WO2024094004A1 - Fully human antibody targeting cd123 and use thereof - Google Patents

Abstract

The invention belongs to the technical field of cell immune engineering, and particularly relates to a fully human antigen binding fragment and single chain fragment variable targeting CD123, and a use thereof. The single chain fragment variable sequence provided by the invention is completely derived from a human antibody gene library. Compared with a murine antibody, a chimeric antibody and a humanized antibody, the immunogenicity of the single chain fragment variable is greatly reduced, and safety can be ensured to the maximum extent in clinical application.

Description

Fully human antibodies targeting CD123 and their applications

This application claims priority to the Chinese patent application filed with the China Patent Office on November 3, 2022, with application number 202211370708.7, and invention name “Fully human antibodies targeting CD123 and their applications”, and the Chinese patent application filed with the China Patent Office on November 3, 2022, with application number 202211370723.1, and invention name “Fully human antibodies targeting CD123 and their applications”, the entire contents of which are incorporated by reference into this application.

Technical Field

The present invention belongs to the technical field of cell engineering, and in particular relates to CD123 antibodies and applications thereof.

Background technique

Adoptive cell therapy (ACT) refers to the separation of immune-competent cells from cancer patients, their expansion and functional identification in vitro, and then their return to the patient, thereby directly killing the tumor or stimulating the body's immune response to kill tumor cells.

Immunotherapy is the fourth type of cancer treatment after surgery, radiotherapy and chemotherapy. Chimeric antigen receptor (CAR) T cell technology is a rapidly developing cellular immunotherapy technology. CAR is an artificially synthesized fusion receptor, which structurally includes an extracellular antigen binding region, a transmembrane region, an intracellular signal transduction region and a co-stimulatory signal region. The extracellular region is a monoclonal antibody sequence (single chain variable fragment, scFv) that recognizes tumor-associated antigens. The transmembrane region connects the extracellular region and the intracellular region. Commonly used transmembrane region molecules are selected from the transmembrane regions and variants of genes such as CD3, CD4, CD8 and CD28. The intracellular region has an immune receptor tyrosine-based activation motif (ITAM), the most commonly used of which are the T cell receptor TCR/CD3ζ chain and the immunoglobulin FC receptor FCεRIγ, which are mainly responsible for signal transduction function; people use molecular cloning methods to recombinate the above elements in vitro to form recombinant plasmids, and then transduce the recombinant plasmids into T cells through viral vectors, electroporation and other means, followed by in vitro culture and amplification, and finally back into the patient's body to treat tumors. This genetically modified and transformed T cell is called CAR-T cell. CAR-T cells recognize and bind to tumor antigens through the scFv segment, and then activate T cells through the CD3ζ chain and co-stimulatory signal region. The modified CAR-T cells recognize and kill tumor cells with two major characteristics: ① Targetedness; ② Overcoming MHC restriction means no longer relying on MHC, thus overcoming the problem of tumor cells escaping immune function by downregulating MHC expression.

In addition to T cells, NK cells also play an important role in tumor treatment. The CAR structure can also form the corresponding CAR-

NK cells and CAR-NK cells are not restricted by HLA matching and can achieve allogeneic adoptive immune therapy. In addition, NK cells have a wide range of sources, including hematopoietic stem cells, peripheral blood, umbilical cord blood, induced pluripotent stem cells, etc. Therefore, CAR-NK can also be used as one of the important methods of immunotherapy.

Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem/progenitor cells. For a long time, anthracyclines and cytarabine chemotherapy have been used to treat mild patients, and hematopoietic stem cell transfer has been used to treat medium- and high-risk patients. Among them, elderly patients (≥60 years old) and patients with poor clinical manifestations have a high mortality rate, and the 5-year overall survival rate is about 10%. Due to the heterogeneity of acute myeloid leukemia, the current treatment of AML patients is still a challenge. In recent years, researchers have found that CD33,

Immune targeted therapy targeting CD123 and CLEC12A is effective in treating AML.

CD123 is a subunit of the heterodimeric interleukin-3 receptor (IL-3R), which is a member of the β co-receptor family. This family of membrane receptors plays an important role in regulating the growth, proliferation, survival and differentiation of hematopoietic cells, as well as immune and inflammatory responses. CD123 is widely expressed in various hematological malignancies. Currently, many monoclonal antibody drugs also use CD123 as a target antigen, so the development of a CD123 antibody for use in adoptive immune cell therapy has also become a research hotspot.

Summary of the invention

The present invention provides a CD123 antibody and a chimeric antigen receptor targeting CD123.

Specifically, the CD123 antibody is an anti-CD123 scFv, the anti-CD123 scFv comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

The L-CDR1, L-CDR2 and L-CDR3 are respectively the following sequences: QSVSSN (SEQ ID NO.1), GAS (SEQ ID NO.2) and QQRSNWPPALT (SEQ ID NO.3);

The H-CDR1, H-CDR2 and H-CDR3 are the following sequences respectively: GYSFTSYW (SEQ ID NO.4), IYPGDSDT (SEQ ID NO.5) and ARIRFDKEQLFYYYYGMDV (SEQ ID NO.6).

Furthermore, the anti-CD123 scFv comprises the amino acid sequence shown in SEQ ID NO.10 or its functional variant.

The present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the anti-CD123 scFv as described above.

Furthermore, the light chain variable region includes, in addition to the above-mentioned high mutation regions of L-CDR1, L-CDR2 and L-CDR3, a low mutation region (also called skeleton region or framework region).

Furthermore, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a functional variant thereof; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.8 or a functional variant thereof.

Furthermore, the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', bispecific antibody and multispecific antibody.

Furthermore, the antibody or antigen-binding fragment thereof is ScFv.

Furthermore, the arrangement of the ScFv is: VL-Linker-VH or VH-Linker-VL.

Furthermore, the amino acid sequence of the Linker is shown in SEQ ID NO.9.

Furthermore, the chimeric antigen receptor comprises an extracellular domain, a hinge region, a transmembrane region and an intracellular signaling domain.

The hinge region is derived from CD4, CD28, CD3, CD40, 4-1BB, OX40, CD84, CD166, CD8α, CD8β, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, CD7, IgG1, IgG4 or the CH2/CH3 domain in immunoglobulin or other protein molecules with equivalent functions. The amino acid sequence of the hinge derived from CD8α is shown in SEQ ID NO.11 or its functional variant, and the sequences of the hinge regions of the above-mentioned other proteins that can be used as hinges can be used. The sequences that can be queried in NCBI.

The nucleotide sequence of the IgG4 hinge is as follows: gcaccacctcgggccagcgccctgcctgca ccacccaccggctccgccctgccagaccctcagacagcatctgccctgccagatcctccagcagcaagcgccctgccc (SEQ ID NO. 23);

The nucleotide sequence of CD7 hinge is as follows: gcaccacctcgggccagcgccctgcctgcaccacccaccggctccgccctgccagaccctcagacagcatctgccctgccagatcctccagcagcaagcgccctgccc (SEQ ID NO.24).

