Preparation and use of camelid nanobodies targeting human cd38 molecules

Abstract

The application provides camelid nanobody preparation and application targeting human CD38 molecules. Specifically, the application provides five camelid-derived nanobodies targeting human CD38 molecules and application thereof. Specifically, the application relates to a specific binding nanobody sequence obtained by screening a camelid natural nanobody phage display library against an extracellular segment of a human CD38 molecule, and an E. coli expression system of a corresponding anti-human CD38 nanobody protein is constructed. The nanobody has the advantages of small molecular weight, easy modification, easy combination with other antibodies to construct a bispecific antibody, easy construction of a CAR-T, easy preparation of a conjugate with other drugs, and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedicine or biopharmaceutical technology, and discloses the preparation and application of camel-family nanobodies targeting human CD38 molecules. Background Technology

[0002] CD38, also known as ADP-ribose cyclase or cyclic ADP-ribose hydrolase, is a single-transmembrane glycoprotein with a molecular weight of 45 kDa. It was discovered in 1980 by E.L. Reinherz and colleagues in their analysis of human lymphocytes. CD38 is primarily expressed in T cells, dendritic cells, natural killer (NK) cells, and hematopoietic stem cells, playing a crucial role in the immune process, such as mediating cytokine production, regulating T lymphocyte proliferation, and protecting mature B lymphocytes and dendritic cells from apoptosis. Furthermore, CD38 is also expressed on immunosuppressive cells, such as regulatory T cells, regulatory B cells, and bone marrow-derived suppressor cells. Therefore, CD38 expression can be used to define a suppressive subset of regulatory T cells that reduces the anti-tumor activity of the immune system. When normal plasma cells degenerate, the expression level of CD38 on their cell surface significantly increases; therefore, CD38 is one of the biomarkers for B-lymphocytic tumors.

[0003] Clinical studies have shown that over 90% of malignant plasma cells in multiple myeloma (MM) patients highly express CD38 molecules, making CD38 a primary target for targeted therapy development in MM patients. Currently, two CD38-specific therapeutic antibodies, Daratumumab and Isatuximab, have been approved by the FDA. Several other CD38 antibody-related drugs are in preclinical and clinical trials, such as chimeric antigen receptor T-cell (CAR-T) and CD38 antibody-drug conjugates (ADCs). These drugs can exert anti-tumor activity through multiple effector mechanisms. However, the aforementioned marketed and clinically tested CD38 antibodies are all fully human or humanized antibodies, which have drawbacks such as large molecular weight, poor tissue penetration, complex manufacturing processes, and high production costs, limiting their therapeutic efficacy to some extent. Therefore, developing novel CD38-targeting antibody molecules in combination with other treatment methods for CD38-related tumors could significantly expand the application scope and therapeutic effects of CD38 antibodies.

[0004] Immunoglobulins in mammalian serum are tetramers composed of two identical heavy and light chains, with a molecular weight of approximately 150 kDa. In 1993, Belgian scientists Hamers-Cazterman et al. discovered a naturally occurring heavy-chain antibody (HcAbs) lacking the light chain in the serum of camels. This HcAb structure possesses a single-domain variable structure that binds to the antigen, with a relative molecular mass of approximately 15 kDa, only about 1 / 10 that of traditional antibodies. It is called a nanobody or VHH antibody. VHH consists of four conserved framework regions and three hypervariable complementarity-determining regions, and is currently the smallest naturally occurring functional antibody structure discovered.

[0005] Compared with currently marketed traditional antibody drugs, nanobodies exhibit several unique advantages:

[0006] (1) The CDR3 of nanobodies presents a prominent long ring shape with the corresponding human antibody region, which can bind to hidden antigenic epitopes, slit-like or pocket-like antigenic epitopes that traditional antibodies cannot bind to. For the currently mature targeted cancer therapy, nanobodies can also be used to develop new antibody drugs; (2) Their sequence has high homology with the fully human antibody sequence and weak immunogenicity; (3) Their molecular weight is small and they are not easy to aggregate in vivo, and they have stronger tissue penetration compared with traditional antibodies; (4) They can maintain good stability in harsh environments, such as high temperature, strong acid, strong alkali or in the presence of proteases. They are easy to store and transport, and can be prepared into finished drug products with various administration methods; (5) They are easy to link with other molecules and can be prepared into various antibody drug forms such as multivalent antibodies, bispecific antibodies, targeted immunotoxins, ADC drugs, etc., expanding their application scope in cancer diagnosis and treatment.

[0007] In 2018, the EMA approved the nanobody drug Cablivi for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults, making Cablivi the world's first marketed nanobody drug. Nanobody-based therapies hold great promise for research in cancer treatment.

[0008] Currently, many marketed monoclonal antibodies targeting human CD38 are either fully human or humanized antibodies. These antibodies have drawbacks such as large molecular weight, poor tissue penetration, high production costs, and difficulty in combining with other target antibodies to create bispecific antibodies. In contrast, camel-derived nanobodies targeting human CD38 offer advantages such as small molecular weight, ease of modification, ease of combining with other antibodies to construct bispecific antibodies, ease of constructing CAR-T therapy, and ease of preparation as conjugates with other drugs.

[0009] In summary, there is an urgent need in this field to develop a camel-family nanobody that targets human CD38. Summary of the Invention

[0010] The purpose of this invention is to provide a camel-like nanobody that targets human CD38.

[0011] In a first aspect of the invention, an anti-CD38 nanobody is provided, the nanobody being capable of specifically binding to CD38, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is selected from one or more of the following:

[0012] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3;

[0013] (2) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12;

[0014] (3) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:20;

[0015] (4) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28;

[0016] (5) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:36.

[0017] In another preferred embodiment, any of the amino acid sequences described above further includes a derived sequence which has optionally been added, deleted, modified and / or substituted at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and retains the ability to bind to CD38 with high affinity.

[0018] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0019] In another preferred embodiment, the VHH chain further includes a frame region FR, wherein the frame region FR is selected from one or more of the following:

[0020] (1) FR1 shown in SEQ ID NO:4, FR2 shown in SEQ ID NO:5, FR3 shown in SEQ ID NO:6, and FR4 shown in SEQ ID NO:7 (corresponding to FR of nanobody 2E);

[0021] (2) FR1 shown in SEQ ID NO:13, FR2 shown in SEQ ID NO:14, FR3 shown in SEQ ID NO:15, and FR4 shown in SEQ ID NO:7 (corresponding to FR of nanobody 7G);

[0022] (3) FR1 shown in SEQ ID NO:21, FR2 shown in SEQ ID NO:22, FR3 shown in SEQ ID NO:23, and FR4 shown in SEQ ID NO:7 (corresponding to FR of nanobody 1E);

[0023] (4) FR1 shown in SEQ ID NO:29, FR2 shown in SEQ ID NO:30, FR3 shown in SEQ ID NO:31, and FR4 shown in SEQ ID NO:32 (corresponding to FR of nanobody 5G);

[0024] (5) FR1 shown in SEQ ID NO:29, FR2 shown in SEQ ID NO:30, FR3 shown in SEQ ID NO:6, and FR4 shown in SEQ ID NO:7 (corresponding to FR of nanobody 8A).

