Completely human antibody targeting CD33

Abstract

The invention provides a fully humanized antibody targeting CD33 and an application of the fully humanized antibody. Specifically, the invention provides a CD33 targeting antibody with a brand new sequence, a chimeric antigen receptor constructed based on the antibody or an antigen binding fragment thereof, and an immune cell for expressing the chimeric antigen receptor. The antibody can be highly specifically combined with a CD33 antigen, has very high affinity and remarkable anti-tumor activity, and can be used for preparing medicines for treating diseases related to CD33.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and in particular to a fully human antibody that specifically binds to human CD33 antigen protein and CD33 antigen in a natural state on the surface of a cell membrane. Background Art

[0002] CD33 is a member of the sialic acid-binding immunoglobulin-like lectin family and is a 67 kDa glycosylated transmembrane protein. It is expressed on most myeloid and monocytic leukemia cells in addition to myelomonocytic and erythroid progenitor cells. CD33 is not expressed on early multipotent stem cells, mature granulocytes, lymphoid cells, or non-hematopoietic cells. CD33 contains two tyrosine residues on its cytoplasmic tail, each followed by a hydrophobic residue, similar to the immunoreceptor tyrosine-based inhibitory motif (ITIM) seen in many inhibitory receptors.

[0003] Monoclonal antibody (mAb)-based therapy has become an important treatment modality for cancer. Leukemia is very suitable for monoclonal antibody (mAb)-based therapy due to the accessibility of malignant cells in the blood, bone marrow, spleen and lymph nodes. In addition, the clear immunophenotypes of different lineages and stages of hematopoietic differentiation allow the identification of target antigens. The vast majority of studies on acute myeloid leukemia (AML) are targeting CD33.

[0004] In two randomized trials, unconjugated anti-CD33 lintuzumab had modest anti-AML activity when combined with conventional standard chemotherapy, but failed to improve patient outcomes. In May 2000, the FDA approved gemtuzumab ozogamicin (GO) for the treatment of AML. GO is a humanized anti-CD33 monoclonal antibody-drug conjugate that was subsequently withdrawn from the market due to toxic side effects demonstrated in clinical trials. In addition, three phase I studies of an anti-CD33-maytansine conjugate (AVE9633; huMy9-6-DM4) were conducted in patients with AML. The maximum tolerated dose (MTD) was determined in only one phase I study (dosing schedule day 1 / 8), as the other two studies were discontinued before reaching the MTD due to lack of clear signs of activity at doses above the saturation dose. AVE9633 had modest activity on day 1 / 8 of the phase I dosing schedule.

[0005] Therefore, it is of great significance to develop safe and effective antibodies targeting CD33 for the diagnosis and treatment of CD33-related diseases, such as cancer. Summary of the invention

[0006] The purpose of the present invention is to provide a safe and effective antibody targeting CD33, and a preparation method and application thereof.

[0007] In a first aspect of the present invention, there is provided a heavy chain variable region targeting CD33, wherein the heavy chain variable region comprises a CDR selected from the following group:

[0008] (Z1) the amino acid sequence of HCDR1 is shown in SEQ ID NO:15, the amino acid sequence of HCDR2 is shown in SEQ ID NO:16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:17; or

[0009] (Z2) the amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:21, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:22;

[0010] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD33 binding affinity.

[0011] In another preferred example, the heavy chain variable region further includes a framework region (FR), and the framework region is a human framework region.

[0012] In another preferred example, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 3 or 6, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the above sequence.

[0013] In another preferred example, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3 or 6.

[0014] In another preferred embodiment, the CD33 includes human CD33 and cynomolgus monkey CD33, preferably, human CD33.

[0015] The second aspect of the present invention provides an antibody targeting CD33, wherein the antibody comprises the heavy chain variable region as shown in the first aspect of the present invention.

[0016] In another preferred embodiment, the antibody further comprises a light chain variable region, and the light chain variable region comprises a CDR selected from the following group:

[0017] (Z1) the amino acid sequence of LCDR1 is as shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is EDS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:14; or

[0018] (Z2) the amino acid sequence of LCDR1 is shown in SEQ ID NO:18, the amino acid sequence of LCDR2 is DNN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:19;

[0019] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD33 binding affinity.

[0020] In another preferred example, the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 2 or 5, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the above sequence.

[0021] In another preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region, and the CDR of the heavy chain variable region is as follows:

[0022] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 15, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 17; and the CDR of the light chain variable region is as follows:

[0023] The amino acid sequence of LCDR1 is shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is EDS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:14.

[0024] In another preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region, and the CDR of the heavy chain variable region is as follows:

[0025] The amino acid sequence of HCDR1 is shown in SEQ ID NO: 20, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 21, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 22; and the CDR of the light chain variable region is as follows:

[0026] The amino acid sequence of LCDR1 is shown in SEQ ID NO:18, the amino acid sequence of LCDR2 is DNN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:19.

[0027] In another preferred example, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:3, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:2.

[0028] In another preferred example, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:5.

[0029] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence which is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD33 binding affinity.

[0030] In another preferred embodiment, the ratio of the affinity F1 of the derived antibody for binding to CD33 to the affinity F0 of the corresponding non-derivatized antibody for binding to CD33 (F1 / F0) is 0.5-2, preferably 0.7-1.5, and more preferably 0.8-1.2.

[0031] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0032] In another preferred embodiment, the antibody includes a single domain antibody (sdAb), a single chain antibody (scFv), or a monoclonal antibody (mAb).

[0033] In another preferred embodiment, the antibody includes a monovalent antibody or a multivalent antibody (eg, a bivalent, trivalent or tetravalent antibody).

[0034] In another preferred embodiment, the antibody is a single domain antibody (sdAb) having a heavy chain variable region shown in SEQ ID NO: 3 and / or 6.

[0035] In another preferred embodiment, the antibody is a single-chain antibody (scFv) having an amino acid sequence as shown in SEQ ID NO: 1 or 4.

[0036] In another preferred embodiment, the antibody is a monoclonal antibody (mAb) having a heavy chain variable region and a light chain variable region as described above;

[0037] Furthermore, the antibody also includes a constant region.

[0038] In another preferred example, the constant region is a human constant region.

[0039] In another preferred embodiment, the CD33 includes human CD33 and cynomolgus monkey CD33, preferably, human CD33.

[0040] In another preferred embodiment, the antibodies include fully human antibodies, chimeric antibodies, animal-derived antibodies, and humanized antibodies.

[0041] In another preferred embodiment, the antibody is a fully human antibody.

[0042] In another preferred embodiment, the antibody targets the IgC domain and / or IgV domain of CD33.

[0043] The third aspect of the present invention provides a recombinant protein, comprising:

[0044] (a) the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, or the antibody targeting CD33 as described in the second aspect of the present invention;

[0045] (b) Optional tag sequence to facilitate expression and / or purification.

[0046] In a fourth aspect, the present invention provides a chimeric antigen receptor (CAR), the antigen binding domain of which comprises a heavy chain variable region targeting CD33 as described in the first aspect of the present invention, or an antibody targeting CD33 as described in the second aspect of the present invention.

[0047] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula I below:

[0048] LAH-TM-C-CD3ζ-PS(I)

[0049] in,

[0050] L is none or a signal peptide sequence;

[0051] A is the antigen binding domain targeting CD33;

[0052] H is the hinge region;

[0053] TM is the transmembrane domain;

[0054] C is a co-stimulatory signal molecule;

[0055] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ;

[0056] P is none or a connecting peptide;

[0057] S is no or enhanced element.

[0058] In another preferred embodiment, the antigen binding domain targeting CD33 is a scFv targeting CD33.

[0059] In another preferred embodiment, the scFv targeting CD33 has an amino acid sequence as shown in SEQ ID NO: 1 or 4.

[0060] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0061] In another preferred embodiment, the L is a signal peptide derived from CD8a.

[0062] In another preferred example, the amino acid sequence of L is shown as SEQ ID NO:23; in another preferred example, the nucleotide sequence of L is shown as SEQ ID NO:46.

[0063] In another preferred embodiment, the H is the hinge region of a protein selected from the following group: CD8a, CD28, IgG1, IgG4, or a combination thereof.

[0064] In another preferred embodiment, the H is the hinge region derived from CD8a.

[0065] In another preferred example, the amino acid sequence of H is shown as SEQ ID NO:24; in another preferred example, the nucleotide sequence of H is shown as SEQ ID NO:47.

[0066] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: CD8a, CD28, ICOS, CD3epsilon, CD45, CD4, CD5, CD9, CD16, GD2, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0067] In another preferred embodiment, the TM is a transmembrane region derived from CD8a or CD28, preferably, a transmembrane region derived from CD8a.

[0068] In another preferred example, the amino acid sequence of TM is shown as SEQ ID NO:25; in another preferred example, the nucleotide sequence of TM is shown as SEQ ID NO:48.

[0069] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: 4-1BB (CD137), CD28, OX40, 2B4, DAP10, DAP12, ICOS, CD2, CD7, CD27, CD30, CD40, CD70, CD134, PD1, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0070] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from 4-1BB, CD28, 2B4, OX40, DAP10 or DAP12.

[0071] In another preferred example, the amino acid sequence of C is shown in any one of SEQ ID NOs: 26-31; in another preferred example, the nucleotide sequence of C is shown in any one of SEQ ID NOs: 49-54.

[0072] In another preferred example, the amino acid sequence of CD3ζ is shown as SEQ ID NO:32; in another preferred example, the nucleotide sequence of CD3ζ is shown as SEQ ID NO:55.

[0073] In another preferred embodiment, the P is a self-cleaving peptide; preferably, the P is a 2A self-cleaving peptide, and its amino acid sequence is shown in SEQ ID NO: 33;

[0074] In another preferred example, the nucleotide sequence of the 2A self-cleaving peptide is shown in SEQ ID NO:56.

[0075] In another preferred embodiment, the enhancing element is used to enhance the cell killing effect of CAR.

