Antibody targeting CD33 and use thereof

Abstract

Provided are an antibody targeting CD33, and a multispecific antibody, chimeric polypeptide, antibody conjugate, pharmaceutical composition and kit containing same, and the use thereof in the diagnosis, treatment and prevention of diseases associated with CD33 expression.

Description

Antibodies targeting CD33 and uses thereof

[0001] Cross-reference to related applications

[0002] The present application claims priority to the Chinese patent application No.CN202411162527.4, filed on August 23, 2024, and entitled“Antibodies targeting CD33 and uses thereof”, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present application belongs to the field of immunotherapy. More specifically, the present application relates to antibodies targeting CD33, and uses thereof in the prevention, treatment and diagnosis of diseases. BACKGROUND

[0004] CD33 (Sialic acid-binding Ig-like lectin 3, Siglec-3) is a transmembrane glycoprotein on the surface of myeloid cells, which is widely expressed in subsets of myeloid cells such as monocytes, macrophages and granulocytes. CD33 is highly expressed in a variety of myeloid malignancies, especially in acute myeloid leukemia (AML), and thus becomes an important target for targeted therapy. In addition, CD33 is also associated with other hematological diseases and certain inflammatory diseases, such as myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and certain autoimmune diseases. In recent years, antibodies, antibody-drug conjugates and cell therapy products targeting CD33 have shown potential application value in the treatment of these diseases.

[0005] However, existing CD33-targeted therapies still face many challenges due to problems such as immunogenicity, targeting, drug resistance and safety. The present application aims to provide an antibody targeting CD33, and uses thereof in the prevention, treatment and diagnosis of diseases. SUMMARY

[0006] In a first aspect, the present application 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, 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, CDR-H3 as shown in SEQ ID NO: 6.

[0007] In some embodiments, the light chain variable region has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to an 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%, or 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO: 8. Preferably, the CD33 antibody comprises a light chain variable region selected from SEQ ID NO: 7 and a heavy chain variable region selected from SEQ ID NO: 8.

[0008] 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 to an amino acid sequence set forth in SEQ ID NO: 9 or 10. Preferably, the antibody comprises an amino acid sequence as set forth in SEQ ID NO: 9 or 10.

[0009] In some embodiments, the antibody of the application is a murine, chimeric, humanized or human antibody.

[0010] In one aspect, the application also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising an anti-CD33 antibody or antigen binding fragment thereof as described above, which further comprises one or more second antibodies that specifically bind to other antigens or antigen epitopes.

[0011] As used herein, the term "multispecific" refers to an antigen binding protein having polyepitopic specificity (i.e., capable of specifically binding to two, three or more different epitopes on one biological molecule or capable of specifically binding to epitopes on two, three or more different biological molecules). As used herein, the term "bispecific" denotes an antigen binding protein having two different antigen binding specificities.

[0012] In some embodiments, the second antibody can be in any antibody or antibody fragment format, such as a full-length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, minibody, diabody, or sdAb.

[0013] Thus, in some embodiments, the second antibody targets an antigen selected from the group consisting of 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, Claudin 18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2 ETS fusion gene, ETV6-AML, EMR2, EGP2, FAP, FcRH5, FCRL5, FCRH2, FCRH1, FCRH4, FCRH5, FCRH6, FCRH7, FCRH10, FCRH11, FCRH12, FCRH3, FCRH8, FCRH9, FCRH4, FCRH5, FCRH6, FCRH7, FCRH10, FCRH11, FCRH12, FCRH3, FCRH8, FCRH9, FOLR1, FcRH2, FcRH5, FcRH1, FcRH4, FcRH5, FcRH6, FcRH7, FcRH10, FcRH11, FcRH12, FcRH3, FcRH8, FcRH9, FcRH4, FcRH5, FcRH6, FcRH7, FcRH10, FcRH11, FcRH12, FcRH3, FcRH8, FcRH9, FcRH4, FcRH5, FcRH6, FcRH7, FcRH10, FcRH11, FcRH12, FcRH3, FcRH8, FcRH9, FcRH4, FcRH5, FcRH6, FcRH7, FcRH10, FcRH11, FcRH12, FcRH3, FEGP40, 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-1 Gag, HLA-A1, HLA-A2, IL6R, IL-11 Ra, IL-13 Ra, 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-beta, PCTA-1 / Galectin 8, p53, p53 mutants, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutants, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp 17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCR alpha, TCR beta, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2 VEGFR, WT1, XAGE1, 5T4, 8H9, alpha v beta 6 integrin, CA9, folate receptor alpha, ephrinB2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor beta, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase reverse transcriptase / hTERT, androgen receptor, intestinal carboxyl esterase, cyclin B1, fibronectin, tenascin, carcinoembryonic variant of the tumor necrosis zone, 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 alpha, CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, and any combination thereof, more preferably from CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, BCMA, and any combination thereof.

[0014] In another embodiment, the second antibody targets an additional antigenic epitope of CD33.