[0025] In another preferred embodiment, the VHH chain of the anti-CD38 nanobody is selected from one or more of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:33 or SEQ ID NO:37.

[0026] In a second aspect of the invention, an anti-CD38 antibody is provided, the antibody comprising one or more anti-CD38 nanobodies as described in the first aspect of the invention.

[0027] In another preferred embodiment, the anti-CD38 antibody comprises one or more amino acid sequences such as the VHH chain shown in SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:33 or SEQ ID NO:37.

[0028] In another preferred embodiment, the antibody may be a monomer, a bivalent antibody, and / or a multivalent antibody.

[0029] In a third aspect of the invention, a polynucleotide is provided that encodes a protein selected from the group consisting of: nanobodies as described in the first aspect of the invention, or anti-CD38 antibodies as described in the second aspect of the invention.

[0030] In another preferred embodiment, the nucleotide sequence of the polynucleotide comprises one or more of SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID NO:38.

[0031] In another preferred embodiment, the polynucleotide is RNA, DNA, or cDNA.

[0032] In a fourth aspect of the invention, an expression vector is provided that expresses the polynucleotide described in the third aspect of the invention.

[0033] 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 includes viral vectors, such as lentiviruses, adenoviruses, AAV viruses, retroviruses, or combinations thereof.

[0034] In a fifth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the fourth aspect of the invention, or having the polynucleotide described in the third aspect of the invention integrated into its genome.

[0035] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0036] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli and yeast cells.

[0037] In a sixth aspect of the invention, a method for generating anti-CD38 nanobodies is provided, comprising the steps of:

[0038] (a) Culturing the host cells described in the fifth aspect of the invention under conditions suitable for generating nanobodies, thereby obtaining a culture containing the anti-CD38 nanobodies; and

[0039] (b) Isolating or recovering the anti-CD38 nanobody from the culture; and optionally...

[0040] (c) Purification and / or modification of the CD38 nanobody obtained in step (b).

[0041] In another preferred embodiment, the anti-CD38 nanobody has an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:33 or SEQ ID NO:37.

[0042] In a seventh aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0043] (a) a nanobody as described in the first aspect of the invention, or an anti-CD38 antibody as described in the second aspect of the invention; and operably linked

[0044] (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins, or combinations thereof.

[0045] In another preferred embodiment, the radionuclide includes:

[0046] (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 combinations thereof; and / or

[0047] (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 combinations thereof.

[0048] In another preferred embodiment, the coupling portion is a drug or toxin.

[0049] In another preferred embodiment, the drug is a cytotoxic drug.

[0050] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0051] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule 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).

[0052] In another preferred embodiment, the toxin is selected from the group consisting of:

[0053] Otostatins (e.g., otostatin E, otostatin F, MMAE, and MMAF), chlortetracycline, tebuconazole, pectin, pectin A-chain, cobustatin, docalimicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, pseudomonas exotoxin (PE) A, PE40, absinthecin, absinthecin A-chain, saccharin A-chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachiin, Sapaonaria (Officialis) inhibitors, glucocorticoids, or combinations thereof.

[0054] In another preferred embodiment, the coupling portion is a detectable marker.

[0055] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0056] In another preferred embodiment, the coupling portion is a microtubule inhibitor DM1.

[0057] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) anti-CD38 nanobody as described above, or an anti-CD38 antibody as described above.

[0058] In another preferred embodiment, the polyvalent means that the amino acid sequence of the immunoconjugate contains a plurality of repeating anti-CD38 nanobodies as described above, or anti-CD38 antibodies as described above.

[0059] In another preferred embodiment, the immunoconjugate is used for the diagnosis or prognosis of cancer, particularly for tumors expressing CD38 (i.e., CD38-positive tumors).

[0060] In another preferred embodiment, the immunoconjugate is used for the diagnosis and / or treatment of tumors expressing the CD38 protein.

[0061] In an eighth aspect of the invention, the use of nanobodies as described in the first aspect of the invention, or anti-CD38 antibodies as described in the second aspect of the invention, is provided for the preparation of (a) reagents for detecting CD38 molecules; and (b) medicaments for treating tumors.

[0062] In another preferred embodiment, the detection is either in vivo or in vitro.

[0063] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0064] In a ninth aspect of the present invention, a pharmaceutical composition comprising:

[0065] (i) the 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, and / or the immunoconjugate as described in the seventh aspect of the present invention;

[0066] (ii) Pharmaceutically acceptable carriers.

[0067] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0068] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors, such as cytotoxic drugs.

[0069] In another preferred embodiment, the pharmaceutical composition is used to treat tumors that express the CD38 protein (i.e., CD38 positive).

[0070] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors, wherein the tumors are selected from the group consisting of: myeloma, gastric cancer, liver cancer, leukemia, kidney tumors, lung cancer, small bowel cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumors, or bladder tumors.

[0071] In another preferred embodiment, the myeloma is multiple myeloma (MM).

[0072] In a tenth aspect of the invention, one or more uses are provided, including nanobodies as described in the first aspect of the invention, or anti-CD38 antibodies as described in the second aspect of the invention, immunoconjugates as described in the seventh aspect of the invention, or pharmaceutical compositions as described in the ninth aspect of the invention.

[0073] (a) Used to detect CD38 molecules;

[0074] (b) Used for flow cytometry detection;

[0075] (c) Used for cellular immunofluorescence detection;

[0076] (d) Used to treat tumors;

[0077] (e) Used for tumor diagnosis.

[0078] (f) Used in the preparation of cell therapy products that specifically bind to human CD38;

[0079] (g) Used in the preparation of antibody drugs for treating tumors, immunotoxins targeting human CD38, and antibody-drug conjugate products;

[0080] (h) Used to prepare cell therapy products for treating tumors.

[0081] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0082] In an eleventh aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0083] (i) the 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; and

[0084] (ii) Optional tag sequences to assist in expression and / or purification.

[0085] In another preferred embodiment, the tag sequence includes a 6His tag and an HA tag.

[0086] In another preferred embodiment, the recombinant protein specifically binds to the CD38 protein.

[0087] In a twelfth aspect of the invention, the use of nanobodies as described in the first aspect of the invention or anti-CD38 antibodies as described in the second aspect of the invention, immunoconjugates as described in the seventh aspect of the invention, or pharmaceutical compositions as described in the ninth aspect of the invention, for the preparation of pharmaceuticals, reagents, detection plates, or kits is provided.

[0088] The reagents, detection plates, or kits are used to detect CD38 protein in samples.

[0089] The drug is used to treat or prevent tumors that express CD38.

[0090] In another preferred embodiment, the tumor includes: myeloma, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small bowel cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.

[0091] In a thirteenth aspect of the invention, a kit is provided containing a nanobody as described in the first aspect of the invention, an anti-CD38 antibody as described in the second aspect of the invention, an immunoconjugate as described in the seventh aspect of the invention, or a pharmaceutical composition as described in the ninth aspect of the invention.

