO:55、109、163、31、34、37、40、43、46、49、52、58、61、64、67、70、73、76、79、82、85、88、91、94、97、100、103、106、112、115、118、121、124、127、130、133、136、139、142、145、148、151、154、157、160、166、169、172、175、178、181、184、187、190、228;各个CDR1、CDR2和CDR3为根据KABAT、Chothia或IMGT的通行分析方法编码;优选地,所述替换为保守氨基酸的替换。
- 权利要求1所述的抗体或抗原结合片段,其特征在于,所述CDR1、CDR2和CDR3分别包含选自SEQ ID NO:11~28或193~223任一项所示VHH结构域中的CDR1、CDR2和CDR3;优选地,根据KABAT、Chothia或IMGT编号系统,(1)所述CDR1选自SEQ ID NO:53、107、161,所述CDR2选自SEQ ID NO:54、108、162、230,所述CDR3选自SEQ ID NO:55、109、163;(2)所述CDR1选自SEQ ID NO:32、86、140,所述CDR2选自SEQ ID NO:33、87、141,所述CDR3选自SEQ ID NO:34、88、142;(3)所述CDR1选自SEQ ID NO:35、89、143、226、227,所述CDR2选自SEQ ID NO:36、90、144,所述CDR3选自SEQ ID NO:37、91、145;(4)所述CDR1选自SEQ ID NO:38、92、146,所述CDR2选自SEQ ID NO:39、93、147,所述CDR3选自SEQ ID NO:40、94、148;(5)所述CDR1选自SEQ ID NO:41、95、149,所述CDR2选自SEQ ID NO:42、96、150、229,所述CDR3选自SEQ ID NO:43、97、151、228;(6)所述CDR1选自SEQ ID NO:44、98、152,所述CDR2选自SEQ ID NO:45、99、153,所述CDR3选自SEQ ID NO:46、100、154;(7)所述CDR1选自SEQ ID NO:47、101、155,所述CDR2选自SEQ ID NO:48、102、156,所述CDR3选自SEQ ID NO:49、103、157;(8)所述CDR1选自SEQ ID NO:50、104、158,所述CDR2选自SEQ ID NO:51、105、159,所述CDR3选自SEQ ID NO:52、106、160;(9)所述CDR1选自SEQ ID NO:29、83、137,所述CDR2选自SEQ ID NO:30、84、138,所述CDR3选自SEQ ID NO:31、85、139;;(10)所述CDR1选自SEQ ID NO:56、110、164,所述CDR2选自SEQ ID NO:57、111、165,所述CDR3选自SEQ ID NO:58、112、166;(11)所述CDR1选自SEQ ID NO:59、113、167,所述CDR2选自SEQ ID NO:60、114、168,所述CDR3选自SEQ ID NO:61、115、169;(12)所述CDR1选自SEQ ID NO:62、116、170,所述CDR2选自SEQ ID NO:63、117、171,所述CDR3选自SEQ ID NO:64、118、172;(13)所述CDR1选自SEQ ID NO:65、119、173,所述CDR2选自SEQ ID NO:66、120、174,所述CDR3选自SEQ ID NO:67、121、175;(14)所述CDR1选自SEQ ID NO:68、122、176,所述CDR2选自SEQ ID NO:69、123、177,所述CDR3选自SEQ ID NO:70、124、178;(15)所述CDR1选自SEQ ID NO:71、125、179,所述CDR2选自SEQ ID NO:72、126、180,所述CDR3选自SEQ ID NO:73、127、181;(16)所述CDR1选自SEQ ID NO:74、128、182,所述CDR2选自SEQ ID NO:75、129、183,所述CDR3选自SEQ ID NO:76、130、184;(17)所述CDR1选自SEQ ID NO:77、131、185,所述CDR2选自SEQ ID NO:78、132、186,所述CDR3选自SEQ ID NO:79、133、187;(18)所述CDR1选自SEQ ID NO:80、134、188,所述CDR2选自SEQ ID NO:81、135、189,所述CDR3选自SEQ ID NO:82、136、190;或,(19)与上述(1)~(18)序列组合相比具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列组合;优选地,所述替换为保守氨基酸的替换。
