Antibodies targeting CD33 and uses thereof

Abstract

The present invention provides antibodies targeting CD33, as well as multispecific antibodies, chimeric polypeptides, antibody conjugates, pharmaceutical compositions and kits comprising the same, and their use in the diagnosis, treatment and prevention of diseases associated with CD33 expression.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy. More specifically, this invention relates to antibodies targeting CD33, and their uses in the prevention, treatment, and diagnosis of diseases. Background Technology

[0002] CD33 (Sialic acid-binding Ig-like lectin 3, Siglec-3) is a transmembrane glycoprotein on the surface of myeloid cells, widely expressed in myeloid cell subsets such as monocytes, macrophages, and granulocytes. CD33 is highly expressed in various myeloid malignancies, particularly acute myeloid leukemia (AML), making it an important target for targeted therapy. In addition, CD33 is associated with various other hematologic disorders and certain inflammatory diseases, such as myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), and certain autoimmune diseases. In recent years, antibodies targeting CD33, antibody-drug conjugates, and cell therapy products have shown potential application value in the treatment of these diseases.

[0003] However, existing CD33-targeting therapies still face many challenges due to issues such as immunogenicity, targeting, drug resistance, and safety. This invention aims to provide an antibody targeting CD33 and its applications in disease prevention, treatment, and diagnosis. Summary of the Invention

[0004] In a first aspect, the present invention provides an antibody targeting CD33, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6.

[0005] In some embodiments, the light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence selected from SEQ ID NO: 7, and the heavy chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence selected from SEQ ID NO: 8. Preferably, the CD33 antibody comprises the light chain variable region selected from SEQ ID NO: 7 and the heavy chain variable region selected from SEQ ID NO: 8.

[0006] In some embodiments, the antibody comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 9 or 10. Preferably, the antibody comprises the amino acid sequence shown in SEQ ID NO: 9 or 10.

[0007] In some embodiments, the antibody of the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0008] In one aspect, the present invention also provides a multispecific antibody (preferably a bispecific or trispecific antibody) comprising the anti-CD33 antibody or an antigen-binding fragment thereof as described above, further comprising one or more second antibodies that specifically bind to other antigens or epitopes.

[0009] As used herein, the term "multispecific" refers to an antigen-binding protein having multiple epitope specificities (i.e., the ability to specifically bind to two, three, or more different epitopes on a single biomolecule or the ability to specifically bind to epitopes on two, three, or more different biomolecules). As used herein, the term "bispecific" indicates that an antigen-binding protein has two different antigen-binding specificities.

[0010] In some implementations, the second antibody may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibody, biantibody, or sdAb.

[0011] Therefore, in some embodiments, the second antibody targets antigens selected from the following: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD 138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2,EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galactoglobulin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, hepatin B2, tyrosinase, fucose GM1, o-acetyl-GD2, folate receptor β, polysialic acid, spermatin 17, survivin and telomerase, sarcoma translocation breakpoint, human telomere reverse transcriptase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tendinin, carcinoembryonic variants of tumor necrosis regions, and any combination thereof. Preferably,The second antigen is selected from CD7, CD19, CD20, CD22, CD30, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor α, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, and any combination thereof, more preferably selected from CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, BCMA, and any combination thereof.

[0012] In another embodiment, the second antibody targets other antigenic epitopes of CD33.

[0013] The term "heavy chain" refers to the larger of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule, and it usually determines the antibody's class. The term "light chain" refers to the smaller of the two types of polypeptide chains present in the naturally occurring conformation of an antibody molecule. Kappa (κ) and lambda (λ) light chains refer to the two main isotypes of antibody light chains.

[0014] The term "complementarity-determining region" or "CDR" refers to the amino acid sequence within the variable region of an antibody that confers antigen specificity and binding affinity. For example, generally, three CDRs exist in each heavy chain variable region (e.g., CDR1-H, CDR2-H, and CDR3-H), and three CDRs exist in each light chain variable region (CDR1-L, CDR2-L, and CDR3-L). The precise amino acid sequence boundaries of a CDR can be determined using any of many well-known schemes, including: the Kabat numbering scheme, the Chothia numbering scheme, the IMGT numbering scheme, the AHo numbering scheme, and the AbM numbering scheme. The precise amino acid sequence of a given CDR or FR may vary depending on the chosen numbering scheme. It should be understood that a "CDR" or "FR" for a given antibody or its region (such as its variable region) encompasses the CDR or FR defined by any of the above-mentioned schemes or other known schemes. Where a specified CDR or FR contains a given amino acid sequence, it should be understood that such a CDR or FR may also have the sequence of the corresponding CDR or FR defined by any of the above-mentioned schemes or other known schemes. The numbering scheme used in this paper to define the boundaries between CDR and FR is the Chothia scheme.