[0015] The term "heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation, and generally determines the class of the antibody. The term "light chain" refers to the smaller of the two types of polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa (K) and lambda (l) light chains refer to the two major antibody light chain isotypes.

[0016] The term "complementarity determining region" or "CDR" refers to amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity. For example, generally, there are three CDRs in each heavy chain variable region (e.g., CDR1-H, CDR2-H, and CDR3-H), and three CDRs in each light chain variable region (CDR1-L, CDR2-L, and CDR3-L). The precise amino acid sequence boundaries of CDRs can be determined using any of a number of well-known schemes, including: the Kabat numbering scheme, the Chothia numbering scheme, the IMGT numbering scheme, the AHo numbering scheme, the AbM numbering scheme. The precise amino acid sequence of a given CDR or FR can differ depending on the numbering scheme chosen, it is understood that "CDRs" or "FRs" of a given antibody or region thereof (such as a variable region thereof) encompass CDRs or FRs defined by any of the above schemes or other known schemes, and where a given CDR or FR contains a given amino acid sequence, it is understood that such CDR or FR can also have the sequence of the corresponding CDR or FR defined by any of the above schemes or other known schemes. The numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.

[0017] 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 contiguous (e.g. connected via a linker) and capable of being expressed in a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv herein can have the VL and VH in any order (e.g. with respect to the N-terminus and C-terminus of the polypeptide), the scFv can comprise VL-linker-VH from N- to C-terminus, or it can comprise VH-linker-VL. The term "linker" refers to a sequence of molecules that connects 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 not antigenic and does not elicit an immune response. In some embodiments, the linker can 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 linker has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22 or 23.

[0018] As used herein, the term "sequence identity" indicates the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of the same sequence have 100% identity. The skilled person is aware 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 ClustalW.

[0019] Nucleic acid, vector, host cell

[0020] In another aspect, the present application relates to nucleic acid molecules encoding the anti-CD33 antibodies or multispecific antibodies of the present application. The nucleic acids of the present application can be RNA, DNA or cDNA.

[0021] The nucleic acids of the application can also be in the form of, can be present in, and / or can be part of a vector, such as a plasmid, cosmid, or YAC. The vector can be, inter alia, an expression vector, i.e., a vector that can provide for expression of the anti-CD33 antibody in vitro and / or in vivo, i.e., in a suitable host cell, host organism, and / or expression system. The expression vector typically comprises at least one nucleic acid molecule of the application operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). Selection of the control elements and their sequences for expression in a particular host is well known to those skilled in the art. Particular examples of control elements and other elements useful or necessary for expression of the anti-CD33 antibody of the application include, but are not limited to, promoters, enhancers, terminators, integration factors, selectable markers, leader sequences, reporter genes.

[0022] In another aspect, the application also provides host cells expressing the anti-CD33 antibodies, multispecific antibodies of the application and / or containing the nucleic acids or vectors of the application. Preferred host cells of the application are bacterial cells, fungal cells, or mammalian cells.

[0023] Suitable bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0024] Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus; or of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0025] Suitable mammalian cells include, e.g., HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0026] However, the present application can also use amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing heterologous proteins.

[0027] Chimeric polypeptides

[0028] In another aspect, the present application also provides a chimeric polypeptide comprising an anti-CD33 antibody as described above, such as a recombinant T cell receptor, a chimeric antigen receptor, a T cell fusion protein, or a T cell antigen coupler, preferably a chimeric antigen receptor.

[0029] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen (e.g., tumor antigen) binding domain (e.g., an antibody or a ligand for an antigen), a transmembrane domain, and a primary signaling domain, optionally, further including a costimulatory domain. The various domains are connected by linkers. CARs are capable of redirecting the specificity and reactivity of 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 application is a chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain, and a primary signaling domain, optionally, further comprising one or more costimulatory 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.

[0030] As used herein, the term "T cell fusion protein" or "TFP" refers to a recombinant polypeptide derived from each component of TCR, usually consisting of TCR subunit and antibody connected therewith and expressed on cell surface. Among them, the TCR subunit includes at least part of TCR extracellular domain, transmembrane domain, TCR intracellular signal domain.

[0031] As used herein, the term "T cell antigen coupler" or "TAC" includes three functional domains: a tumor targeting domain, including a single-chain antibody (e.g., an anti-FasL antibody, an anti-IRP60 antibody, or an anti-SIRPα antibody of the present application), 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; a transmembrane region and an intracellular region of CD4 co-receptor, wherein the intracellular region connects protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motif (ITAM) of TCR complex as the initial step of T cell activation.

[0032] As used herein, the term "T cell receptor" or "TCR" is a characteristic marker of the surface of T cells that forms a complex with CD3 in a non-covalent bond. Antigen presenting cells present an antigen peptide to T cells through major histocompatibility complex molecules (MHC) and bind to the TCR complex to induce a series of intracellular signaling. The TCR consists of six peptide chains that form heterodimers, respectively, which are generally divided into αβ type and γδ type. Each peptide chain includes a constant region and a variable region, in which the variable region is responsible for binding specific antigen and MHC molecules. The term "recombinant TCR receptor" refers to a T cell receptor artificially constructed further comprising an antigen (e.g., tumor antigen) binding domain.

