Anti-CD38 nano antibody as well as preparation method and application thereof

Abstract

The invention provides an anti-CD38 nano antibody as well as a preparation method and application thereof. Specifically, the invention provides an anti-CD38 nano antibody, an anti-CD38 antibody, a corresponding humanized antibody, a chimeric antigen receptor, a fusion protein, a recombinant protein, polynucleotide, an expression vector, a host cell and the like, as well as a preparation method and application of the anti-CD38 nano antibody and the anti-CD38 antibody. The anti-CD38 nano antibody provided by the invention can reach a protein concealment epitope which cannot be approached by a conventional antibody, can reach higher affinity compared with the prior art, is superior to a positive control Darumumab, and has higher patent medicine value and potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-CD38 nano antibody and a preparation method and application thereof. Background Art

[0002] CD38, also known as cyclic ADP ribose hydrolase, is a transmembrane glycoprotein. CD38 is expressed at very low levels in normal lymphocytes and bone marrow cells and some tissues of non-hematopoietic origin, and is expressed in various malignant blood disease cells, including but not limited to multiple myeloma cells, B-cell chronic lymphocytic leukemia cells, and B-cell acute lymphocytic leukemia cells. A variety of monoclonal antibodies have been developed around CD38, which play a role in killing MM cells through multiple mechanisms such as mediating Fc receptor-dependent immune effector mechanisms, immunomodulatory effects, and direct cross-linking reactions. Therefore, for this type of CD38-positive tumor disease, anti-CD38 antibodies can be used as detection agents or therapeutic agents.

[0003] Daratumumab, a humanized IgG1 CD38 monoclonal antibody developed by Johnson & Johnson, is the first CD38 antibody to be marketed. Its initial approval as a monotherapy for severe myeloma was based on the Phase 1 / 2 GEN501 and SIRIUS trials, and it was approved by the U.S. Food and Drug Administration in 2015 for the treatment of multiple myeloma patients who have received at least three treatment regimens. Daratumumab has a high affinity for malignant cells expressing CD38 and induces tumor cell death through multiple mechanisms of action.

[0004] There are no reports of CD38 nano-antibodies on the market at home and abroad. Nano-antibodies can reach hidden epitopes of proteins that are inaccessible to conventional antibodies and have high affinity. In addition, compared with traditional antibodies, nano-antibodies have the following advantages: high stability and refolding ability under adverse conditions of pH, temperature and even protein exposure; the absence of light chain contact surfaces and the presence of hydrophilic residues in FR2 increase solubility and make it less likely to aggregate; the high sequence similarity between camel-derived VHH and human VH domains results in low immunogenicity; it is easy to express and prepare in microbial systems, and antibody modification is easy.

[0005] Because CD38 expression is widespread and non-specific, soluble CD38 will also reduce the effectiveness of its targeted therapy (this is the main reason for its poor effect in treating multiple myeloma). Therefore, there is an urgent need in this field to screen and develop high-affinity CD38 nanoantibodies to increase their effectiveness in treating malignant blood tumors. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-CD38 nanobody and a preparation method and application thereof.

[0007] Another object of the present invention is to provide antibodies, chimeric antigen receptors, fusion proteins, recombinant proteins and their encoding nucleic acids, expression vectors, host cells, immunoconjugates, etc. based on anti-CD38 nanobodies, and to provide pharmaceutical compositions comprising the above active ingredients.

[0008] Another object of the present invention is to provide a method for preventing and / or treating diseases with high CD38 expression, and a method for diagnosing diseases with high CD38 expression.

[0009] In the first aspect of the present invention, an anti-CD38 nanobody is provided, wherein the anti-CD38 nanobody has one or more complementary determining regions (CDRs) selected from the following group:

[0010] (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:7;

[0011] (2) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:11;

[0012] (3) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 18;

[0013] (4) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:25;

[0014] (5) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:32;

[0015] (6) CDR1 shown in SEQ ID NO:35, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:39;

[0016] (7) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45;

[0017] (8) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:50;

[0018] (9) CDR1 shown in SEQ ID NO:35, CDR2 shown in SEQ ID NO:54, and CDR3 shown in SEQ ID NO:56;

[0019] (10) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:61, and CDR3 shown in SEQ ID NO:63;

[0020] (11) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:68, and CDR3 shown in LNY;

[0021] (12) CDR1 shown in SEQ ID NO:72, CDR2 shown in SEQ ID NO:74, and CDR3 shown in SEQ ID NO:76;

[0022] (13) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:79, and CDR3 shown in SEQ ID NO:81;

[0023] (14) CDR1 shown in SEQ ID NO:84, CDR2 shown in SEQ ID NO:86, and CDR3 shown in SEQ ID NO:88;

[0024] (15) CDR1 shown in SEQ ID NO:91, CDR2 shown in SEQ ID NO:93, and CDR3 shown in SEQ ID NO:95;

[0025] (16) CDR1 shown in SEQ ID NO:98, CDR2 shown in SEQ ID NO:99, and CDR3 shown in SEQ ID NO:101;

[0026] (17) CDR1 shown in SEQ ID NO: 104, CDR2 shown in SEQ ID NO: 106, and CDR3 shown in SEQ ID NO: 108;

[0027] (18) CDR1 shown in SEQ ID NO: 111, CDR2 shown in SEQ ID NO: 113, and CDR3 shown in SEQ ID NO: 115;

[0028] (19) CDR1 shown in SEQ ID NO: 118, CDR2 shown in SEQ ID NO: 120, and CDR3 shown in SEQ ID NO: 122;

[0029] (20) CDR1 shown in SEQ ID NO: 125, CDR2 shown in SEQ ID NO: 126, and CDR3 shown in SEQ ID NO: 128;

[0030] (21) CDR1 shown in SEQ ID NO: 130, CDR2 shown in SEQ ID NO: 131, and CDR3 shown in SEQ ID NO: 133;

[0031] (22) CDR1 shown in SEQ ID NO:136, CDR2 shown in SEQ ID NO:138, and CDR3 shown in SEQ ID NO:140.

[0032] In another preferred embodiment, any one of the above amino acid sequences also includes a derivative sequence that is optionally added, deleted, modified and / or substituted with at least one (such as 1-3, preferably 1-2, and more preferably 1) amino acid and can retain the ability to specifically bind to CD38.

[0033] In another preferred embodiment, the derivative sequence that has been added, deleted, modified and / or substituted at least one amino acid and can retain the ability to specifically bind to CD38 is an amino acid sequence with a homology or sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0034] In another preferred embodiment, the CDR1, CDR2 and CDR3 are respectively separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0035] In another preferred embodiment, the anti-CD38 nanobody has a structure shown in formula (I):

[0036] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 formula (I)

[0037] Wherein, the "-"s are each independently a connecting peptide or a peptide bond.

[0038] In another preferred embodiment, there is an inter-loop disulfide bond between the CDR1 region and the CDR3 region of the anti-CD38 nanobody.

[0039] In another preferred example, the anti-CD38 nanobody also includes a framework region FR.

[0040] In another preferred embodiment, the framework region FR is one or more selected from the following group:

[0041] (1) FR1 shown in SEQ ID NO: 2, FR2 shown in SEQ ID NO: 4, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 8;

[0042] (2) FR1 shown in SEQ ID NO: 10, FR2 shown in SEQ ID NO: 4, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 8;

[0043] (3) FR1 shown in SEQ ID NO: 13, FR2 shown in SEQ ID NO: 15, FR3 shown in SEQ ID NO: 17, and FR4 shown in SEQ ID NO: 8;

[0044] (4) FR1 shown in SEQ ID NO:20, FR2 shown in SEQ ID NO:22, FR3 shown in SEQ ID NO:24, and FR4 shown in SEQ ID NO:8;

[0045] (5) FR1 shown in SEQ ID NO:27, FR2 shown in SEQ ID NO:29, FR3 shown in SEQ ID NO:31, and FR4 shown in SEQ ID NO:8;

[0046] (6) FR1 shown in SEQ ID NO:34, FR2 shown in SEQ ID NO:36, FR3 shown in SEQ ID NO:38, and FR4 shown in SEQ ID NO:8;

[0047] (7) FR1 shown in SEQ ID NO:41, FR2 shown in SEQ ID NO:43, FR3 shown in SEQ ID NO:6, and FR4 shown in SEQ ID NO:8;

[0048] (8) FR1 shown in SEQ ID NO:47, FR2 shown in SEQ ID NO:15, FR3 shown in SEQ ID NO:49, and FR4 shown in SEQ ID NO:8;

[0049] (9) FR1 shown in SEQ ID NO:52, FR2 shown in SEQ ID NO:53, FR3 shown in SEQ ID NO:55, and FR4 shown in SEQ ID NO:8;

[0050] (10) FR1 shown in SEQ ID NO:58, FR2 shown in SEQ ID NO:60, FR3 shown in SEQ ID NO:62, and FR4 shown in SEQ ID NO:8;

[0051] (11) FR1 shown in SEQ ID NO:65, FR2 shown in SEQ ID NO:67, FR3 shown in SEQ ID NO:69, and FR4 shown in SEQ ID NO:8;