[0076] In another preferred embodiment, the enhancing element is selected from: tEGFR, IL-15, IL-2, IL-7, IL-18, IL-21, CCL19, CXCR2, CCR7, TGF-βDNR, c-Jun, hnCD16 or a combination thereof.

[0077] In another preferred example, the amino acid sequence of the tEGFR is shown as SEQ ID NO:57; in another preferred example, the nucleotide sequence of the tEGFR is shown as SEQ ID NO:34.

[0078] In another preferred example, the amino acid sequence of the IL-15 is shown as SEQ ID NO:58; in another preferred example, the nucleotide sequence of the IL-15 is shown as SEQ ID NO:35.

[0079] In another preferred embodiment, the chimeric antigen receptor includes, from N-terminus to C-terminus, a CD8a signal peptide, an antigen binding domain targeting CD33, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and CD3ζ.

[0080] In another preferred embodiment, the chimeric antigen receptor includes, from N-terminus to C-terminus, a CD8a signal peptide, an antigen binding domain targeting CD33, a CD8a hinge region, a CD8a transmembrane domain, a CD28 co-stimulatory domain, CD3ζ, a P2A self-cleavage peptide, and IL-15.

[0081] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is shown in any one of SEQ ID NOs: 38, 39, 42, and 43.

[0082] The fifth aspect of the present invention provides a polynucleotide encoding the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, or the antibody targeting CD33 as described in the second aspect of the present invention, or the recombinant protein as described in the third aspect of the present invention, or the chimeric antigen receptor as described in the fourth aspect of the present invention.

[0083] In another preferred example, the polynucleotide encodes the heavy chain variable region targeting CD33, and its nucleotide sequence is shown in SEQ ID NO: 9 or 12.

[0084] In another preferred example, the polynucleotide encodes the antibody targeting CD33, and its nucleotide sequence is shown in SEQ ID NO: 7 or 10.

[0085] In another preferred example, the polynucleotide encodes the chimeric antigen receptor, and its nucleotide sequence is shown in any one of SEQ ID NO: 36, 37, 40, and 41.

[0086] The sixth aspect of the present invention provides an expression vector comprising the polynucleotide as described in the fifth aspect of the present invention.

[0087] In another preferred embodiment, the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0088] In another preferred embodiment, the viral vector includes a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, and the like.

[0089] The seventh aspect of the present invention provides a host cell, which contains the expression vector as described in the sixth aspect of the present invention, or the polynucleotide as described in the fifth aspect of the present invention is integrated into its genome.

[0090] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0091] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli and yeast cells.

[0092] The eighth aspect of the present invention provides an engineered immune cell, the cell surface expressing the chimeric antigen receptor as described in the fourth aspect of the present invention.

[0093] In another preferred embodiment, the immune cells are T cells or NK cells.

[0094] The ninth aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0095] (a) an antibody portion, which is selected from the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, or the antibody targeting CD33 as described in the second aspect of the present invention, or the recombinant protein as described in the third aspect of the present invention;

[0096] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0097] In another preferred embodiment, the coupling part is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0098] The tenth aspect of the present invention provides a pharmaceutical composition, comprising: (a) the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, or the antibody targeting CD33 as described in the second aspect of the present invention, or the recombinant protein as described in the third aspect of the present invention, or the engineered immune cell as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention; and

[0099] (b) a pharmaceutically acceptable carrier.

[0100] In another preferred embodiment, the pharmaceutical composition is prepared as an injection form.

[0101] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating a disease associated with CD33.

[0102] In another preferred embodiment, the disease associated with CD33 includes autoimmune disease or tumor.

[0103] In another preferred embodiment, the tumor is a blood tumor, such as leukemia.

[0104] In another preferred embodiment, the tumor is acute myeloid leukemia (AML).

[0105] The eleventh aspect of the present invention provides a method for producing an antibody targeting CD33, comprising the steps of:

[0106] (i) culturing the host cell according to the seventh aspect of the present invention under conditions suitable for protein expression, thereby obtaining a culture containing the antibody targeting CD33; and

[0107] (ii) isolating or recovering the antibody targeting CD33 from the culture.

[0108] The twelfth aspect of the present invention provides a method for producing the engineered immune cells as described in the eighth aspect of the present invention, the method comprising the steps of:

[0109] (a) providing immune cells to be modified; and

[0110] (b) transducing the polynucleotide molecule as described in the fifth aspect of the present invention or the vector as described in the sixth aspect of the present invention into the immune cell, thereby obtaining the engineered immune cell.

[0111] In another preferred embodiment, the method further comprises the step of testing the function and effectiveness of the obtained engineered immune cells.

[0112] In a thirteenth aspect, the present invention provides a use of the CD33-targeting heavy chain variable region as described in the first aspect of the present invention, the CD33-targeting antibody as described in the second aspect of the present invention, the recombinant protein as described in the third aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, for preparing a reagent, a detection plate or a kit for detecting CD33 protein;

[0113] In another preferred embodiment, the detection includes flow cytometry detection and ELISA detection.

[0114] In another preferred embodiment, the reagent, detection plate or kit is used to diagnose a disease associated with CD33.

[0115] In a fourteenth aspect, the present invention provides a use of the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, the antibody targeting CD33 as described in the second aspect of the present invention, the recombinant protein as described in the third aspect of the present invention, the engineered immune cell as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention for preparing a medicament for treating a disease associated with CD33.

[0116] In another preferred embodiment, the disease associated with CD33 includes autoimmune disease or tumor.

[0117] In another preferred embodiment, the tumor is a blood tumor, such as leukemia.

[0118] In another preferred embodiment, the tumor is acute myeloid leukemia (AML).

[0119] A fifteenth aspect of the present invention provides a method for detecting CD33 protein in a sample, comprising the steps of:

[0120] (i) contacting the sample with the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, the antibody targeting CD33 as described in the second aspect of the present invention, the recombinant protein as described in the third aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention;

[0121] (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD33 protein in the sample.

[0122] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0123] In another preferred embodiment, the method is an in vitro method.

[0124] In a sixteenth aspect, the present invention provides a method for treating a disease associated with CD33, the method comprising: administering to a subject in need thereof the heavy chain variable region targeting CD33 as described in the first aspect of the present invention, the antibody targeting CD33 as described in the second aspect of the present invention, the recombinant protein as described in the third aspect of the present invention, the engineered immune cell as described in the eighth aspect of the present invention, the immunoconjugate as described in the ninth aspect of the present invention, or the pharmaceutical composition as described in the tenth aspect of the present invention.

[0125] In another preferred embodiment, the subject in need thereof includes a human or a non-human mammal.

[0126] In another preferred embodiment, the disease associated with CD33 includes autoimmune disease or tumor.

[0127] In another preferred embodiment, the tumor is a blood tumor, such as leukemia.

[0128] In another preferred embodiment, the tumor is acute myeloid leukemia (AML).

[0129] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 The basic process of the present invention for screening specific antibodies targeting CD33 from a phage antibody library is shown.

[0131] Figure 2 The results of enzyme-linked immunosorbent assay (ELISA) of some selected phage monoclones with target antigen and control antigen are shown.

[0132] Figure 3 The results of flow cytometric analysis of the binding of some selected phage monoclones to MM.1S-CD33 and MM.1S cells are shown. Control 1 is a negative control for phage, Control 2 is a negative control with only the first antibody (Anti-M13 phagemouse Ab) and the second antibody (anti-mouse HRP Ab), Control 3 is a negative control with only the second antibody (anti-mouse HRP Ab), and Control 4 is a positive control with the addition of CD33 antibody.

[0133] Figure 4 Shown are the binding results of the detection antibody HIM3-4 to various cell lines used in this protein characterization.

[0134] Figure 5 The results of flow cytometric analysis of the screened antibody clones binding to various cell lines are shown. APC-antihuman IgG Ab is a negative control with only the second antibody added, cell only is a negative control without the antibody added, and GO is a CD33 positive control antibody.

[0135] Figure 6The results of ELISA analysis of the screened antibody clones with various CD33 antigen proteins and non-related antigens are shown. GO / anti-human IgG HRP Ab is a CD33 positive control antibody (gemtuzumabozogamicin, GO), Anti-Human IgG HRP Ab is a negative control with only a secondary antibody added; Anti-his HRP Ab is an antibody for detecting antigen tags; the bar graphs corresponding to each test antibody and control group represent the test results with reagents Acro-CD33-His-Bio, SB-CD33-His-Bio, SB-cyno CD33 his, SB-mouse CD33 his, KACTUS-BAFFR-his-Bio, and SA from left to right.

[0136] Figure 7 The degranulation function of CAR-T cells constructed by screening specific clones under different target cell stimulation is shown. Among them, the target cells are U937 (CD33 positive, RS12459419 CT genotype), HL60 (CD33 positive, RS12459419 CC genotype), NB4 (CD33 positive, RS12459419 CC genotype), NB4-D2 (CD33 positive, gene edited to RS12459419 TT genotype), K562 (CD33 weakly positive, RS12459419 TT genotype), NB4-CD33KO (CD33 negative, CD33 gene knockout), and Nalm6 (CD33 negative).

[0137] Figure 8 The results show that the CAR-T cells constructed by screening specific clones have the ability to kill different target cells in vitro.

[0138] Fig. 9 The degranulation function of CAR-NK cells constructed by screening specific clones under stimulation of different target cells is shown.

[0139] Fig.10 The results show that the CAR-NK cells constructed by screening specific clones have the ability to kill different target cells in vitro.

[0140] Fig.11 The anti-tumor efficacy of some CAR-NK cells was shown in the human acute myeloid leukemia Molm-13 immunodeficient tumor-bearing mouse model. DETAILED DESCRIPTION

[0141] After extensive and in-depth research, the inventors used a large-capacity phage antibody library to screen fully human CD33-specific antibodies, and evaluated the binding specificity of these antibodies at the protein level through ELISA and FACS experiments. Finally, several fully human antibody clones with good specificity were obtained.