[0015] The term "single-chain antibody" or "scFv" refers to a fusion protein comprising at least one light chain variable region (VL) and at least one heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are adjacent (e.g., linked via a linker) and are capable of being expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the complete antibody from which it originated. Unless otherwise stated, the scFv herein may have the VL and VH in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may comprise VL-linker-VH or VH-linker-VL from the N-terminus to the C-terminus. The term "linker" refers to a molecular sequence that links two molecules or two sequences on the same molecule. In some embodiments, the linker is a peptide linker. Preferably, the linker does not adversely affect the expression, secretion, or biological activity of the polypeptide. Furthermore, the linker is preferably non-antigenic and does not elicit an immune response. In some embodiments, the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a GS-rich sequence), or a non-peptide chemical linker. In some embodiments, the adapter has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:22 or 23.

[0016] As used herein, the term sequence “identity” refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0017] Nucleic acid, vector, host cell

[0018] In another aspect, the present invention relates to nucleic acid molecules encoding the anti-CD33 antibody or multispecific antibody of the present invention. The nucleic acid of the present invention may be RNA, DNA, or cDNA.

[0019] The nucleic acids of the present invention may also be in vector form, and may be present in and / or part of a vector, such as plasmids, sticky-terminal plasmids, or YACs. The vector may be, in particular, an expression vector, providing a means for expressing anti-CD33 antibodies in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). This expression vector typically contains at least one nucleic acid molecule of the present invention operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of said regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the anti-CD33 antibodies of the present invention include, but are not limited to, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0020] In another aspect, the present invention also provides host cells expressing the anti-CD33 antibody, multispecific antibody, and / or containing the nucleic acid or vector of the present invention. Preferred host cells of the present invention are bacterial cells, fungal cells, or mammalian cells.

[0021] Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0022] Suitable fungal cells include cells from species of the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells from species of the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichiapastoris* and *Pichia methanolica*), and *Hansenula*.

[0023] Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0024] However, the present invention may also use amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0025] Chimeric Peptides

[0026] In another aspect, the present invention also provides chimeric polypeptides comprising the anti-CD33 antibody as described above, such as recombinant T-cell receptors, chimeric antigen receptors, T-cell fusion proteins or T-cell antigen couplers, preferably chimeric antigen receptors.

[0027] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding domain (e.g., a tumor antigen) (e.g., a ligand of an antibody or antigen), a transmembrane domain, and a primary signaling domain, optionally also including a co-stimulatory domain. The domains are linked by a linker. CARs can specifically and reactively redirect T cells and other immune cells to a selected target in a non-MHC-restricted manner. In some embodiments, the chimeric polypeptide of the present invention is a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and a primary signaling domain, optionally also comprising one or more co-stimulatory domains. In some embodiments, the chimeric antigen receptor further comprises one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, etc.

[0028] As used herein, the term "T-cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from the components of the TCR, typically composed of TCR subunits and antibodies linked to them, and expressed on the cell surface. The TCR subunits include at least a portion of the TCR extracellular domain, transmembrane domain, and intracellular signaling domain.

[0029] As used herein, the term “T-cell antigen coupler” or “TAC” includes three functional domains: a tumor-targeting domain, including single-chain antibodies (e.g., the anti-FasL antibody, anti-IRP60 antibody, or anti-SIRPα antibody of the present invention), a designed ankyrin repeat protein (DARPin), or other targeting groups; an extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor close to the TCR receptor; and an intracellular domain of the transmembrane region and the CD4 co-receptor, wherein the intracellular domain connects to a protein kinase LCK, which catalyzes phosphorylation of the immune receptor tyrosine activation motif (ITAM) of the TCR complex as an initial step in T-cell activation.

[0030] As used herein, the term "T-cell receptor" or "TCR" is a characteristic marker on the surface of T cells that binds nonvalently to CD3 to form a complex. Antigen-presenting cells present antigenic peptides to T cells via the major histocompatibility complex (MHC) molecule, which bind to the TCR complex to induce a series of intracellular signaling pathways. The TCR consists of six polypeptide chains that form heterodimers, generally classified as αβ and γδ types. Each polypeptide chain includes a constant region and a variable region, where the variable region is responsible for binding specific antigens and MHC molecules. The term "recombinant TCR receptor" refers to an artificially constructed T-cell receptor that further includes an antigen-binding domain (e.g., a tumor antigen).