[0052] (12) FR1 shown in SEQ ID NO:71, FR2 shown in SEQ ID NO:73, FR3 shown in SEQ ID NO:75, and FR4 shown in SEQ ID NO:8;

[0053] (13) FR1 shown in SEQ ID NO:78, FR2 shown in SEQ ID NO:29, FR3 shown in SEQ ID NO:80, and FR4 shown in SEQ ID NO:8;

[0054] (14) FR1 shown in SEQ ID NO:83, FR2 shown in SEQ ID NO:85, FR3 shown in SEQ ID NO:87, and FR4 shown in SEQ ID NO:8;

[0055] (15) FR1 shown in SEQ ID NO:90, FR2 shown in SEQ ID NO:92, FR3 shown in SEQ ID NO:94, and FR4 shown in SEQ ID NO:8;

[0056] (16) FR1 shown in SEQ ID NO:97, FR2 shown in SEQ ID NO:73, FR3 shown in SEQ ID NO:100, and FR4 shown in SEQ ID NO:8;

[0057] (17) FR1 shown in SEQ ID NO: 103, FR2 shown in SEQ ID NO: 105, FR3 shown in SEQ ID NO: 107, and FR4 shown in SEQ ID NO: 8;

[0058] (18) FR1 shown in SEQ ID NO: 110, FR2 shown in SEQ ID NO: 112, FR3 shown in SEQ ID NO: 114, and FR4 shown in SEQ ID NO: 8;

[0059] (19) FR1 shown in SEQ ID NO: 117, FR2 shown in SEQ ID NO: 119, FR3 shown in SEQ ID NO: 121, and FR4 shown in SEQ ID NO: 8;

[0060] (20) FR1 shown in SEQ ID NO: 124, FR2 shown in SEQ ID NO: 4, FR3 shown in SEQ ID NO: 127, and FR4 shown in SEQ ID NO: 8;

[0061] (21) FR1 shown in SEQ ID NO:27, FR2 shown in SEQ ID NO:29, FR3 shown in SEQ ID NO:132, and FR4 shown in SEQ ID NO:8;

[0062] (22) FR1 shown in SEQ ID NO:135, FR2 shown in SEQ ID NO:137, FR3 shown in SEQ ID NO:139, and FR4 shown in SEQ ID NO:8.

[0063] In another preferred example, the amino acid sequence of the VHH chain of the anti-CD38 nanobody is selected from one or more of SEQ ID NO: 1, 9, 12, 19, 26, 33, 40, 46, 51, 57, 64, 70, 77, 82, 89, 96, 102, 109, 116, 123, 129, and 134.

[0064] In another preferred embodiment, the anti-CD38 nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0065] In the second aspect of the present invention, an anti-CD38 antibody is provided, wherein the antibody comprises one or more anti-CD38 nanobodies as described in the first aspect of the present invention.

[0066] In another preferred embodiment, the antibody comprises one or more VHH chains of the anti-CD38 nanobody as described in the first aspect of the present invention.

[0067] In another preferred example, the amino acid sequence of the VHH chain of the anti-CD38 nanobody is selected from one or more of SEQ ID NO: 1, 9, 12, 19, 26, 33, 40, 46, 51, 57, 64, 70, 77, 82, 89, 96, 102, 109, 116, 123, 129, and 134.

[0068] In another preferred embodiment, the anti-CD38 antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0069] In another preferred embodiment, the anti-CD38 antibody is a bivalent antibody.

[0070] In another preferred embodiment, the bivalent antibody further comprises an antigen binding domain targeting a second target.

[0071] In another preferred embodiment, the second target is selected from the following group: BCMA, CD16a, CD19, CD20, CD138, CD3, CD47, CD5, or a combination thereof.

[0072] In the third aspect of the present invention, a humanized antibody is provided, wherein the humanized antibody is obtained from the anti-CD38 nanobody as described in the first aspect of the present invention or the anti-CD38 antibody as described in the second aspect of the present invention.

[0073] In another preferred example, the humanized antibody has the amino acid sequence shown in SEQ ID NO:141.

[0074] In another preferred embodiment, the humanized antibody has the amino acid sequence shown in SEQ ID NO: 141, and has a mutation at position 5, 13, 14, 44, 75, 79, 87, 88 or 126; preferably at position 44, 75, 79, 87, 88 or 126; most preferably at position 44, 79 or 87.

[0075] In another preferred embodiment, the humanized antibody has the amino acid sequence shown in SEQ ID NO: 141, and has a mutation selected from the following group: L5Q, Q13R, P14A, G44E, S75A, L79V, R87K, A88P or T126Q; preferably, it has a mutation selected from the following group: G44E, S75A, L79V, R87K, A88P or T126Q; most preferably, it has a mutation selected from the following group: G44E, L79V or R87K.

[0076] In the fourth aspect of the present invention, a chimeric antigen receptor (CAR) is provided, wherein the CAR contains an extracellular domain, wherein the extracellular domain comprises the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, or the humanized antibody as described in the third aspect of the present invention.

[0077] In another preferred embodiment, the extracellular domain also includes a signal peptide.

[0078] In another preferred embodiment, the extracellular domain also includes other exogenous proteins.

[0079] In another preferred embodiment, the CAR has a structure shown in Formula Ia:

[0080] L-Nb-H-TM-C-CD3ζ (Ia)

[0081] In the formula,

[0082] L is none or a signal peptide sequence;

[0083] Nb is the specific binding domain;

[0084] H is none or hinge region;

[0085] TM is the transmembrane domain;

[0086] C is the co-stimulatory signaling domain;

[0087] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type, or mutant / modified form thereof);

[0088] The "-" is a connecting peptide or a peptide bond.

[0089] In another preferred embodiment, the L is selected from the signal peptides of the following proteins: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0090] In another preferred embodiment, the Nb targets CD38.

[0091] In another preferred embodiment, the Nb is an anti-CD38 nanobody.

[0092] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0093] In another preferred embodiment, the H is a human IgG1 Fc hinge region.

[0094] In another preferred embodiment, the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or their mutants / modified forms, or their combinations.

[0095] In another preferred embodiment, the C is selected from the co-stimulatory domains of the following proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0096] In another preferred embodiment, the CAR includes the intracellular domain of a cytokine.

[0097] In another preferred embodiment, the cytokine includes: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony stimulating factor (CSF), growth factor, chemokine, or a combination thereof; preferably, the cytokine is interleukin.

[0098] In another preferred embodiment, the CAR includes the intracellular domain of interleukin.

[0099] In another preferred embodiment, the interleukin is selected from the following group: IL-12, IL-2, IL-15, IL-21, or a combination thereof; preferably IL-12.

[0100] In a fifth aspect of the present invention, a fusion protein is provided, wherein the fusion protein has:

[0101] (Z1) a first protein, comprising: the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, or an active fragment thereof;

[0102] (Z2) a second protein comprising a cytokine; and

[0103] (Z3) optionally a linker between the first protein and the second protein.

[0104] In another preferred embodiment, the cytokines include: interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony stimulating factor (CSF), growth factor, chemokine, or a combination thereof.

[0105] In another preferred embodiment, the second protein is interleukin.

[0106] In another preferred embodiment, the first protein is the anti-CD38 nanobody as described in the first aspect of the present invention, and the second protein is interleukin.

[0107] In another preferred embodiment, the interleukin is selected from the following group: IL-12, IL-2, IL-15, IL-21, or a combination thereof.

[0108] In another preferred embodiment, the interleukin is IL-12.

[0109] In a sixth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0110] (i) the anti-CD38 nanobody as described in the first aspect of the invention, the anti-CD38 antibody as described in the second aspect of the invention, the humanized antibody as described in the third aspect of the invention, the fusion protein as described in the fifth aspect of the invention, or an active fragment thereof; and

[0111] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0112] In another preferred example, the tag includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0113] In another preferred embodiment, the recombinant protein specifically binds to CD38.

[0114] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0115] In another preferred embodiment, the tag is an Fc tag.

[0116] In the seventh aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or a combination thereof.

[0117] In another preferred embodiment, the polynucleotide is RNA, DNA or cDNA.

[0118] In the eighth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide as described in the seventh aspect of the present invention.

[0119] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0120] In another preferred embodiment, the expression vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0121] In another preferred embodiment, the expression vector is a pcDNA3.1 vector, a pMES4 vector, or a pABG1 vector (including a pABG1-Fc vector).

[0122] In another preferred embodiment, the expression vector further includes a member selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

[0123] In the ninth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector as described in the eighth aspect of the present invention, or the polynucleotide as described in the seventh aspect of the present invention is integrated into its genome.

[0124] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0125] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0126] In the tenth aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell contains the expression vector as described in the eighth aspect of the present invention or an exogenous polynucleotide as described in the seventh aspect of the present invention is integrated into its genome, or expresses the chimeric antigen receptor as described in the fourth aspect of the present invention.

[0127] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0128] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0129] (ii) chimeric antigen receptor γδ T cells (CAR-T cells);

[0130] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0131] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0132] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0133] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0134] In the eleventh aspect of the present invention, a method for producing an anti-CD38 nanobody is provided, the method comprising the steps of:

[0135] (a) culturing the host cell as described in the ninth aspect of the present invention under conditions suitable for the production of nanobodies, thereby obtaining a culture containing anti-CD38 nanobodies;

[0136] (b) separating and / or recovering said anti-CD38 Nanobody from said culture; and

[0137] (c) Optionally, purifying and / or modifying the anti-CD38 Nanobody obtained in step (b).