[0142] Specifically, the inventors used different antibody libraries, and after recombinant CD33 protein panning and protein / cell alternating panning, a total of 364 monoclones were selected for enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) detection screening, of which 322 clones specifically bound to CD33-his-Bio protein and CD33-positive cells MM.1S-CD33, but not to control protein SA and CD33-negative cells MM.1S (protein panning, protein / cell alternating panning, ELISA and FACS screening). Different monoclonal sequences were obtained after sequencing. These clones were then expressed as IgG antibodies, and the binding specificity of these antibodies to various CD33-positive (MM.1S-CD33, NB4, 293CT-CD33) and negative cell lines (NB4-KO CD33, MM.1S, 293CT) was analyzed by FACS at the protein level. The binding specificity with CD33 proteins from different companies (Acro-CD33-His-Bio, SB-CD33-His-Bio), species-crossing proteins (SB-cyno CD33-His, SB-mouse CD33-His) and non-related proteins (KACTUS-BAFFR-his-Bio, SA) were analyzed by ELISA. These clones showed good binding and specificity on multiple cell lines and multiple protein antigens. The acquisition of these clones laid the foundation for the subsequent development of fully human CD33CAR-T products or antibody drugs. The overall project process is as follows Figure 1 shown.

[0143] the term

[0144] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0145] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0146] The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (called kappa and lambda) based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of the different classes of immunoglobulins are well known in the art.

[0147] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). This segment is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs in between are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chains constitute the antigen binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0148] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0149] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are from different animal species, such as a chimeric antibody having a variable region from a monoclonal antibody from a mouse, and a constant region from a human immunoglobulin (see, for example, U.S. Pat. No. 4,816,567 and U.S. Pat. No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Pat. No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0150] In the present invention, the antibodies may be monospecific, bispecific, trispecific, or more multispecific.

[0151] In the present invention, the antibody of the present invention also includes its conservative variants, which refers to a polypeptide formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present invention. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table A.

[0152] Table A

[0153] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0154] In the present invention, the antibody is an antibody targeting CD33. The present invention provides a high-specificity and high-affinity antibody against CD33, comprising a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) amino acid sequence, and the light chain comprises a light chain variable region (VL) amino acid sequence.

[0155] In one embodiment of the present invention, the heavy chain variable region (VH) of the antibody has the following complementarity determining regions (CDRs):

[0156] HCDR1 shown in SEQ ID NO:15, HCDR2 shown in SEQ ID NO:16, and HCDR3 shown in SEQ ID NO:17; and

[0157] The light chain variable region (VL) of the antibody has the following complementarity determining regions (CDRs):

[0158] LCDR1 shown in SEQ ID NO:13, LCDR2 having an amino acid sequence of EDS, and LCDR3 shown in SEQ ID NO:14;

[0159] In another embodiment of the present invention, the heavy chain variable region (VH) of the antibody has the following complementarity determining regions (CDRs):

[0160] HCDR1 shown in SEQ ID NO:20, HCDR2 shown in SEQ ID NO:21, and HCDR3 shown in SEQ ID NO:22; and

[0161] The light chain variable region (VL) of the antibody has the following complementarity determining regions (CDRs):

[0162] LCDR1 shown in SEQ ID NO:18, LCDR2 having an amino acid sequence of DNN, and LCDR3 shown in SEQ ID NO:19;

[0163] Preferably, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2; or

[0164] The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:6, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:5;

[0165] Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD33 binding affinity.

[0166] In another preferred embodiment, the sequence formed by adding, deleting, modifying and / or replacing at least one amino acid sequence is preferably an amino acid sequence with a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% (e.g. 96%, 97%, 98%, 99%, 100%).

[0167] Preferably, the antibody of the present invention is one or more of a full-length antibody protein, an antigen-antibody binding domain protein fragment, a bispecific antibody, a multispecific antibody, a single chain antibody (scFv), a single domain antibody (sdAb) and a single region antibody (Signle-domain antibody), as well as a monoclonal antibody or a polyclonal antibody prepared from the above antibodies. The monoclonal antibody can be prepared by a variety of approaches and techniques, including hybridoma technology, phage display technology, single lymphocyte gene cloning technology, etc. The mainstream is to prepare monoclonal antibodies from wild-type or transgenic mice by hybridoma technology.

[0168] The full-length antibody protein is a conventional full-length antibody protein in the art, which includes a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region. The heavy chain variable region and light chain variable region of the protein and the human heavy chain constant region and the human light chain constant region constitute a full-length human antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3 or IgG4.

[0169] The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from an animal-derived antibody, a chimeric antibody, a humanized antibody, a fully human antibody, and more preferably a fully human antibody.

[0170] The antibody derivatives of the present invention can be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0171] The single-chain antibody is a conventional single-chain antibody in the art, which includes a heavy chain variable region, a light chain variable region and a short peptide of 15 to 20 amino acids. Preferably, the single-chain antibody has an amino acid sequence as described in SEQ ID NO: 1 or 4.

[0172] The antibody of the present invention targets CD33, specifically, targets the IgC domain and / or IgV domain of CD33.

[0173] Chimeric Antigen Receptor (CAR)

[0174] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0175] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0176] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting CD33. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be caused to die or otherwise affected, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of a 4-1BB or CD28 signaling domain and a CD3ζ signaling domain.

[0177] As used herein, "antigen binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab') fragments, etc. 2 Fragment, or single Fv fragment. Fv antibody contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv. The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the antigen binding domain comprises an antibody that specifically recognizes CD33, preferably a single-chain antibody.

[0178] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0179] Preferably, the CAR of the present invention further comprises an enhancing element for enhancing the cell killing effect of CAR. The enhancing element includes (but is not limited to): tEGFR, IL-15, IL-2, IL-7, IL-18, IL-21, CCL19, CXCR2, CCR7, TGF-βDNR, c-Jun, hnCD16 or a combination thereof.

[0180] In a preferred embodiment of the present invention, the chimeric antigen receptor includes, from N-terminus to C-terminus, a CD8a signal peptide, an antigen binding domain targeting CD33, a CD8a hinge region, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and CD3ζ.

[0181] In another preferred embodiment, the chimeric antigen receptor includes, from N-terminus to C-terminus, a CD8a signal peptide, an antigen binding domain targeting CD33, a CD8a hinge region, a CD8a transmembrane domain, a CD28 co-stimulatory domain, CD3ζ, a P2A self-cleavage peptide, and IL-15.

[0182] Preferably, the amino acid sequence of the chimeric antigen receptor is as shown in any one of SEQ ID NO: 38, 39, 42, and 43.

[0183] Polynucleotide

[0184] The present invention also provides a polynucleotide encoding the above-mentioned antibody or its active fragment or its recombinant protein, or the CAR.

[0185] In another preferred example, the nucleotide sequence of the polynucleotide encoding the heavy chain variable region is shown in SEQ ID NO:9 or 12; and / or, the nucleotide sequence of the polynucleotide encoding the light chain variable region is shown in SEQ ID NO:8 or 11.

[0186] More preferably, the nucleotide sequence of the polynucleotide encoding the heavy chain variable region is shown as SEQ ID NO:9; and the nucleotide sequence of the polynucleotide encoding the light chain variable region is shown as SEQ ID NO:12.

[0187] In another preferred example, the nucleotide sequence of the polynucleotide encoding the single-chain antibody is as shown in SEQ ID NO: 7 or 10.

[0188] In another preferred example, the nucleotide sequence of the polynucleotide encoding the CAR is shown in any one of SEQ ID NO: 36, 37, 40, and 41.

[0189] The method for preparing the polynucleotide is a conventional method in the art, and preferably comprises the following steps: obtaining a polynucleotide molecule encoding the above protein by gene cloning technology, or obtaining a polynucleotide molecule encoding the above protein by artificial full sequence synthesis.

[0190] Those skilled in the art know that the base sequence encoding the amino acid sequence of the above-mentioned protein can be appropriately introduced with substitution, deletion, change, insertion or addition to provide a polynucleotide homologue. The polynucleotide homologue of the present invention can be prepared by replacing, deleting or adding one or more bases of the gene encoding the protein sequence within the range of maintaining the antibody activity.

[0191] Carrier

[0192] The present invention also provides a recombinant expression vector comprising the polynucleotide.

[0193] The recombinant expression vector can be obtained by conventional methods in the art, that is, the polynucleotide molecule of the present invention is connected to various expression vectors. The expression vector is any conventional vector in the art, as long as it can carry the aforementioned polynucleotide molecule. The vector preferably includes various plasmids, cosmids, phages or virus vectors, etc.

[0194] The present invention also provides a recombinant expression transformant comprising the recombinant expression vector.

[0195] Wherein, the preparation method of the recombinant expression transformant is a conventional preparation method in the art, preferably: the above-mentioned recombinant expression vector is transformed into a host cell to obtain it. The host cell is various host cells conventional in the art, as long as it can satisfy the above-mentioned recombinant expression vector to stably replicate itself, and the polynucleotide carried can be effectively expressed. Preferably, the host cell is E.coli TG1 or E.coli BL21 cell (expressing single-chain antibody or Fab antibody), or HEK293 or CHO cell (expressing full-length IgG antibody). The aforementioned recombinant expression plasmid is transformed into a host cell to obtain a preferred recombinant expression transformant of the present invention. Wherein the transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0196] Chimeric Antigen Receptor T Cells (CAR-T Cells)

[0197] As used herein, the terms "CAR-T cell", "CAR-T", and "CAR-T cell of the present invention" include the CAR-T cell comprised in the third aspect of the present invention.

[0198] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction targeting a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; and (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0199] Chimeric Antigen Receptor NK Cells (CAR-NK Cells)

[0200] As used herein, the terms "CAR-NK cells", "CAR-NK", and "CAR-NK cells of the present invention" all refer to the CAR-NK cells included in the third aspect of the present invention. The CAR-NK cells of the present invention can be used to treat tumors with high expression of CD33, such as AML.

[0201] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxic effects.

[0202] Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are very easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0203] Antibody preparation

[0204] The sequence of the DNA molecule of the antibody of the present invention or its fragment can be obtained by conventional techniques, such as PCR amplification or genomic library screening. In addition, the coding sequences of the light chain variable region and the heavy chain variable region can be fused together to form a single-chain antibody.