Further, the transmembrane region is derived from CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD7, CD8α, CD8β, CD16, CD27, CD28, CD28H, CD40, CD80, CD84, CD86, CD134, CD137, CD166, CD278, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44, , NKp46, NKG2C, NKG2D, CS1, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or the transmembrane region of T cell receptor polypeptide SIRPβ1 or NKp44, or other protein molecules with equivalent functions; wherein the transmembrane amino acid sequences derived from CD8α, SIRPβ1, and NKp44 are respectively as shown in SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14, or their functional variants, and the sequences of the other protein transmembrane regions that can be used as transmembrane regions can be sequences that can be queried in public databases such as NCBI, ENA, and DDBJ.

Furthermore, the intracellular signaling domain is derived from CD28, OX40, CD27, DAP10, CD3γ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, OX40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), PD1L, B7-H3, KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, LFA-1 (CD11a/CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), CD137, 2B4, CD3ζ, DAP10 functional domains, ICAM-1, CD7, CD83, CD86 and CD127 other equivalent functional protein molecules. The amino acid sequence of the functional domain of CD137 is shown in SEQ ID NO.15 or its functional variant, the amino acid sequence of the functional domain of 2B4 is shown in SEQ ID NO.16 or its functional variant, the amino acid sequence of CD3ζ is shown in SEQ ID NO.17 or its functional variant, the amino acid sequence of the functional domain of DAP10 is shown in SEQ ID NO.18 or its functional variant. Its functional variants and other intracellular functional domains of proteins that can serve as intracellular signaling domains can adopt sequences that can be searched in public databases such as NCBI, ENA, and DDBJ.

The nucleotide sequence of CD28 also includes the following sequence: aggagcaagcggagcagaggcggccacagcgactacatgaacatgaccccccggaggcctggccccacccggaagcactaccagccctacgcccctcccagggacttcgccgcctaccggagc (SEQ ID NO.25).

Furthermore, the chimeric antigen receptor also includes a signal peptide, which is derived from the signal peptide functional domain of CD8α or GM-CSF or GM-CSFR, and the amino acid sequences of the signal peptides derived from CD8α and GM-CSFR are respectively shown in SEQ ID NO.19 and SEQ ID NO.20 or their functional variants.

The chimeric antigen receptor comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR. The NKR component can be a transmembrane domain, hinge domain, or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIR), such as KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; natural cytotoxicity receptors (NCR), for example, NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, for example, CD48, CD1239, 2B4, CD84, NTB-A, CRA, BLAME and CD2F-10; Fc receptors (FcR), for example, CD16, and CD64; and Ly49 receptors, for example, LY49A, LY49C. The NKR-CAR molecules can interact with adapter molecules or intracellular signaling domains (e.g., DAP12).

Further, the chimeric antigen receptor can be a multi-target CAR-T, preferably a dual-target CAR-T, comprising the antibody of the present invention and the recognition of CD123 antigen and the antibody of the present invention without competing for binding antibodies or other tumor surface molecules and/or immune checkpoint molecules that recognize non-CD123 antigens. The dual-target CAR structure can be CAR1-connector peptide-CAR2, or ScFv1-connector peptide-ScFv2-hinge region (hinge)-transmembrane region (TM)-intracellular signaling region (including costimulatory domains and/or intracellular activation domains), or VL1-VL2-VH2-VH1-hinge region (hinge)-transmembrane region (TM)-intracellular signaling region (including costimulatory domains and/or intracellular activation domains) of light and heavy chain cross structures, or any other CAR structure that can play a variety of antigen functions.

Furthermore, the chimeric antigen receptor can reverse tumor suppressor molecules, cytokines, Fusion protein combination/combination of T cell activation molecules, etc., the molecule that reverses the tumor suppression signal can be PD1, and the fusion protein structure can be PD1 extracellular segment-hinge-transmembrane-intracellular signal region, or it can be a fusion protein/polypeptide form of any structure such as PD1 extracellular segment and membrane region-intracellular signal region. The fusion protein has various forms. The structures of different fusion proteins will not affect the function of the chimeric antigen receptor of the present invention to recognize CD123. Therefore, it is easy for those skilled in the art to combine the chimeric antigen receptor of the present invention with a fusion protein/polypeptide of any structure. In addition to the above-mentioned PD1 molecules, the tumor suppression molecules that reverse can also be CTLA-4, PD-L1, LAG-3, TIM-3, BTLA, CD47, etc.; cytokines can be membrane-bound IL-15, membrane-bound IL12, membrane-bound IL21, etc.; T cell activation molecules can be CD80, CD86, etc. Further, the chimeric antigen receptor contains secretable cytokines, antibodies or other polypeptides. Furthermore, the chimeric antigen receptor comprises a recognizable marker molecule, which may be a truncated receptor such as truncated EGFR (EGFRt), truncated HER2 (HER2t), truncated CD34 (CD34t) and truncated CD19 (CD19t).

Furthermore, the chimeric antigen receptor can be combined with immune checkpoint inhibitors, which include but are not limited to: CTLA-4 inhibitors, PD-1/PD-L1 inhibitors, LAG-3 (Lymphocyte Activation Gene-3) inhibitors, TIM-3 (T cell immunoglobulin-3), TIGIT (T cell immunoglobulin and ITIM domain protein), and BTLA inhibitors. In some embodiments, the inhibitors are small molecule drugs, and in some embodiments, they can be antibodies, active peptides, etc.

In certain embodiments, the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', bispecific antibody and multispecific antibody, and can be directly or indirectly coupled/bound to a cytotoxic agent to form an immunoconjugate or antibody ADC drug. Antibody-drug conjugates are a class of targeted biological agents that connect cytotoxic drugs to monoclonal antibodies through covalent compound linking molecules. In some embodiments, the cytotoxic agent can be a microtubule inhibitor and/or a DNA damaging agent, such as α-amanitin (selective RNA polymerase II inhibitor), DM1, PBD, MMAE (monomethyl auristatin E), N-acetyl-gamma-calicheamicin, mitomycin C, anthracyclines, methotrexate, camptothecin derivatives, SN-38 and other toxin molecules.