[0033] In some embodiments, the present application 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 signaling domain.

[0034] As used herein, the term "transmembrane domain" refers to a polypeptide structure capable of expressing a chimeric antigen receptor on the surface of an immune cell (e.g., a T cell, an NK cell, or an NKT cell) and directing a cellular response of the immune cell against a target cell. The transmembrane domain can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to a target antigen. A transmembrane domain particularly suitable for use in the present application can be derived from, for example, a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, a CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and functional fragments thereof. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from a CD8 alpha chain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0035] As used herein, the term "intracellular signaling domain" refers to the portion of a protein that transduces a signal of an effector function and directs the cell to perform the specified function. In some embodiments, the intracellular signaling domain comprised by the chimeric antigen receptors of the present application can be the intracellular region sequences of T cell receptors and co-receptors that act in concert to initiate signaling upon antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence having the same or similar function. The intracellular signaling domain can comprise a number of immunoreceptor tyrosine-based activation motifs (ITAMs). Non-limiting examples of intracellular signaling domains of the present application include, but are not limited to, the intracellular regions of FcRy, FcRP, CD3y, CD3d, CD3s, CD3z, CD22, CD79a, CD79b, and CD66d, among others. In preferred embodiments, the signaling domain of the CARs of the present application can comprise a CD3z intracellular region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15 or 16.

[0036] In some embodiments, the chimeric antigen receptors of the present application can further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligo- or polypeptide that functions to link the transmembrane domain to the antibody. In particular, the hinge region serves to provide greater flexibility and accessibility to the antibody. The hinge region can comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region can be derived in whole or in part from a natural molecule, such as the extracellular region of CD8, CD4, or CD28, in whole or in part, or from an antibody constant region, in whole or in part. Alternatively, the hinge region can be a synthetic sequence corresponding to a naturally occurring hinge sequence, or can be a completely synthetic hinge sequence. In preferred embodiments, the hinge region comprises a hinge region portion of CD8a, CD28, FcyRIIIa receptor, IgG4, or IgGl, more preferably a CD8a, CD28, or IgG4 hinge having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 19-21.

[0037] In some embodiments, the chimeric antigen receptor can further comprise one or more costimulatory domains. A costimulatory domain can be an intracellular functional signaling domain from a costimulatory molecule, which comprises the entire intracellular portion of the costimulatory molecule or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds with a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of costimulatory domains of the present application include, but are not limited to, costimulatory signaling 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 costimulatory domain of the CAR of the present application is from 4-1BB and / or CD28. In some embodiments, the CD28 costimulatory domain 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 set forth in SEQ ID NO: 13. In some embodiments, the 4-1BB costimulatory domain 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 set forth in SEQ ID NO: 14.

[0038] In some embodiments, the CAR of the application can also comprise a signal peptide, such that when it is expressed in a cell, e.g., a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of a signal peptide can contain a long stretch of hydrophobic amino acids with a tendency to form a single a-helix. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptidase can cleave during or after translocation to produce a free signal peptide and a mature protein. The free signal peptide is then digested by specific proteases. Signal peptides useful in the application are well known to those skilled in the art, e.g., signal peptides derived from B2M, CD8a, IgGl, GM-CSFRa, etc. In some embodiments, the signal peptide useful in the application is from B2M, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the signal peptide useful in the application is from CD8a, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18.

[0039] In some embodiments, the CAR contains an anti-CD33 antibody or antigen binding fragment thereof as provided herein or a multispecific antibody containing the anti-CD33 antibody, a CD8a or CD28 transmembrane region, a CD28 and / or 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain. In this embodiment, the CAR can further comprise a signal peptide from B2M, CD8a, IgGl, or GM-CSFRa.

[0040] The application also provides nucleic acid molecules encoding a chimeric antigen receptor targeting CD33 as defined above, as well as vectors comprising said nucleic acid molecules.

[0041] As used herein, the term "vector" is a nucleic acid molecule that serves as a vehicle for the transfer of (exogenous) genetic material into a host cell, where it can, for example, replicate and / or be expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a vehicle for delivery of an isolated nucleic acid to the interior of a cell, by, for example, homologous recombination or use of a hybrid recombinase enzyme that specifically targets sequences at a site. An "expression vector" is a vehicle for the transcription of a heterologous nucleic acid sequence (such as those encoding the chimeric antigen receptor polypeptides of the application) in a suitable host cell, and the translation of their mRNA. Suitable vectors useful in the application are known in the art, and many are commercially available. In some embodiments, vectors of the application include, but are not limited to, plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc.), bacteriophages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). Vectors themselves are generally nucleic acid molecules, typically DNA sequences comprising an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. Engineered vectors often also contain an origin of replication for autonomous replication in a host cell (if stable expression of the polynucleotide is desired), a selection marker, and restriction enzyme cleavage sites (such as a multiple cloning site, MCS). Vectors can additionally contain elements such as promoters, polyadenylation tails (polyA), 3'UTRs, enhancers, terminators, insulators, operators, selection markers, reporter genes, targeting sequences, and / or protein purification tags. In one particular embodiment, the vector is an in vitro transcribed vector.