[0205] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0206] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0207] At present, the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0208] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0209] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S, HEK-293 cells.

[0210] Typically, the transformed host cells are cultured under conditions suitable for the expression of the antibodies of the present invention, and then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, etc., conventional separation and purification means well known to those skilled in the art to obtain the antibodies of the present invention.

[0211] The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0212] The antibodies of the present invention can be expressed in cells, on cell membranes, or secreted outside cells. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0213] Pharmaceutical composition

[0214] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein or its immunoconjugate or corresponding immune cells, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the disease to be treated.

[0215] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0216] The antibody of the present invention can also be expressed in cells by nucleotide sequences for cell therapy, for example, the antibody is used for chimeric antigen receptor T cell immunotherapy (CAR-T) or chimeric antigen receptor NK cell immunotherapy (CAR-NK), etc.

[0217] The pharmaceutical composition of the present invention is a pharmaceutical composition for preventing and / or treating diseases associated with abnormal CD33 expression or function.

[0218] The pharmaceutical composition of the present invention can be directly used to bind to CD33 protein molecules, and thus can be used to prevent and treat diseases such as tumors.

[0219] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned monoclonal antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0220] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0221] In the present invention, preferably, the amount of the pharmaceutical composition administered is an effective amount, which is an amount that can alleviate or delay the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the results sought. Those skilled in the art can determine the effective amount by using the above factors on an individual basis and using experiments that do not exceed routine.

[0222] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 20 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0223] The present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating a disease associated with abnormal expression or function of CD33. Preferably, the disease associated with abnormal expression or function of CD33 is an autoimmune disease or a tumor.

[0224] application

[0225] The antibodies or immunoconjugates thereof of the present invention can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.

[0226] In the present invention, the sample (specimen) used includes cells, tissue samples and biopsy specimens. The term "biopsy" used in the present invention should include all types of biopsies known to those skilled in the art. Therefore, the biopsy used in the present invention can include, for example, a resection sample of a tumor, a tissue sample prepared by endoscopic methods or puncture or needle biopsy of an organ. The sample used in the present invention includes a fixed or preserved cell or tissue sample.

[0227] The antibodies or immunoconjugates thereof of the present invention or their corresponding immune cells (CAR-T or CAR-NK cells) can be used for therapeutic and / or preventive applications, and for the preparation of drugs for the prevention and / or treatment of diseases associated with abnormal CD33 expression or function. Preferably, the diseases associated with abnormal CD33 expression or function are autoimmune diseases or tumors.

[0228] The beneficial effects of the present invention include:

[0229] (1) The CD33-targeting antibody of the present invention has strong specificity and does not bind to CD33-negative cell lines or irrelevant proteins.

[0230] (2) Since the CD33 splice isoform lacks the IgV domain, antibodies targeting the CD33 IgV domain (e.g., gemtuzumab) cannot bind to CD33. However, the clone 74 antibody provided by the present invention can bind to all CD33s because its binding site is in the CD33 IgC domain.

[0231] (3) The fully human antibodies provided by the present invention specifically bind to CD33 with high affinity and have lower immunogenicity compared with heterologous antibodies; they have great application potential in the development of antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.) and cell therapy drugs (including CAR-T, CAR-NK, etc.); in addition, the fully human antibodies can also be used in the development of detection reagents.

[0232] (4) The present invention uses a protein panning method in the antibody screening process, which can efficiently enrich antibodies that bind to recombinant CD33 protein, greatly reducing the difficulty of subsequent antibody screening and improving efficiency.

[0233] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0234] Example 1. Enrichment of specific antibody clones targeting CD33 protein from phage antibody library by affinity panning

[0235] Appropriate negative and positive panning strategies are used to enrich the desired specific antibody clones from the phage antibody library.

[0236] Construction of phage antibody library

[0237] The phage antibody library constructed by the present invention includes a natural library, a semi-synthetic library and a single domain library. The semi-synthetic phage antibody library, used together with the natural library, solves the problem that the natural library may lack CD33 high-affinity antibody clones. The single-domain phage antibody library is an antibody library composed only of variable region amino acids of heavy chain antibodies, with a molecular weight of only 12-15kDa, but has similar or higher specificity and affinity than traditional antibodies. In addition, single-domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies. CD33 is an antigen expressed by normal cells in the human body. For this type of antigen, the body will inactivate those cells that can express CD33 antibodies during development through the mechanism of clonal screening, resulting in a lack of high-affinity antibodies for this type of antigen in the normal human body. Clonal screening is a normal self-recognition and self-protection mechanism of the body. However, the most commonly used phage antibody library form is a natural library, which is constructed by a method of directly cloning antibody genes in healthy human lymphocytes, and is likely to lack antibody clones for normally existing antigens in the human body such as CD33. For this reason, the inventors not only constructed a natural library when constructing an antibody library, but also constructed a semi-synthetic antibody library. The semi-synthetic antibody library is composed of a light chain from a natural antibody sequence and a heavy chain FR1-FR3 and an artificially designed heavy chain CDR3, which can greatly increase antibody diversity and improve the chance of screening for high-affinity antibodies for normally existing antigens in the body.

[0238] CD33 protein panning

[0239] Multiple rounds of panning were performed using CD33-His as the positive panning protein to obtain a phage pool enriched with the target antibody clone. The experimental steps are briefly described as follows:

[0240] 1) Block the SA magnetic beads with blocking solution for 2 hours, and then bind the target antigen (CD33-His) to the blocked SA magnetic beads;

[0241] 2) Add phage library (containing 5x10 12 phage particles) and a clean SA magnetic bead were incubated together to remove the phage antibody clones that non-specifically bound to the SA magnetic beads;

[0242] 3) After incubation, the supernatant is transferred to SA magnetic beads bound to the target antigen and incubated further to allow the phage to bind to the target antigen;

[0243] 4) Wash the magnetic beads with washing solution to remove unbound phages;

[0244] 5) Elute the positive phage from the target antigen with an elution solution and add a neutralization solution to neutralize it;

[0245] 6) Re-infect the host bacteria XL1-blue with the eluted phages to amplify the recovered phages. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phages;

[0246] 7) Repeat steps 1) to 6), usually 3 to 4 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0247] The enriched phage pool can be used for subsequent single clone selection and ELISA / FACS screening.

[0248] MM.1S-CD33 / MM.1S cell selection

[0249] Multiple rounds of panning were performed using CD33-negative MM.1S cells as negative panning cells and CD33-positive MM.1S-CD33 cells as positive panning cells to obtain a phage pool enriched with the target antibody clone.

[0250] The brief experimental steps are as follows:

[0251] 1) The phage pool (containing 5x10 11 phage particles) and 1x10 7 Mix the negative selection cells MM.1S and incubate at room temperature for 2 hours on a rotating mixer to allow the antibody clones that bind to the negative selection cell line to fully bind to these cells;

[0252] 2) Centrifuge at 1500 rpm for 5 minutes to pellet the cells, transfer the supernatant to a new tube, and mix with 1x10 7MM.1S-CD33 cells (CD33 positive cells) were mixed and allowed to bind on a rotating mixer at room temperature for 2 hours;

[0253] 3) Wash the cells 6 times with PBS, discard the supernatant each time, resuspend and centrifuge at 1500 rpm for 5 minutes to remove unbound phages;

[0254] 4) Elute the positive phage from the target antigen with an elution solution and add a neutralization solution to neutralize it;

[0255] 5) Use the eluted phage to re-infect the host bacteria and amplify the recovered phage. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phage;

[0256] 6) Repeat steps 1) to 5), usually 2 to 3 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0257] The enriched phage pool can be used for the next step of monoclonal selection and ELISA / FACS screening.

[0258] Main materials and reagents:

[0259] Fully human phage antibody library, including natural library, semi-synthetic library and single domain library;

[0260] Helper phage KO7, Thermo / Invitrogen, 18311019;

[0261] Biotinylated Human Siglec-3 / CD33 Protein,Avitag TM ,His Tag,ACRObiosystem,CD3-H82E7;

[0262] BeaverBeads TM Streptavidin, Beaver Bio, 22307-10;

[0263] High binding ELSIA plate,Costar,#3590

[0264] Blocking solution: PBS + 3% BSA

[0265] Rinse solution: PBS + 0.1% Tween20

[0266] Eluent: 0.2M Glycine, pH 2.2

[0267] Neutralization solution: 1M Tris, pH 9.1

[0268] Experimental results:

[0269] Using different antibody libraries, three rounds of protein panning were performed, and a significant increase in the recovery rate was observed in each panning (Table 1), demonstrating that the antibody clones were effectively enriched.

[0270] Table 1 Protein panning experimental results

[0271]

[0272] It can be seen that after three rounds of panning, different antibody libraries have been enriched (the recovery rate in the third round was significantly higher than that in the previous round). However, in subsequent FACS experiments, the clones selected from these phage pools were less bound to the MM.1S-CD33 cell line that highly expressed the CD33 antigen, that is, they could not recognize the CD33 antigen in its natural state on the cell surface. Therefore, in subsequent experiments, the method of alternating protein and cell panning was used to enrich specific antibody clones that can simultaneously bind to CD33 protein and CD33 in its natural state on the cell surface from the phage antibody library. Table 2 shows the results of combined panning using recombinant CD33 protein and MM.1S-CD33 / MM.1S cell line. In terms of recovery rate, all four pannings were enriched and can be used for the next step of selecting single clones.

[0273] Table 2 Protein / cell selection experiment results

[0274]

[0275]

[0276] Example 2. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) from enriched phage Pool screening for specific clones

[0277] Purpose and Principle: The phage pool enriched by affinity panning contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. In order to obtain specific clones, it is necessary to separate monoclonal clones, package them into monoclonal phages, and screen a large number of monoclonal clones by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), and select monoclonal clones that specifically bind to CD33 protein and CD33-positive cell line MM.1S-CD33 at the same time. The specific monoclonal clones are further sequenced by DNA to determine the unique antibody sequence contained therein.