[0031] In some embodiments, the present invention provides a chimeric antigen receptor comprising an anti-CD33 antibody as described above or a multispecific antibody containing the anti-CD33 antibody, a transmembrane domain, and an intracellular signal transduction domain.

[0032] As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric antigen receptor on the surface of immune cells (e.g., T cells, NK cells, or NKT cells) and guides the cellular response of immune cells against target cells. Transmembrane domains can be natural or synthetic and can be derived from any membrane-binding or transmembrane protein. When a chimeric antigen receptor binds to a target antigen, the transmembrane domain enables signal transduction. Transmembrane domains particularly suitable for use in this invention can be derived from, for example, the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, transmembrane domains can be synthetic and may primarily contain hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from the CD8α chain and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:11.

[0033] As used herein, the term "intracellular signaling domain" refers to a protein portion that transduces effector functional signals and directs the cell to perform a specified function. In some embodiments, the intracellular signaling domain of the chimeric antigen receptor of the present invention may be an intracellular region sequence of a T-cell receptor and a co-receptor that, upon antigen receptor binding, act together to initiate signal transduction, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar functions. The intracellular signaling domain may contain a number of immune receptor tyrosine activation motifs (ITAMs). Non-limiting embodiments of the intracellular signaling domain of the present invention include, but are not limited to, intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR of the present invention may contain a CD3ζ intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 15 or 16.

[0034] In some embodiments, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect the transmembrane domain to the antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antibody. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. The hinge region may be wholly or partially derived from natural molecules, such as the extracellular regions of CD8, CD4, or CD28, or wholly or partially derived from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or it may be a fully synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a hinge region portion of CD8α, CD28, FcγRIIIα receptor, IgG4, or IgG1, more preferably a CD8α, CD28, or IgG4 hinge, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in any of SEQ ID NO:19-21.

[0035] In some embodiments, the chimeric antigen receptor may further comprise one or more co-stimulatory domains. A co-stimulatory domain may be an intracellular functional signaling domain derived from a co-stimulatory molecule, comprising the entire intracellular portion of the co-stimulatory molecule or a functional fragment thereof. A "co-stimulatory molecule" refers to a homologous binding partner that specifically binds to a co-stimulatory ligand on a T cell, thereby mediating a co-stimulatory response (e.g., proliferation) of the T cell. Co-stimulatory molecules include, but are not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. Non-limiting embodiments of the co-stimulatory domains of the present invention include, but are not limited to, co-stimulatory signal transduction domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, B7-H3, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the co-stimulatory domain of the CAR of the present invention is derived from 4-1BB and / or CD28. In some embodiments, the CD28 co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:13. In some embodiments, the 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:14.

[0036] In some embodiments, the CAR of the present invention may further comprise a signal peptide such that, when expressed in cells such as T cells, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long, hydrophobic amino acid segment with a tendency to form a single α-helix. At the end of the signal peptide, there is typically an amino acid segment that is recognized and cleaved by a signal peptidase. The signal peptidase may cleave the peptide during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, etc. In some embodiments, the signal peptide that can be used in the present invention is derived from B2M and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:17. In some embodiments, the signal peptide used in this invention is derived from CD8α, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:18.

[0037] In some embodiments, the CAR contains an anti-CD33 antibody or its antigen-binding fragment as provided herein, or a multispecific antibody containing the anti-CD33 antibody, a CD8α or CD28 transmembrane region, a CD28 and / or 4-1BB co-stimulatory domain, and a CD3ζ intracellular signal transduction domain. In this embodiment, the CAR may further contain a signal peptide from B2M, CD8α, IgG1, or GM-CSFRα.

[0038] The present invention also provides a nucleic acid molecule encoding a chimeric antigen receptor targeting CD33 as defined above, and a vector comprising said nucleic acid molecule.

[0039] As used herein, the term "vector" is a medium nucleic acid molecule used to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule may, for example, be replicated and / or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids into the cell interior by, for example, homologous recombination or using a hybrid recombinase with a specific target site sequence. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences (e.g., those encoding the chimeric antigen receptor polypeptide of the present invention) in a suitable host cell and for the translation of their mRNA. Suitable vectors for use in the present invention are known in the art and many are commercially available. In some embodiments, the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia virus, Raul's sarcoma virus (RSV, polyomavirus, and adeno-associated virus (AAV) etc.), bacteriophages, phage particles, granules, and artificial chromosomes (including BAC and YAC). The vector itself is typically a nucleic acid molecule, usually consisting of a DNA sequence containing an insert (transgenic) and a larger sequence serving as the vector's "backbone." Engineered vectors typically also include an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selection marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally include elements such as a promoter, polyA tail, 3'UTR, enhancer, terminator, insulator, operon, selection marker, reporter gene, target sequence, and / or protein purification tag. In one specific embodiment, the vector is an in vitro transcription vector.