[0092] In a fourteenth aspect of the present invention, a method for detecting CD38 protein in a sample is provided, the method comprising the steps of:

[0093] (1) Contact the sample with the nanobody described in the first aspect of the present invention;

[0094] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD38 protein in the sample.

[0095] In another preferred embodiment, the method is an in vitro method.

[0096] In another preferred embodiment, the method is a non-therapeutic and non-diagnostic method.

[0097] In a fifteenth aspect of the invention, a method for treating CD38-related diseases is provided, the method comprising administering to a subject in need a nanobody as described in a first aspect of the invention or an anti-CD38 antibody as described in a second aspect of the invention, an immunoconjugate as described in a seventh aspect of the invention, or a pharmaceutical composition as described in a ninth aspect of the invention.

[0098] In a sixteenth aspect of the invention, a CAR-T cell is provided, the CAR-T cell expressing a chimeric antigen receptor CAR, wherein the antigen-binding domain of the CAR has a nanobody as described in the first aspect of the invention.

[0099] In a seventeenth aspect of the invention, a formulation is provided comprising the CAR-T cells described in the sixteenth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0100] In another preferred embodiment, the formulation is a liquid formulation.

[0101] In another preferred embodiment, the dosage form of the preparation includes an injection.

[0102] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0103] Figure 1 Flow cytometry analysis was used to analyze the binding of anti-human CD38 nanobody to CD38+-CHO and CD38--CHO cells. a) Analysis of the binding of anti-human CD38 nanobody to MM1S cells; b) Analysis of the binding of anti-human CD38 nanobody to CD38--CHO cells; c) Analysis of the binding of anti-human CD38 nanobody to CD38+-CHO cells; PC) Control of anti-human CD38 positive human ScFv antibody.

[0104] Figure 2 SDS-PAGE electrophoresis image of purified anti-human CD38 nanobody.

[0105] Figure 3 Flow cytometry analysis of affinity for anti-human CD38 nanobodies.

[0106] Figure 4 Three-dimensional structural simulation of the interaction between anti-human CD38 nanobody and CD38.

[0107] Figure 5 Flow cytometry was used to evaluate the endocytosis rate of anti-human CD38 nanobodies.

[0108] Figure 6 Detection of in vitro cytotoxic activity of anti-human CD38 nanobody (FA-2E)-DM1 conjugate against cells. Detailed Implementation

[0109] Through extensive and in-depth research and numerous screenings, the inventors successfully obtained a group of anti-CD38 nanobodies. Specifically, using biologically active, high-purity human CD38 extracellular protein, and employing phage display technology based on camel-derived nanobodies, candidate nanobodies targeting human CD38 were obtained via solid-phase screening. After analysis at the protein and cellular levels using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FCM), five nanobodies specifically binding to the human CD38 extracellular protein and capable of binding to CD38+ cells were ultimately obtained. These human CD38-specific nanobodies have broad application prospects in the development of therapeutic antibody drugs targeting human CD38. This invention was completed based on this foundation.

[0110] In addition, the present invention provides five camel-derived natural nanobodies targeting the extracellular domain of human CD38, along with the nucleotide and amino acid sequences of these nanobodies, and vectors and host cells containing the coding sequences.

[0111] the term

[0112] As used herein, the term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0113] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0114] As used herein, the terms "nanobody" (single domain antibody, sdAb, or VHH) and "nanobody" have the same meaning: a nanobody constructed by cloning the variable region of an antibody heavy chain, consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0115] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 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 the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. 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; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0116] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are 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.

[0117] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions 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)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0118] As those skilled in the art will know, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the described anti-CD38 protein antibody or fragments thereof.

[0119] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably.H "They can be used interchangeably."

[0120] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0121] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0122] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0123] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to the CD38 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0124] 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, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0125] 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 frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the 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 regions.

[0126] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein.

[0127] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0128] As used herein, the terms “fragment,” “derivative,” and “analyte” 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 conserved or non-conserved amino acid residues (preferably conserved 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 substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0129] The antibody of this invention refers to a polypeptide containing the aforementioned CDR region and having CD38 protein-binding activity. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0130] The variant forms of the polypeptide include: homologous sequences, conserved 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 severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0131] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies 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.

[0132] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0133] Table A

[0134]

[0135]

[0136] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0137] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0138] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0139] The present invention also relates to polynucleotides that hybridize with the above-described 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 hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0140] The VHH chain of the nanobody provided by this invention comprises CDR1, CDR2, and CDR3 selected from the following combinations:

[0141] (1) CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3 (corresponding to the CDR of nanobody 2E);

[0142] (2) CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12 (corresponding to the CDR of nanobody 7G);

[0143] (3) CDR1 shown in SEQ ID NO:18, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:20 (corresponding to the CDR of nanobody 1E);

[0144] (4) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:27, and CDR3 shown in SEQ ID NO:28 (corresponding to the CDR of nanobody 5G);

[0145] (5) CDR1 shown in SEQ ID NO:26, CDR2 shown in SEQ ID NO:35, and CDR3 shown in SEQ ID NO:36 (corresponding to the CDR of nanobody 8A).

[0146] In another preferred embodiment, the amino acid sequence of the nanobody of the present invention is shown in SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:33 or SEQ ID NO:37; and its nucleotide sequence is shown in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID NO:38, respectively. Further, the nanobody of the present invention also includes a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the above sequences.

[0147] In another preferred embodiment, the nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34 or SEQ ID NO:38 may have one or more nucleotide sequences added, substituted, deleted or inserted to obtain a derived sequence that retains the ability to bind to CD38 with high affinity.

[0148] By utilizing affinity maturation and computer simulation techniques, amino acid sequences can be modified on the nanobody sequences of this invention to obtain new nanobody sequences.

[0149] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0150] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0151] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0152] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0153] This invention provides an expression system for expressing the above-mentioned human CD38 nanobody, wherein a host cell contains the above-mentioned expression vector. The host cell is preferably *Escherichia coli*.

[0154] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0155] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0156] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0157] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0158] This invention provides a method for preparing nanobodies targeting human CD38, specifically including the following steps: first, synthesizing human CD38 extracellular fragment protein and CD38-highly expressing cell lines; then, coupling the CD38 protein onto an ELISA plate to display the spatial conformation of human CD38; screening nanobodies using a camel-derived natural nanobody phage display library technology to obtain nanobodies that specifically bind to human CD38; and finally, transferring the candidate nanobody gene into *E. coli* to establish an expression system that can efficiently express nanobodies in *E. coli*.

[0159] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0160] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0161] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. drug-activated enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 9. therapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0162] Immunoconjugates

[0163] The present invention also provides an immunoconjugate comprising:

[0164] (a) the VHH chain of the anti-CD38 nanobody as described in the first aspect of the present invention, or the anti-CD38 nanobody as described in the second aspect of the present invention; and

[0165] (b) The coupling part selected from the following group: radionuclides, enzyme antibodies, cells, or combinations thereof.