[0042] Engineered immune cells

[0043] In one aspect, the application also provides engineered immune cells expressing the chimeric polypeptides described herein, such as recombinant TCR receptors or chimeric antigen receptors.

[0044] As used herein, the term "immune cell" refers to any cell of the immune system having one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). For example, an immune cell can be a T cell, a macrophage, a neutrophil, a dendritic cell, a monocyte, an NK cell, and / or an NKT cell. In some embodiments, an immune cell is derived from a stem cell, such as an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, an induced pluripotent stem cell, a totipotent stem cell, or a hematopoietic stem cell, among others. Preferably, the immune cell is a T cell. The T cell can be any T cell, such as a T cell cultured in vitro, e.g., a primary T cell, or a T cell from a T cell line cultured in vitro, e.g., Jurkat, SupTl, and the like, or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. The T cell can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. The T cell can also be concentrated or purified. The T cell can be at any stage of development, including but not limited to, CD4+CD8+T cells, CD4+T cells (e.g., Thl and Th2 cells), CD8+T cells (e.g., cytotoxic T cells), CD4 - CD8 - T cells, tumor infiltrating cells, memory T cells, naive T cells, gamma delta-T cells, alpha beta-T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. The T cell can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.

[0045] In some embodiments, to reduce the risk of graft versus host disease, the engineered immune cell further comprises at least one gene selected from the group consisting of CD52, GR, dCK, TCR / CD3 genes (e.g. TRAC, TRBC, CD3y, CD35, CD3s, CD3z), 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, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, 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 cell further comprises at least one gene selected from the group consisting of 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.

[0046] Methods of inhibiting gene expression or silencing a gene are well known to those skilled in the art. For example, antisense RNA, RNA decoy, RNA aptamer, siRNA, shRNA / miRNA, trans dominant negative protein (TNP), chimeric / antibody conjugate, chemokine ligand, anti-infectious cellular protein, intracellular antibody (sFv), nucleoside analog (NRTI), non-nucleoside analog (NNRTI), integrase inhibitor (oligonucleotide, dinucleotide, and chemical agent), and protease inhibitor can be used to inhibit the expression of a gene. In addition, a gene can also be silenced by, for example, DNA breakage mediated by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system.

[0047] In some embodiments, the engineered immune cell further comprises a second chimeric polypeptide targeting other antigens or antigen epitopes. The other antigens targeted by the second chimeric polypeptide can 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, CD138, CD151, CD171, CD179a, CD300LF, CDH16, CSPG4, CS1, Claudin 6, Claudin 18.1, Claudin 18.2, CEA, CEACAM6, CLL1, c-Met, CAIX, CXORF61, CA125, CYP1B1, CS1, ELF2M, EGFR, EPCAM, EGFRvIII, EphA2, ERG / TMPRSS2 ETS fusion gene, ETV6-AML, EMR2, EGP2, FAP, FcRH5, FCRL5, FCRH2, FCRH1, FCRH3, FCRH4, FCRH5, FCRH6, FCRH7, FCRH8, FCRH9, FCRH10, FCRH11, FCRH12, FCRH13, FCRH14, FCRH15, FCRH16, FCRH17, FCRH18, FCRH19, FCRH20, FCRH21, FCRH22, FCRH23, FCRH24, FCRH25, FCRH26, FCRH27, FCRH28, FCRH29, FCRH30, FCRH31, FCRH32, FCRH33, FCRH34, FCRH35, FCRH36, FCRH37, FCRH38, FCRH39, FCRH40, FCRH41, FCRH42, FCRH43, FCRH44, FCRH45, FCRH46, FCRH47, FCRH48, FCRH49, FCRH50, FCRH51, FCRH52, FCRH53, FCRH54, FCRH55, FCRH56, FCRH57, FCRH58, FCRH59, FCRH60, FCRH61, FCRH62, FCRH63, FCRH64, FCRH65, FCRH66, FCRH67, FCRH68, FCRH69, FCRH70, FCRH71, FCREGP40, 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-1 Gag, HLA-A1, HLA-A2, IL6R, IL-11 Ra, IL-13 Ra, 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-beta, PCTA-1 / Galectin 8, p53, p53 mutants, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutants, RhoC, RU1, RU2, Robol, SSEA-4, SSX2, SART3, Sp 17, TSHR, Tn Ag, TGS5, TEM1 / CD248, TEM7R, TARP, TCR alpha, TCR beta, TGFBR1, TGFBR2, TNFRSF4, TWEAK-R, TLR7, TLR9, TAG72, TROP-2, Tie 2, TRP-2, TNFR1, TNFR2, TEM1, UPK2 VEGFR, WT1, XAGE1, 5T4, 8H9, alpha v beta 6 integrin, CA9, folate receptor alpha, ephrinB2, tyrosinase, fucosyl GM1, o-acetyl-GD2, folate receptor beta, polysialic acid, sperm protein 17, survivin and telomerase, sarcoma translocation breakpoints, human telomerase reverse transcriptase / hTERT, androgen receptor, intestinal carboxyl esterase, cyclin B1, fibronectin, tenascin, carcinoembryonic variant of the tumor necrosis zone, and any combination thereof. Preferably,The second antigen is selected from the group consisting of CD7, CD19, CD20, CD22, CD30, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor alpha, 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 the group consisting of CD7, CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, BCMA, and any combination thereof.