[0138] In the twelfth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0139] (a) the anti-CD38 nanobody as described in the first aspect of the invention, the anti-CD38 antibody as described in the second aspect of the invention, the humanized antibody as described in the third aspect of the invention, the fusion protein as described in the fifth aspect of the invention, or the recombinant protein as described in the sixth aspect of the invention; and

[0140] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0141] In another preferred embodiment, the part (a) is the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, or the humanized antibody as described in the third aspect of the present invention.

[0142] In another preferred embodiment, the (a) part and the coupling part are coupled via a chemical bond or a linker.

[0143] In another preferred embodiment, the radionuclide includes:

[0144] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0145] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0146] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0147] In another preferred embodiment, the drug is a drug for targeted treatment of diseases with high CD38 expression.

[0148] In another preferred embodiment, the diseases with high CD38 expression include but are not limited to: solid tumors, blood tumors, immune diseases, or a combination thereof.

[0149] In another preferred embodiment, the CD38 high expression disease includes but is not limited to: multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, large intestine cancer, bile duct cancer, cervical cancer, lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma), esophageal cancer, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, psoriasis, or a combination thereof.

[0150] In another preferred embodiment, the drug is a cytotoxic drug.

[0151] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0152] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, Auristatins, Camptothecins, Duocarmycins, Etoposides, Maytansines and Maytansinoids (e.g., DM1 and DM4), Taxanes, Benzodiazepines or Benzodiazepine containing drugs (e.g., Pyrrolo[1,4]benzodiazepines (PBDs), Indolinobenzodiazepines and Oxazolidinobenzodiazepines), Vinca alkaloids, or combinations thereof.

[0153] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansyl, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyproline, succinimidyl, tadalafil, tadalafil, succinimidyl ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, Mitogellin, Retstrictocin, phenomycin, enomycin, curcin, crotonin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids, or a combination thereof.

[0154] In another preferred embodiment, the coupling moiety is a detectable label.

[0155] In another preferred embodiment, the coupling portion is selected from the following group: 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)) or any form of nanoparticles.

[0156] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) VHH chain of the anti-CD38 nanobody as described in the first aspect of the present invention.

[0157] In another preferred embodiment, the multivalency refers to the inclusion of multiple repeated identical or different VHH chains of the anti-CD38 nanobody as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0158] In the thirteenth aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is selected from the group consisting of the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof, wherein the active ingredient is used to prepare:

[0159] (a) Drugs for preventing and / or treating diseases with high CD38 expression;

[0160] (b) Reagents for detecting diseases with high CD38 expression.

[0161] In another preferred embodiment, the reagent is a diagnostic reagent, preferably, the diagnostic reagent is a detection sheet or a detection plate.

[0162] In another preferred embodiment, the diagnostic reagent is used to detect CD38 protein or a fragment thereof in a sample.

[0163] In another preferred embodiment, the diseases with high CD38 expression include but are not limited to: solid tumors, blood tumors, immune diseases, or a combination thereof.

[0164] In another preferred embodiment, the CD38 high expression disease includes but is not limited to: multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, large intestine cancer, bile duct cancer, cervical cancer, lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma), esophageal cancer, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, psoriasis, or a combination thereof.

[0165] In the fourteenth aspect of the present invention, a method for detecting CD38 protein or a fragment thereof in a sample in vitro is provided, the method comprising the steps of:

[0166] (1) in vitro, contacting the sample with the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof;

[0167] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD38 protein or its fragment in the sample.

[0168] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0169] In the fifteenth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0170] (i) the anti-CD38 nanobody as described in the first aspect of the invention, the anti-CD38 antibody as described in the second aspect of the invention, the humanized antibody as described in the third aspect of the invention, the chimeric antigen receptor as described in the fourth aspect of the invention, the fusion protein as described in the fifth aspect of the invention, the recombinant protein as described in the sixth aspect of the invention, the host cell as described in the ninth aspect of the invention, the engineered immune cell as described in the tenth aspect of the invention, the immunoconjugate as described in the twelfth aspect of the invention, or a combination thereof as an active ingredient; and

[0171] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0172] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized agent.

[0173] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutically acceptable carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0174] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1×10 3 -1×10 8 cells / mL, preferably 1×10 4 -1×10 7 cells / mL.

[0175] A sixteenth aspect of the present invention provides a kit, comprising:

[0176] (1) a first container, wherein the first container contains the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof; and / or

[0177] (2) a second container, wherein the second container contains a secondary antibody against the contents of the first container;

[0178] or,

[0179] The kit contains a detection plate, which includes: a substrate (support plate) and a test strip, wherein the test strip contains the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof.

[0180] In another preferred embodiment, the kit further comprises an instruction manual, according to which the kit is used for non-invasively detecting CD38 expression in a test subject.

[0181] In another preferred embodiment, the kit is used for detecting diseases with high CD38 expression.

[0182] In another preferred embodiment, the diseases with high CD38 expression include but are not limited to: solid tumors, blood tumors, immune diseases, or a combination thereof.

[0183] In another preferred embodiment, the CD38 high expression disease includes but is not limited to: multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, large intestine cancer, bile duct cancer, cervical cancer, lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma), esophageal cancer, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, psoriasis, or a combination thereof.

[0184] In the seventeenth aspect of the present invention, a method for preventing and / or treating a disease with high CD38 expression is provided, the method comprising: administering to a subject in need thereof the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, the pharmaceutical composition as described in the fifteenth aspect of the present invention, or a combination thereof.

[0185] In another preferred embodiment, the subject includes mammals, such as humans.

[0186] In another preferred embodiment, the diseases with high CD38 expression include but are not limited to: solid tumors, blood tumors, immune diseases, or a combination thereof.

[0187] In another preferred embodiment, the CD38 high expression disease includes but is not limited to: multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, large intestine cancer, bile duct cancer, cervical cancer, lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma), esophageal cancer, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, psoriasis, or a combination thereof.

[0188] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells (autologous cells) derived from the subject.

[0189] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0190] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0191] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0192] In the eighteenth aspect of the present invention, a method for diagnosing a disease with high anti-CD38 expression is provided, comprising the steps of:

[0193] (i) obtaining a sample from a diagnostic subject, and contacting the sample with the anti-CD38 nanobody as described in the first aspect of the invention, the anti-CD38 antibody as described in the second aspect of the invention, the humanized antibody as described in the third aspect of the invention, the chimeric antigen receptor as described in the fourth aspect of the invention, the fusion protein as described in the fifth aspect of the invention, the recombinant protein as described in the sixth aspect of the invention, the host cell as described in the ninth aspect of the invention, the engineered immune cell as described in the tenth aspect of the invention, the immunoconjugate as described in the twelfth aspect of the invention, or a combination thereof; and

[0194] (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of a disease with high CD38 expression.

[0195] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

[0196] In another preferred embodiment, the diseases with high CD38 expression include but are not limited to: solid tumors, blood tumors, immune diseases, or a combination thereof.

[0197] In another preferred embodiment, the CD38 high expression disease includes but is not limited to: multiple myeloma, acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, large intestine cancer, bile duct cancer, cervical cancer, lymphoma (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma), esophageal cancer, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, psoriasis, or a combination thereof.

[0198] In the nineteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, wherein the recombinant polypeptide is the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or a combination thereof, the method comprising:

[0199] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for expression; and

[0200] (b) isolating the recombinant polypeptide from the culture.

[0201] 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

[0202] Figure 1 SDS-PAGE gel electrophoresis was used to identify the CD38 antigen protein.

[0203] Figure 2 Titer determinations of multiple rounds of immune sera are shown.

[0204] Figure 3 The results of RNA extraction from alpaca lymphocytes are shown, wherein lane 1 is the RNA band after extraction; lane 2 is the marker.

[0205] Figure 4 The agarose gel verification result of PCR amplified CALL fragment is shown, wherein lane 1 is the marker and lanes 2-5 are the CALL fragments.

[0206] Figure 5 The agarose gel verification results of PCR amplified VHH fragments are shown, wherein lane 1 is the marker and lanes 2-12 are the VHH fragments.

[0207] Figure 6 The agarose gel verification results of the enzyme-digested PMES4 vector gel recovery are shown, wherein lane 1 is the undigested plasmid, lane 2 is the marker, and lanes 3-4 are the enzyme-digested plasmids.

[0208] Figure 7 The results of sequencing and verifying the positive rate of positive monoclonal samples are shown, where lane 1 is the marker and lanes 2-24 are the VHH bands after bacterial P.

[0209] Figure 8The binding level of each nanobody to CD38 protein is shown.

[0210] Fig. 9 The binding levels of each serially diluted nanobody to CD38 protein and the EC50 value are shown.

[0211] Fig.10 The binding levels of serially diluted Fc-carrying nanobodies and the positive control Daratumumab to CD38 protein are shown.