[0166] This invention provides a conjugate of a nanobody targeting human CD38 and DM1, wherein the anti-CD38 nanobody is conjugated with the microtubule inhibitor DM1 to obtain a drug conjugate based on the anti-human CD38 nanobody, and the cytotoxic effect of this drug conjugate on CD38+ cells is detected by CCK-8 assay.

[0167] The nanobody targeting human CD38 and the conjugate of DM1 provided by this invention have a significant in vitro killing effect on CD38+ myeloma cells.

[0168] The immunoconjugate of the present invention can be used for non-invasive detection of CD38 expression in the test subject. It has small size and high specificity, is suitable for systemic detection that simultaneously targets primary and metastatic tumors, has high accuracy, and low radiation dose.

[0169] Cytotoxic agents

[0170] The conjugation portion constituting the antibody-drug conjugates of the present invention includes: a toxin, such as a small molecule toxin or enzyme-active toxin of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, tebuconazole, pyrethroid toxin, pyrethroid toxin A-chain, cobustatin, docalimicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxetine dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrinogen, abrinogen A-chain, and arbuscular mycotoxin A-chain. Antibodies can be conjugated with α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitors, glucocorticoids and other chemotherapeutic agents, as well as radioactive isotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32, and radioactive isotopes of Lu, including Lu177. Antibodies can also be conjugated to anticancer prodrug activating enzymes capable of converting prodrugs to their active forms.

[0171] The preferred small molecule drug is a compound with high cytotoxicity, preferably monomethylauristatin, galicariin, maytansine, or combinations thereof; more preferably selected from: monomethylauristatin-E (MMAE), monomethylauristatin-D (MMAD), monomethylauristatin-F (MMAF), or combinations thereof.

[0172] Pharmaceutical Composition

[0173] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or immunoconjugate, and a pharmaceutically acceptable carrier. Typically, these substances are 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 may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0174] The pharmaceutical compositions of the present invention can be directly used to bind to CD38 protein molecules, and therefore can be used to treat tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0175] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or conjugate thereof) 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 formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. 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 nanograms / kg body weight to about 50 milligrams / kg body weight per day, more preferably 50 nanograms / kg body weight to about 1 milligram / kg body weight, or 10 micrograms / kg body weight to about 10 milligrams / kg body weight per day.

[0176] Furthermore, the polypeptides or their conjugates of the present invention can also be used with other therapeutic agents (such as antitumor agents or immunomodulators).

[0177] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals, wherein this safe and effective amount is generally at least about 10 nanograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably about 50 nanograms per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skill of a skilled physician.

[0178] Labeled nanobodies

[0179] In a preferred embodiment of the invention, the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0180] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, anti-CD38 nanobodies are labeled with colloidal gold to obtain colloidal gold-labeled nanobodies.

[0181] The anti-CD38 nanobody of the present invention has excellent specificity and high potency.

[0182] CAR-T cells

[0183] As used herein, the terms "CAR-T cell", "CAR-T", and "CAR-T cell of the present invention" all refer to the CAR-T cell described in the nineteenth aspect of the present invention.

[0184] As used herein, a chimeric antigen receptor (CAR) comprises an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. The extracellular domain includes an optional signal peptide and a target-specific binding element (also known as an antigen-binding domain). The intracellular domain includes a co-stimulatory molecule and a ζ-chain portion. The co-stimulatory signal transduction region refers to a portion of the intracellular domain containing the co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, not an antigen receptor or its ligands.

[0185] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically scFv (single-chain variable fragment). Single-chain antibodies are preferably a single amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the invention, the scFv comprises the VHH chain described in the first aspect of the invention, or the nanobody described in the second aspect of the invention.

[0186] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0187] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0188] When CARs are expressed in T cells, their extracellular domain recognizes a specific antigen, which is then transduced through an intracellular domain. This transduction induces cell activation and proliferation, cytotoxicity, and the secretion of cytokines such as IL-2 and IFN-γ, affecting tumor cells and leading to tumor cell atrophy, induced death, or other adverse effects, ultimately resulting in tumor burden reduction or elimination in patients. The antigen-binding domain is preferably fused with one or more intracellular domains derived from co-stimulatory molecules and the ζ chain.

[0189] Detection methods

[0190] The present invention also relates to a method for detecting CD38 protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of CD38 protein in the dissolved samples.

[0191] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0192] Reagent test kit

[0193] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0194] This invention also provides a detection kit for detecting CD38 levels, comprising an antibody that recognizes the CD38 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0195] The present invention also provides a kit containing the immunoconjugate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, an isotope tracer, and one or more reagents selected from the group consisting of contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0196] Preferably, the kit of the present invention is an in vivo diagnostic kit for non-invasive detection of CD38 expression in a test subject.

[0197] application

[0198] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of CD38-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of CD38.

[0199] The present invention also provides the application of the aforementioned nanobody targeting human CD38 and conjugate of DM1 in the preparation of tumor drugs.

[0200] The main advantages of this invention include

[0201] (1) The anti-human CD38 nanobody of the present invention has a small molecular weight, is easy to modify, is easy to combine with other antibodies to construct bispecific antibodies, is easy to construct CAR-T, and is easy to prepare as a conjugate with other drugs.

[0202] (2) The nanobody of the present invention can be expressed using a prokaryotic expression system, thereby reducing production costs and simplifying production, thus expanding the application scope of therapeutic antibodies targeting human CD38.

[0203] (3) The anti-CD38 nanobody drug conjugate of the present invention has a smaller molecular weight than conventional full-size antibody-drug conjugates, which theoretically will have better tumor tissue penetration, and therefore may have unique advantages in the treatment of solid tumors. Nanobodies have significant advantages in production, transportation and administration methods.

[0204] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0205] The antibody sequence involved in this invention

[0206] Table 1. Nanobodies of the present invention and their sequences

[0207]

[0208]

[0209]

[0210] Example 1: Screening of nanobodies targeting human CD38

[0211] (1) Screening was performed using solid-phase screening. Human CD38 protein was diluted to 10 μg / mL with PBS, and 100 μL was coated in a 96-well microplate and incubated overnight at 4°C.

[0212] (2) On the second day, discard the protein solution and block with 10% MBPS at room temperature for 2 hours.

[0213] (3) After 2 hours, add 100 μL of natural camel family nanoantibody phage library and shake at room temperature for 2 hours to bind.

[0214] (4) After 2 hours, wash with PBST 10 times, then wash with PBS 10 times to remove unbound phages.

[0215] (5) Add 200 μL of 0.1M glycine hydrochloric acid solution (0.1M Gly-HCl, pH 3.0), shake and elute for 20 min, then add 60 μL of 1M Tris-HCl to neutralize, gently pipette to obtain 520 μL of eluent.

[0216] (6) The bacteriophages that were eluted and collected and bound to CD38 were transferred into Escherichia coli TG1 cells in the logarithmic growth phase, cultured at 37°C with shaking for 1 h, and then plated on 2YT plates (1.6% (W / V) tryptone, 1% (W / V) yeast extract, 0.5% (W / V) sodium chloride) containing a final concentration of 100 μg / mL ampicillin (Amp) resistance, and cultured at 37°C overnight.