[0040] Engineered immune cells

[0041] In one aspect, the present invention also provides engineered immune cells expressing the chimeric polypeptides described herein, such as recombinant TCR receptors or chimeric antigen receptors.

[0042] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell-killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, immune cells can be T cells, macrophages, neutrophils, dendritic cells, monocytes, NK cells, and / or NKT cells. In some embodiments, immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Preferably, the immune cell is a T cell. T cells can be any type of T cell, such as cultured T cells, like primary T cells, or T cells derived from cultured T cell lines such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and their transgenic species. T cells can be obtained from a variety of sources, including peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be at any developmental stage, including but not limited to CD4+CD8+ T cells, CD4+ T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), CD4+ T cells, ... - CD8-T cells, tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. Various techniques known to those skilled in the art, such as Ficoll isolation, can be used to obtain T cells from the subject's blood.

[0043] In some embodiments, to reduce the risk of graft-versus-host disease, the engineered immune cells further comprise at least one gene selected from the following whose expression is suppressed or silenced: CD52, GR, dCK, TCR / CD3 genes (e.g., TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA), and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTP. N22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFBRRI, SMAD2, SMAD 3. SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. Preferably, the engineered immune cells further comprise at least one gene selected from the following whose expression is suppressed or silenced: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.

[0044] Methods for suppressing gene expression or silencing genes are well known to those skilled in the art. For example, gene expression can be suppressed using antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA / miRNA, trans dominant-negative proteins (TNPs), chimeric / antibody conjugates, chemokine ligands, anti-infective cellular proteins, intracellular antibodies (sFv), nucleoside analogs (NRTIs), non-nucleoside analogs (NNRTIs), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents), and protease inhibitors. Alternatively, gene silencing can also be achieved by mediating DNA breaks through, for example, broad-spectrum nucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.

[0045] In some embodiments, the engineered immune cells further comprise a second chimeric polypeptide that targets other antigens or antigenic epitopes. The other antigens targeted by the second chimeric polypeptide may be selected from, for example, ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD137, CD 138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin 6, Claudin18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2ETS fusion gene, ETV6-AML, EMR2, EGP2,EGP40, FAP, FAR, FBP, FLT3, FOSL1, FCRL5, FCAR, Flt3, Flt4, Frizzled, GD2, GD3, gp100, gp130, GM3, GPC2, GPC3, GPRC5D, GPR20, GloboH, GHRHR, GHR, GITR, Her2, HER3, HER-4, HMWMAA, HAVCR1, HPV E6,E7, HVEM, HIV-1Gag, HLA-A1, HLA-A2, IL6R, IL-11Ra, IL-13Ra, IGF-I receptor, LTPR, LIFRP, LRP5, IGLL1, IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, MSLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA / MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY-ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR-β, PCTA-1 / galactoglobulin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCRα, TCRβ, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2VEGFR, WT1, XAGE1, 5T4, 8H9, αvβ6 integrin, CA9, folate receptor α, hepatin B2, tyrosinase, fucose GM1, o-acetyl-GD2, folate receptor β, polysialic acid, spermatin 17, survivin and telomerase, sarcoma translocation breakpoint, human telomere reverse transcriptase / hTERT, androgen receptor, intestinal carboxylesterase, cyclin B1, fibronectin, tendinin, carcinoembryonic variants of tumor necrosis regions, and any combination thereof. Preferably,The second antigen is selected from CD7, CD19, CD20, CD22, CD30, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor α, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, and any combination thereof, more preferably from CD7, CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, BCMA, and any combination thereof.

[0046] In some embodiments, multiple immune cells are provided, each engineered to express one or more chimeric antigen receptors. For example, in some embodiments, one immune cell is engineered to express a chimeric antigen receptor that binds to and / or targets CD33 (e.g., a CAR containing the anti-CD33 antibody described in this invention), and another cell is engineered to express a chimeric antigen receptor that binds to and / or targets other antigens or epitopes. In some embodiments, the immune cells may also express multispecific chimeric antigen receptors that target one or more antigens, including CD33. For example, such multispecific chimeric antigen receptors may contain a multispecific antibody targeting CD33, or simultaneously contain the anti-CD33 antibody described in this invention and an antibody targeting other antigens or epitopes. In such embodiments, the multiple engineered immune cells may be administered together or separately. In some embodiments, the multiple immune cells may be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.