[0278] In the ELISA primary screening, the biotinylated target protein (CD33-his-Bio) is closer to the antigen conformation of the natural state in the reaction solution through the binding of streptavidin and biotin. Those that only bind to CD33-his-Bio but not to streptavidin are identified as specific clones. The FACS primary screening is carried out using the positive cell line MM.1S-CD33 with high expression of CD33 and the CD33-negative cell line MM.1S. Those that only bind to MM.1S-CD33 cells and not to MM.1S cells are identified as specific clones. Through the two primary screenings of ELISA and FACS, candidate antibodies that can bind to recombinantly expressed CD33 protein and recognize CD33 molecules in the natural state on the cell surface can be obtained for subsequent further screening.

[0279] Brief steps of ELISA experiment:

[0280] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0281] 2) Dilute Strepavidin to 2 μg / mL with PBS, add 100 μL / well to the high binding ELISA plate, and bind for 2 h at room temperature;

[0282] 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C;

[0283] 4) Wash the plate twice with 250 μL of rinse solution;

[0284] 5) Dilute the biotin-tagged target protein to 2 μg / mL with PBS, add 100 μg / well to the ELISA plate pre-coated with Strepavidin, and bind at room temperature for 1 hour;

[0285] 6) Wash the plate twice with 250 μL of rinse solution;

[0286] 7) Add 100 μL of the cultured phage supernatant in step 1) to the wells coated with the target antigen and allow to bind for 2 h at room temperature;

[0287] 8) Wash the plate 4 times with 250 μL of rinse solution;

[0288] 9) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, and incubate at room temperature for 45 min;

[0289] 10) Wash the plate 4 times with 250 μL of rinse solution;

[0290] 11) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL / well, and incubate at room temperature for 45 min;

[0291] 12) Wash the plate 6 times with 250 μL of rinse solution;

[0292] 13) Add 100 μL TMB colorimetric substrate and allow color to develop for 5 to 10 minutes;

[0293] 14) Add 100 μL 2M H 2 SO 4 Stop the reaction and read the results on a microplate reader.

[0294] Brief steps of FACS screening experiment:

[0295] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0296] 2) MM.1S-CD33 and MM.1S cells were washed twice with PBS and resuspended in PBS to 1x10 7 / mL concentration, and dispensed into 96-well deep-well plates at 50 μL;

[0297] 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and allow to bind at 4°C for 2 h;

[0298] 4) Wash twice with 200 μL PBS;

[0299] 5) Add 1:2000 diluted mouse anti-M13 primary antibody, 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0300] 6) Wash twice with 200 μL PBS;

[0301] 7) Add 1:300 diluted FITC horse anti mouse-IgG (H+L), 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0302] 8) Wash twice with 200 μL PBS; finally resuspend the cells with 200 μL PBS;

[0303] 9) Detect the fluorescence intensity of the sample FITC channel on a flow cytometer and analyze the results.

[0304] Main materials and reagents:

[0305] Helper phage KO7, Thermo / Invitrogen, 18311019

[0306] Streptavidin,Pierce,21125

[0307] Biotinylated Human Siglec-3 / CD33 Protein,Avitag TM ,His Tag,ACRObiosystem,CD3-H82E7;

[0308] High binding ELSIA plate,Costar,#3590

[0309] Corning 96Well Clear Round Bottom TC-Treated Microplate,Costar,#3799

[0310] Blocking solution: PBS + 3% BSA

[0311] Rinse solution: PBS + 0.1% Tween20

[0312] Soluble one-component TMB substrate solution, Tiangen, PA-107-02

[0313] Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225

[0314] HRP Goat anti-mouse IgG(minimal x-reactivity)Antibody,Biolegend,405306

[0315] Fluorescein(FITC)AffiniPure Goat Anti-Mouse IgG(H+L),JacksonImmunoReseach,115-095-003

[0316] Experimental results:

[0317] Monoclonal clones were randomly selected from the enriched phage antibody pool, packaged into phage, and then tested for binding of monoclonal phage to CD33-his-Bio protein and SA protein by phage ELISA to find CD33-specific phage antibody clones. The ELISA results of some example clones are shown in Figure 1. Figure 2 As shown in the figure, clones H1, H2, H3, H4, H5, H6, and H7 bind well to the target antigen CD33 (CD33-his-Bio) and do not bind to the control antigen streptavidin, showing good specificity.

[0318] Negative Control is a negative control phage antibody clone. Anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative control with only the first antibody (Anti-M13 phage mouse Ab) and the second antibody (anti-mouse HRP Ab) added without phage; anti-mouse HRP Ab is a negative control with only the second antibody (anti-mouse HRPAb) added, which has no reaction with both antigens, indicating that the antibody has no non-specific binding; mouse anti-humanCD33 Ab is a positive control with the addition of CD33 detection antibody, which binds well to the target antigen and does not bind to SA, indicating that the antigen activity of this experiment is good.

[0319] The FACS screening results of some sample clones are as follows: Figure 3 As shown. Control 1 is the negative control of phage, Control 2 is the negative control of adding only the first antibody (Anti-M13 phage mouse Ab) and the second antibody (anti-mouse HRP Ab), Control 3 is the negative control of adding only the second antibody (anti-mouse HRP Ab), and Control 4 is the positive control of adding CD33 antibody. Among them, H1, H4, H6 and H7 clones do not bind to MM.1S, but bind to MM.1S-CD33 cells, and are specific clones; other clones are non-specific (bind to both cells) or negative clones (do not bind to both cells).

[0320] Based on the preliminary screening of ELISA and FACS, the exemplary clones that were double positive by ELISA and FACS were considered to be positive specific clones.

[0321] Antibodies targeting CD33 antigen were enriched from phage antibody library by affinity panning and clones binding to CD33 antigen were obtained through preliminary screening. However, after the antibody molecules expressed in prokaryotic system were converted into IgG antibody molecules expressed in eukaryotic system, their binding ability and specificity needed to be further confirmed. To this end, IgG expression plasmids of these clones were prepared, expressed by transient transfection of 293 cells, and purified from Protein A to antibodies. Then, the binding specificity of monoclonal was further determined by ELISA and FACS.

[0322] Example 3. Obtaining the binding specificity of antibodies to multiple cell lines using FACS analysis at the protein level Experimental purpose and principle:

[0323] Antibodies used for treatment must have very good target specificity, only binding to the target antigen, and not binding to any unrelated antigens; on the other hand, the amino acid sequence of the same antigen on different cell lines may be different (isomers or mutants) or the bound ligands may be different, and it is also necessary to examine whether the antibodies of the present invention can bind to cells that are positive for various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, the specificity of the initial screening clones was further evaluated by flow cytometry. In this experiment, a variety of CD33-positive cell lines and a variety of CD33-negative cell lines were used to react with these monoclonal phage antibodies to analyze whether these clones can bind to the CD33 antigen on different cell lines, and whether there is any non-specific binding with other cell lines that do not express CD33. Through this experiment, several clones with excellent specificity were obtained.

[0324] Experimental method: Same as the FACS primary screening in Example 2;

[0325] Main samples and reagents:

[0326] BD OptiBuild TM BV421 Mouse Anti-Human CD33, BD, 744350, HIM3-4;

[0327] Alexa 647 AffiniPure Goat Anti-Human IgG,Fcγfragmentspecific,Jackson ImmunoReseach,109-605-008

[0328] MM.1S-CD33 cell line, a cell line overexpressing full-length CD33, with high-density expression;

[0329] MM.1S cell line, a CD33-negative cell line;

[0330] NB4, CD33 full-length expression cell line, low-density expression;

[0331] NB4 KO CD33 cell line, CD33-negative cell line;

[0332] 293CT-CD33, CD33 C domain overexpressing cell line;

[0333] 293CT cell line, CD33-negative cell line;

[0334] The remaining reagents were the same as those for the FACS primary screening.

[0335] Experimental results:

[0336] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, the specific clones obtained in Example 2 were identified by enzyme-linked immunosorbent assay and flow cytometry on more antigens and cell lines. The results of flow cytometry detection of cell binding are shown in Figure 4-5 middle, Figure 4 To test the results of antibody HIM3-4 staining on various cell lines used in this experiment, the binding epitope of this detection antibody is on the CD33 C domain, so it can bind to both full-length and Cdomain-expressing cells. The staining results with NB4, 293CT-CD33, and MM.1S-CD33 are positive, and the staining results with NB4-KO CD33, 293CT, and MM.1S are negative; Figure 5 The flow cytometry results of the binding of the cloned proteins screened in the present invention (the concentration of the whole antibody is 2ug / mL, and the concentration of the single domain antibody (SdAb) is 1ug / mL) to each cell line: APC-anti human IgG Ab is a negative control with only the second antibody added, cell only is a negative control without the addition of antibody, and GO / APC anti-human IgG Ab is a CD33 positive control antibody (gemtuzumab ozogamicin, GO). The MM.1S-CD33 cell line overexpresses the full-length CD33 antigen with a high expression density, and the 7 clones in the example (clones 2, 4, 23, 66, 74, 76 and 82) can all bind; the NB4 cell line naturally expresses the full-length CD33 antigen with a relatively low expression density, clone 66SdAb binds weakly, and the other 6 example clone antibodies can bind strongly; 293CT-CD33 cells overexpress the CD33C domain antigen, and clone 82SdAb can bind strongly, indicating that the epitope of clone 82SdAb antibody is on the C domain; the flow cytometry results of the three cells with negative CD33 antigen expression, NB4-CD33 KO, MM.1S, and 293CT, were all negative, indicating that the 7 example clone antibodies screened can specifically recognize and bind to cells expressing CD33.

[0337] Example 4. Obtaining the binding specificity of antibodies to different antigens using ELISA analysis at the protein level

[0338] Purpose and principle of the experiment: In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, the specificity of the initial screening clones was further evaluated by enzyme-linked immunosorbent assay (ELISA). In this experiment, CD33 antigens purchased from different companies and a variety of CD33-unrelated antigens were used to react with these monoclonal antibodies to analyze whether these clones can bind to different CD33 antigens, whether they have cross-species activity, and whether they have any non-specific binding with other CD33-unrelated antigens. Through this experiment, several clones with excellent specificity were obtained.