[0217] (7) The next day, take 20 μL of overnight bacterial culture obtained by scraping and add it to 10 mL of 2YT-Amp culture medium. Culture at 37°C with shaking until the logarithmic growth phase. Add an appropriate amount of helper phage M13KO7. Infect for 1 hour and then culture at 37°C with shaking overnight.

[0218] (8) The next day, the phage supernatant was collected by centrifugation and used for the next round of solid-phase screening. The same screening was repeated 3 times to gradually obtain enrichment.

[0219] Example 2: Screening for specific monoclonal antibodies using phage enzyme-linked immunosorbent assay (ELISA).

[0220] (1) Monoclonal phage ELISA detection: From the TG1 cell colonies containing phage obtained after the fourth round of screening, single colonies were randomly selected and inoculated into 100 μL of 2YT-Amp medium in a 96-well plate. The plates were cultured at 37°C with shaking until the logarithmic growth phase. An appropriate amount of helper phage M13KO7 was added, and after 1 hour of infection, the plates were cultured overnight at 37°C with shaking. The next day, the phage supernatant was collected by centrifugation, blocked with MPBST at room temperature for 2 hours, and transferred to an antigen-coated ELISA plate (prepared with MPBST blocking at room temperature for 2 hours) that had been incubated overnight at 4°C. The plate was reacted at room temperature for 2 hours. Unbound phage was washed away with PBST, and HRP-anti-M13 antibody was added. The plate was reacted at room temperature for 1 hour. Unbound antibody was washed away again with PBST, and TMB chromogenic solution was added. The plate was reacted at room temperature for 10 minutes, and the reaction was terminated with 2M H2SO4. The absorbance was read at 450 nm using a microplate reader. A phage monoclonal antibody is considered positive when its OD450 reading is greater than 1.2 for binding to the CD38-Fc antigen and less than 0.5 for binding to the IgG4-Fc tag protein. Colonies testing positive are sent to a sequencing company for sequencing. The sequencing results are compared using DNAMAN analysis software, and candidate nanobodies with identical sequences are merged.

[0221] (2) Crude extraction of antibody periplasmic protein: The TG1 nanobody strains that obtained positive results in the above screening were cultured at 37°C with shaking until the logarithmic growth phase. IPTG was added to a final concentration of 1 mM, and the culture was carried out overnight at 30°C with shaking. The expressed bacterial cells were collected by centrifugation the next day. The precipitate was resuspended with 1 mg / mL lysozyme solution and digested on ice for 20 to 30 min. The crude periplasmic protein sample of the antibody expression supernatant was collected by high-speed refrigerated centrifugation.

[0222] (3) Periplasmic protein cell binding assay: After culturing, the cell samples to be tested were digested and collected, and resuspended in 2% FPBS and washed twice. 200 μL of different periplasmic protein samples were added and incubated at 4℃ for 40 min. The cells were washed twice with 2% FPBS. Secondary antibody incubation: 100 μL of anti-Flag-APC secondary antibody was added to each tube and incubated at 4℃ in the dark for 20–30 min. After washing the cells with 2% FPBS, they were resuspended in 400 μL of 2% FPBS, filtered, and transferred to flow cytometry tubes for FCM analysis. Based on the flow cytometry results, cells binding to CD38 were screened. + Candidate nanobodies that bind to Daudi cells (human B-cell lymphoma cells).

[0223] Example 3: Expression and purification of candidate target human CD38 nanobody in E. coli host.

[0224] (1) The plasmids carrying the above different nanobody expression sequences were transformed into BL21 / DE3 Escherichia coli competent cells, plated in 2YT-Amp culture plates, and cultured overnight at 37°C.

[0225] (2) The next day, pick single colonies from the plate and add them to 2YT culture medium (1.6% (w / v) tryptone, 1% (w / v) yeast extract, 0.5% (w / v) sodium chloride), and incubate at 37°C with shaking. 600 The value was 0.6–0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM, and expression was induced overnight at 30°C (approximately 16 h).

[0226] (3) Collect the bacterial cells by centrifugation, prepare a lysozyme solution with a final concentration of 1 mg / mL using PBS buffer, resuspend the centrifuged bacterial cell pellet, and incubate on ice for 20-30 min.

[0227] (4) Centrifuge at 4℃ to collect the supernatant after sterilization treatment for subsequent purification.

[0228] (5) Purification of nanobodies using His-Ni column affinity chromatography: The supernatant was loaded onto the Ni-NTA column at a flow rate of approximately 1 mL / min. The Ni-NTA column was washed with 5 column volumes of NP-10 (10 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl); subsequently, the Ni-NTA column was washed with 10 column volumes of NP-20 (20 nM imidazole). The target protein was eluted with NP-250 (250 nM imidazole), and the eluted protein solution was collected. The protein purity was determined by SDS-PAGE electrophoresis.

[0229] (6) Replace the nanobody buffer system with ultrafiltration and concentrate the protein sample concentration. Transfer the nanobody sample eluted by affinity chromatography into an ultrafiltration tube, centrifuge at 4°C at low speed, and when the sample volume is concentrated to about 1 mL, add the same volume of pre-cooled PBS and concentrate to 1 mL. Repeat 3 times.

[0230] (7) Collect the nanobody samples after concentration of replacement buffer and store them at -80℃.

[0231] Example 4: Specificity detection of candidate target human CD38 nanobodies

[0232] 4.1. Enzyme-linked immunosorbent assay (ELISA) detection:

[0233] (1) Dilute human CD38 and BSA protein with PBS to 1 μg / ml, and coat 100 μL / well in a 96-well microplate. Incubate overnight at 4°C.

[0234] (2) The next day, discard the coating solution. Wash three times with 300 μL / well PBST and block with 5% MPBST at room temperature for 1 h. At the same time, block the nanobody samples with 5% MPBST for 1 h.

[0235] (3) Discard the blocking solution and wash three times with 300 μL / well PBST. Add the purified nanobody sample and incubate at room temperature for 1 h.

[0236] (4) Discard the nanobody sample solution and wash three times with 300 μL / well PBST. Add anti-Flag-HRP secondary antibody and incubate at room temperature in the dark for 1 h.

[0237] (5) Discard the secondary antibody and wash 6 times with 300 μL / well PBST. Add TMB chromogenic solution and detect the OD value at 450 nm after 10 min. According to the reading results, nanobodies that bind to CD38-Fc protein but do not bind to the protein tag IgG1-Fc are judged as positive candidate antibodies.

[0238] 4.2. Flow cytometry (FCM) detection and human CD38 + Cell-specific binding candidate nanobodies:

[0239] (1) The positive nanobodies obtained in Example 4.1 were further subjected to cell binding detection. Adherent myeloma cell line MM1S (CD38) was cultured... + ), self-built CHO (CD38) + ) and CHO (CD38) - Cells were digested with trypsin, centrifuged and collected, and washed twice with 2% FPBS.