- 权利要求1或2所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含SEQ ID NO:11~28或193~223中的CDR1、CDR2和CDR3序列组合。
- 根据权利要求1~3任一项所述的抗体或抗原结合片段,其特征在于,其包含与所述CDR1、CDR2和/或CDR3相比具有至少80、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的序列。
- 根据权利要求1~4任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含SEQ ID NO:11~28或193~223任一项所示VHH结构域中的FR区;可选地,所述抗体或抗原结合片段包含与SEQ ID NO:11~28或193~223任一项所示VHH结构域中的FR区相比具有至少80、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的序列;或,可选地,所述抗体或抗原结合片段包含与SEQ ID NO:11~28或193~223任一项所示VHH结构域中的FR区相比发生至多15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变 的序列;所述突变可选自插入、缺失和/或替换,所述替换优选为保守氨基酸的替换。
- 根据权利要求1~5任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含SEQ ID NO:11~28或193~223任一项所示序列;可选地,所述抗体或抗原结合片段包含与SEQ ID NO:11~28或193~223任一项所示序列相比具有至少80、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%同一性的序列;或,可选地,所述抗体或抗原结合片段包含与SEQ ID NO:11~28或193~223任一项所示序列相比发生至多20个、19个、18个、17个、16个、15个、14个、13个、12个、11个、10个、9个、8个、7个、6个、5个、4个、3个、2个或1个突变的序列;所述突变可选自插入、缺失和/或替换,所述替换优选为保守氨基酸的替换。
- 根据权利要求1~6任一项所述的抗体或抗原结合片段,其特征在于,其与人CD33结合的解离常数(KD)不大于100nM;与猴CD33结合的解离常数(KD)不大于100nM。
- 根据权利要求1~7任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段包含或不包含抗体重链恒定区;可选的,所述抗体重链恒定区可选自人、羊驼、小鼠、大鼠、兔或羊;可选地,所述抗体重链恒定区可选自IgG、IgM、IgA、IgE或IgD,所述IgG可选自IgG1,IgG2,IgG3或IgG4;可选地,所述重链恒定区可选自Fc区、CH3区、不存在CH1片段的重链恒定区或完整重链恒定区;优选地,所述重链恒定区为人Fc区,更优选具有如SEQ ID NO:191所示氨基酸序列;优选地,所述抗体或抗原结合片段为重链抗体。
- 根据权利要求1~8任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段为:(1)嵌合抗体或其片段;(2)人源化抗体或其片段;或,(3)全人源抗体或其片段。
- 根据权利要求1~9任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段还偶联有治疗剂或示踪剂;优选地,所述治疗剂选自放射性同位素、细胞毒性剂或免疫调节剂,所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂;更优选地,所述细胞毒性剂选自生物碱类(alkaloids)、甲氨蝶呤(methotrexate)、蒽环类抗生素(doxorubicin)或紫杉烷类(taxanes)。
- 根据权利要求1~9任一项所述的抗体或抗原结合片段,其特征在于,所述抗体或抗原结合片段还连接有另一功能性分子,所述功能性分子可选自以下一种或多种:信号肽、蛋白标签、或细胞因子。
- 一种多特异性抗体,其特征在于,所述多特异性抗体包含权利要求1~11任一项所述的抗体或抗原结合片段;优选地,所述多特异性抗体进一步包含特异性结合CD33以外 的抗原或结合与权利要求1~11任一项所述抗体或抗原结合片段不同的CD33表位的抗体或抗原结合片段。
- 根据权利要求12的多特异性抗体,其特征在于,所述CD33以外的抗原可选自:CD3,优选CD3ε;CD16,优选CD16A;CS32B;PD-1;PD-2;PD-L1;NKG2D;CD19;CD20;CD40;CD47;4-1BB;CD137;EGFR;EGFRvIII;TNF-alpha;MSLN;HER2;HER3;HSA;CD5;CD27;EphA2;EpCAM;MUC1;MUC16;CEA;Claudin18.2;叶酸受体;Claudin6;WT1;NY-ESO-1;MAGE3;ASGPR1或CDH16。