[0339] Experimental method: Same as the ELISA primary screening in Example 2;

[0340] Main samples and reagents:

[0341]

[0342] The remaining reagents are the same as those for the ELISA primary screening.

[0343] Experimental results:

[0344] Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, multiple specific clones obtained in Example 2 were identified using enzyme-linked immunosorbent assay (ELISA) on multiple antigens. The results are shown in Figure 6 In the table, GO / anti-human IgG HRP Ab is the CD33 positive control antibody (gemtuzumab, GO), Anti-Human IgG HRP Ab is the negative control with only the second antibody added; Anti-his HRP Ab is the antibody for detecting the antigen tag, indicating that the coated antigens are correctly bound to the plate. Clones 2, 4, 23, 66, 74, 76 and 82 bind to both CD33 antigens (Acro-CD33-His-Bio, Kactus-CD33-his), clones 74 and 82 antibodies bind to SB-cyno CD33-His antigen and have monkey cross-reactivity. None of these 7 antibodies bind to SB-mouse CD33-His antigen, indicating that none of them has mouse cross-reactivity, and none of them bind to two unrelated antigens (KACTUS-BAFF R-bio-his, SA), indicating that these 7 antibodies have good specificity.

[0345] Based on the identification results of both Example 3 and Example 4, clones 2, 4, 23, 66, 74, 76, and 82 were considered to be specific binding clones.

[0346] In summary, the present invention uses fully human phages for antibody screening to directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, the difficult step of humanizing mouse antibodies is omitted, and fully human antibodies have lower immunogenicity than humanized mouse antibodies, and have better potential in applications such as antibody drugs, CAR-T, and detection reagents.

[0347] During the antibody screening process, the inventors found that the antibody clones directly screened using the recombinantly expressed CD33 protein had less binding to the cell line MM.1S-CD33 that highly expressed CD33. This may be due to the fact that the recombinantly expressed CD33 protein antigen and the natural state CD33 on the cell membrane surface have great differences in conformation and accessible antigen epitopes. In order to overcome this problem, the protein / cell line alternating panning method was used to enrich phage antibodies that can simultaneously bind to the recombinantly expressed CD33 protein and MM.1S-CD33 cells, and monoclonal antibodies that can specifically bind to the CD33 antigen on the cell membrane surface were screened.

[0348] Example 5. In vitro functional evaluation of CD33CAR-T

[0349] Experimental purpose and principle:

[0350] Acute myeloid leukemia (AML) is a type of blood system malignancy that seriously threatens human health. Currently, the treatment efficacy of AML other than acute promyelocytic leukemia is not optimistic, and the prognosis is mostly poor. Traditional chemotherapy drugs cannot fundamentally solve the huge problems of relapse and drug resistance, while hematopoietic stem cell transplantation is expensive and risky, and there is also the possibility of relapse after transplantation.

[0351] Chimeric antigen receptor T cells (CAR-T) refer to T cells that can recognize specific target antigens in an MHC-unrestricted manner and continuously activate and proliferate after genetic modification. Biological immune cell therapy represented by CAR-T has become the fourth means of treating tumors in addition to surgery, radiotherapy, and chemotherapy, and will become an essential means of tumor treatment in the future. CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve the survival of patients. Chimeric antigen receptor (CAR) is the core component of CAR-T, usually a tumor antigen-associated antigen (TAA) binding region (usually derived from scFv or VH of monoclonal antibodies), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Using scFv or single-domain antibodies derived from the CD33-specific antibodies obtained in the above examples, CAR molecules are constructed for in vitro functional evaluation of CAR-T cells, and CD33CAR-T cells that may be used for AML treatment are obtained.

[0352] Experimental methods:

[0353] (1) Construction and preparation of lentiviral vectors

[0354] Taking the second-generation CAR molecular structure as an example, first, the following nucleotide sequences are artificially synthesized: Kozak (whose nucleotide sequence is GCCGCCACC), CD8a signal peptide (whose amino acid sequence is SEQ ID NO: 23), scFv or VH (including scFv of clones 76 and 74, VH of clone 82 (VH82), VH of clone 66 (VH66), VH of clones 4 and 23 (VH23+VH04), as well as scFv of Gemtuzumab, scFv of HIM3-4), hinge region (whose amino acid sequence is SEQ ID NO: 24), transmembrane region (whose amino acid sequence is SEQ ID NO: 25), co-stimulatory factor (whose amino acid sequence is SEQ ID NO: 26), CD3ζ intracellular signaling domain (whose amino acid sequence is SEQ ID NO: 32), 2A cleavage peptide (whose amino acid sequence is SEQ ID NO: 33), tEGFR (whose amino acid sequence is SEQ ID NO: 57, and the encoding nucleotide sequence is shown in SEQ ID NO: 34). The nucleotide sequence of the above elements can be optimized according to the codon usage, but the encoded amino acid sequence remains unchanged. The synthesized CAR molecule sequence is loaded into a lentiviral vector, such as pLKO or pCDH, by a molecular cloning method to obtain a lentiviral vector shuttle plasmid (Transfer).

[0355] Then, the shuttle plasmid and other packaging plasmids (encoding Gag-pol, Rev and VSV-G, respectively) are co-transfected into HEK293T cells to prepare the packaging of the lentiviral vector. Specific operation steps are as follows:

[0356] HEK293T cells were revived and cultured in DMEM medium containing 10% FBS. After 2-3 generations of cell expansion, 6×10 4 Pieces / cm 2 The density was inoculated into a ten-layer cell factory. Three days after cell inoculation, plasmid transfection was performed. The plasmid transfection solution was prepared with Opti-MEM, and the final concentration of the plasmid was 10μg / mL, which contained a suitable proportion of shuttle plasmid, pMDLg / pRRE plasmid (encoding Gag-pol), pRSV-Rev plasmid (encoding Rev) and pMD2.G plasmid (encoding VSV-G). In addition, PEI with a final concentration of 30μg / mL was added to the plasmid transfection solution, mixed thoroughly, and used after incubation at room temperature for 30 minutes. Each cell factory was transfected with 100mL of plasmid transfection solution.

[0357] After 72 hours, collect the supernatant into a centrifuge tube and centrifuge at 3000g for 10 minutes at 4℃. Filter the supernatant with a 0.45μm filter. Centrifuge the filtered supernatant at 27000g for 4 hours at 4℃. Discard the supernatant after centrifugation and resuspend the virus in PBS precooled at 4℃. Aliquot the resuspended virus and store at -80℃ for later use.

[0358] (2) Preparation of CAR-T cells

[0359] In this example, healthy donor cells were used to prepare CAR-T. The preparation process of CAR-T is as follows: On the first day, peripheral blood of healthy donors was collected, PBMCs were separated using Ficoll, and T cells were further sorted using CD3 MicroBeads. The sorted T cells were activated using CD3 / CD28 Dynabeads. After about 24 hours of activation (the second day), the prepared lentiviral vectors were added for transduction (MOI = 3). The T cell density during transduction was about 1.5×10 6 cells / mL. On day 3, the transduced T cells were replaced with a culture medium. After that, the cell density was maintained at (0.6-2.0)×10 6 The cell culture time is usually about 10-14 days, and the obtained CAR-T cells are used for in vitro functional evaluation or animal experiments.

[0360] (3) CD107a degranulation experiment

[0361] CD107a is a marker of intracellular microvesicles. When microvesicles loaded with granzymes fuse with the cell membrane, CD107a on the cell membrane will increase. When monesin (purchased from BioLegend) is used to block its recovery, the intensity of microvesicle release can be quantitatively reflected. Therefore, when CAR-T cells are stimulated by target cell surface antigens to produce degranulation effects, the positive rate of CD107a on the surface of CAR-T cells can be detected by flow cytometry to determine the activation of CAR-T cells.

[0362] Specific operation steps: (a) Centrifuge the target cells at room temperature and 300g for 5 min; discard the supernatant and resuspend them in T cell culture medium to 2×10 5cells / mL; (b) According to the CAR positive rate and E:T value (usually 0.3:1) of the CAR-T cells to be tested, the CAR-T cells were resuspended to an appropriate density, and monensin and PE / Cy7 mouse anti-humanCD107a antibodies were added; (c) In a U-bottom 96-well plate, 100 μL / well of the CAR-T cells to be tested and 100 μL / well of the target cells were added, mixed evenly, and then placed in an incubator (37°C, 5% CO 2 ) and incubate for 3 h; (d) after incubation, centrifuge at 4°C, 600g for 5 min, discard the supernatant, and wash the cells twice with 200 μL / well DPBS + 1% HSA; (e) resuspend the cells with 20 μL / well DPBS + 1% HSA, add APC mouse anti-human CD8 antibody and Alexa Fluor 488 anti-human EGFR antibody (or FITC-CD33 protein), mix the cells and incubate on ice in the dark for 20 min; (f) after incubation, wash the cells three times with 200 μL / well DPBS + 1% HSA, then resuspend the cells with 200 μL / well DPBS + 1% HSA and perform flow cytometry detection.

[0363] (4) In vitro cell killing assay

[0364] The target cells used to evaluate the in vitro cell killing function of CAR-T are usually cell lines that can stably express firefly luciferase (ffLuc) after being transformed by lentiviral vector transduction. In the in vitro cell killing experiment, CAR-T cells and target cells are co-cultured at different effector-target ratios (E:T). When the target cells are killed by CAR-T cells, luciferase is released and quickly inactivated. If the target cells are not killed or inhibited by CAR-T cells, more luciferase will be produced as the target cells proliferate and the luciferase continues to be expressed. Therefore, the killing of target cells by CAR-T can be detected by the activity of luciferase.