[0240] (2) Add 100 μL of the candidate positive nanobody obtained in step 4 to each tube and incubate at room temperature for 1 h. Centrifuge to collect cells, discard the antibody incubation solution, and wash the cells three times with 2% FPBS. Simultaneously, the ScFv region of the fully human anti-CD38 antibody (US9951144B2 patent number) was used as a positive control. Figure 1 The sample, labeled PC, was synthesized using the corresponding nucleotide sequence of the antibody to construct a recombinant plasmid. This plasmid was then transformed into the E. coli BL21(DE3) prokaryotic expression system. After IPTG induction, the plasmid was purified by His-nickel column affinity chromatography for later use.

[0241] (3) Add 100 μL of the anti-Flag-APC secondary antibody targeting the candidate nanobody and the positive control recombinant antibody Flag tag to each tube and incubate in the dark for 30 min.

[0242] (4) Centrifuge and discard the supernatant, resuspend in 2% FPBS, wash 3 times, and centrifuge to collect cells.

[0243] (5) Resuspend in 400 μL of 2% FPBS, filter and transfer into a flow cytometer for FCM analysis.

[0244] Based on the flow cytometry results ( Figure 1 ), will be with CD38 +MM1S and CD38 + -CHO cells bind, but not to CD38. - -CHO-bound candidate nanobodies were identified as those capable of binding to CD38. + Cell-bound positive nanobodies. Five nanobody sequences specifically targeting CD38 were ultimately screened.

[0245] The numbers are 2E, 7G, 1E, 5G, and 8A. Their nucleotide sequences are SEQ ID NO:9, SEQ ID NO:17, SEQ ID NO:25, SEQ ID NO:34, and SEQ ID NO:38; the corresponding amino acid sequences are SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:33, and SEQ ID NO:37.

[0246] The purification diagrams of the five CD38 nanobodies are shown below. Figure 2 The results showed that the purity of nanobodies could reach over 90% after affinity purification.

[0247] Example 5: Evaluation of Nanobody Affinity

[0248] The interaction between different concentrations of nanobodies and myeloma cell line MM1S (CD38) was detected by flow cytometry (FCM). + The average fluorescence intensity of the binding was used to reflect the affinity of the nanobody to the CD38 protein, and the affinity was compared with that of the positive control PC (PC control is the ScFv sequence segment of the anti-CD38 antibody sequence in Sorrento's CD38 CAR-T project, and the plasmid expressing the ScFv segment was constructed as PC positive control).

[0249] (1) The positive nanobodies obtained in Example 5 were serially diluted three times starting from 100 μg / μl. 100 μL of the antibody sample was added to myeloma cells MM1S resuspended in 2% FPBS. The cells were incubated at room temperature for 1 h.

[0250] (2) Centrifuge to collect cells, discard antibody incubation solution, and wash cells 3 times with 2% FPBS.

[0251] (3) Add 100 μL of anti-Flag-APC secondary antibody to each tube and incubate in the dark for 30 min.

[0252] (4) Centrifuge and discard the supernatant, resuspend in 2% FPBS, wash 3 times, and centrifuge to collect cells.

[0253] (5) Resuspend in 400 μg / μl 2% FPBS, filter and transfer into a flow cytometer for FCM analysis.

[0254] (6) The collected data were analyzed using FlowJo 7.6 and Graphpad Prism 5 analysis software to calculate the affinity value of the nanobody.

[0255] The results show (see) Figure 3 Nanobody 2E showed better affinity than the positive control antibody, while 7G showed an affinity level comparable to the control antibody.

[0256] Example 6 Evaluation and analysis of the interaction between nanobody and CD38

[0257] (1) The structures of known CD38 antibody-CD38 complexes were searched in the Protein Structure Database (PDB) (using the CD38-antibody complex with PDB database number 4cmh as the interaction structure analysis template) to analyze the binding of CD38 to the antibody. Figure 4 A is a spatial structure analysis diagram of the interaction between CD38 and antibody (surface model) in the 4cmh database of PBD. The red structural area is the structural part of the interaction between CD38 and 4cmh.

[0258] (2) Multiple protein three-dimensional structure modeling software such as I-TASSER, FR-t5-M, and FALCON were used to predict the three-dimensional protein structure of candidate nanobodies.

[0259] (3) Using ZDOCK 3.0.2, combined with the template interaction structure obtained in step (1) and the nanobody spatial structure predicted in step (2), the docking prediction of CD38 and candidate nanobodies was performed. In addition, based on the evaluation of docking conformations by tools such as MEFTop and QIPI, the optimal docking model was found through comparison and selection.

[0260] Figure 4 Results B show that the protein interaction regions (red areas) between CD38 and the three nanobodies FA-2E, FB-1E, and FB-5G are relatively far apart. However, the interaction regions (red areas) between FA-7G and FB-8A and CD38 are similar to the docking structural regions of the CD38-antibody complex in the known 4cmh model, and the binding regions of these two nanobodies to CD38 are significantly different from those of FA-2E, FB-1E, and FB-5G.

[0261] Example 7: Preparation of anti-human CD38 antibody-DM1 conjugate

[0262] 7.1. Conjugation and analysis of nanobodies with maytansin DM1:

[0263] (1) According to the molar ratio of antibody to SMCC-DM1 of 1:18, 100 μL of nano antibody solution (10 mg / mL) was mixed with the corresponding proportion of SMCC-DM1 (5 mM / L) and reacted at room temperature for 2 to 4 hours with stirring.

[0264] (2) The reaction solution was then transferred to a 0.5 mL centrifugal filter (10 kDa filter membrane) and 300 μL PBS was added. The mixture was centrifuged at 5000 g for 15 min, 2 to 3 times, to remove excess SMCC-DM1 in the reaction system.

[0265] (3) Collect the coupling products and package them separately, and store them at -80℃.

[0266] (4) Mass spectrometry analysis of the prepared nanobody-DM1 conjugate: Dilute the nanobody solution and the corresponding conjugate solution to 100 pM / L with PBS. Take 1 μL of the diluted antibody solution and ADC solution respectively, add 1 μL of 2,5-dihydroxybenzoic acid (DHB) and mix thoroughly, then spot them onto the sample target plate and air dry. Place the sample target plate into the MALDI-TOF target plate slot. Set the parameters, adjust the laser intensity, and collect data.

[0267] 7.2. Flow cytometry detection of endocytosis of anti-human CD38 nanobody:

[0268] (1) The FA-2E and FA-7G nanoantibodies with the highest affinity among the candidate anti-human CD38 nanoantibodies were incubated with target cells MM1S for 30 min and unbound antibodies were washed away.

[0269] (2) The control group was placed at 4℃, and the experimental group was placed at 37℃ to induce cell endocytosis. Samples were taken at 1h, 2h, 3h, and 4h and incubated with anti-Flag-APC secondary antibody. The median fluorescence intensity (MFI) of APC was detected. The endocytosis rate of the antibody was calculated using the following formula: Antibody endocytosis rate = (Surface binding amount at 4℃ - Surface residue at different time points at 37℃ - Dissociation amount from the surface in each group) / Surface binding amount at 4℃ × 100%.