[0048] In some embodiments, a plurality of immune cells is provided, each of which is 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 and / or targets CD33 (e.g., a CAR comprising an anti-CD33 antibody described herein), and another cell is engineered to express a chimeric antigen receptor that binds and / or targets another antigen or antigenic epitope. In some embodiments, an immune cell can also express a multi-specific chimeric antigen receptor that targets one or more antigens, including CD33. For example, such a multi-specific chimeric antigen receptor can comprise a multi-specific antibody that targets CD33, or both an anti-CD33 antibody described herein and an antibody that targets another antigen or antigenic epitope. In such embodiments, the plurality of engineered immune cells can be administered together or separately. In some embodiments, the plurality of immune cells can be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.

[0049] Antibody conjugates

[0050] In one aspect, the present application provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a second functional structure, wherein the second functional structure is selected from the group consisting of an Fc, a radioisotope, a half-life extending moiety, a detectable label, and a drug.

[0051] In some embodiments, the present application provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and an Fc. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen binding fragment produced by digestion of an intact antibody, whether in monomeric form or in multimeric form. The source of the immunoglobulin from which the native Fc is produced is preferably derived from a human. The native Fc fragment is composed of monomeric polypeptides that can be linked into dimeric or multimeric form by covalent (e.g., disulfide bonds) and noncovalent linkages. Depending on the class (e.g., IgG, IgA, IgE, IgD, IgM) or subclass (e.g., IgGl, IgG2, IgG3, IgAl, IgGA2) of the antibody, there are 1-4 intermolecular disulfide bonds between the monomeric subunits of the native Fc molecule. One example of a native Fc is the disulfide-linked dimer produced by digestion of IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). The term "native Fc" as used herein refers generally to monomeric, dimeric, and multimeric forms. "Variant Fc" refers to an amino acid sequence that differs from the amino acid sequence of a "native" or "wild type" Fc due to at least one "amino acid modification" as defined herein, also referred to as "Fc variant". Thus, "Fc" also includes single chain Fc (scFc), i.e., a single chain Fc composed of two Fc monomers connected by a polypeptide linker, which is capable of folding into a functional dimeric Fc region naturally. In some embodiments, the Fc is preferably an Fc of a human immunoglobulin, more preferably an Fc of human IgGl.

[0052] In some embodiments, the present application provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a radioisotope. Examples of radioisotopes useful in the present application 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.

[0053] In some embodiments, the present application provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a half-life extending moiety selected from the group consisting of a binding structure of albumin, a binding structure of transferrin, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and a white polypeptide (including an antibody) that binds human serum albumin.

[0054] In some embodiments, the present application provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a detectable label. The term "detectable label" means herein a compound that generates a detectable signal. For example, the detectable label can be an MRI contrast agent, a scintigraphic contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent. Examples of detectable labels include fluorophores (such as fluorescein, Alexa or cyanine), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as horseradish peroxidase, glucose-6-phosphatase, beta-galactosidase), antibiotic (e.g. kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes and contrast agents (such as nanoparticles or gadolinium). The skilled person can select a suitable detectable label depending on the detection system used.

[0055] In some embodiments, the present application provides an antibody conjugate comprising an anti-CD33 antibody as defined herein and a drug, e.g., a cytotoxin or an immunomodulatory agent, coupled to the anti-CD33 antibody (i.e., an antibody drug conjugate). Typically the drug is linked to the antibody covalently, and often depends on a linker. In some embodiments, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulatory agent. Examples of cytotoxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-fluorouracil, dacarbazine, mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methyl- l-nitrosourea, cyclophosphamide, nitrogen mustards, busulfan, decarbazine, cis-dichlorodiamine platinum (II) (DDP), cisplatin, carboplatin, dactinomycin, doxorubicin, detorubicin, daunomycin, daunorubicin, idarubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, calicheamicin, duocarmycin, anthramycin (AMC), vincristine, vinblastine, paclitaxel, ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, teniposide, colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (o, p'-(DDD)), interferons, and combinations thereof. Examples of immunomodulatory agents include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and analogs thereof, cytokines, stem cell growth factors, lymphotoxin, 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-alpha, interferon-beta, and interferon-gamma), stem cell growth factor named "S1 factor", erythropoietin, and thrombopoietin, or combinations thereof.