[0047] Antibody conjugates

[0048] In one aspect, the present invention provides an antibody conjugate comprising an anti-CD33 antibody as defined in the present invention and a second functional structure, wherein the second functional structure is selected from Fc, a radioisotope, a structural portion for extending half-life, a detectable marker, and a drug.

[0049] In some embodiments, the present invention provides an antibody conjugate comprising an anti-CD33 antibody and an Fc as defined herein. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, including native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether in monomeric or multimeric form, generated by digesting an intact antibody. The immunoglobulin source that generates native Fc is preferably derived from humans. Native Fc fragments consist of monomeric polypeptides that can be linked covalently (e.g., disulfide bonds) and non-covalently into dimer or multimer forms. Depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subtype (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), the monomeric subunits of the native Fc molecule have 1-4 intermolecular disulfide bonds. An example of a natural Fc is a disulfide-linked dimer produced by digesting IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). As used herein, the term “natural Fc” generally refers to monomeric, dimeric, and polymeric forms. A “variant Fc” is an amino acid sequence that differs from the amino acid sequence of a “natural” or “wild-type” Fc due to at least one “amino acid modification” as defined herein, also referred to as an “Fc variant.” Therefore, “Fc” also includes single-chain Fc (scFc), i.e., a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, capable of naturally folding into a functional dimer Fc region. In some embodiments, the Fc is preferably a human immunoglobulin Fc, more preferably a human IgG1 Fc.

[0050] In some embodiments, the present invention provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a radioactive isotope. Examples of radioactive isotopes that can be used in the present invention include, but are not limited to, At. 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 , 99m Tc, 123 I, 18 F and 68 Ga.

[0051] In some embodiments, the present invention provides an antibody conjugate comprising an anti-CD33 antibody as defined in the present invention and a structural portion for extending half-life, wherein the structural portion for extending half-life is selected from albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides (including antibodies) that bind to human serum albumin.

[0052] In some embodiments, the present invention provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a detectable marker. The term "detectable marker" herein refers to a compound that generates a detectable signal. For example, a detectable marker may be an MRI contrast agent, a scintillation scanning contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, or an optical imaging contrast agent. Examples of detectable markers include fluorophores (such as fluorescein, Alexa, or anthocyanins), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as horseradish peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotic resistance genes (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.), and contrast agents (such as nanoparticles or gadolinium). Those skilled in the art can select appropriate detectable markers based on the detection system used.

[0053] In some embodiments, the present invention provides an antibody-drug conjugate comprising an anti-CD33 antibody as defined herein and a drug, such as a cytotoxin or immunomodulator, conjugated to said anti-CD33 antibody (i.e., an antibody-drug conjugate). Typically, the drug is covalently linked to the antibody and is usually dependent on a linker. In some embodiments, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulator. Examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin, cis-dichlorodiamineplatin(II) (DDP), cisplatin, carboplatin, zolrubicin, doxorubicin, detoxin, carminoxetine, idarubicin, epirubicin, mitoxantrone, and actinomycin. Bleomycin D, bleomycin, salinomycin, sclerosomycin, atrazocin (AMC), vincristine, vinblastine, paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthradinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferon, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vorciclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their analogues, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony-stimulating factors (e.g., G-CSF and GM-CSF), interferons (e.g., interferon-α, interferon-β, and interferon-γ), stem cell growth factors named "S1 factor," erythropoietin, and thrombopoietin, or combinations thereof.

[0054] reagent kits and pharmaceutical compositions

[0055] In another aspect, the present invention also provides a detection kit comprising the antibodies, multispecific antibodies, antibody-drug conjugates, engineered immune cells, or chimeric antigen receptors described in the present invention.

[0056] In another aspect, the present invention also provides a pharmaceutical composition comprising the antibody, chimeric antigen receptor, multispecific antibody, engineered immune cell or antibody conjugate described herein, and one or more pharmaceutically acceptable excipients.

[0057] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adhesion agents, flow aids, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension modifiers. Those skilled in the art know how to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients used in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. Typically, the selection of a suitable excipient depends in particular on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0058] The pharmaceutical compositions according to the invention are suitable for administration via a variety of routes. Typically, administration is performed via parenteral delivery. Parenteral delivery methods include local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0059] The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly as ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for the desired method of administration. Processes known in this invention for manufacturing pharmaceuticals may include, for example, conventional mixing, dissolving, granulation, coating, grinding, emulsification, encapsulation, embedding, or lyophilization processes. Pharmaceutical compositions containing, for example, immune cells as described herein, are generally provided in solution form and preferably contain pharmaceutically acceptable buffers.