In certain embodiments, the antibody of the present invention may be a multispecific antibody, preferably a bispecific antibody. Bispecific monoclonal antibody (BsAb). Another targeting molecule of the bispecific antibody may be a surface marker of an immune cell, and the surface marker molecule of the immune cell may be a T cell surface marker, which forms a bifunctional antibody (BispecificT cell engager, BiTE) with the antibody of the present invention in which T cells participate. In certain embodiments, the T cell surface marker molecule is CD3. In certain embodiments, the bispecific antibody comprises an antibody against CD123 described in the present invention, and further comprises an antibody against an immune checkpoint, and the surface molecule targeted by the immune checkpoint antibody may be: PD1, PDL1, CTLA4, CD47, TIGHT, TIM3, BTLA, LMTK3, etc. In certain embodiments, the bispecific antibody comprises the antibody against CD123 described in the present invention, and further comprises an antibody against a tumor surface molecule, wherein the tumor surface molecule includes but is not limited to: CD19, CD20, CD123, CD33, CLL-1 (CLEC12A), CD7, CD5, CD70, CD123, CEACAM5, CEACAM6, CEACAM7, Mesothelin, MUC1, CLDN18.2, CDH17, Trop2, BCMA, NKG2D, PDL1, EGFR, EGFRVIII, PSCA, PSMA, MUC16, CD133, GD2, IL13R2, B7H3, Her2, CD30, SLAMF7, CD38, GPC3, WT1 or TAG-72.

Furthermore, the antibody or its antigen-binding fragment is selected from ScFv, Fab, Fab', bispecific antibody and multispecific antibody, and can be combined with immune checkpoint inhibitors, including but not limited to: CTLA-4 inhibitors, PD-1/PD-L1 inhibitors, LAG-3 (Lymphocyte Activation Gene-3) inhibitors, TIM-3 (T cell immunoglobulin-3), TIGIT (T cell immunoglobulinand ITIM domain protein), BTLA inhibitors. In some embodiments, the inhibitor is a small molecule drug, and in some embodiments, it can be an antibody, active peptide, etc.

Furthermore, the ScFv, Fab, Fab', bispecific antibodies and multispecific antibodies of the present invention can be combined with chemical drugs, such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cyclophosphamide, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228.

The term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.; the linker does not need to affect the function or properties of the antibody itself, and any of the previously disclosed linkers can be used.

The present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof as described above, or comprising a nucleotide sequence encoding the chimeric antigen receptor as described above.

The nucleic acid molecule encodes the chimeric antigen receptor as described in any one of the above, and the nucleotide sequence encoding the light chain variable region of the 1h13 antibody is as shown in SEQ ID NO.21 or its functional variant; wherein the nucleotide sequence encoding the heavy chain variable region of the 1h13 antibody is as shown in SEQ ID NO.22 or its functional variant.

The relationship between CAR in the 202211370708.7 document and CAR in the present invention is as follows:

The present invention also provides an expression vector, which comprises the nucleic acid molecule described above.

Furthermore, the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, a DNA vector, an RNA vector, and a plasmid.

Further, the lentiviral vector is selected from the group consisting of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).

Further, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a chimeric antigen receptor encompassed herein, and a right (3') retroviral LTR.

Furthermore, the chimeric antigen receptor comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

Furthermore, the promoter of the 5'LTR is replaced by a heterologous promoter.

Furthermore, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter or a simian virus 40 (SV40) promoter.

Furthermore, the 5'LTR or 3'LTR is a lentiviral LTR.

Furthermore, the 3'LTR is a self-inactivating (SIN) LTR.

The present invention also provides an engineered cell, wherein the cell is transduced with the above-mentioned nucleic acid molecule or the above-mentioned expression vector, or is obtained by transfecting an immune cell with the above-mentioned recombinant plasmid, or the immune engineered cell comprises any of the above-mentioned chimeric antigen receptors.

Furthermore, the cell is a T cell, a T cell precursor, a macrophage, a NKT cell, a γδT cell or a NK cell.

The present invention also provides a cell product, which comprises the engineered cells as described above.

The chimeric antigen receptor as described in any one of the above or the nucleic acid molecule as described above or Use of the above-mentioned expression vector, the above-mentioned engineered cell, or the above-mentioned cell product in the preparation of anti-tumor drugs.

Alternatively, the present invention also provides the use of any of the above-mentioned antigens or antigen-binding fragments, any of the above-mentioned nucleic acid molecules, any of the above-mentioned recombinant plasmids, any of the above-mentioned chimeric antigen receptors, and any of the above-mentioned immune-engineered cells in the preparation of anti-tumor/autoimmune inflammatory drugs.

Furthermore, the tumor/autoimmune inflammation is B-cell lymphoma, diffuse large B-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), acute myeloid leukemia (AML), acute lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, systemic lupus erythematosus.

Furthermore, the anti-tumor drug is a drug for tumors expressing CD123.

Furthermore, the drugs against CD123-expressing tumors include drugs against acute lymphoid leukemia, drugs against chronic lymphocytic leukemia, drugs against chronic myeloid leukemia, drugs against non-Hodgkin's lymphoma, drugs against Hodgkin's lymphoma and drugs against acute myeloid leukemia.

The CD123scFv in the present invention is applicable to all CAR structures, not just limited to those structures listed in the following examples.

In the present invention, the term "functional variant" generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the chimeric antigen receptor) and having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity therewith. In certain embodiments, the variant of the amino acid sequence has substantially the same function therewith.

The CD123 sequence in the present invention can be used together with all other currently disclosed target sequences to construct a dual-target chimeric antigen receptor, such as a dual-target chimeric antigen receptor together with all currently disclosed scFvs such as CD19, CLL-1, CD33, etc.

The cell preparations of the present invention also include other active agents that can enhance the expression activity of CAR.

In certain embodiments, the agent that enhances the activity of CAR-expressing cells can be an agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to initiate immune effector responses in some embodiments. Inhibitory molecules include PD1, PD -L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as PD1CAR. In certain specific embodiments, the activating agent that enhances the activity of CAR-expressing cells can also be an activating agent of a cytokine receptor chemokine receptor. The cytokine receptor may be derived from a type I cytokine receptor, such as IL-2, IL-4, IL-7, IL-9, IL-13, IL-15 or IL-21.

Further, a variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab, pembrolizumab, pidilizumab, and atezolizumab.

In particular, additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, imatinib, cetuximab, panitumumab, catumaxomab, ibritumomab, tositumomab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib ), sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib , tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib b), pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, and denileukin.

Furthermore, the cell product can also be used in combination with some therapeutic means, and the therapeutic means can be surgery, chemotherapy, and radiation.

The invention also provides a pharmaceutical composition, which includes the antigen or antigen-binding fragment described in any one of the above, the nucleic acid molecule described in any one of the above, the recombinant plasmid described in any one of the above, the chimeric antigen receptor described in any one of the above, the immune engineered cell described in any one of the above, and the cell product described in any one of the above.

The present invention also provides a use, which is the use of any one of the above antibodies or antigen-binding fragments thereof in the preparation of a detection reagent/detection kit.

In the present invention, the antibody or antigen-binding fragment thereof in the present invention can be linked to a detectable label. The detectable label in the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g., ), calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidins (e.g., streptavidin) modified with the above labels.

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a subject in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used. body.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Vols. 1-4, Cold Spring Harbor Press, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

In the prior art, the method for preparing genetically modified immune cells includes at least one of the following steps: ① taking a blood sample, ② enrichment, ③ sorting, ④ activation, ⑤ transfection, and ⑥ re-enrichment.