[0212] Fig.11 The results of the cellular level binding experiment of 2C7, 2G5, and 2H2 nanobodies to Daudi cells (highly expressing CD38 protein) are shown.

[0213] Fig.12 The results of the cell-level binding experiment between 2C7+Fc nanobody and the positive control Daratumumab and Daudi cells (highly expressing CD38 protein) and the EC50 value are shown.

[0214] Fig.13 The binding ability of 2C7+Fc+BCMA-VHH bispecific nanoantibody to BCMA and CD38 as well as the EC50 value are shown.

[0215] Fig.14 The binding specificity detection of 2C7+Fc nanobody to various antigens is shown.

[0216] Fig.15 It shows that the nanobody of the present invention can induce ADCC effect. Compared with the positive control Daratumumab, the nanobody of the present invention induces a stronger ADCC effect.

[0217] Fig.16 The temperature stability of the nanobody of the present invention is shown. Compared with the positive control Daratumumab, the temperature stability of the nanobody of the present invention is higher.

[0218] Fig.17 The affinity level of the nanobody 2C7+Fc of the present invention is better than 10 -9 M level.

[0219] Fig.18 ELISA results for humanized Nanobodies of the invention are shown.

[0220] Fig.19The ELISA results of mutant antibodies after back mutation of humanized nanobodies are shown. A. ELISA results after back mutation of positions 44 / 79 / 87; B. ELISA results after back mutation of positions 75 / 88 / 126; C. ELISA results after back mutation of positions 5 / 13 / 14. DETAILED DESCRIPTION

[0221] After extensive and in-depth research and a large number of screenings, the inventors unexpectedly obtained anti-CD38 nano antibodies with high affinity and high specificity for the first time. Specifically, the present invention develops nano antibody drugs for CD38 targets, and screens nano antibodies that specifically bind to human CD38 by preparing CD38 protein, immunizing alpacas, and using phage library display technology. Protein level and cell level binding detection show that the anti-CD38 nano antibody of the present invention has a good binding ability with CD38 protein, and is better than the positive control Daratumumab, has a higher drug value and potential, and provides better treatment and diagnosis means for diseases with high CD38 expression. The present invention was completed on this basis.

[0222] the term

[0223] In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning given below.

[0224] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0225] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0226] The terms "nanoantibody", "single domain antibody", "antibody of the present invention", "nanoantibody of the present invention" and the like are used interchangeably and refer to the nanoantibody as described in the first aspect of the present invention that specifically recognizes and binds to CD38 protein (including human CD38 protein).

[0227] The antibody numbers of the Nanobodies of the present invention and the corresponding sequence numbers are shown in Table A below.

[0228] Table A

[0229]

[0230]

[0231] Note: Each numerical value in the table represents a sequence number, that is, "1" represents "SEQ ID NO: 1", and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the numbers of their amino acid sequences.

[0232] 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.

[0233] As used herein, the terms "single domain antibody", "VHH", "Nanobody", "single domain antibody (sdAb, or nanobody)" have the same meaning and can be used interchangeably, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0234] 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 parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial b-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, Vol. I, pp. 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0235] As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (Cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments of the present invention to form conjugates. The present invention also includes cell surface markers or antigens combined with the nano antibodies or fragments thereof against CD38.

[0236] As used herein, the terms "hypervariable region", "hypervariable region", "complementarity determining region" and "complementarity determining region (CDR)" are used interchangeably.

[0237] In a preferred embodiment of the present invention, the heavy chain variable region of the Nanobody or antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

[0238] In a preferred embodiment of the present invention, the heavy chain of the Nanobody or antibody comprises the above-mentioned heavy chain variable region and heavy chain constant region.

[0239] In the present invention, the terms "nanoantibodies of the present invention", "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably and refer to polypeptides that specifically bind to CD38 proteins, such as proteins or polypeptides having heavy chain variable regions. They may or may not contain an initial methionine.

[0240] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0241] Generally, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four framework regions (FR). 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 chain 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.

[0242] The heavy chain variable regions of the nanobodies or antibodies of the invention are of particular interest because they are at least partially involved in binding to antigen. Thus, the invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0243] The term "humanized antibody" as used herein has the meaning conventionally understood by those skilled in the art, and refers to an antibody obtained by humanizing alpaca source, thereby reducing the immunogenicity of alpaca source antibodies. Based on the anti-CD38 nanobody of the present invention, those skilled in the art know how to perform humanization to obtain a humanized antibody. In a specific embodiment, the sequence of the humanized antibody of the present invention is shown in SEQ ID NO: 141.

[0244] To improve the affinity of the humanized antibody, those skilled in the art can perform back mutation on the humanized antibody, thereby obtaining a humanized antibody with improved affinity. In a specific embodiment, the amino acid at position 5, 13, 14, 44, 75, 79, 87, 88 or 126 in SEQ ID NO: 141; preferably at position 44, 75, 79, 87, 88 or 126; most preferably at position 44, 79 or 87 is back mutated. In a preferred embodiment, the humanized antibody of the present invention has the sequence shown in SEQ ID NO: 141, and has a mutation selected from the group consisting of L5Q, Q13R, P14A, G44E, S75A, L79V, R87K, A88P or T126Q; preferably, has a mutation selected from the group consisting of G44E, S75A, L79V, R87K, A88P or T126Q; most preferably, has a mutation selected from the group consisting of G44E, L79V or R87K.

[0245] 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.

[0246] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0247] The antibody of the present invention refers to a polypeptide having CD38 protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0248] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0249] The present invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0250] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides 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 having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table B.

[0251] Table B

[0252] 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

[0253] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0254] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0255] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0256] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0257] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.

[0258] 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 proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0259] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) 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.

[0260] 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.

[0261] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0262] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0263] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0264] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. 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 a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-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.

[0265] The Nanobodies or antibodies or humanized antibodies of the invention can be used alone or in combination or coupled to a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety or a combination of any of these.

[0266] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0267] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0268] Multiple Myeloma and CD38 Nanobodies

[0269] Existing treatment strategies have significantly improved the prognosis of patients with multiple myeloma (MM), but most MM patients still relapse repeatedly. In recent years, some new drugs, such as proteasome inhibitors, immunomodulatory drugs and monoclonal antibody drugs, together with high-dose treatment and autologous hematopoietic stem cell transplantation, have been committed to deeper and more lasting clinical responses. However, most MMs still relapse due to the development of drug resistance. Therefore, there is an urgent need to develop new treatment strategies for MM in this field.

[0270] As a new type of genetically engineered antibody, nanobodies are the smallest antigen-binding antibody fragments to date, with a molecular weight of about 15kD. Nanobodies are small in size but have longer CDR3 regions than ordinary monoclonal antibodies, so they are more able to reach hidden protein epitopes and high affinity that are inaccessible to conventional antibodies. In addition, nanobodies have the following advantages: they exhibit high stability and refolding ability under adverse conditions of pH, temperature, and even protein exposure; the absence of light chain contact surfaces and the presence of hydrophilic residues in FR2 increase solubility and make it less likely to aggregate; the high sequence similarity between camel-derived VHH and human VH domains results in low immunogenicity; they are easy to express and prepare in microbial systems, and antibody modification is easy. In general, these advantages make nanobodies valuable in cancer diagnosis and immunotherapy.

[0271] The expression of CD38 is widespread and non-specific, and soluble CD38 will also reduce the effect of its targeted therapy, which is the main reason for its poor effect in treating multiple myeloma. Therefore, the inventors aim to screen out CD38 nanoantibodies with high affinity, and perform modifications such as coupling Fc and fusing other related target antibodies to increase its therapeutic effect.

[0272] Anti-CD38 nanoantibodies and anti-CD38 antibodies and humanized antibodies thereof

[0273] In the present invention, the anti-CD38 nanobody and the anti-CD38 antibody and humanized antibody thereof may include: a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0274] For bivalent antibodies and multivalent antibodies, in addition to the antigen binding domain targeting the first target CD38, they may also include antigen binding domains targeting other antigens or proteins (secondary target, tertiary target, fourth target, etc.).

[0275] Based on the anti-CD38 nanobodies and antibodies and humanized antibodies provided by the present invention, those skilled in the art can easily construct: multispecific antibodies that simultaneously target CD38 and any other antigens or proteins commonly found in the art. Other antigens or proteins (other targets) can be, but are not limited to: BCMA, CD16a, CD19, CD20, CD138, CD3, CD47, CD5, etc., or a combination thereof.

[0276] Labeled Antibodies

[0277] In a preferred embodiment of the present invention, the nanobody or antibody or humanized antibody thereof may carry a detectable marker. More preferably, the marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.

[0278] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-CD38 nanobody or antibody or its humanized antibody is labeled with colloidal gold to obtain a colloidal gold-labeled antibody. The anti-CD38 nanobody or antibody or its humanized antibody of the present invention can effectively bind to the CD38 protein.

[0279] Detection Methods

[0280] The present invention also relates to a method for detecting CD38 protein or its fragments. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of CD38 protein in the dissolved sample.

[0281] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.