[0060] The pharmaceutical compositions according to the invention can also be administered in combination with one or more other pharmaceutical agents suitable for treating and / or preventing the disease to be treated. Preferred examples of pharmaceutical agents suitable for combination include known anticancer drugs, such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, and orlistatine E. E) vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, DNases and RNases; radionuclides, such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225 and astatine-213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. Furthermore, the pharmaceutical compositions of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

[0061] Therapeutic / Preventive / Diagnostic Uses

[0062] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing diseases associated with CD33 expression, comprising administering to a subject an antibody, chimeric peptide, multispecific antibody, antibody-drug conjugate, engineered immune cell, or pharmaceutical composition as described above.

[0063] In some implementations, diseases associated with CD33 expression include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) and solid tumors. Hematologic malignancies are cancers of the blood or bone marrow, including but not limited to acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Solid tumors include: lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, T-lymphoblastic leukemia / lymphoma (T-ALL / LBL), early pro-T lymphoblastic leukemia (ETP-ALL), extranodal NK / T-cell lymphoma, small lymphoblastic lymphoma (SLL), and spinal dysplasia. Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas; they can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, pancreatic cancer, ovarian cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small bowel cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, gastric cancer, testicular cancer, colon cancer, esophageal cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, thyroid cancer, and ureteral cancer.

[0064] In some implementations, the disease associated with CD33 expression is leukemia, preferably selected from acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphoblastic leukemia, chronic myeloid leukemia, and myelodysplastic syndrome.

[0065] In other embodiments, the disease associated with CD33 expression is lymphoma, preferably selected from multiple myeloma, non-Hodgkin lymphoma and Burkitt lymphoma.

[0066] The present invention will now be described in detail with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are merely illustrative and do not constitute any limitation on the present invention. Where there is no contradiction, the embodiments and features described in this application can be combined with each other.

[0067] The present invention will now be described in detail with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are merely illustrative and do not constitute any limitation on the present invention. Where there is no contradiction, the embodiments and features described in this application can be combined with each other. Attached Figure Description

[0068] Figure 1 Expression levels of CAR-CD33 pro binding in CAR33 CAR-T cells as determined by flow cytometry.

[0069] Figure 2 The killing effect of CAR-T cells on target cells. Two-way ANOVA was used for analysis, and the T-test was used for statistical analysis. * indicates that the P-value is less than 0.05, which is statistically significant.

[0070] Figure 3 : IL-2 (A) and IFNγ (B) release levels of CAR-T cells after co-culturing with target cells and non-target cells, respectively. Detailed Implementation

[0071] Example 1: Screening for anti-CD33 antibodies

[0072] Age-appropriate Balb / c mice were immunized with Human CD33 Protein (ACRO, catalog number: CD3-H5257), followed by repeated immunizations every 2–3 weeks for a total of 4 times. Then, mouse spleen lymphocytes were harvested and mixed with SP2 / 0 myeloma cells and electrofused to prepare hybridoma cells. Hybridoma clones binding to CD33 were screened using a CD33-overexpressing cell line (CHO-CD33 cells) via ELISA or flow cytometry. After multiple rounds of screening, one antibody clone specifically binding to CD33 was obtained. The obtained antibodies were sequenced, and the results showed that the amino acid sequence of antibody CDR-L1 is shown in SEQ ID NO:1, the amino acid sequence of CDR-L2 is shown in SEQ ID NO:2, the amino acid sequence of CDR-L3 is shown in SEQ ID NO:3, the amino acid sequence of CDR-H1 is shown in SEQ ID NO:4, the amino acid sequence of CDR-H2 is shown in SEQ ID NO:5, the amino acid sequence of CDR-H3 is shown in SEQ ID NO:6, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO:7, and the amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO:8.

[0073] Example 2: Construction of CAR T cells

[0074] The following coding sequences were synthesized and sequentially cloned into the pGEM-T Easy vector (Promega, catalog number A1360): CD8α signal peptide (SEQ ID NO: 18), anti-CD33 scFv (SEQ ID NO: 10), CD8α hinge region (SEQ ID NO: 19), CD8α transmembrane region (SEQ ID NO: 11), 4-1BB co-stimulatory domain (SEQ ID NO: 14), and CD3ζ intracellular signal transduction domain (SEQ ID NO: 15) to obtain a conventional CAR33-CAR. The correct insertion of the target sequence was confirmed by sequencing.

[0075] Add 3 ml of Opti-MEM (Gibco, catalog number 31985-070) to a sterile tube to dilute the plasmid. Then, add the packaging vector psPAX2 (Addgene, catalog number 12260) and the envelope vector pMD2.G (Addgene, catalog number 12259) in a plasmid:viral packaging vector:viral envelope vector ratio of 4:2:1. Next, add 120 μL of X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001), mix immediately, and incubate at room temperature for 15 min. Then, add the plasmid / vector / transfection reagent mixture dropwise to a culture flask of 293T cells. Collect the virus at 24 and 48 hours, combine them, and then ultracentrifuge (25000g, 4℃, 2.5 h) to obtain concentrated lentivirus.