[0365] Specific operation steps: (a) The target cells expressing ffLuc were centrifuged at room temperature and 300 g for 5 min, the supernatant was discarded, and then resuspended in T cell complete medium to 2×10 5 cells / mL; 100 μL / well of target cells were added to a bottom-transparent 96-well plate; (b) according to the CAR positive rate and E:T value (e.g., 2:1, 1:1, 0.5:1) of the CAR-T cells to be tested, 100 μL / well of CAR-T cells were added to the 96-well plate, mixed with the target cells, and placed in an incubator (37°C, 5% CO 2) and incubate for 24 h; (c) after incubation, centrifuge at room temperature and 800 g for 5 min, and collect 100 μL / well supernatant as a sample for cytokine detection (stored at -80°C); (d) the luciferase activity in each well of the remaining cells after sampling was detected using a luciferase detection kit.

[0366] Experimental results:

[0367] (1) CD107a degranulation function of CD33CAR-T cells

[0368] The target cells used for the evaluation of degranulation function included U937 (CD33 positive, RS12459419 CT genotype), HL60 (CD33 positive, RS12459419 CC genotype), NB4 (CD33 positive, RS12459419CC genotype), NB4-D2 (CD33 positive, gene-edited to RS12459419 TT genotype), K562 (CD33 weakly positive, RS12459419 TT genotype), NB4-CD33KO (CD33 negative, CD33 gene knockout), and Nalm6 (CD33 negative) cell lines.

[0369] After CAR-T cells and target cells were co-incubated, the positive rate of CD107a in CAR-T cells was detected. Figure 7 As shown; among them, the scFv of PXL1696 is derived from the monoclonal antibody Gemtuzumab, and the antigen binding site is located in the IgV domain of the CD33 protein (expressed in both RS12459419 CC / CT genotypes, but the CD33 protein in the TT genotype lacks the IgV domain); the scFv of PXL2874 is derived from the monoclonal antibody HIM3-4, and the antigen binding site is located in the IgC domain of the CD33 protein (expressed in all RS12459419 genotypes). After co-incubation with different target cells, the degranulation functions of three CAR-Ts, PXL2812 (antigen binding domains are two single domain antibodies VH23 and VH04 formed by linker tandem), PXL2912 (antigen binding domain is VH66 single domain antibody), and PXL2922 (antigen binding domain is clone 76 scFv antibody, the sequence is shown in SEQ ID NO: 41 and 43) are similar to or better than PXL1696, while the degranulation functions of two CAR-Ts, PXL2883 (antigen binding domain is VH82 single domain antibody) and PXL2920 (antigen binding domain is clone 74 scFv antibody, the sequence is shown in SEQ ID NO: 40 and 42) are similar to or better than PXL2874, which is correlated with the antigen binding specificity data of antibodies in CAR molecules (Examples 3 and 4).

[0370] (2) In vitro cell killing function of CD33CAR-T

[0371] The target cells for the in vitro cell killing experiment of CD33CAR-T were U937, HL60, NB4, NB4-D2, K562, NB4-CD33KO, Nalm6, etc. These cell lines were genetically modified to overexpress ffLuc protein. After co-incubation of CAR-T cells and target cells, the in vitro cell killing function was detected. Figure 8 As shown in the figure; all cloned CAR-T cells have strong killing effects on U937 and NB4 cells, and the killing ability is not much different; PXL2920, PXL2922, PXL2883, and PXL2812 have stronger killing ability against HL60 than the two controls PXL1696 and PXL2874; only PXL2883, PXL2920, and PXL2874 have specific killing ability against NB4-D2, and the other clones have no specific killing ability against NB4-D2 because they target the IgV domain of CD33; all clones have no specific killing ability against CD33-negative NB4-CD33KO and Nalm6 and CD33-weakly positive K562;

[0372] Therefore, the two clones targeting the IgC domain of CD33 (PXL2883 and PXL2920) have good in vitro function for most CD33-positive targets and are not affected by the RS12459419 genotype. The two clones targeting the IgV domain of CD33 (PXL2912 and PXL2922) and the clones of two single-domain antibodies in tandem (PXL2812) also have good in vitro function for CD33-positive target cells other than the RS12459419TT genotype.

[0373] Example 6. In vitro functional evaluation of CD33CAR-NK

[0374] Experimental purpose and principle:

[0375] NK cells are a type of lymphocytes that have a strong killing effect on tumor cells and are independent of the major histocompatibility complex (MHC). The recognition of tumor cells by NK cells mainly depends on the cross-regulation of their surface activating receptors and inhibitory receptors. After identifying tumor cells, NK cells kill tumor cells by releasing killing mediators perforin and granzymes to cause target cell apoptosis, expressing membrane tumor necrosis factor (TNF) family molecules to induce target cell apoptosis, and antibody-dependent cytotoxicity. Therefore, NK cells are regarded as highly potential effector cells that can also enhance their anti-tumor ability through CAR modification due to their special mechanism of identifying target cells, short physiological cycles, and extensive tumor killing ability. Therefore, this embodiment evaluates the in vitro function of CAR molecules constructed using the above-mentioned CD33-specific antibody scFv or single-domain antibody in CAR-NK cells.

[0376] Experimental methods:

[0377] (1) Construction and preparation of retroviral vectors

[0378] Taking the second-generation CAR molecular structure as an example, first, the following nucleotide sequences are artificially synthesized: Kozak (whose nucleotide sequence is GCCGCCACC), CD8a signal peptide (whose amino acid sequence is SEQ ID NO: 23), scFv or VH (including scFv of clones 76 and 74, VH of clone 82 (VH82), VH of clone 66 (VH66), VH of clones 4 and 23 (VH23×04), as well as scFv of gemtuzumab, scFv of HIM3-4), hinge region (whose nucleotide sequence is SEQ ID NO: 24), transmembrane region (whose nucleotide sequence is SEQ ID NO: 25), co-stimulatory factor (whose amino acid sequence is SEQ ID NO: 27), CD3ζ intracellular signaling domain (whose amino acid sequence is SEQ ID NO: 32), 2A cleavage peptide (whose amino acid sequence is SEQ ID NO: 33), IL-15 (whose amino acid sequence is SEQ ID NO: 58, and the encoding nucleotide sequence is SEQ ID NO:35), eGFP (whose amino acid sequence is SEQ ID NO:45, and its encoding nucleotide sequence is SEQ ID NO:44). Fig. 9 The CAR structure involved is LAH-TM-C-CD3ζ-eGFP, Fig.10The CAR structure is LAH-TM-C-CD3ζ-IL-15. The nucleotide sequence of the above elements can be optimized according to the codon usage, but the encoded amino acid sequence remains unchanged. The synthesized CAR molecule sequence is loaded into a retroviral vector, such as a vector such as pMSCV, by a molecular cloning method to obtain a retroviral vector shuttle plasmid (Transfer).

[0379] Then, the shuttle plasmid and other packaging plasmids (encoding Gag-pol and RD114-TR, respectively) are co-transfected into HEK293T cells to prepare the packaging of the retroviral vector. Specific operation steps are as follows:

[0380] HEK293T cells were revived and cultured in DMEM medium containing 10% FBS. After 2-3 generations of cell expansion, 3×10 6 Cells were inoculated into 10 cm culture dishes. On the second day after cell inoculation, plasmid transfection was performed. The plasmid transfection solution was prepared with Opti-MEM, and the final concentration of the plasmid was 10 μg / mL, which contained the shuttle plasmid, pUMVC plasmid (encoding Gag-pol) and pCMV-RD114TR plasmid (encoding RD114-TR) in appropriate proportions. In addition, 35 μl / mL of FugeneHD was added to the plasmid transfection solution, mixed thoroughly, and used after incubation at room temperature for 15 minutes. Each 10 cm culture dish was transfected with 1 mL of plasmid transfection solution.

[0381] After 48 hours, the supernatant was collected into a centrifuge tube and centrifuged at 400g and 4°C for 10 min. The supernatant was aliquoted and stored at -80°C for later use.

[0382] (2) Preparation of CAR-NK cells

[0383] In this embodiment, healthy donor cells are used to prepare CAR-NK. The preparation process of CAR-NK is as follows: on the first day, peripheral blood from healthy donors is collected, PBMC is separated using Ficoll, and NK cells are further sorted using NK Cell MicroBeads. According to the ratio of Feeder:NK=2:1, irradiated trophoblasts are added and cultured in NK cell culture medium containing 40IU / ml IL-2. On the 6th day, 400IU / ml IL-2 is added to the NK culture medium for NK cell activation. On the 7th day, retroviral supernatant is added to the six-well plate coated with retronectin, and the supernatant is discarded after centrifugation at 2000g and 4℃ for 60min; then NK cells are added, and after centrifugation at 300g again and room temperature for 5min, the transduced NK cells are placed in the incubator for further culture. The cell culture time is usually about 14 days, and the obtained CAR-NK cells are used for in vitro functional evaluation or animal experiments.

[0384] (3) CD107a degranulation experiment

[0385] Specific operation steps: (a) Centrifuge the target cells at room temperature and 300g for 5 min; discard the supernatant and resuspend them in NK cell culture medium to 2×10 5 cells / mL; (b) According to the E:T ratio of total NK cells and target cells in the sample to be tested (usually 1:1), the CAR-NK cells were resuspended to an appropriate density, and monensin, Fc block and PE / Cy7 mouse anti-human CD107a antibody were added; (c) 100 μL / well of the CAR-NK cells to be tested and 100 μL / well of the target cells were added to a U-bottom 96-well plate, mixed evenly, and then placed in an incubator (37°C, 5% CO 2 ) and incubate for 3 h; (d) after incubation, centrifuge at 4°C, 600g for 5 min, discard the supernatant, and wash the cells twice with 200 μL / well DPBS + 1% HSA; (e) resuspend the cells with 20 μL / well DPBS + 1% HSA, add APC mouse anti-human CD56 antibody and FITC-CD33 protein, mix the cells and incubate on ice in the dark for 20 min; (f) after incubation, wash the cells three times with 200 μL / well DPBS + 1% HSA, then resuspend the cells with 200 μL / well DPBS + 1% HSA and perform flow cytometry detection.