[0282] Reagent test kit

[0283] The present invention also provides a kit containing the anti-CD38 nanobody or antibody or humanized antibody (or its fragment) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0284] The present invention also provides a detection kit for detecting CD38 protein levels, which includes an antibody that recognizes CD38 protein, a lysis medium for dissolving a sample, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0285] Pharmaceutical composition

[0286] The present invention also provides a pharmaceutical composition. It contains the anti-CD38 nanobody as described in the first aspect of the present invention, the anti-CD38 antibody as described in the second aspect of the present invention, the humanized antibody as described in the third aspect of the present invention, the chimeric antigen receptor as described in the fourth aspect of the present invention, the fusion protein as described in the fifth aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, the host cell as described in the ninth aspect of the present invention, the engineered immune cell as described in the tenth aspect of the present invention, the immunoconjugate as described in the twelfth aspect of the present invention, or a combination thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0287] Typically, 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 substance being formulated and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intraperitoneal, intravenous, or topical administration.

[0288] 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 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 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0289] 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 dose is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dose 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.

[0290] application

[0291] As described above, the nanobodies and antibodies of the present invention have a wide range of biological and clinical application values, and their applications involve diagnosis and treatment of diseases related to CD38 protein, basic medical research, biological research, etc. A preferred application is for clinical diagnosis, prevention and treatment of CD38 protein.

[0292] The present invention also provides a method for stimulating an immune response mediated by T cells targeting a tumor cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.

[0293] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach makes the probability of graft-versus-host reaction extremely low, and the antigen is recognized by the T cell in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0294] In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo expansion and can last for months to years. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-T cells can induce a specific immune response to tumor cells that highly express the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the present invention cause a specific immune response to tumor cells that highly express CD38.

[0295] Treatable cancers include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. The types of cancers treated with the CAR of the present invention include, but are not limited to, breast cancer, gastric cancer, colorectal cancer, ovarian cancer, lung cancer, prostate cancer, liver cancer, kidney tumors, small intestine cancer, colon cancer, bile duct cancer, cervical cancer, lymphoma, esophageal cancer, etc.

[0296] Generally, cells activated and expanded as described herein can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, which comprises administering a therapeutically effective amount of CAR-T cells of the present invention to a subject in need of treatment.

[0297] The CAR-T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell populations. In short, the pharmaceutical composition of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0298] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or can be determined by clinical trials.

[0299] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. The pharmaceutical composition comprising the T cells described herein can be administered in an amount of 104 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 7 The T cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0300] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of the T cell can be directly injected into the tumor, lymph node or infection site.

[0301] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, simultaneously or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0302] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 5 to 1×10 10 The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0303] Main advantages of the present invention

[0304] 1. The existing CD38 recombinant antibodies have a large molecular weight and are not easy to bind to hidden epitopes, while the CD38 nanoantibodies of the present invention can reach hidden protein epitopes that are inaccessible to conventional antibodies.

[0305] 2. The existing CD38 nanoantibodies have low cell binding ability and low drug-making potential, while the CD38 nanoantibodies of the present invention can achieve higher affinity than the existing technology, are better than the positive control Daratumumab, and have higher drug-making value and potential.

[0306] 3. The existing CD38 nanoantibodies only have a single targeting effect and cannot change the problems such as drug resistance caused by target loss and reduced antigen expression. The CD38 nanoantibody of the present invention can be used to form bispecific antibodies or multispecific antibodies with other targets (including but not limited to the malignant blood tumor target BCMA), and can be used to improve recurrent multiple myeloma and drug resistance caused by antigen loss and target shedding.

[0307] 4. The CD38 nanobody of the present invention can also be linked to Fc or CD16a antibody (NK cell-related target) to have an ADCC effect and be used to kill tumor cells.

[0308] 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.

[0309] Sequence Listing

[0310]

[0311]

[0312]

[0313]

[0314]

[0315] Example 1: CD38 antigen expression The CD38 gene is from NCBI (ACCESSION: NC_000004; VERSION: NC_000004.12), only the extracellular segment (46-300aa) is expressed, and the His tag is connected. The gene fragment was synthesized by BGI and connected to pCDNA3.1+ after synthesis. After the plasmid was synthesized, it was shaken in LB and the plasmid was extracted according to the instructions for plasmid extraction. It was transfected into 293 cells using PEI and cultured at 37℃ for 72h before the sample was collected.

[0316] Centrifuge and take the supernatant for SDS test. The result is as follows Figure 1 As shown: Compared with the negative control (i.e., lane 3: negative), there is an obvious band at about 40KD in the supernatant (i.e., lane 4: supernatant), which is consistent with the theoretical molecular weight of CD38 protein. After large-scale expression, it was purified using a Ni column, and the buffer used a PBS solution with a pH of 7.4. Preliminary experiments showed that the elution effect was best at a concentration of 10mM imidazole. After purification, the buffer was replaced with PBS by ultrafiltration, and a total of 10mg of purified CD38 protein was obtained. The purity was verified to be more than 95% by running gel (i.e., lane 6: purification).

[0317] Example 2: CD38 antigen immunization

[0318] Take 1 ml of the purified CD38 protein prepared in Example 1 at 1 mg / ml, take the same amount of Freund's reagent and mix them in a ratio of 1:1, and inject them subcutaneously at multiple points into adult healthy alpacas. Immunize once every two weeks for a total of four times. Take jugular vein peripheral blood 7 days after immunization to measure the titer.

[0319] The results are as follows Figure 2 As shown, the triple-immune vaccine has the highest titer, which is 1:72900.

[0320] Example 3: Lymphocyte extraction

[0321] Use a 50ml peripheral blood lymphocyte separation tube, add 10ml of three-free alpaca blood to each tube, centrifuge at 800×g for 15 minutes. Carefully suck out the milky white ring with a pipette tip, add 10ml PBS, centrifuge at 250×g for 10 minutes to wash, repeat the wash three times, resuspend and count with a small amount of PBS, and use RNAkeeper to dilute the cell density to 5×10 ^ 6 cells / ml, frozen at -80℃.

[0322] Example 4: Library Construction

[0323] 1. Use the NoviZan RNA extraction kit to extract RNA from lymphocytes, catalog number RC101-01:

[0324] Centrifuge at 6000 rpm, 4°C for 5 min and discard the supernatant;

[0325] Check whether 1% mercaptoethanol is added to Buffer RL1 (provides the environment required for animal tissue and cell lysis) before use;

[0326] Take 500 μl Buffer RL1 to resuspend the cells, transfer the resuspended solution into gDNA-Fiter columns, centrifuge at 12000 rpm, 4°C for 2 min, and collect the filtrate;

[0327] Add 1.6 times the volume of Buffer RL2 (800 μl) to the filtrate (providing an RNA-specific column loading environment);

[0328] Take 700 μl of the filtrate from the previous step and put it into RNAPure Colums, centrifuge at 12000 rpm, 4°C for 1 min, discard the waste liquid, and then draw the remaining liquid to pass through the column;

[0329] Add 500 μl Buffer RW1 (to remove impurities such as proteins and DNA in RNA), centrifuge at 12000 rpm, 4°C for 1 min, and discard the waste liquid;

[0330] Add 700 μl Buffer RW2 (to remove residual salt ions in RNA), centrifuge at 12000 rpm, 4°C for 1 min, and discard the waste liquid;

[0331] 65 μl RDD + 5 μl DNAseI were added to the column and allowed to stand at room temperature for 20 min;

[0332] Add 500 μl Buffer RW1, centrifuge at 12000 rpm, 4°C for 1 min and discard the waste liquid;

[0333] Add 700 μl Buffer RW2, centrifuge at 12000 rpm, 4°C for 1 min and discard the waste liquid;

[0334] Add 700 μl Buffer RW2, centrifuge at 12000 rpm, 4°C for 1 min and discard the waste liquid;

[0335] 2min at room temperature, 2min in air-drying;

[0336] Add 80 μl of preheated RNAse-free H2O (to elute the total RNA on the purification column membrane), centrifuge for 1 min, and measure the concentration.

[0337] After RNA extraction, a portion was immediately subjected to electrophoresis, and the rest was immediately subjected to reverse transcription.

[0338] The results are as follows Figure 3 As shown, the extracted RNA subunits are intact and not degraded, and can be used for the next experiment.

[0339] 2. Reverse transcription into cDNA, using Novizan reverse transcription kit, Hiscriptr III ISt strand cDNAsynthesis Kit (tgrna wiper), catalog number R312-02:

[0340] Before this experiment, you need to prepare a metal bath at 65°C, ice, a water bath at 42°C, and a PCR instrument.

[0341] The RNA concentration and usage must meet the requirements of the kit for each time range of 10pg-5μg. If they meet the requirements, they will be denatured directly.

[0342] Denaturation: Take 80 μl of the extracted RNA, incubate at 65°C for 5 min, and then incubate on ice for 2 min.

[0343] Genome removal: After standing for 2 min, add 20 μl 5×g DNA wiper to the RNA and stand at 42°C for 2 min.

[0344] Prepare the first-strand cDNA synthesis reaction solution: prepare the system according to the table below and dispense 20 μl / tube after preparation.

[0345] name Volume (μl) Previous step mixture 100 10×RTmix 20 Hiscriptr III enzyme Mix 20 Oligo(dt)20VN 10 <![CDATA[RNAse-freeH2O]]> 50

[0346] Procedure: Incubate at 37°C for 45 min, then at 85°C for 5 s. Measure concentration, aliquot and freeze.