[0076] T cells were activated using DynaBeads CD3 / CD28 CTSTM (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 1 day. Then, virus was added, and the cells were cultured for 3 days to obtain CD33-targeting CAR-T cells (CAR33 CAR-T cells).

[0077] After culturing at 37℃ and 5% CO2 for 9 days, the binding level of CAR to CD33 pro on CAR33 CAR-T cells was detected by flow cytometry using Human CD33 pro, Fc Tag (Acro, catalog number CD3-H5257) as the primary antibody. The results are as follows: Figure 1 As shown (NT is unmodified wild-type T cells).

[0078] The results show that the CAR33 CAR-T cells of the present invention can effectively express CAR33 and bind to CD33 pro.

[0079] Example 3: Killing effect of CAR T cells on target cells and cytokine release

[0080] 3.1 Killing effect of CAR-T cells on HL60 cells

[0081] When T cells kill target cells, the number of target cells decreases. When T cells are co-cultured with fluorescently labeled target cells, both the number of target cells and the fluorescence intensity decrease. Therefore, the detected fluorescence intensity can reflect the T cells' ability to kill target cells.

[0082] To test the killing ability of CAR-T cells against target cells, we first used 2x10 4 HL60 cells were seeded into 96-well plates. Then, CAR33 CAR-T cells and untransfected T cells (negative control) were seeded into 96-well plates at a 5:1 effector-to-target ratio (i.e., effector T cells to target cells) for co-culture. The culture medium was RPMI 1640 + Xvivo 15 (1:1) + 10% FBS. Fluorescence values ​​were measured using a microplate reader after 24 hours. The killing efficiency was calculated using the formula: (mean fluorescence value of target cells - mean fluorescence value of sample cells) / mean fluorescence value of target cells × 100%. The results are as follows. Figure 2 As shown.

[0083] It can be seen that, compared with the control group, CAR33 CAR-T cells have a significant killing effect on HL60 cells.

[0084] 3.2 Cytokine Release from CAR-T Cells

[0085] When T cells kill target cells, the number of target cells decreases while cytokines such as IL2 and IFN-γ are released. Following the steps below, enzyme-linked immunosorbent assay (ELISA) was used to determine the release levels of cytokines IL2 and IFN-γ during Fite-CARX T cell killing of target cells.

[0086] (1) Collect cell co-culture supernatant

[0087] With 1x10 5 Target cells (HL60) were seeded in 96-well plates, and then CAR33CAR-T and NT cells (negative control) were co-cultured with the target cells at a 1:1 ratio. The cell co-culture supernatant was collected after 18-24 hours.

[0088] (2) ELISA detection of IFNγ secretion in supernatant

[0089] The 96-well plate was coated with the capture antibody Purified anti-human IFN-γ Antibody (Biolegend, catalog number 506502) and incubated overnight at 4°C. The antibody solution was then removed, and 250 μL of PBST (containing 0.1% Tween in 1X PBS) containing 2% BSA (Sigma, catalog number V900933-1kg) was added. The plate was incubated at 37°C for 2 hours. The plate was then washed three times with 250 μL of PBST (containing 0.1% Tween in 1X PBS). 50 μL of cell co-culture supernatant or standards was added to each well, and the plate was incubated at 37°C for 1 hour. The plate was then washed three times with 250 μL of PBST (containing 0.1% Tween in 1X PBS). Then, add 50 μL of the detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, catalog number ab25017) to each well, incubate at 37°C for 1 hour, and wash the plate three times with 250 μL of PBST (containing 0.1% Tween in 1X PBS). Add HRP Streptavidin (Biolegend, catalog number 405210), incubate at 37°C for 30 minutes, discard the supernatant, add 250 μL of PBST (containing 0.1% Tween in 1X PBS), and wash five times. Add 50 μL of TMB substrate solution to each well. Allow the reaction to proceed in the dark at room temperature for 30 minutes, then add 50 μL of 1 mol / L H2SO4 to each well to stop the reaction. Within 30 minutes of stopping the reaction, measure the absorbance at 450 nm using a microplate reader, and calculate the cytokine levels according to the standard curve (plotted based on the readings and concentrations of the standards). The results are as follows: Figure 3 As shown.