In certain embodiments, the CAR-T preparation method used is a rapid preparation method, and the steps are as follows: 1. Peripheral blood nuclear cell separation: the collected whole blood or single-collected nucleated cells can be frozen; 2. Peripheral blood nuclear cell activation 0-72h: the separated or resuscitated mononuclear cells are sorted by immunomagnetic beads to obtain T lymphocytes or NK cells, which can be frozen; specifically, the sorted T lymphocytes or NK cells or the resuscitated T lymphocytes or NK cells are contacted with antibodies (which may contain magnetic beads) or small molecules of the complex and/or antibodies (which may contain magnetic beads) to co-stimulatory molecules on the surface of stimulating cells; 3. Transfection 0-72h: T lymphocytes or NK cells are contacted with lentiviral vectors; 4. Enrichment 14-72h: The finally obtained CAR-T/CAR-NK cells are collected.

Density gradient centrifugation can be used in any step of the above-mentioned rapid preparation, and the solution used can be a solution with a continuous or discontinuous density difference, specifically, polysucrose-diatrizoate with a specific gravity of 1.077±0.001, and the resuspension solution and the washing solution in the centrifugation step can be a buffer solution for maintaining the osmotic pressure of the cells, such as containing Na ions (such as physiological saline or physiological saline containing 0.5wt% to 2wt% HSA) or K ions (such as KCL solution or KCL aqueous solution containing 0.5wt% to 2wt% HSA) or containing protein components (such as different concentrations of human albumin, different concentrations of human autologous plasma).

The beneficial effects of the present invention are:

1) The variable region of the single-chain antibody provided by the present invention is derived from a natural fully human antibody, and the sequence is completely derived from the human antibody gene library. Compared with mouse antibodies, chimeric antibodies, and humanized antibodies, its immunogenicity is greatly reduced, avoiding the immunogenicity of mouse antibodies and ensuring safety to the greatest extent in clinical applications.

2) The single-chain antibody provided by the present invention can specifically recognize the human CD123 antigen and can be applied to immunotherapy of blood or solid tumors targeting CD123 targets.

3) After the CD123 chimeric antigen receptor provided by the present invention binds to CD123 on tumor cells, it shows obvious anti-tumor activity, wherein the CD123-scFv is a fully humanized antibody with high affinity for human CD123 antigen.

4) The single-chain antibody provided by the present invention has good affinity performance and can bind to CD123 positive cells in flow cytometry detection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow cytometry staining image of 293T cells with fully human ScFv FK-22-C08;

Figure 2 is a flow cytometry staining image of 293T-CD123 cells with fully human ScFv FK-22-C08;

FIG3 shows the binding kinetics of fully human ScFvFK-22-C08CD123 to CD123 antigen;

Figure 4 shows the in vitro killing efficiency of CAR-1-CAR-3;

Figure 5 shows the in vitro killing efficiency of CAR-1, CAR-2, and CAR-4;

Figure 6 shows the secretion of CAR-1, CAR-2, and CAR-4 factors;

Figure 7 shows the in vitro killing efficiency of CAR-1, CAR-2, CAR-5, and CAR-6;

Figure 8 shows the secretion of CAR-1, CAR-2, CAR-5, and CAR-6 factors;

Figure 9 shows the in vitro killing efficiency of CAR-2, CAR-6, CAR-7, and CAR-8;

Figure 10 shows the secretion of CAR-2, CAR-6, CAR-7, and CAR-8 factors;

Figure 11 shows the in vitro killing efficiency of CAR-1 and CAR-2 modified T cells;

Figure 12 shows the secretion of CAR-1 and CAR-2 factors;

Figure 13 is the in vivo efficacy evaluation of NK cells modified with CAR-1 and CAR-2;

Figure 14 shows the in vitro killing efficiency of CAR-1, CAR-8 and CAR-9;

FIG. 15 shows the factor secretion of CAR-1, CAR-8 and CAR-9.

Detailed ways

The embodiments are provided to better illustrate the present invention, but the present invention is not limited to the embodiments. Non-essential improvements and adjustments to the implementation scheme still fall within the scope of protection of the present invention.

The specific implementation process of ELISA detection in the process of the present invention is as follows:

a. Coating: Dilute CD123 antigen to 1 μg/mL with 50 mM carbonate coating buffer (pH 9.6), add 100 μL/well to a 96-well plate, and cover and incubate overnight at 4°C;

b. Obtaining phage samples: centrifuge the overnight cultured phage monoclonal recombinant bacterial solution at 4°C and 8000 rpm for 10 min, and take the supernatant as the test sample;

c. Blocking: After the antigen-coated plate was washed 3 times with PBS on a plate washer, 200 μL of 5% skim milk powder blocking solution was added to each well and blocked at 37°C for 2 hours;

d. Antibody incubation: Wash the plate 3 times with PBS in a plate washer, add 100 μL of the phage monoclonal recombinant solution to be tested to each well, and incubate at 37°C for 1 hour;

e. Add secondary antibody: Wash the plate 3 times with PBS in a plate washer, add 100 μL of 1:5000 Anti-M13-HRP secondary antibody to each well, and incubate at 37°C for 1 hour;

f. Color development: Wash the plate 6 times with a plate washer, add 100 μL of TMB color development solution to each well, and place at room temperature away from light for 25 minutes for color development;

g. Termination: Add 50 μL 2M H2SO4 to each well to terminate the reaction;

h. Detection: Place the test plate in a microplate reader to detect the OD450 absorbance. Phage clones that are 2.5 times higher than the negative control are positive clones.

Example 1 Preparation of CD123 scFv

1. Construction of a fully human single-chain antibody library

Ficoll separation solution is used for PBMC separation, and the Ficoll separation solution is slowly added to normal human blood so that the Ficoll separation solution and normal human blood maintain a clear separation interface. A 50mL centrifuge tube containing the blood and the separation solution is centrifuged at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, wherein at the junction of the upper and middle layers, there is a narrow band of white cloud layer mainly composed of PBMC, i.e., the PBMC cell layer. Carefully suck off the upper plasma mixture with a sterile Pasteur pipette, and then suck PBMC with a new sterile Pasteur pipette to obtain separated PBMC.

2. Preparation of Phage Display Fully Human Single-chain Antibody Library

Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Bradbury, A. (1998) A definitive set of oligonucleotide primers for amplifying human V regions. Immunotechnology 3, 271-278. Primers were designed using the method in the article, wherein VL was in front or in the back, VH was in the back or in the front, and they were connected by a flexible linker in the middle. The heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody were obtained by PCR. The scFv nucleic acid fragment was amplified by a conventional overlapping PCR method (the PCR method was referred to in Molecular Cloning: A Laboratory Manual (3rd edition), Joe Sambrook, David Russell, Science Press, USA). The scFv nucleic acid fragment was connected to the phagemid vector pComb3XSS, and the product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

Add the library bacterial solution to fresh LB liquid medium for recovery, culture to OD600≈0.5, then add VSCM13 helper phage at a multiplicity of infection of VCSM13: bacteria = 50:1, mix thoroughly, let stand and continue to culture in a shaker. Centrifuge the culture and discard the supernatant, resuspend the precipitate in ampicillin and kanamycin double resistance SOB medium and culture overnight. Centrifuge the bacterial solution for 20 minutes, collect the supernatant and add PEG 8000 2.5mol/L NaCl solution, incubate on ice for 1 hour, centrifuge again for 30 minutes, resuspend the precipitated phage with PBS and filter with a 0.22μm filter membrane.