[0056] Kits and pharmaceutical compositions

[0057] In another aspect, the present application also provides a test kit comprising an antibody, multispecific antibody, antibody conjugate, engineered immune cell, or chimeric antigen receptor as described herein.

[0058] In another aspect, the present application also provides a pharmaceutical composition comprising an antibody, chimeric antigen receptor, multispecific antibody, engineered immune cell, or antibody conjugate as described herein, and one or more pharmaceutically acceptable excipients.

[0059] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is compatible, in pharmacology and / or physiology, with the subject and the active ingredient (i.e., capable of producing the desired therapeutic effect without causing any undesirable local or systemic effects) and is well known in the art (see, e.g., 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, coating agents, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, cosolvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, encapsulating agents, isotonic agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of a suitable excipient to prepare the desired pharmaceutical composition of the present application is known to one skilled in the art. Exemplary excipients for use in the pharmaceutical compositions of the present application include saline, buffered saline, dextrose, and water. Generally, the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0060] The pharmaceutical compositions according to the present application can be suitable for administration by a variety of routes. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0061] The pharmaceutical compositions according to the present application can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or fluidextracts, or in a form particularly suitable for the desired method of administration. Processes known to the present application for the production of medicaments can include, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions comprising, for example, the immune cells described herein are typically provided in solution form, and preferably comprise a pharmaceutically acceptable buffer.

[0062] The pharmaceutical composition according to the present application can also be administered in combination with one or more other agents useful in the treatment and / or prevention of the disease to be treated. Preferred examples of 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, auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; 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 stimulatory protein and the like; 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. In addition, the pharmaceutical composition of the present application can also be used in combination with other therapeutic method(s) such as chemotherapy, radiotherapy.

[0063] Therapeutic / Prophylactic / Diagnostic Uses

[0064] In another aspect, the present application also provides a method of treating and / or preventing and / or diagnosing a disease associated with CD33 expression, comprising administering to a subject an antibody, a chimeric polypeptide, a multispecific antibody, an antibody conjugate, an engineered immune cell or a pharmaceutical composition as described above.

[0065] In some embodiments, the disease associated with CD33 expression includes non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas) and solid tumors. Hematological tumors are cancers of the blood or bone marrow, including but not limited to acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt's lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, T lymphoblastic leukemia / lymphoma (T-ALL / LBL), early T-precursor lymphoblastic leukemia (ETP-ALL), extranodal NK / T cell lymphoma, small lymphocytic lymphoma (SLL), and myelodysplasia. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas, which can be benign or malignant. Different types of solid tumors are named for the type of cell that forms 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, renal cancer, skin cancer, small bowel cancer, melanoma, renal cancer, laryngeal cancer, soft tissue cancer, stomach 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, neck cancer, gallbladder cancer, pleural cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, thyroid cancer, and ureteral cancer.

[0066] In some embodiments, the disease associated with CD33 expression is a leukemia, preferably selected from acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myelodysplastic syndrome.

[0067] In other embodiments, the disease associated with CD33 expression is a lymphoma, preferably selected from multiple myeloma, non-Hodgkin's lymphoma, and Burkitt's lymphoma.

[0068] The present application will be described in detail below with reference to the attached drawings and examples. It is to be understood that the drawings and examples of the present application are only for the purpose of illustration and are not to be construed as limiting the present application in any way. The examples and features of the examples in the present application can be combined with each other without contradiction, if necessary.

[0069] The present application will be described in detail below with reference to the accompanying drawings and examples. It should be noted that those skilled in the art should understand that the accompanying drawings and examples of the present application are only for illustration and cannot constitute any limitation on the present application. The examples in the present application and the features in the examples can be combined with each other without contradiction. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1: Expression level of CAR33 CAR-T cell CAR binding to CD33 pro determined by flow cytometry.

[0071] Figure 2: Killing effect of CAR-T cells on target cells. Analyzed by Two-way ANOVA and statistically analyzed by T test. * indicates P value less than 0.05, reaching a significant level.

[0072] Figure 3: IL-2 (A) and IFNγ (B) release levels after co-culture of CAR-T cells with target cells and non-target cells, respectively. DETAILED DESCRIPTION

[0073] Example 1: Screening of anti-CD33 antibodies

[0074] Human CD33 Protein protein (ACRO, item number: CD3-H5257) was used to immunize suitable age Balb / c mice, and then repeated immunization injection every 2-3 weeks, a total of 4 times. Then, the mouse spleen lymphocytes were mixed with SP2 / 0 myeloma cells and electrofused to prepare hybridoma cells. CD33 overexpressing cell line (CHO-CD33 cells) was used to screen hybridoma clones binding to CD33 by ELISA or flow cytometry. After several rounds of screening, one antibody clone that can specifically bind to CD33 was obtained. Sequencing of the obtained antibody showed that the amino acid sequence of CDR-L1 was as shown in SEQ ID NO: 1, the amino acid sequence of CDR-L2 was as shown in SEQ ID NO: 2, the amino acid sequence of CDR-L3 was as shown in SEQ ID NO: 3, the amino acid sequence of CDR-H1 was as shown in SEQ ID NO: 4, the amino acid sequence of CDR-H2 was as shown in SEQ ID NO: 5, the amino acid sequence of CDR-H3 was as shown in SEQ ID NO: 6, the amino acid sequence of light chain variable region VL was as shown in SEQ ID NO: 7, and the amino acid sequence of heavy chain variable region VH was as shown in SEQ ID NO: 8.