- 根据权利要求12或13的多特异性抗体,其特征在于,所述多特异性抗体可为双特异性抗体、三特异性抗体或四特异性抗体,所述多特异性抗体可为二价、四价或六价。
- 一种嵌合抗原受体(CAR),其特征在于,所述嵌合抗原受体至少包含细胞外抗原结合结构域、跨膜结构域和胞内信号传导结构域,所述细胞外抗原结合结构域包含权利要求1~11任一项所述抗体或抗原结合片段。
- 一种免疫效应细胞,其特征在于,所述免疫效应细胞表达权利要求15所述的嵌合抗原受体,或包含编码权利要求15所述嵌合抗原受体的核酸片段;优选地,所述免疫效应细胞选自T细胞、NK细胞(natural killer cell)、NKT细胞(natural killer T cell)、DNT细胞(double negative T cell)、单核细胞、巨噬细胞、树突状细胞或肥大细胞,所述T细胞优选自细胞毒性T细胞、调节性T细胞或辅助性T细胞;优选地,所述免疫效应细胞为自体免疫效应细胞或同种异体免疫效应细胞。
- 一种分离的核酸片段,其特征在于,所述核酸片段编码权利要求1~11任一项所述的抗体或抗原结合片段,或权利要求12~14任一项所述多特异性抗体,或权利要求15所述的嵌合抗原受体。
- 一种载体(vector),其特征在于,所述载体包含权利要求17所述的核酸片段。
- 一种宿主细胞,其特征在于,所述宿主细胞包含权利要求18所述的载体;优选地,所述细胞为原核细胞或真核细胞,例如细菌(大肠杆菌)、真菌(酵母)、昆虫细胞或哺乳动物细胞(CHO细胞系或293T细胞系)。
- 一种制备权利要求1~11任一项所述抗体或抗原结合片段或权利要求12~14任一项所述多特异性抗体的方法,其特征在于,所述方法包括培养权利要求19所述细胞,以及分离所述细胞表达的抗体或抗原结合片段,或分离所述细胞表达的多特异性抗体。
- 一种制备权利要求16所述免疫效应细胞的方法,其特征在于,所述方法包括将编码权利要求15所述CAR的核酸片段导入所述免疫效应细胞,可选地,所述方法还包括启动所述免疫效应细胞表达权利要求15所述CAR。
- 一种药物组合物,其特征在于,所述药物组合物包含权利要求1~11任一项所述的抗体或抗原结合片段,或权利要求12~14任一项所述的多特异性抗体,或权利要求16所述免疫效应细胞,或权利要求17所述的核酸片段,或权利要求18所述载体;或权利要求20~21任一项所述方法制备获得的产品;可选地,所述药物组合物还包含药学上可接受的运载体(carrier)、稀释剂或助剂;可选地,所述药物组合物还包含额外的抗肿瘤剂。
- 权利要求1~11任一项所述的抗体或抗原结合片段,或权利要求12~14任一项所述的多特异性抗体,或权利要求16所述免疫效应细胞,或权利要求17所述的核酸片段,或权利要求18所述载体;或权利要求20~21任一项所述方法制备获得的产品;或权利要求22所述药物组合物在制备预防和/或治疗肿瘤的药物中的用途;所述肿瘤优选骨髓增生异常综合症(MDS),急性髓性白血病(AML),慢性髓性白血病(CML)和前髓细胞性白血病(PML)。
- 一种预防和/或治疗肿瘤的方法,包含向有此需要的患者施用有效量的权利要求1~11任一项所述的抗体或抗原结合片段,或权利要求12~14任一项所述的多特异性抗体,或权利要求16所述免疫效应细胞,或权利要求17所述的核酸片段,或权利要求18所述载体,或权利要求20~21任一项所述方法制备获得的产品,或权利要求22所述药物组合物;所述肿瘤优选骨髓增生异常综合症(MDS),急性髓性白血病(AML),慢性髓性白血病(CML)和前髓细胞性白血病(PML)。
- 权利要求1~11任一项所述的抗体或抗原结合片段,或权利要求12~14任一项所述的多特异性抗体,或权利要求16所述免疫效应细胞,或权利要求17所述的核酸片段,或权利要求18所述载体,或权利要求20~21任一项所述方法制备获得的产品,或权利要求22所述药物组合物,其特征在于,用于预防和/或治疗肿瘤;所述肿瘤优选骨髓增生异常综合症(MDS),急性髓性白血病(AML),慢性髓性白血病(CML)和前髓细胞性白血病(PML)。
- 一种试剂盒,其包含权利要求1~11任一项所述的抗体或抗原结合片段,或权利要求12~14任一项所述的多特异性抗体,或权利要求16所述免疫效应细胞,或权利要求17所述的核酸片段,或权利要求18所述载体,或权利要求20~21任一项所述方法制备获得的产品,或权利要求22所述药物组合物。
- 一种体外抑制表达CD33细胞增殖或迁移的方法,其特征在于,在权利要求1~11任一项所述的抗体或抗原结合片段与CD33之间能够形成复合物的条件下,使所述细胞与权利要求1~11任一项所述的抗体或抗原结合片段接触。
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