[0386] (4) In vitro cell killing assay

[0387] Specific operation steps: (a) The target cells expressing ffLuc were centrifuged at room temperature and 300 g for 5 min, the supernatant was discarded, and then resuspended in T cell complete medium to 2×10 5 cells / mL; 100 μL / well of target cells were added to a bottom-transparent 96-well plate; (b) according to the CAR positive rate and E:T value of the CAR-NK cells to be tested (for example: 8:1, 4:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, 0:1), 100 μL / well of CAR-NK cells were added to the 96-well plate, mixed with the target cells, and placed in an incubator (37°C, 5% CO 2 ) and incubated for 4 h; (c) after completion of the incubation, the cells were centrifuged at room temperature and 800 g for 5 min, and the luciferase activity in each well was detected using a luciferase detection kit.

[0388] Experimental results:

[0389] (1) CD107a degranulation function of CD33CAR-NK cells

[0390] The target cells used for the evaluation of degranulation function included HL60 (CD33 positive, RS12459419 CC genotype), HL60-CD33KO (CD33 negative, CD33 gene knockout), NB4 (CD33 positive, RS12459419CC genotype), NB4-D2 (CD33 positive, gene-edited to RS12459419 TT genotype), NB4-CD33KO (CD33 negative, CD33 gene knockout), K562 (CD33 weakly positive, RS12459419 TT genotype) and other cell lines.

[0391] After CAR-NK cells and target cells were co-incubated, the positive rate of CD107a in CAR-NK cells was detected. Fig. 9 As shown; Different from the CAR-T degranulation function experiment in Example 5, since NK cells themselves have innate receptors that can recognize tumor cells, all target cells in this experiment can stimulate CAR-NK cells to produce degranulation function to a certain extent; Since the CAR molecule binds to the corresponding CD33 antigen, it can further stimulate CAR-NK cells. Therefore, different target cells will have a certain degree of difference in the strength of stimulating CAR-NK cells to produce CD107a (CD107a MFI).

[0392] For example, the scFv of 3024 is derived from the monoclonal antibody gemtuzumab (P67.6), and the antigen binding site is located in the IgV domain of the CD33 protein (expressed in both CC / CT genotypes of RS12459419, but the CD33 protein in the TT genotype lacks the IgV domain). Therefore, HL-60 and NB4 cells can stimulate CAR-NK to produce a strong CD107a degranulation effect (both CD107% and MFI are high), but the CD107a degranulation effect produced by CAR-NK stimulated by HL60-CD33KO, NB4-D2 and NB4-CD33KO cells is relatively weak. Different from 3024, the scFv of 3026 is derived from the monoclonal antibody HIM3-4, and the antigen binding site is located in the IgC domain of the CD33 protein (expressed by all RS12459419 genotypes). Therefore, HL-60, NB4 and NB4-D2 cells can stimulate CAR-NK to produce a strong CD107a degranulation effect, while the CD107a degranulation effect produced by HL60-CD33KO and NB4-CD33KO cells stimulating CAR-NK is relatively weak. The three CAR-NK cells, 3025 (antigen binding domains are two single domain antibodies, VH23 and VH04, formed by linker tandem), 3028 (antigen binding domain is VH66 single domain antibody), and 3030 (antigen binding domain is clone 76 scFv antibody), which also bind to the CD33 IgV domain, produce CD107a degranulation effects similar to the control 3024 when stimulated by different target cells; the two CAR-NK cells, 3027 (antigen binding domain is VH82 single domain antibody) and 3029 (antigen binding domain is clone 74 scFv antibody), which bind to the CD33 IgC domain, produce CD107a degranulation effects similar to the control 3026 when stimulated by different target cells ( Fig. 9 ).

[0393] (2) In vitro cell killing function of CD33CAR-NK

[0394] Take HL60 and K562 as two target cells as examples: Since K562 cells hardly express CD33 protein, the killing effect of CAR-NK cells on K562 mainly comes from the activation of NK cells' own intrinsic receptors, so the killing ability of NK cells and all CAR-NK cells on K562 cells is almost the same; since CAR-NK cells can bind to HL60 cells through CAR molecules, most CAR-NK cells have a stronger killing effect on HL60 than NK cells; due to the different recognition and activation abilities of different CAR molecules, the killing ability of 2997 (antigen binding domain is scFv76, sequence as shown in SEQ ID NO: 37 and 39) and 2996 (antigen binding domain is scFv74, sequence as shown in SEQ ID NO: 36 and 38) CAR-NK cells on HL60 is similar to the control 2962 (Gemtuzumab, P67.6) ( Fig.10 ); In the figure, 2927 (CD5 CAR) was used as an "irrelevant CAR molecule" control.

[0395] Therefore, combined with the degranulation function and in vitro cell killing function data of CAR-NK cells, the two CAR molecules constructed from scFv clones 76 and 74 have better functions on CAR-NK cells.

[0396] Example 7. Evaluation of the efficacy of CD33CAR-NK in animals

[0397] Experimental purpose and principle:

[0398] This example evaluates the anti-tumor activity of CD33CAR-NK cells obtained through the screening process of Examples 1-6 in animals. The Molm-13 cell line is derived from the peripheral blood of a 20-year-old male patient with acute myeloid leukemia. After being labeled with the ffLuc reporter gene, the Molm-13 cell line can be cultured as a xenograft in immunodeficient mice as a model for simulating human acute myeloid leukemia (AML). The Molm-13 cell line also expresses CD33 protein, which can be used to detect the specific anti-tumor effect of CD33CAR-NK cells in animals.

[0399] Experimental methods:

[0400] Six-week-old female NPG mice were inoculated with 1x10 6 Molm13-ffLuc cells. Four days after tumor implantation, the mice were divided into groups (G1-G4 groups, 5 mice in each group) for administration of CAR-NK cell test products based on the results of bioluminescence imaging. Each mouse was administered 1×10 7CAR-NK cells / 200ul, or control NK cells / 200ul, or 200ul PBS as vehicle control. After administration, the health status of mice was monitored daily, including body weight measurement twice a week and tumor burden monitoring by bioluminescence imaging every week.

[0401] Experimental results:

[0402] The CD33CAR-NK cells obtained in Examples 1-6 were evaluated in the human acute myeloid leukemia Molm-13 immunodeficient tumor-bearing mouse model. Fig.11 Taking CAR-NK cells as an example, the anti-tumor effect of CD33CAR-NK cells in animals was evaluated and compared. Fig.11 The mean bioluminescence (+ / -SEM) of tumor cells shows the disease burden in intact animals. The PBS control group that did not receive any NK cells showed the baseline tumor growth kinetics of Molm-13 cells in NPG mice; due to the rapid progression of Molm-13 tumor cells in the animals, all animals died on Day 16 due to tumor progression. Compared with the PBS group, the NK cell control group failed to significantly inhibit the proliferation of Molm-13 cells in NPG mice, but was able to significantly prolong the survival of animals, which shows that NK cells also have a certain tumor inhibitory effect. The CAR-NK cell administration group 2997 (scFv76) can delay tumor progression to a certain extent and prolong animal survival.

[0403] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A heavy chain variable region targeting CD33, It is characterized in that The heavy chain variable region comprises a CDR selected from the group consisting of: (Z1) the amino acid sequence of HCDR1 is shown in SEQ ID NO:15, the amino acid sequence of HCDR2 is shown in SEQ ID NO:16, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:17; or (Z2) the amino acid sequence of HCDR1 is shown in SEQ ID NO:20, the amino acid sequence of HCDR2 is shown in SEQ ID NO:21, and the amino acid sequence of HCDR3 is shown in SEQ ID NO:22; Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD33 binding affinity.

2. An antibody targeting CD33, It is characterized in that The antibody comprises the heavy chain variable region as shown in claim 1.

3. The antibody according to claim 2, It is characterized in that The antibody further comprises a light chain variable region comprising a CDR selected from the group consisting of: (Z1) the amino acid sequence of LCDR1 is as shown in SEQ ID NO:13, the amino acid sequence of LCDR2 is EDS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:14; or (Z2) the amino acid sequence of LCDR1 is shown in SEQ ID NO:18, the amino acid sequence of LCDR2 is DNN, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:19; Among them, any one of the above amino acid sequences also includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain CD33 binding affinity.

4. A recombinant protein, It is characterized in that The recombinant protein comprises: (a) the heavy chain variable region targeting CD33 according to claim 1, or the antibody targeting CD33 according to claim 2 or 3; (b) Optional tag sequence to facilitate expression and / or purification.

5. A chimeric antigen receptor (CAR), It is characterized in that The antigen binding domain of the chimeric antigen receptor comprises the heavy chain variable region targeting CD33 as claimed in claim 1, or the antibody targeting CD33 as claimed in claim 2 or 3.

6. A polynucleotide encoding the heavy chain variable region targeting CD33 as claimed in claim 1, or the antibody targeting CD33 as claimed in claim 2 or 3, or the recombinant protein as claimed in claim 4, or the chimeric antigen receptor as claimed in claim 5.

7. An engineered immune cell, It is characterized in that The cell surface expresses the chimeric antigen receptor according to claim 5.

8. An immunoconjugate, It is characterized in that The immunoconjugate contains: (a) an antibody portion, which is selected from the heavy chain variable region targeting CD33 as described in claim 1, or the antibody targeting CD33 as described in claim 2 or 3, or the recombinant protein as described in claim 4; (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

9. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises: (a) the heavy chain variable region targeting CD33 as claimed in claim 1, or the antibody targeting CD33 as claimed in claim 2 or 3, or the recombinant protein as claimed in claim 4, or the engineered immune cell as claimed in claim 7, or the immunoconjugate as claimed in claim 8; and (b) a pharmaceutically acceptable carrier.

10. Use of the CD33-targeting heavy chain variable region according to claim 1, the CD33-targeting antibody according to claim 2 or 3, the recombinant protein according to claim 4, the engineered immune cell according to claim 7, or the immunoconjugate according to claim 8 for preparing a medicament for treating a disease associated with CD33.

Images