[0075] Example 2: Construction of CAR T cells

[0076] The following coding sequences were synthesized and cloned into pGEM-T Easy vector (Promega, Cat. No. A1360) in turn: CD8a signal peptide (SEQ ID NO: 18), anti-CD33 scFv (SEQ ID NO: 10), CD8a hinge region (SEQ ID NO: 19), CD8a transmembrane region (SEQ ID NO: 11), 4-1BB costimulatory domain (SEQ ID NO: 14), CD3 zeta intracellular signaling domain (SEQ ID NO: 15), to obtain a conventional CAR33-CAR, and the correct insertion of the target sequence was confirmed by sequencing.

[0077] After diluting the above plasmid with 3ml Opti-MEM (Gibco, Cat. No. 31985-070) in a sterile tube, add packaging vector psPAX2 (Addgene, Cat. No. 12260) and envelope vector pMD2.G (Addgene, Cat. No. 12259) according to the ratio of plasmid: virus packaging vector: virus envelope vector = 4:2:1. Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15min, then add the plasmid / vector / transfection reagent mixture dropwise into the culture bottle of 293T cells. Collect the virus at 24 hours and 48 hours, combine them, and obtain concentrated lentivirus by ultracentrifugation (25000g, 4℃, 2.5 hours).

[0078] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured at 37℃ and 5% CO2 for 1 day. Then, add the virus, and after 3 days of continuous culture, obtain CAR-T cells targeting CD33 (CAR33 CAR-T cells).

[0079] After 9 days of culture at 37℃ and 5% CO2, the binding level of CAR on CAR33 CAR-T cells to CD33 pro was detected by flow cytometry using Human CD33 pro, Fc Tag (Acro, Cat. No. CD3-H5257) as the primary antibody, and the results are shown in Figure 1 (NT is unmodified wild-type T cells).

[0080] As can be seen from the results, the CAR33 CAR-T cells of the application can effectively express CAR33 and bind to CD33 pro.

[0081] Example 3: Killing effect of CAR T cells on target cells and cytokine release

[0082] 3.1 CAR-T cell killing effect on HL60 cells

[0083] When T cells kill target cells, the number of target cells will decrease. After co-culturing T cells and target cells with fluorescent markers, the number of target cells decreases, and the fluorescence intensity also decreases. Therefore, the detected fluorescence intensity can reflect the killing ability of T cells on target cells.

[0084] To detect the killing ability of CAR-T cells on target cells, first, HL60 cells were plated in a 96-well plate at 2x10 4 cells / well, and then CAR33 CAR-T cells and untransfected T cells (negative control) were co-cultured in a 96-well plate at an effector-to-target ratio of 5:1 (i.e., the ratio of effector T cells to target cells), and the culture medium was RPMI1640+Xvivo15 (1:1)+10% FBS. After 24 hours, the fluorescence value was measured using a microplate reader. According to the calculation formula: (target cell fluorescence mean value-sample fluorescence mean value) / target cell fluorescence mean value x 100%, the killing efficiency was calculated, and the results are shown in Figure 2.

[0085] It can be seen that, compared with the control group, CAR33 CAR-T cells have a significant killing effect on HL60 cells.

[0086] 3.2 Cytokine release of CAR-T cells

[0087] When T cells kill target cells, the number of target cells decreases, and the target cells also release cytokines such as IL2 and IFN-γ. According to the following steps, enzyme-linked immunosorbent assay (ELISA) was used to determine the release level of cytokines IL2 and IFNγ when Fite-CARX T cells kill target cells.

[0088] (1) Collecting cell co-culture supernatant

[0089] Target cells (HL60) were plated in a 96-well plate at 1x10 5 cells / well, and then CAR33 CAR-T and NT cells (negative control) were co-cultured with target cells at a ratio of 1:1, and cell co-culture supernatant was collected after 18-24 hours.