[0347] 3. Use cDNA as template to PCR amplify the CALL fragment and recover the band of about 700 bp:

[0348] Preparation system:

[0349] name Dosage cDNA 50μg 2×master Mix 425μl Upstream primer 35μl Downstream primer 35μl water to 850μl

[0350] Mix well and dispense into 50 μl / tube. In addition, the negative control group was prepared by using the above system without adding cDNA template.

[0351] PCR procedure:

[0352]

[0353] After running the PCR, run the gel on 1.5% agarose gel for 40 minutes. The results are as follows Figure 4As shown, the CALL fragment has a band of about 700 bp, which is completely amplified and can be subsequently cut and recovered using the Tiangen Bio-Gel Recovery Kit.

[0354] 4. Amplify the VHH fragment using CALL as a template and recover the band of about 450 bp:

[0355] Homologous recombination primers were used in this amplification to facilitate subsequent seamless cloning with the vector.

[0356] Preparation system:

[0357] name Dosage 2×Mix 2ml Upstream primer 100μl Downstream primer 100μl template 450ng water to 4ml

[0358] Mix well and dispense into 50 μl / tube. In addition, the negative control group was prepared by using the above system without adding cDNA template.

[0359] PCR procedure:

[0360]

[0361] 2% agarose run for 40 min, the results are as follows Figure 5 As shown, the VHH fragment is about 450 bp, and the band is clear and single, which can be used for subsequent gel recovery. The Tiangen Bio Gel Recovery Kit was used for gel recovery.

[0362] 5. Use PstI and BstEII enzymes to digest the PMES4 vector and verify:

[0363] The enzyme digestion system is as follows:

[0364] name Dosage PMES4 10 μg BsE 4μl PsI 4μl 10×cutsmart 4μl water to 40μl

[0365] The above system was digested overnight at 37°C. After digestion, the gel was run and recovered. The results were as follows Figure 6 As shown, it shows that the plasmid recovered by enzyme digestion is complete and can be used for subsequent ligation experiments. The plasmid recovered by gel was ligated and transformed using solutionI enzyme, and cultured in a 37°C incubator overnight. No bacteria were observed to grow on the ampicillin plate the next day, which also shows that the plasmid recovered by enzyme digestion is complete and can be used for subsequent ligation experiments.

[0366] 6. Connect the electroporator:

[0367] The recovered VHH and PMES4 were ligated in small amounts using homologous recombinase.

[0368] Connection system:

[0369] name Dosage Carrier 900ng VHH 300ng Homologous recombination enzyme 25μl water to 50μl

[0370] The above ligation system was connected at 50°C for 30 min. The reaction product was directly converted or frozen at -20°C.

[0371] After the transformation bacteria P was successfully verified, a large amount of them were connected and recovered by column.

[0372] After determining the concentration of the recovered product, preheat the SOC medium at 37°C. Wash the electroporation cup with ultrapure water, dry it, and irradiate it with ultraviolet light for 30 minutes. Cool it on ice for 5 minutes. Take the competent state on ice for 5 minutes, add the ligation product to the competent state, and let it stand on ice for 10 minutes. Add the mixture to the electroporation cup without bubbles. Select the preset program bacteria column of the electroporator, 2.5KV, 25μF, 200Ω, the competent state volume is 50μl, and the electroporation cup is 2mm. Electroporation. Immediately add 1ml of SOC medium after electroporation and shake it in a 15ml centrifuge tube at 37°C for 2h.

[0373] Take 500 μl of bacteria and add 500 μl of glycerol to freeze. Take 100 μl of bacteria and dilute 10 ^ After 6 times, 100 μl was taken to plate, and the number of monoclonal clones on the next day was 217. Therefore, the library capacity of this construction was 2.17×10 ^ 9cfu / ml.

[0374] Calculated storage capacity = dilution factor × number of colonies × 10.

[0375] Bacteria P verification positive rate: 23 monoclonal clones were selected for bacteria P, and the results were as follows Figure 7 As shown, the positive rate of 23 monoclonal clones was 100%.

[0376] From the above positive single clones, 12 clones before bacterial P were randomly selected for sequencing to test diversity, and the diversity was 100%. The library capacity was 2.17×10 ^ 9cfu / ml.

[0377] Take another 500μl of the electroporated bacteria and add it to 10ml of 2YT medium containing 2% glucose and 1 / 1000 ampicillin, shake it to OD 0.6-0.8, add M13K07 at a ratio of 20:1, let it infect for 1h, centrifuge at 4000rpm for 10min to remove the supernatant, resuspend it in 10ml of 2YT medium without glucose, culture it at 37℃, 220rpm for 1h, transfer it to 100ml of 2YT medium without glucose, add ampicillin, kanamycin, and IPTG at 1:1000; 1:1000; 1:2000 respectively. Shake it at 37℃ for 1h and then transfer it to 30℃ for overnight culture.

[0378] Example 5: Screening Library

[0379] 1. Precipitate phage:

[0380] The bacterial solution in the above step was centrifuged at 1000rpm for 10min, the supernatant was collected, and PEG / NaCl was added at a ratio of 4:1 and placed on ice for 2h. Then the phage was centrifuged at 4000rpm, 4℃, for 15min, the supernatant was discarded, the water was drained, 1ml PBS was added to resuspend, and the titer was measured after passing through a 0.45μm filter membrane. The titer of the phage after amplification was determined to be 2×10 ^ 13pfu / ml.

[0381] 2. Select phage antibodies on solid-phase antigens:

[0382] Coat the immunotube with 1 ml of 50 μg / ml CD38 antigen dissolved in CBS. Make another tube and do the same as below except that phage is not added. Coat overnight at 4°C

[0383] The tubes were washed three times with 2 ml PBS and blocked with 2 ml 2% BSA at 37°C for 2 h. At 1.5 h, the phages were diluted with 1% BSA to a titer of 10 ^ 13, incubate at 37°C for 0.5h. Then wash the test tube 3 times with PBS, add the incubated phage and incubate at 37°C for 1h.

[0384] After incubation, wash 10 times with PBST-0.05%. Use 1 ml of 0.2 M Gly-HCl at pH 2.2 to elute at room temperature for 10 min, and immediately add 20 μl of Tris-HCl at pH 9.0 to neutralize to pH 7.0. Pass through a 0.45 μm filter membrane for later use.

[0385] Take 10 μl of the eluted phage to infect 200 μl of fresh TG1 (using glucose 2YT medium), incubate at 37°C for 1 hour, take 100 μl for 10-fold, 100-fold, and 1000-fold dilutions and plate to measure the titer (the plate is ampicillin 2YT solid medium with glucose).

[0386] Take 500 μl of eluted phage and add it to 4 ml of glucose 2YT medium, let it stand for infection for 1 hour, then add helper phage at a ratio of 20:1, let it stand for superinfection for 1 hour. Centrifuge to remove the supernatant and glucose, resuspend in 10 ml of 2YT medium without glucose, add ampicillin according to the proportion, shake at 37°C for 1 hour, add to 50 ml of 2YT medium without glucose, add ampicillin and IPTG, and shake at 30°C overnight.

[0387] The next day, the phages were precipitated again and the above library screening operation was repeated. The specific conditions and results are shown in Table 1 below.

[0388] Table 1

[0389]

[0390] 3. Monoclonal ELISA:

[0391] 470 monoclones were picked from the titer measurement plate and subjected to monoclonal ELISA.

[0392] Pick a single clone and transfer it to 200 μl 2YT-ampicillin medium. Culture it at 37°C, 200 rpm for 2-3 h until the OD reaches about 0.6.

[0393] Take 50 μl and add 50 μl glycerol to preserve the bacteria at 4°C.

[0394] 10 ^ Add M13K07 to 9 / well, 100μl per well, let it stand for 30min, then add kanamycin IPTG, prepare it first and then add 200μl / well (90μl kanamycin, 50μl IPTG, 40ml culture medium), 37℃ for 1h and then change to 30℃ overnight. Pack 5 96-well plates, 100μl / well, 2μg / ml, and pack 5 96-well plates with 2% BSA 100μl / well as a negative control.

[0395] Block with 2% BSA, 200 μl / well, at 37°C for 2 h. Centrifuge the selected single clones and collect the supernatant.

[0396] After blocking, wash the plate, take 100μl of the supernatant and add it to three 96-well plates, and mark them. Do the same for the three BSA-coated plates. Also make negative controls of M13K07 and TG1 supernatants. Incubate at 37℃ for 1h, wash the plate, add the secondary antibody against M13, incubate for 1h, add 100μl / well of TMB colorimetric solution, stop the reaction with sulfuric acid at room temperature and avoid light for 10min, and read the OD at 450nm.

[0397] Single clones with OD values ​​greater than 1 and P / N>2.1 were sent for sequencing, and a total of 179 clones were sent for sequencing.

[0398] Example 6: Nanobody expression and purification

[0399] Comparing the sequencing results, expressing and purifying the nanobodies, 22 nanobodies with different amino acid sequences were obtained: 1C7, 1D7, 2C7, 2G5, 2H2, 4F11, 5A5, 5A6, 5A7, 5B4, 5B5, 5B6, 5C4, 5C5, 5C6, 5D4, 5D5, 5E3, 5F4, 5F6, 5G4, 5H4. The sequence information is shown in the sequence table and Table A.