3. Antigen Panning

The CD123 protein with the His-Fc tag is co-incubated with proteinG magnetic beads to prepare CD123-proteinG coupled magnetic beads, which are then drawn into the prepared fully human single-chain antibody library phage panning. After 3-4 rounds of co-incubation, washing and elution, specific monoclonal antibodies against the antigen can be enriched.

The specific implementation process of antigen panning in the present invention is as follows:

a. Blocking: Dissolve 5% skim milk powder in PBS, filter through a 0.45μm filter membrane and use as blocking solution. Resuspend the phage and CD123-proteinG-coupled magnetic beads with appropriate amount of blocking solution, and mix by rolling at 160rpm for 1h at room temperature.

b. Co-incubation: Place CD123-proteinG coupled magnetic beads on a magnetic rack, discard the supernatant and resuspend the magnetic beads with phage, and incubate at room temperature at 160 rpm for 1 hour;

c. Washing: Place the magnetic bead-phage mixture on a magnetic rack, discard the supernatant, and add an appropriate volume of 0.1% Tween-80 PBST washing solution to wash the magnetic beads. The number of washes was determined as follows: the first round of panning consisted of 5 washes with PBST and 5 washes with PBS, the second round of panning consisted of 6 washes with PBST and 6 washes with PBS, and the third round of panning consisted of 3 washes with 5% FBS and 1 wash with PBS;

d. Elution: Place the washed magnetic beads on a magnetic rack, absorb the supernatant, add 400 μL of pH 2.2 Gly-HCl, mix well, incubate at room temperature for 7 minutes, then add about 20 μL of pH 9.0 Tris-HCl to adjust the pH to near neutral, finally place the mixture on a magnetic rack, transfer the phage supernatant to a new 1.5 mL EP tube to complete a round of panning;

e. Enrichment: The phages were inoculated into TGI bacterial solution with an OD600 of about 0.5 for infection, and then centrifuged after standing at 37°C for 30 minutes. 200 μL of culture medium was reserved to resuspend the precipitate, which was then spread on a 2YTAG plate and inverted for overnight culture.

f. Plate washing: Wash the plaques from the plates incubated overnight with the culture medium, which will be used as the seed solution for the next round of library packaging. The specific process is the same as that for the preparation of the single-chain antibody library.

4. Screening of positive clones

After the panning, the monoclonal plaques finally released from the library were picked for ELISA detection and screening, and the phage clones binding to the CD123 antigen were obtained for downstream research. The following scFv was obtained by panning, numbered: FK-22-C08; it has the specific binding ability to the human CD123 antigen.

5. Expression and purification of ScFV

100ng of pComb3xss plasmid of positive phage clone was mixed with 100μL competent Rosetta gami (DE3) bacteria and placed in an ice bath, heat shock for 90s, and then spread on an LB plate containing ampicillin resistance after ice bath, and placed in a 37℃ constant temperature incubator for overnight culture; pick the formed monoclonal plaque and place it in LB medium, and after shaking culture, transfer the bacterial solution to 200mL LB medium, culture at 37℃ 250rpm, and add IPTG when the bacterial solution OD reaches 0.5-1.0 and adjust the final concentration to 1mM to induce expression. Collect the bacteria by centrifugation and add PBS to resuspend the precipitate, ultrasonically break it for 2min, centrifuge the lysate, discard the precipitate, and collect the supernatant for protein purification.

The supernatant of bacterial lysis was filtered through 0.22 μm, diluted with an equal volume of PBS, and then enriched with GE Ni Sep harose excel purification column. After 5 column volumes of PBS were washed, impurities were removed by washing with 5 column volumes of imidazole-containing PBS solution. The protein was eluted with imidazole-containing PBS solution, and the washing solution was collected and concentrated with a 3KDa ultrafiltration tube. The sample was loaded on a GE molecular exclusion chromatography column, washed with PBS and the ultraviolet absorption peak was collected. After SDS-PAGE to identify the purification effect of scFv, flow staining was performed. Sex detection.

After the panning, the monoclonal plaques finally released from the library were picked for chemiluminescence detection screening, and then re-tested with ELISA detection to obtain phage clones that bind to the CD123 antigen for use in downstream research. Antibodies were screened and numbered h13 and h20 respectively.

VI. Mouse Humanization

Referring to the methods in patents 201811125919.8 and 201811125906.0 applied by our company, the scFv numbered 1h7 was finally prepared.

7. Flow Cytometry Staining Specificity Identification

293T and 293T-CD123 cells were each divided into 1.5mL Eppendorf tubes, with 1×106 cells in each tube as target cells. All cells were centrifuged at 400g for 5min, and the supernatant was discarded. The cells were resuspended with 50μL 15μg/mL FK-22-C08 ScFv solution and incubated at 4℃ in the dark. After 30min, 1mL PBS was added to resuspend the cells, washed at 400g for 5min, and the supernatant was discarded. 30μL Anti-His-647 fluorescent secondary antibody for detection was added and the cells were resuspended, and incubated at 4℃ in the dark for 30min; the cells were resuspended with 1mL PBS and washed twice at 400g for 5min, and the supernatant was discarded and resuspended with 200μL PBS. All cell tubes were tested for the positive rate of flow staining and the specificity of flow staining of ScFv was evaluated. The staining results of the selected CD123 ScFv on 293T cells are shown in Figure 1, and the staining results on 293T-CD123 are shown in Figure 2.

8. Detection of Kinetic Parameters of Antigen-Antibody Binding

The ProA biosensor was subjected to baseline 1 for 60s in the analysis buffer, loading for 120s in a 30ug/mL CD12 3-hFc solution for ligand protein immobilization, transferred to baseline 2 in the analysis buffer for 120s, transferred to a gradient diluted analyte (CD123 ScFv) solution for association for 120s, and finally dissociation for 300s in the analysis buffer. The change in the response value R is used to determine whether specific binding exists. Generally speaking, specific binding exists when the maximum response value Rmax after subtracting the reference is greater than 0.05nm and there is concentration dependence and dissociation. The Data Analysis software that comes with the Fortebio Octet K2 instrument can provide the kinetic parameters represented by the binding-dissociation curve, such as the binding constant (Kon), the dissociation constant (Kdis), and the equilibrium dissociation constant (KD). The unit of Kon is 1/Ms, which is used to express the rate of antigen-antibody binding. The higher the Kon, the faster the antibody-antigen binding to form a complex. The unit of Kdis is 1/s, which is used to express the rate of antigen-antibody dissociation. The higher the Kdis, the faster the antibody-antigen binding to form a complex. The faster the dissociation of the antigen-antibody complex; KD is the ratio of Kdis to Kon, which is used to comprehensively describe the difficulty of antigen-antibody binding. The smaller the KD, the higher the antibody affinity is generally considered. The binding kinetics of the CD123-specific ScFv obtained by panning and the CD123 antigen are shown in Figure 3.