[0270] The fewer antibodies that bind to the cell surface during antibody endocytosis, the weaker the fluorescence of the secondary antibody that binds to them.

[0271] Figure 5 Figure a shows that, compared with the control group, the MFI of FA-2E and FA-7G in the experimental group gradually decreased over time, indicating that the surface fluorescently labeled antibodies were gradually endocytosed into the target cells. Figure 5The statistical results in Figure b show that the ratio of FA-2E and FA-7G nanobodies endocytosis in target cells increases with time. Among them, the endocytosis effect of FA-2E nanobodies is more obvious, so they are subsequently prepared as DM1 conjugates.

[0272] 7.3. Detection of cytotoxic effects of anti-human CD38 antibody-DM1 conjugate:

[0273] (1) Dilute myeloma cells MM1S with culture medium to 2×10⁻⁶. 5 Inoculate 50 μl / well into a 96-well plate at a concentration of 1 / mL.

[0274] (2) FA-2E and FA-2E-DM were serially diluted to a gradient concentration and added to a 96-well plate that had been seeded with MM1S cells. Each concentration was replicated in 3 places. Control wells without naked antibody or conjugate samples were set up. After incubation at 37°C for 72 hours, the samples were detected using CCK-8 assay.

[0275] Figure 6 The results showed that the naked antibody FA-2E treatment group had an effect on MM1S cells (CD38). + The control group did not exhibit any killing effect, while the FA-2E-DM1 administration group showed concentration-gradient-dependent cytotoxic activity. Experiments show that the anti-human CD38 antibody-DM1 conjugate (FA-2E-DM1) possesses targeted tumor cell killing activity. The killing effect of FA-2E-DM1 is based on the targeting and endocytosis of FA-2E nanobodies targeting CD38 on the cell surface, thereby allowing DM1 to enter the cell and exert its anti-tumor activity.

[0276] discuss

[0277] Currently, clinical research targeting CD38 encompasses monoclonal antibodies, antibody-drug conjugates (ADCs), bispecific antibodies, and chimeric antigen receptor T cells. In November 2015, the FDA approved Daratumumab as a monotherapy, becoming the world's first approved humanized full-length monoclonal antibody targeting CD38 for the treatment of multiple myeloma (MM). In March 2020, the FDA approved isatuximab, developed by Sanofi, for marketing, in combination with IMiD for the treatment of relapsed / relapsed MM patients. In addition, several CD38 antibodies are in preclinical research stages (e.g., TAK-169, Dara-DM4, etc.). Antibody drug development targeting CD38 extends beyond monoclonal antibodies, also involving CAR-T and ADC drugs. Examples include Sorrento's CD38 CAR-T project and Ohio State University's CD38 ADC project. However, there are currently no effective cases in China utilizing CD38 nanobodies to construct CAR-T cell therapy and ADC drug therapy.

[0278] In summary, currently used human CD38-targeting antibodies in immunotherapy are all fully human or humanized antibodies, and their application is limited to the treatment of hematological malignancies. Existing antibodies suffer from problems such as large molecular weight, relatively poor tissue penetration, and difficulties in constructing and expressing bispecific antibodies against other targets, thus limiting the application and therapeutic scope of CD38 antibody drugs. Therefore, the search for novel antibodies with smaller molecular weights that are easy to prepare into various types, including bispecific antibodies, antibody-drug conjugates, and antibody immunotoxins, will expand the application scope and therapeutic efficacy of CD38-targeting therapeutic antibodies.