[0400] The obtained plasmid was transformed into BL21. After a single colony was selected for sequencing and confirmed, 800 ml of IPTG was used to induce expression. The next day, TES was used to extract the periplasmic protein, which was purified using a Ni column and then run on a protein gel.

[0401] Example 7: Nanobody Binding and Affinity Detection

[0402] 1. Protein level binding:

[0403] 4μg / ml CD38-Fc was coated overnight at 4℃, and the plates were washed and blocked the next day. Nano antibodies were added at 200μg / ml, 100μl / well, as parallel samples, incubated at 37℃ for 1h, and Anti-His-HRP was added after washing, and incubated at 37℃ for 45min. TMB colorimetric solution was added after washing, and color development was terminated with 1M sulfuric acid, and OD was read at 450nm.

[0404] The results are as follows Figure 8 As shown, six antibodies, 1D7, 2C7, 2G5, 2H2, 5A5, and 5G4, have good binding, followed by 5B5. The binding force to CD38 protein is ranked as follows: 2G5>2C7>1D7>2H2>5A5>5G4>5B5.

[0405] Select 1D7, 2C7, 2G5, 2H2, 5A5, and 5G4 to continue the experiment. 4μg / ml CD38-Fc was coated overnight at 4℃, and the plates were washed and blocked the next day. The nano antibody concentration was 15μg / ml, diluted 5 times, and added at 100μl / well for parallel samples. Incubated at 37℃ for 1h, Anti-His-HRP was added after washing, and incubated at 37℃ for 45min. Wash the plates and add TMB colorimetric solution, stop the color development with 1M sulfuric acid, and read the OD at 450nm.

[0406] The results are as follows Fig. 9 As shown, 2C7, 2G5, and 2H2 antibodies have good binding, 2G5 is the best, followed by 2C7 and 2H2.

[0407] 2C7, 2G5, and 2H2 were connected to Fc to construct nanobodies with Fc, and then expressed and purified, and the binding at the protein level was detected by ELISA.

[0408] The results are shown in Table 2. Fig.10 As shown, the EC50 (μg / ml) of nanobody 2C7+Fc is significantly better than that of the positive antibody (positive control Daratumumab), and the EC50 (μg / ml) of nanobody 2G5+Fc is also slightly better than that of the positive antibody (positive control Daratumumab).

[0409] In addition, as shown in Table 3, the EC50 (pm) of Nanobody 2C7+Fc was significantly better than that of the positive antibody (positive control Daratumumab).

[0410] Table 2

[0411] 2C7+Fc 2G5+Fc 2H2+Fc Positive Control EC50(μg / ml) 0.003398 0.008465 0.2557 0.01064

[0412] Table 3

[0413] 2C7+Fc Positive Control EC50(pm) 42.475 70.9333

[0414] 2. Cell level binding:

[0415] Based on the above ELISA data, 2C7-His, 2G5-His, and 2H2-His were selected for cell-level binding experiments.

[0416] Treat cells: Collect Daudi cells (highly expressing CD38 protein) in the logarithmic growth phase and centrifuge at 300g / 5min, discard the supernatant. Plate cells: Resuspend cells with FACS buffer (2% FBS in PBS), adjust the cell density to 3E6 / ml, and plate 100μl per well.

[0417] Incubation with primary antibody: dilute the primary antibody with FACS buffer, centrifuge the cells at 300g / 5min and discard the supernatant, resuspend the cells with primary antibody at a concentration of 100μg / ml (100μl / well), and incubate at 4℃ for 60min.

[0418] Wash the primary antibody: wash twice with 1×PBS 200 μl / well, centrifuge at 300g / 5min and discard the supernatant.

[0419] Incubation with secondary antibody: dilute the secondary antibody with FACS buffer, centrifuge the cells at 300g / 5min and discard the supernatant, resuspend the cells with 100μl / well of secondary antibody, and incubate at 4℃ for 60min.

[0420] Wash the secondary antibody: wash twice with 1×PBS 200 μl / well, centrifuge at 300g / 5min and discard the supernatant.

[0421] Detection: Resuspend cells with 100-200 μl / well PBS and detect on the instrument.

[0422] The results are as follows Fig.11 As shown, the three antibodies can bind to cells expressing CD38 protein, among which 2C7 has the strongest signal intensity.

[0423] Comprehensively select 2C7 to continue the experiment.

[0424] 2C7+Fc was diluted five-fold from 500 μg / ml, and the positive antibody (positive control Daratumumab) was diluted five-fold from 100 μg / ml, and flow cytometry experiments were performed.

[0425] The results are as follows Fig.12 As shown, the binding ability of nanobody 2C7+Fc at the cellular level is better than that of positive antibody.

[0426] Example 8: Functional Experiment

[0427] 1. Construction of 2C7+Fc+BCMA-VHH:

[0428] To verify the feasibility of subsequent bispecific antibodies, a 2C7+Fc+BCMA-VHH bispecific nanoantibody was constructed (BCMA-VHH uses the US2022 / 0265710 A1 sequence). The constructed molecular plasmid was transfected into 293F, and ELISA was performed after expression and purification. BCMA-His and CD38-His were used to package the plates, and the antibody concentration for BCMA was diluted 5 times from 1.5 mg / ml, and the antibody concentration for CD38 was diluted 5 times from 0.3 mg / ml for ELISA experiments.

[0429] The results are as follows Fig.13 As shown, the binding ability of the 2C7+Fc+BCMA-VHH bispecific nanobody to CD38 (ie, EC50: 0.004290 μg / ml) is not significantly reduced compared to 2C7+Fc (ie, EC50: 0.003398 μg / ml), indicating that the nanobody screened by the present invention can be used for the assembly of bispecific antibodies and does not affect the binding ability of bispecific antibodies to CD38. In addition, the 2C7+Fc+BCMA-VHH bispecific nanobody can also specifically bind to BCMA. It is proved that the 2C7+Fc+BCMA-VHH bispecific nanobody can simultaneously bind to BCMA and CD38 dual targets.

[0430] 2. Use ELISA method to detect whether the nanoantibody binds to other irrelevant antigens except CD38 and antigens used to immunize alpacas:

[0431] like Fig.14 As shown, it can be seen that 2C7+Fc only reacts with CD38 and does not bind to other unrelated proteins, which shows that the screened nanoantibodies have high specificity.

[0432] 3.ADCC:

[0433] (1) Daudi cells were adjusted to 1E5 cells / ml, and the culture plate was pre-cultured in an incubator for 10 min (at 37° C., 5% CO 2 ). 100 μl of the cell suspension was added to a 96-well plate.

[0434] (2) Add 100 μl of sterile primary antibody (primary antibody is 2C7+Fc or positive control) at different concentrations to the culture plate, starting from 10 μg / ml, 9 gradients, the last well is the well with 0 antibody concentration, 2 duplicate wells, 5-fold dilution.

[0435] (3) NK cells were added at an effector-target ratio of NK:Daudi = 10:1, 50 μl / well, and the NK concentration was 2E6 cells / ml; the culture plate was incubated in an incubator at 37°C for 2 h.

[0436] Set up control wells:

[0437] A: 100 μl of Daudi medium without Daudi cells; B: 100 μl of Daudi medium containing Daudi cells; C: 100 μl of Daudi + 50 μl of NK; D: 100 μl of the nanobody of the present invention or the positive control Daratumumab antibody + 100 μl of Daudi + 50 μl of NK.

[0438] (4) Add 10 μl of CCK-8 solution to each well (be careful not to create bubbles in the wells as they will affect the OD reading).

[0439] (5) Incubate the culture plate in an incubator at 37°C for 4 h.

[0440] (6) Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0441] The results are as follows Fig.15 As shown, the killing effect of the nanoantibody of the present invention is better than that of the positive control Daratumumab, and can induce a stronger ADCC effect.

[0442] 4. Temperature stability:

[0443] 10μg / ml, 100μl / well coated antigen at 4℃, 2C7+Fc and positive control were diluted to 5μg / mL with PBS, and incubated at 4℃, 25℃, 37℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 30min, respectively, and then ELISA was performed.

[0444] The results are as follows Fig.16 As shown, the temperature stability of the Nanobody of the present invention is better than that of the positive control Daratumumab.

[0445] 5. Multi-concentration affinity test:

[0446] 2C7+Fc, 2H2+Fc, and positive control Daratumumab were selected for affinity test with CD38 protein. The parameters of affinity test were selected as flow rate 30 μL / min, injection time 60 s, and dissociation time 600 s.

[0447] The results are as follows Fig.17 As shown, the affinity of the nanobody 2C7+Fc of the present invention is better than 10 -9 M level.

[0448] Example 9: Humanization transformation

[0449] 2C7 was selected for humanization to reduce immunogenicity. MOE software was used to model homologous molecules of 2C7, and then the following humanization scheme was designed, as shown in Table 4. The sequence was synthesized by BGI, and the plasmid was extracted using a plasmid extraction kit after synthesis. The plasmid was transfected into 293F cells using PEI reagent, and the supernatant was collected by centrifugation after 72 hours, filtered through a 0.45μm filter membrane, and the protein was purified using a Ni column. After purification, the buffer was replaced with PBS by ultrafiltration and SDS-PAGE was performed.