Example 2 Construction of CAR plasmid targeting CD123

The scFv sequence was obtained by PCR amplification, and then the sequence was connected to a lentiviral vector containing different hinges, different transmembrane regions, and different co-stimulatory signals by restriction endonuclease digestion, thereby obtaining CAR plasmids targeting CD123 with different structures. The structures are as follows (8h in the following structure indicates that the hinge region is derived from the hinge region of human CD8α, 8TM indicates that the transmembrane region is derived from the transmembrane region of human CD8α, BB indicates that the intracellular signaling domain is derived from human CD137, z indicates that the CD3ζ chain, and 134 indicates that the intracellular Signaling domain, 2B4leader indicates the signal peptide domain derived from 2B4, 2B4 indicates the intracellular signaling domain of the 2B4 molecule, 2B4h indicates the hinge region is the extracellular hinge region derived from 2B4, 2B4TM indicates the transmembrane region is the transmembrane region derived from 2B4, NKp44TM indicates the transmembrane region is the transmembrane region derived from NKp44, SIRPβ1TM indicates the transmembrane region is the transmembrane region derived from SIRPβ1TM, GMR indicates the signal peptide of GM-CSFR, and 2A is the self-cleaving polypeptide from P2A):

CAR-1: CD8α-CD123 scFv(1h7)-8h-8TM-BBz

CAR-2: CD8α-CD123 scFv(h13)-8h-8TM-BBz

CAR-3: CD8α-CD123 scFv(h20)-8h-8TM-BBz

CAR-4: CD8α-CD123 scFv(1h7)-8h-8TM-2B4z

CAR-5: GMR-CD123 scFv(1h7)-8h-8TM-2B4z

CAR-6: GMR-CD123 scFv(h13)-8h-8TM-2B4z

CAR-7: GMR-CD123 scFv(h13)-8h-SIRPβ1TM-2B4z

CAR-8: GMR-CD123 scFv(h13)-8h-NKp44TM-2B4z

CAR-9: GMR-CD123 scFv(h13)-8h-NKp44TM-2B4-DAP10

Table 1: Nucleotide sequences

Table 2 Amino Acid Sequence Listing

Example 3 Preparation of lentivirus and infection of T/NK lymphocytes

In this embodiment, CAR-1 to CAR-9 are transfected into NK cells, and CAR-1 and CAR-2 are transfected into T cells.

In this embodiment, the calcium phosphate method is used to package the lentivirus, specifically: 293T cells are cultured with DMEM culture medium containing 10% FBS (w/v) to an optimal state, the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5mL centrifuge tube in a certain proportion, CaCl2 and 2×H BS are added, and the mixture is allowed to stand at room temperature and then added to the treated 293T cell culture medium. After 3-5 hours, the medium is replaced again with 10mL DMEM culture medium containing 10% FBS. After 48 hours or 72 hours, the cell supernatant is collected, the virus is purified, and the titer is measured.

The packaging cell line for NK cells was prepared as follows: the CAR-1 to CAR-9 target plasmids were mixed with three lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G in a ratio of 3:1:1:1, and the lentiviral packaging cell line was transfected by calcium transfection. After 6 hours of transfection, fresh 5% FBS-DMEM culture medium was replaced, and the crude virus extract was harvested 48 hours after transfection.

The prepared lentivirus titer was determined by using CHO cells. After 1e5/well of CHO cells were infected with the test virus for 48 hours, the total CAR expression was detected with Protein-L, and the positive rate was calculated. The positive rate calculation formula is: titer (Tu/ml) = 1e5 × positive rate × dilution factor × 1000 ÷ virus volume (uL). The titer of the above CAR structure virus is shown in Table 3.

Table 3 CD123-CAR lentivirus titer list

Lymphocytes were separated by gradient centrifugation. After centrifugation, the second white lymphocyte layer was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. The obtained PBMC cells were activated with anti-CD3 and CD28 monoclonal antibodies for 24 hours, and then infected with the activated PBMC at a certain multiplicity of infection (MOI). The positivity rate of CAR-T was detected on the 12th day after virus infection. The detection method was flow cytometry and the antibody was Protein-L-PE. Protein-L could recognize the light chain of antibody. The light chain of the scFv sequence in the CAR antigen recognition region could be recognized by Protein-L. Therefore, Protein-L could be used to detect the positivity rate and expression intensity of CAR.

For NK cells, after obtaining PBMC, CD56 antibody-labeled Microbeads need to be used for magnetic sorting to obtain CD56-positive NK cells. NK cells can be transduced with lentivirus 24 hours after activation. Three days after transduction, the positive rate of CAR-NK needs to be detected using CD123-6×His (ACRO-Biosystems) or monoclonal antibodies targeting the corresponding CD123-scFv. The results of the CAR-T/NK positive rate test using CD3-PE/Cy7 and CD56-BV510 (BioLegend) labeled NK cells are shown in Table 4. As shown in the table below, all constructed structures can be expressed well.

Table 4 CD123-CAR-NK/T positive rate detection results

Example 4 CAR-1-CAR-3 modified NK cell killing experiment in vitro and cytokine secretion experiment

CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells. CAR-NK cells were plated on target cells at a ratio of 1:1, and killing was detected by luciferase after 24 hours.

Luciferase principle: During the test, the target cells are lysed with lysis buffer, and the luciferase in them will decompose the substrate to emit fluorescence. The fluorescence value analysis result formula is: CAR-NK cell killing rate = 1-(fluorescence value of the experimental group ÷ fluorescence value of the blank control group).

The results are shown in the figure and the following Table 5 and Figure 4. CAR-NK cells modified with the CAR structure CAR-2 composed of CD123(h13)-scFv showed obvious advantages in killing KG1a-Luc-GFP.

Table 5 In vitro killing data of CAR-1, CAR-2 and CAR-3

Example 5 In vitro killing experiment and cytokine secretion experiment of NK cells modified by CAR-1, CAR-2 and CAR-4

CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells, and CAR-NK cells were plated on the target cells at a ratio of 1:1. After 24 hours, killing was detected by luciferase.

Luciferase principle: During the test, the target cells are lysed with lysis buffer, and the luciferase in them will decompose the substrate to emit fluorescence. The fluorescence value analysis result formula is: CAR-NK cell killing rate = 1-(fluorescence value of the experimental group ÷ fluorescence value of the blank control group).