[0279] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> East China Normal University <120> Preparation and application of camel-family nanobodies targeting human CD38 molecules <130> P2021-2053 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <400> 1 Arg Tyr Thr Tyr Asn Ser Asn Trp Val Ala 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <400> 2 Ser Ile Tyr Thr Gly Gly Thr Ser Thr Tyr Tyr Gly 1 5 10 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <400> 3 Ala Ser Gly Leu Leu Tyr Gly Asp Pro Leu Arg Ala Thr Asn Tyr Pro 1 5 10 15 Tyr <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Thr Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 5 <211> 14 <212> PRT <213> Artificial Sequence <400> 5 Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Ala Val Ala 1 5 10 <210> 6 <211> 35 <212> PRT <213> Artificial Sequence <400> 6 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn 1 5 10 15 Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met 20 25 30 Tyr Tyr Cys 35 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <400> 7 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser 1 5 10 <210> 8 <211> 125 <212> PRT <213> Artificial Sequence <400> 8 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Thr Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Tyr Asn Ser Asn 20 25 30 Trp Val Ala Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Ala Val 35 40 45 Ala Ser Ile Tyr Thr Gly Gly Thr Ser Thr Tyr Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ser Gly Leu Leu Tyr Gly Asp Pro Leu Arg Ala Thr Asn Tyr Pro 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser 115 120 125 <210> 9 <211> 375 <212> DNA <213> Artificial Sequence <400> 9 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggac tctgagactc 60 tcctgtgcag cctctagata cacctacaat agcaactggg tggcctggtt ccgccaggct 120 ccagggcagg agcgcgaggc ggtcgcaagt atttatactg gtggtactag cacatactat 180 ggcgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtatat 240 ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gtcgggccta 300 ctatacggcg accctctgcg cgcaacaaac tatccgtact ggggccaggg gacccaggtc 360 accgtctcct caagt 375 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <400> 10 Gly Tyr Thr Tyr Arg Thr Asn Cys Met Ala 1 5 10 <210> 11 <211> 12 <212> PRT <213> Artificial Sequence <400> 11 Gly Ile Val Asn Gly Pro Gly Thr Ile Tyr Met Ser 1 5 10 <210> 12 <211> 18 <212> PRT <213> Artificial Sequence <400> 12 Ala Arg Lys Asp Leu Thr Phe Gly Cys Pro Thr Pro Leu Ser Glu Tyr 1 5 10 15 Thr Tyr <210> 13 <211> 25 <212> PRT <213> Artificial Sequence <400> 13 Ala Val Gln Leu Val Asp Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <400> 14 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val Ala 1 5 10 <210> 15 <211> 35 <212> PRT <213> Artificial Sequence <400> 15 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn 1 5 10 15 Thr Val Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met 20 25 30 Tyr Tyr Cys 35 <210> 16 <211> 126 <212> PRT <213> Artificial Sequence <400> 16 Ala Val Gln Leu Val Asp Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Arg Thr Asn 20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val 35 40 45 Ala Gly Ile Val Asn Gly Pro Gly Thr Ile Tyr Met Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Lys Asp Leu Thr Phe Gly Cys Pro Thr Pro Leu Ser Glu Tyr 100 105 110 Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser 115 120 125 <210> 17 <211> 378 <212> DNA <213> Artificial Sequence <400> 17 gccgtgcagc tggtggattc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtgcag cctctggata cacctatcgt accaactgca tggcgtggtt ccgccaggct 120 ccaggaaagg agcgtgagcg ggtcgcagga atagtcaacg gtccgggcac tatatatatg 180 tccgactccg tgaagggccg attcaccatc tcctcagaca acgccaagaa tacggtctat 240 ctccaaatga acgacctgaa gcctgaggac acggccatgt attactgcgc gcgaaaggat 300 ttgacgttcg gatgtccgac gccactgagc gaatatacgt actggggtca ggggacccag 360 gtcaccgtct cctcaagt 378 <210> 18 <211> 10 <212> PRT <213> Artificial Sequence <400> 18 Gly Ala Thr Phe Ser Tyr Asn Ser Met Ala 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial Sequence <400> 19 Tyr Ile Tyr Ile Leu Asp Gly Thr Thr Ser Tyr Thr 1 5 10 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <400> 20 Ala Ala Ala Ser Val Thr Gly Ser Gly Met Trp Arg Pro Gly Tyr Asn 1 5 10 15 Tyr <210> twenty one <211> 25 <212> PRT <213> Artificial Sequence <400> twenty one Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser 20 25 <210> twenty two <211> 14 <212> PRT <213> Artificial Sequence <400> 22 Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 <210> 23 <211> 35 <212> PRT <213> Artificial Sequence <400> 23 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn 1 5 10 15 Met Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met 20 25 30 Tyr Tyr Cys 35 <210> 24 <211> 125 <212> PRT <213> Artificial Sequence <400> 24 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ala Thr Phe Ser Tyr Asn 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Leu Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Tyr Ile Tyr Ile Leu Asp Gly Thr Thr Ser Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Ala Ser Val Thr Gly Ser Gly Met Trp Arg Pro Gly Tyr Asn 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser 115 120 125 <210> 25 <211> 375 <212> DNA <213> Artificial Sequence <400> 25 gaggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtacag cctctggagc caccttcagt tacaactcca tggcctggtt ccgccaggct 120 ctagggaagg agcgcgaggg ggtcgcatat atttatattc ttgatggtac cacaagctat 180 accgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa tatggtgtat 240 ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc agccgctagt 300 gtaacgggga gtggtatgtg gcgtccgggg tataactact ggggccaggg gacccaggtc 360 accgtctcct caagt 375 <210> 26 <211> 10 <212> PRT <213> Artificial Sequence <400> 26 Gly Tyr Thr Tyr Ser Ser Tyr Cys Met Gly 1 5 10 <210> 27 <211> 12 <212> PRT <213> Artificial Sequence <400> 27 Ala Ile Asn Ser Gly Gly Gly Asp Thr Tyr Tyr Ala 1 5 10 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <400> 28 Ala Ala Arg Arg Gly Tyr Gly Asn Ser Cys Thr Gly Pro Ser Leu 1 5 10 15 <210> 29 <211> 25 <212> PRT <213> Artificial Sequence <400> 29 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 30 <211> 14 <212> PRT <213> Artificial Sequence <400> 30 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala 1 5 10 <210> 31 <211> 35 <212> PRT <213> Artificial Sequence <400> 31 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn 1 5 10 15 Thr Val Tyr Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Thr 20 25 30 Tyr Tyr Cys 35 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <400> 32 Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser Ser 1 5 10 <210> 33 <211> 123 <212> PRT <213> Artificial Sequence <400> 33 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asn Ser Gly Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ala Arg Arg Gly Tyr Gly Asn Ser Cys Thr Gly Pro Ser Leu Trp 100 105 110 Ser Gln Gly Thr Gln Val Thr Val Ser Ser Ser 115 120 <210> 34 <211> 369 <212> DNA <213> Artificial Sequence <400> 34 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtgcag cctctggata cacctacagt agctactgca tgggttggtt ccgccaggct 120 ccagggaagg agcgtgaggg ggtcgcagct attaatagtg gtggtggtga cacatactac 180 gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacggtgtat 240 ctgcaaatga acagcctgca acctgaggac acggccacgt attactgtgc ggctcgtcga 300 gggtacggta atagctgcac ggggccttca ctatggagcc aggggaccca ggtcaccgtc 360 tcctcaagt 369 <210> 35 <211> 12 <212> PRT <213> Artificial Sequence <400> 35 Ala Ile Asn Ser Gly Gly Gly Gly Ser Thr Tyr Tyr Ala 1 5 10 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <400> 36 Ala Ala Gly Arg Arg Ser Tyr Gly Asn Ser Cys Thr Gly Pro Ser Leu 1 5 10 15 <210> 37 <211> 123 <212> PRT <213> Artificial Sequence <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Tyr Ser Ser Tyr 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala Ala Ile Asn Ser Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Gly Arg Ser Tyr Gly Ser Tyr Cys Ser Ala Asn Lys Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ser 115 120 <210> 38 <211> 369 <212> DNA <213> Artificial Sequence <400> 38 caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60 tcctgtgcag cctctggata cacctatagt agctactgca tgggctggtt ccgccaggct 120 ccagggaagg agcgtgaggg ggtcgcagct attaatagtg gtggtggtag cacatactac 180 gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa tacggtgtat 240 ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc agcaggccgg 300 tcgtatggta gttactgtag tgcgaataag tactggggcc aggggaccca ggtcaccgtc 360 tcctcaagt 369

Claims

1. An anti-CD38 nanobody, characterized in that, The nanobody can specifically bind to CD38, and the complementarity-determining region (CDR) of the VHH chain in the nanobody is shown below: CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:

12.

2. The nanobody as described in claim 1, characterized in that, The VHH chain also includes a frame region FR, which is shown below: FR1 shown in SEQ ID NO: 13, FR2 shown in SEQ ID NO: 14, FR3 shown in SEQ ID NO: 15, and FR4 shown in SEQ ID NO:

7.

3. The nanobody as described in claim 1, characterized in that, The VHH chain of the anti-CD38 nanobody is selected from SEQ ID NO:

16.

4. A polynucleotide, characterized in that, The polynucleotide encodes the nanobody as described in claim 1.

5. An expression carrier, characterized in that, The expression vector expresses the polynucleotide of claim 4.

6. A host cell, characterized in that, The host cell contains the expression vector of claim 5, or the polynucleotide of claim 4 is integrated into its genome.

7. A method for generating anti-CD38 nanobodies, characterized in that, Including the following steps: (a) Culturing the host cells of claim 6 under conditions suitable for the production of nanobodies, thereby obtaining a culture containing the anti-CD38 nanobodies; and (b) Isolating or recovering the anti-CD38 nanobody from the culture; and optionally... (c) Purification and / or modification of the CD38 nanobody obtained in step (b).

8. The use of the nanobody as described in claim 1, characterized in that, Reagents used to prepare for the detection of CD38 molecules.

9. A pharmaceutical composition, characterized in that, contain: (i) the nanobody as described in claim 1, and (ii) Pharmaceutically acceptable carriers.

10. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the nanobody as described in claim 1; and (ii) Tag sequences that assist in expression and / or purification.

11. A reagent kit, characterized in that, The kit contains the nanobody as described in claim 1, or the pharmaceutical composition as described in claim 9.

12. A method for detecting CD38 protein in a sample in vitro, non-therapeutic and non-diagnostic manner, characterized in that, The method includes the following steps: (1) Contacting the sample with the nanobody as described in claim 1; and (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD38 protein in the sample.

Images