[0090] (2) ELISA detection of IFNγ secretion in supernatant

[0091] The 96-well plate was coated with a capture antibody, purified anti-human IFN-γ antibody (Biolegend, item number 506502), and incubated at 4°C overnight, then the antibody solution was removed, 250 μL of PBST (0.1% Tween in 1X PBS) containing 2% BSA (sigma, item number V900933-1kg) was added, and incubated at 37°C for 2 hours. Then the plate was washed with 250 μL of PBST (0.1% Tween in 1X PBS) for 3 times. 50 μL of cell co-culture supernatant or standard was added to each well, and incubated at 37°C for 1 hour, then the plate was washed with 250 μL of PBST (0.1% Tween in 1X PBS) for 3 times. Then 50 μL of detection antibody, Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, item number ab25017), was added to each well, and incubated at 37°C for 1 hour, then the plate was washed with 250 μL of PBST (0.1% Tween in 1X PBS) for 3 times. HRP Streptavidin (Biolegend, item number 405210) was added, and incubated at 37°C for 30 minutes, then the supernatant was discarded, 250 μL of PBST (0.1% Tween in 1X PBS) was added, and washed for 5 times. 50 μL of TMB substrate solution was added to each well. The reaction was allowed to occur at room temperature in the dark for 30 minutes, then 50 μL of 1 mol / L H2SO4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, the absorbance at 450 nm was detected using a microplate reader, and the content of the cytokine was calculated according to the standard curve (drawn according to the reading and concentration of the standard), and the results are shown in Figure 3.

[0092] It can be seen that the release of IL2 and IFN-γ of CAR33 CAR-T cells is significantly higher than that of NT cells when killing target cells. In general, the release of cytokines by CAR33 CAR-T cells in the present application is significantly higher than that of NT cells against HL60 cells.

[0093] It should be noted that the above is only a preferred embodiment of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

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 a CDR-L1 as set forth in SEQ ID NO: 1, a CDR-L2 as set forth in SEQ ID NO: 2, a CDR-L3 as set forth in SEQ ID NO: 3, and the heavy chain variable region comprises a CDR-H1 as set forth in SEQ ID NO: 4, a CDR-H2 as set forth in SEQ ID NO: 5, a CDR-H3 as set forth in SEQ ID NO:

6.

2. The antibody of claim 1, wherein the light chain variable region is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7, and the heavy chain variable region is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:

8.

3. The antibody of claim 1, wherein the antibody comprises an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 9 or 10.

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

5. The antibody of any one of claims 1-4, wherein the antibody is selected from the group consisting of an IgG, Fab, Fab', F(ab')2, Fd, Fd', Fv, scFv, sdFv, a linear antibody, and a diabody.

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

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

8. The multispecific antibody of claim 7, wherein the second antibody or antigen binding portion thereof is selected from the group consisting of a full length antibody, Fab, Fab', (Fab')2, Fv, scFv, scFv-scFv, a minibody, a diabody, or a sdAb.

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

10. A host cell expressing the antibody of any one of claims 1-5 or the multispecific antibody of 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, the chimeric polypeptide selected from the group consisting of a recombinant T cell receptor, a chimeric antigen receptor, a T cell fusion protein, and a T cell antigen coupler.

12. The chimeric polypeptide of claim 11, the chimeric polypeptide selected from the group consisting of a chimeric antigen receptor, further comprising a transmembrane domain and a primary signaling domain, and optionally, further comprising a costimulatory domain.

13. The chimeric polypeptide of claim 12, wherein the transmembrane domain is selected from the transmembrane domain of the following proteins: TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, CD3 zeta subunit, CD3 epsilon subunit, CD3 gamma subunit, CD3 delta subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

14. The chimeric polypeptide of claim 12, wherein the primary signaling domain is selected from the intracellular region of the following proteins: FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD7b, and CD66d.

15. The chimeric polypeptide of claim 12, wherein the costimulatory domain is selected from the costimulatory signaling domain 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 of any one of claims 11-15.

17. The engineered immune cell of claim 16, wherein the immune cell is selected from the group consisting of a T cell, an NK cell, an NKT cell, a monocyte, a neutrophil, a macrophage, and a dendritic cell.

18. The engineered immune cell of claim 16, wherein the immune cell is selected from the group consisting of CD4 + CD8 + T cells, CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells, tumor infiltrating cells, memory T cells, naive T cells, gamma delta-T cells, and alpha beta-T cells.

19. The engineered immune cell of claim 16, further comprising a second chimeric polypeptide that targets one or more additional antigens or antigen epitopes.

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

21. An antibody 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 the group consisting of an Fc, a radioisotope, a half-life extending moiety, a detectable label, and a drug.

22. The antibody conjugate of claim 21, wherein the half-life extending moiety is selected from the group consisting of a binding structure of albumin, a binding structure of transferrin, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and a white polypeptide that binds human serum albumin; the detectable marker is selected from the group consisting of a fluorophore, a chemiluminescent compound, a bioluminescent compound, an enzyme, an antibiotic resistance gene, and a contrast agent; and the drug is selected from the group consisting of a cytotoxin and an immunomodulator.

23. A diagnostic 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 cell of any one of claims 16-20, or the antibody conjugate of claim 21 or 22.

24. A pharmaceutical composition 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 cell of any one of claims 16-20, or the antibody conjugate of 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 conjugate of claim 21 or 22, the diagnostic kit of claim 23, or the pharmaceutical composition of claim 24 in the manufacture of a medicament for the treatment and / or prevention and / or diagnosis of a disease associated with CD33 expression.

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