[0090] It can be seen that, when killing target cells, the release levels of IL2 and IFN-γ from CAR33 CAR-T cells were significantly higher than those from NT cells. In summary, the CAR33 CAR-T cells in this invention released significantly more cytokines from HL60 cells than from NT cells.

[0091] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. It is understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An antibody targeting CD33, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3, and the heavy chain variable region comprises CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO:

6.

2. The antibody of claim 1, wherein the light chain variable region has at least 90% identity with the amino acid sequence selected from SEQ ID NO: 7, and the heavy chain variable region has at least 90% identity with the amino acid sequence selected from SEQ ID NO:

8.

3. The antibody of claim 1, wherein the antibody comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO: 9 or 10.

4. The antibody of claim 1, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

5. The antibody according to any one of claims 1-4, wherein the antibody is selected from IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, linear antibodies and dimers.

6. A nucleic acid molecule encoding the antibody according to any one of claims 1-5.

7. A multispecific antibody comprising the antibody according to any one of claims 1-5 and one or more second antibodies or their antigen-binding portions specifically binding to other antigens or antigenic epitopes.

8. The multispecific antibody of claim 7, wherein the second antibody or its antigen-binding portion is selected from full-length antibodies, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, microantibodies, biantibodies, or sdAb.

9. A vector comprising a nucleic acid molecule encoding an antibody according to any one of claims 1-5 or a multispecific antibody according to claim 7 or 8.

10. A host cell expressing the antibody according to any one of claims 1-5 or the multispecific antibody according to claim 7 or 8.

11. A chimeric polypeptide comprising the antibody of any one of claims 1-5 or the multispecific antibody of claim 7 or 8, wherein the chimeric polypeptide is selected from recombinant T-cell receptors, chimeric antigen receptors, T-cell fusion proteins, and T-cell antigen couplers.

12. The chimeric polypeptide of claim 11, wherein the chimeric polypeptide is selected from chimeric antigen receptors, further comprising a transmembrane domain and a primary signal transduction domain, and optionally, further comprising a co-stimulatory domain.

13. The chimeric polypeptide of claim 12, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

14. The chimeric polypeptide of claim 12, wherein the primary signal transduction domain is selected from the intracellular regions of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD7b and CD66d.

15. The chimeric polypeptide of claim 12, wherein the co-stimulatory domain is selected from the co-stimulatory signal transduction domains of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, B7-H3, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70.

16. An engineered immune cell comprising the chimeric polypeptide according to any one of claims 11-15.

17. The engineered immune cell of claim 16, wherein the immune cell is selected from T cells, NK cells, NKT cells, monocytes, neutrophils, macrophages, and dendritic cells.

18. The engineered immune cells of claim 16, wherein the immune cells are selected from CD4+. + CD8 + T cells, CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells, tumor-infiltrating cells, memory T cells, naive T cells, γδ-T cells, and αβ-T cells.

19. The engineered immune cell of claim 16, further comprising a second chimeric polypeptide targeting one or more other antigens or antigenic epitopes.

20. The engineered immune cell according to any one of claims 16-19, further comprising at least one gene selected from the group whose expression is suppressed or silenced: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3 and CTLA4.

21. An antibody-drug conjugate comprising the antibody of any one of claims 1-5 or the multispecific antibody of claim 7 or 8, and a second functional structure, wherein the second functional structure is selected from Fc, radioisotopes, structural portions with extended half-life, detectable markers, and drugs.

22. The antibody-drug conjugate of claim 21, wherein the structural portion for extending the half-life is selected from: albumin-binding structures, transferrin-binding structures, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin, and albumin polypeptides bound to human serum albumin; the detectable marker is selected from fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents; and the drug is selected from cytotoxins and immunomodulators.

23. A detection kit comprising the antibody of any one of claims 1-5, the multispecific antibody of claim 7 or 8, the chimeric polypeptide of any one of claims 11-15, the engineered immune cells of any one of claims 16-20, or the antibody conjugate of claim 21 or 22.

24. A pharmaceutical composition comprising an antibody according to any one of claims 1-5, a multispecific antibody according to claim 7 or 8, a chimeric polypeptide according to any one of claims 11-15, an engineered immune cell according to any one of claims 16-20, or an antibody conjugate according to claim 21 or 22, and one or more pharmaceutically acceptable excipients.

25. Use of the antibody of any one of claims 1-5, the multispecific antibody of claim 7 or 8, the chimeric polypeptide of any one of claims 11-15, the engineered immune cell of any one of claims 16-20, the antibody-drug conjugate of claim 21 or 22, the detection kit of claim 23, or the pharmaceutical composition of claim 24 in the preparation of a medicament for the treatment and / or prevention and / or diagnosis of diseases related to CD33 expression.

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