The results are shown in Figure 5 and Table 6: 24 hours after the addition of CAR-NK cells, the CAR-2 modified with the CAR structure composed of CD123(h13)-scFv still showed obvious functional advantages. Secondly, among the CAR-NK cells modified with the CAR structure composed of CD123(1h7)-scFv, CAR-4 showed better in vitro function.

Table 6 In vitro killing data of CAR-1, CAR-2 and CAR-4

The cell supernatant was collected 24 hours after killing to detect the IFN-γ secretion ability of CAR-NK cells after being stimulated by target cells. The collected supernatant was tested for IFN-γ secretion using ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 6 and Table 7. After antigen stimulation, CAR-2 has a stronger IFN-γ secretion ability, and the CAR structure composed of CD123 (1h7)-scFv The modified CAR-NK and CAR-2 have higher factor secretion levels, therefore, CAR-2 constructed with CD123(h13)-scFv is preferred.

Table 7 Cytokine secretion data of CAR-1, CAR-2, and CAR-4

Example 6 In vitro killing experiment of NK cells and cytokine secretion experiment decorated with CAR-1, CAR-2, CAR-5 and CAR-6

CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells, and CAR-NK cells were plated on the target cells at a ratio of 1:1. After 24 hours, the killing was detected by luciferase. Principle of luciferase: When the target cells are lysed with lysis buffer, the luciferase in them will decompose the substrate and emit fluorescence. The formula for analyzing the results using fluorescence value is: CAR-NK cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group).

The results are shown in Figure 7 and Table 8: 24 hours after the addition of CAR-NK cells, CAR-2 and CAR-6 decorated with the CAR structures composed of CD123(h13)-scFv still showed obvious functional advantages. Secondly, among the CAR-NK modified with the CAR structure composed of CD123(1h7)-scFv, CAR-5 showed better in vitro function.

Table 8 Cytokine secretion data of CAR-1, CAR-2, CAR-5, and CAR-6

The cell supernatant was collected 24 hours after killing, and the IFN-γ secretion ability of CAR-NK cells after stimulation by target cells was detected. The collected supernatant was used to detect the secretion of IFN-γ by ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 8 and Table 9. After antigen stimulation, CAR-2 and CAR-6 have stronger IFN-γ secretion ability, which fully illustrates the advantages of CD123-scFv (h13) in the composition of CAR molecules.

Table 9 CAR-1, CAR-2, CAR-5, CAR-6 factor secretion (unit: pg/ml)

Example 7 In vitro killing experiment and cytokine secretion experiment of NK cells modified by CAR-2, CAR-6, CAR-7, and CAR-8

CD123+KG1a-Luc-GFP, MV-4-11-Luc-GFP, and MOLM-13-Luc-GFP cells were used as positive target cells, and KG-1a-CD123(-)-Luc-GFP (KG1a-Luc-GFP knocked out CD123) was used as negative target cells. CAR-NK cells were plated on target cells at a ratio of 1:1, and killing was detected by luciferase after 24 hours. Luciferase principle: When the target cells are lysed with lysis buffer during the detection, the luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using fluorescence value is: CAR-NK cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group).

The results are shown in Figure 9 and Table 10: 24 hours after the addition of CAR-NK cells, CAR-2, 6, 7, and 8 modified CAR-NK cells composed of CD123 (h13)-scFv CAR structures still showed good in vitro function, among which CAR-2, 6, 7, and 8 all had good in vitro function.

Table 10 In vitro killing efficiency of CAR-2, CAR-6, CAR-7 and CAR-8

The cell supernatant was collected 24 hours after killing, and the IFN-γ secretion ability of CAR-NK cells after being stimulated by target cells was detected. The collected supernatant was used to detect the secretion of IFN-γ by ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 10 and Table 11, and the secretion levels of CAR-2, 6, 7, and 8 factors are comparable.

Table 11 CAR-2, CAR-6, CAR-7, CAR-8 factor secretion

Example 8 CAR-1 and CAR-2 modified T cells in vitro killing experiment and cytokine secretion experiment

CD123+ cells KG1a-Luc-GFP and MOLM-13-Luc-GFP were used as positive target cells, and Raji-Luc-GFP was used as negative target cells. CAR-T cells were plated on target cells at a ratio of 1:1, and killing was detected by luciferase after 24 hours. Luciferase principle: When the target cells are lysed with lysis buffer during the detection, the luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using fluorescence value is: CAR-T cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group).

The results are shown in the figure and the following Table 12 and Figure 11. CAR-1 and CAR-2 modified CAR-T cells have obvious and effective killing against KG1a-Luc-GFP and MOLM-13-Luc-GFP, but the killing level for negative cells Raji-Luc-GFP is extremely low.

Table 12 In vitro killing efficiency of CAR-1 and CAR-2 modified T cells

The cell supernatant was collected 24 hours after killing to detect the IFN-γ secretion ability of CAR-T cells after stimulation by target cells. The collected supernatant was used to detect the secretion of IFN-γ by ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 12 and Table 13. Compared with CAR-1, CAR-2 modified T cells have stronger IFN-γ secretion ability after antigen stimulation.

Table 13 Secretion of CAR-1 and CAR-2 modified T cells factors

Example 9 In vivo efficacy evaluation of CAR-1 and CAR-22 modified NK cells

CD123+ cells MV-4-11-Luc-GFP were used as positive target cells, and NCG mice were pre-tumored by tail vein injection at a dose of 1e6/mouse, and the pre-tumor time was 8 days. After 13 days of in vitro culture, CAR-1 and CAR-2 modified NK cells were injected into NCG mice with pre-tumored tumors at a dose of 6e6/mouse through the tail vein, and NK cells without CAR modification were injected at the same dose and method as the control group. After that, the fluorescent substrate of luciferase was injected intraperitoneally once a day, and the tumor load was detected by in vivo imaging using a small animal in vivo imaging instrument.

The results are shown in Figure 13. CAR-2 modified NK cells have the best in vivo efficacy advantage over CAR-1 modified NK cells and unmodified NK.

Example 10 In vitro killing experiment and cytokine secretion experiment of NK cells modified by CAR-1, CAR-8 and CAR-9

CD123+KG1a-Luc-GFP and MV-4-11-Luc-GFP cells were used as positive target cells, and CAR-NK cells were plated on the target cells at a ratio of 1:1. After 24 hours, the killing was detected by luciferase. Luciferase principle: When the target cells are lysed with lysis buffer during the detection, the luciferase in them will decompose the substrate to emit fluorescence. The formula for analyzing the results using fluorescence value is: CAR-NK cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group).

The results are shown in Figure 14 and Table 14: 24 hours after the addition of CAR-NK cells, CAR-8 modified with the CAR structure composed of CD123(h13)-scFv still showed good in vitro function, and the functions of CAR-8 and CAR-9 were equivalent.