[0450] Table 4

[0451]

[0452] To verify whether the activity of the humanized nanoantibody has decreased, ELISA was performed. 4 μg / mL CD38-Fc antigen was added to 100 μL per well of the ELISA plate, coated overnight at 4°C, and washed 3 times with a plate washer the next day. 200 μL of 5% skim milk powder was added to each well, blocked at 37°C for 2h, and washed 5 times with a plate washer. 2C7-RYH-1, 100 μg / mL, was diluted 5 times as the primary antibody, 100 μL per well was added to the ELISA plate, incubated at 37°C for 1.5h, Anti-His-HRP was diluted 1:5000 with PBST, 100 μL per well was added to the ELISA plate, incubated at 37°C for 45min, and washed 5 times with a plate washer. TMB was equilibrated to room temperature in advance, 100 μL per well was added to the ELISA plate, color was developed for 5min in the dark, and 100 μL per well was added to 0.1M sulfuric acid to stop color development. The absorbance at 450 nm was measured using an enzyme reader.

[0453] ELISA results Fig.18 As shown, the activity decreased after humanization, so the nanobody 2C7-RYH-1 was reverse mutated. The amino acids at positions 5 / 13 / 14 / 44 / 75 / 79 / 87 / 88 / 126 were reverse mutated respectively. The designed point mutation primers are shown in Table 5:

[0454] Table 5

[0455]

[0456]

[0457] The above primers were used for PCR, and the PCR system was as follows:

[0458] name Dosage Template 2C7 1ng 2×master Mix 15μl Upstream primer 1μl Downstream primer 1μl water 13μl

[0459] After PCR, 9 μL was taken out and 1 μL Dpn I enzyme was added to digest overnight at 37°C. The next day, the reaction solution was transformed into DH5α, spread on LB plates containing ampicillin, and cultured overnight at 37°C. The next day, single clones were picked, and 700 μL LB medium containing ampicillin was added to each single clone. The culture was shaken at 37°C and 220 rpm until the OD was about 0.6. 500 μL was taken out for sequencing, and the rest of the bacterial solution was kept for later use.

[0460] Take out 100 μL of the correctly sequenced monoclonal bacterial solution, add it to 100 mL of LB medium for expansion culture, add 100 μL of ampicillin, culture at 37°C, 220 rpm overnight, and extract the plasmid the next day according to the plasmid extraction instructions.

[0461] The plasmid was transfected into 293F using PEI. After 72 hours, the supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane. The protein was purified using a Ni column. After purification, the buffer was replaced with PBS by ultrafiltration and subjected to SDS-PAGE.

[0462] To detect the activity of the protein after the reverse mutation, ELISA was performed. 100 μL of 4 μg / mL CD38-Fc antigen was added to each well of the ELISA plate, coated overnight at 4°C, and washed 3 times with a plate washer the next day. 200 μL of 5% skim milk powder was added to each well, blocked at 37°C for 2 hours, and washed 5 times with a plate washer. 9 antibodies, starting at 100 μg / mL, were diluted 5 times as primary antibodies, and 100 μL of each well was added to the ELISA plate, incubated at 37°C for 1.5 hours. Anti-His-HRP was diluted 1:5000 with PBST, and 100 μL of each well was added to the ELISA plate, incubated at 37°C for 45 minutes, and washed 5 times with a plate washer. TMB was equilibrated to room temperature in advance, and 100 μL of each well was added to the ELISA plate. The color was developed for 5 minutes in the dark, and 100 μL of 0.1M sulfuric acid was added to each well to stop the color development. The absorbance at 450 nm was measured using an ELISA reader. The results are as follows: Fig.19 As shown, there are three mutants whose activities are close to those of the non-humanized sequences ( Fig.19 A), the activity of three lines decreased by about 3 times ( Fig.19 B), and the activity of three other lines decreased by about 5 times ( Fig.19 C).

[0463] 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. An anti-CD38 nanobody, characterized in that: The anti-CD38 nanobody has one or more complementary determining regions (CDRs) selected from the following group: (1) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:7; (2) CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:11; (3) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 18; (4) CDR1 shown in SEQ ID NO:21, CDR2 shown in SEQ ID NO:23, and CDR3 shown in SEQ ID NO:25; (5) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:30, and CDR3 shown in SEQ ID NO:32; (6) CDR1 shown in SEQ ID NO:35, CDR2 shown in SEQ ID NO:37, and CDR3 shown in SEQ ID NO:39; (7) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:44, and CDR3 shown in SEQ ID NO:45; (8) CDR1 shown in SEQ ID NO:14, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:50; (9) CDR1 shown in SEQ ID NO:35, CDR2 shown in SEQ ID NO:54, and CDR3 shown in SEQ ID NO:56; (10) CDR1 shown in SEQ ID NO:59, CDR2 shown in SEQ ID NO:61, and CDR3 shown in SEQ ID NO:63; (11) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:68, and CDR3 shown in LNY; (12) CDR1 shown in SEQ ID NO:72, CDR2 shown in SEQ ID NO:74, and CDR3 shown in SEQ ID NO:76; (13) CDR1 shown in SEQ ID NO:28, CDR2 shown in SEQ ID NO:79, and CDR3 shown in SEQ ID NO:81; (14) CDR1 shown in SEQ ID NO:84, CDR2 shown in SEQ ID NO:86, and CDR3 shown in SEQ ID NO:88; (15) CDR1 shown in SEQ ID NO:91, CDR2 shown in SEQ ID NO:93, and CDR3 shown in SEQ ID NO:95; (16) CDR1 shown in SEQ ID NO:98, CDR2 shown in SEQ ID NO:99, and CDR3 shown in SEQ ID NO:101; (17) CDR1 shown in SEQ ID NO: 104, CDR2 shown in SEQ ID NO: 106, and CDR3 shown in SEQ ID NO: 108; (18) CDR1 shown in SEQ ID NO: 111, CDR2 shown in SEQ ID NO: 113, and CDR3 shown in SEQ ID NO: 115; (19) CDR1 shown in SEQ ID NO: 118, CDR2 shown in SEQ ID NO: 120, and CDR3 shown in SEQ ID NO: 122; (20) CDR1 shown in SEQ ID NO: 125, CDR2 shown in SEQ ID NO: 126, and CDR3 shown in SEQ ID NO: 128; (21) CDR1 shown in SEQ ID NO: 130, CDR2 shown in SEQ ID NO: 131, and CDR3 shown in SEQ ID NO: 133; (22) CDR1 shown in SEQ ID NO:136, CDR2 shown in SEQ ID NO:138, and CDR3 shown in SEQ ID NO:

140.

2. An anti-CD38 antibody, characterized in that: The antibodies include one or more anti-CD38 nanobodies as described in claim 1.

3. A humanized antibody, which is obtained from the anti-CD38 nanobody according to claim 1 or the anti-CD38 antibody according to claim 2.

4. A chimeric antigen receptor (CAR), characterized in that The CAR contains an extracellular domain, which comprises the anti-CD38 nanobody as described in claim 1, the anti-CD38 antibody as described in claim 2, or the humanized antibody as described in claim 3.

5. A fusion protein, characterized in that The fusion protein has: (Z1) a first protein, comprising: the anti-CD38 nanobody according to claim 1, the anti-CD38 antibody according to claim 2, the humanized antibody according to claim 3, or an active fragment thereof; (Z2) a second protein comprising a cytokine; and (Z3) optionally a linker between the first protein and the second protein.

6. A recombinant protein, characterized in that The recombinant protein has: (i) the anti-CD38 nanobody according to claim 1, the anti-CD38 antibody according to claim 2, the humanized antibody according to claim 3, the fusion protein according to claim 5, or an active fragment thereof; and (ii) optionally a tag sequence to facilitate expression and / or purification.

7. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the following group: the anti-CD38 nanobody as described in claim 1, the anti-CD38 antibody as described in claim 2, the humanized antibody as described in claim 3, the chimeric antigen receptor as described in claim 4, the fusion protein as described in claim 5, the recombinant protein as described in claim 6, or a combination thereof.

8. An expression vector, characterized in that The expression vector contains the polynucleotide according to claim 7.

9. A host cell, characterized in that The host cell contains the expression vector according to claim 8, or the polynucleotide according to claim 7 is integrated into its genome.

10. An engineered immune cell, characterized in that: The engineered immune cell contains the expression vector as claimed in claim 8 or has the exogenous polynucleotide as claimed in claim 7 integrated into its genome, or expresses the chimeric antigen receptor as claimed in claim 4.

11. A method for producing anti-CD38 nanobodies, characterized in that: The method comprises the steps of: (a) culturing the host cell as claimed in claim 9 under conditions suitable for the production of nanobodies, thereby obtaining a culture containing anti-CD38 nanobodies; (b) separating and / or recovering said anti-CD38 Nanobody from said culture; and (c) Optionally, purifying and / or modifying the anti-CD38 Nanobody obtained in step (b).

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