Table 14 In vitro killing efficiency of NK cells modified with CAR-1 and CAR-2

The cell supernatant was collected 24 hours after killing, and the IFN-γ secretion ability of CAR-NK cells after being stimulated by target cells was detected. The collected supernatant was used to detect the secretion of IFN-γ by ELISA (enzyme-linked immunosorbent assay). The results are shown in Figure 15 and Table 15. After antigen stimulation, both CAR-8 and CAR-9 modified NK cells have a strong IFN-γ secretion ability.

Table 15 NK cell factor secretion modified by CAR-1, CAR-8, and CAR-9

Claims (23)

An antibody or antigen-binding fragment thereof capable of specifically binding to CD123, characterized in that it comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3; The amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 are shown below: QSVSSN, GAS and QQRSNWPPALT The amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are shown below: GYSFTSYW, IYPGDSDT, and ARIRFDKEQLFYYYYGMDV. The antibody or antigen-binding fragment thereof as described in claim 1, characterized in that the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.7 or a functional variant thereof; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.8 or a functional variant thereof. The antibody or antigen-binding fragment thereof as described in claim 1 or 2, characterized in that the antibody or antigen-binding fragment thereof is selected from ScFv, Fab, Fab', bispecific antibodies and multispecific antibodies. Preferably, the antibody or antigen-binding fragment thereof is ScFv. Preferably, the arrangement of the ScFv is: VL-Linker-VH or VH-Linker-VL. More preferably, the amino acid sequence of the Linker is as shown in SEQ ID NO.9. A chimeric antigen receptor, characterized in that the chimeric antigen receptor comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3. The chimeric antigen receptor as described in claim 4 is characterized in that: the chimeric antigen receptor comprises ScFv or a functional variant thereof as shown in SEQ ID NO.10. The chimeric antigen receptor according to claim 4 or 5, characterized in that the chimeric antigen receptor comprises an extracellular domain, a hinge region, a transmembrane region and an intracellular signaling domain. The chimeric antigen receptor as described in claim 6 is characterized in that: the hinge region is derived from the CH2/CH3 domain in CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8α, CD8β, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, IgG1 or immunoglobulin, and the amino acid sequence of the hinge derived from CD8α is shown in SEQ ID NO.11 or its functional variant. The chimeric antigen receptor according to claim 6, wherein the transmembrane region is The transmembrane region derived from CD2, CD3D, CD3E, CD3G, CD3ζ, CD4, CD8α, CD8β, CD16, CD27, CD28, CD28H, CD40, CD84, CD166, 4-1BB, OX40, ICOS, ICAM-1, CTLA4, PD1, LAG3, 2B4, BTLA, DNAM1, DAP10, DAP12, FcERIγ, IL7, IL12, IL15, KIR2DL4, KIR2DS1, KIR2DS2, NKp30, NKp44,, NKp46, NKG2C, NKG2D, CS1, or T cell receptor polypeptide SIRPβ1 or NKp44, the transmembrane amino acid sequence derived from CD8α, SIRPβ1, NKp44 is shown as SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 respectively, or its functional variant. The chimeric antigen receptor according to claim 6, characterized in that: the intracellular signaling domain is derived from CD28, OX40, CD27, DAP10, CD3γ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRA The signaling functional domains of NCE/RANKL, LFA-1 (CD11a/CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), CD137, 2B4, CD3ζ, and DAP10, the amino acid sequence of the CD137 functional domain is as shown in SEQ ID NO.15 or its functional variant, the amino acid sequence of the 2B4 functional domain is as shown in SEQ ID NO.16 or its functional variant, the amino acid sequence of the CD3ζ functional domain is as shown in SEQ ID NO.17 or its functional variant, and the amino acid sequence of the DAP10 functional domain is as shown in SEQ ID NO.18 or its functional variant. The chimeric antigen receptor as described in any one of claims 4-9 is characterized in that: the chimeric antigen receptor also includes a signal peptide, the signal peptide is derived from CD8α or GM-CSF or GM-CSFR, and the amino acid sequences of the signal peptides derived from CD8α and GM-CSFR are respectively shown in SEQ ID NO.19 and SEQ ID NO.20 or their functional variants. A nucleic acid molecule, characterized in that: It comprises a nucleotide sequence encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, Or comprising a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 4-10. The nucleic acid molecule as described in claim 11 is characterized in that the nucleotide sequence encoding the antibody light chain variable region is as shown in SEQ ID NO.21 or a functional variant thereof; wherein the nucleotide sequence encoding the antibody heavy chain variable region is as shown in SEQ ID NO.22 or a functional variant thereof. The nucleic acid molecule as described in claim 11 is characterized in that the nucleotide sequence encoding the chimeric antigen receptor is shown in any one of SEQ ID NO.26~34. An expression vector, characterized in that the expression vector comprises the nucleic acid molecule according to any one of claims 11 to 13. The expression vector according to claim 14, characterized in that the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, a DNA vector, an RNA vector, and a plasmid. An engineered cell, characterized in that: the cell is transduced with the nucleic acid molecule according to any one of claims 11 to 13 or the expression vector according to claim 14 or 15, preferably, the cell is a T cell, a T cell precursor, a γδT cell or a NK cell. A cell product, characterized in that: the cell product comprises the engineered cells as described in claim 16. A pharmaceutical composition, characterized in that: the pharmaceutical composition comprises: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or comprising the chimeric antigen receptor according to any one of claims 4 to 10, or comprising the nucleic acid molecule according to any one of claims 11 to 13, or comprising the expression vector according to claim 14 or 15, or comprising the engineered cell of claim 16, Or comprising the cell product according to claim 17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the chimeric antigen receptor according to any one of claims 4 to 10, or the nucleic acid molecule according to any one of claims 11 to 13, or the expression vector according to claim 14 or 15, or the engineered cell according to claim 16, or the cell product according to claim 17, or the pharmaceutical composition according to claim 18 in the preparation of anti-tumor and/or anti-autoimmune inflammatory drugs. The use according to claim 19, characterized in that the anti-tumor drug is a drug against tumors expressing CD123. The use as claimed in claim 20 is characterized in that: the drugs against CD123-expressing tumors include anti-acute lymphoid leukemia drugs, anti-chronic lymphocytic leukemia drugs, anti-chronic myeloid leukemia drugs, anti-non-Hodgkin's lymphoma drugs, anti-B-cell lymphoma drugs, anti-diffuse large B-cell lymphoma drugs, anti-blastic plasmacytoid dendritic cell neoplasm (BP DCN) drugs, anti-Hodgkin's lymphoma drugs and anti-acute myeloid leukemia drugs. The use according to claim 19 is characterized in that the anti-autoimmune inflammatory drug is an anti-systemic lupus erythematosus drug. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the chimeric antigen receptor according to any one of claims 4 to 10, or the nucleic acid molecule according to any one of claims 11 to 13, or the expression vector according to claim 14 or 15, or the engineered cell according to claim 16, or the cell product according to claim 17, or the pharmaceutical composition according to claim 18 in the preparation of a detection reagent/detection kit.