Nanobody targeting CD73, nanobody-drug conjugate, method for producing the same, and use thereof

Abstract

The present invention relates to a nanobody (Nb) targeting a novel CD73, a nanobody-drug conjugate (NDC), a method for producing the same, and use thereof. The monoclonal nanobody and corresponding NDC of the present invention efficiently and highly specifically bind to purified CD73 protein, CD73 on the surface of many tumor cells and immune cells, block the catalytic activity of the CD73 enzyme, have high affinity and low immunogenicity, and have a remarkable antitumor effect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular, to nanobodies targeting CD73 (CD73-Nb) and CD73 nanobody-drug conjugates (Nanobody-drug conjugate, CD73-NDC), methods for producing the same, and uses thereof.

Background Art

[0002] In recent years, the treatment of tumors has advanced significantly. Among them, targeted therapy and immunotherapy have provided new ideas for tumor treatment. However, due to the low response rate of patients to drugs and the rapid development of drug resistance, the treatment of tumors still faces great challenges. Therefore, the search for new treatment targets and drugs has become the direction of the treatment of refractory tumors. Recent studies have shown that the progression of tumors and the occurrence of drug resistance are often related to tumor metastasis, recurrence, and immunosuppressive microenvironments, and CD73 is one of the molecular mechanisms closely related thereto.

[0003] CD73 is abnormally expressed in many tumors, including lung cancer, breast cancer, melanoma, and glioblastoma, and is closely related to tumor metastasis, recurrence, drug resistance, and immunosuppression. CD73 is a 5'-nucleotidase anchored to the cell membrane surface and is the main rate-limiting enzyme in the case of dephosphorylating adenosine monophosphate (AMP) by an enzymatic reaction to generate adenosine (ADO). Adenosine binds to the corresponding adenosine receptors (A1AR, A2AR, A2BR, A3AR), thereby forming an immunosuppressive microenvironment, for example, suppressing the proliferation and function of T cells, suppressing the differentiation and maturation of dendritic cells (DCs), affecting the polarization of macrophages, and promoting the release of anti-inflammatory cytokines, such as TGFβ, IL-10, etc., from immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs), and is involved in many physiological and pathological processes in the body. Drug resistance is a major obstacle affecting the efficacy of tumor treatment, and studies have shown that CD73 is involved in the drug resistance of tumor chemotherapeutic drugs, small molecule target drugs, and immune checkpoint inhibitors. In addition, CD73 is involved in processes such as epithelial-mesenchymal transition, angiogenesis, metastasis, and recurrence of tumors. Therefore, targeting CD73, either alone or in combination, can provide a novel strategy for tumor treatment.

[0004] Monoclonal antibody drugs have already brought many improvements to cancer treatment. However, since conventional monoclonal antibodies have a molecular weight of 150 kd, the permeability of solid tumors and the distribution of drugs are limited, and there is still room for further improvement in the therapeutic effect. Nanobodies (Nb) are antibodies composed only of the heavy chains naturally occurring in alpacas. Among them, the target recognition module is composed of a single variable heavy chain region (VHH), with a molecular weight of only 12 - 14 kd, and it is expected to overcome the defect of low permeability of conventional monoclonal antibodies to solid tumors. Nanobodies can be linked to functional domains such as Fc, other nanobodies, or polypeptide tags and toxins. Due to their small size, they have characteristics such as a higher diffusion rate, vascular permeability, and tumor penetration power, and have a more uniform tissue distribution than conventional monoclonal antibodies. These characteristics make them particularly suitable for specific and effective tumor target therapy in the body. And nanobodies also have advantages such as low immunogenicity, high stability, high degradation resistance, low cost of prokaryotic expression production, and easy characterization of products. However, despite many potential advantages, the development of tumor therapeutics based on nanobodies is still in the initial exploratory research stage.

[0005] An antibody-drug conjugate (ADC) utilizes the characteristic that a monoclonal antibody specifically recognizes a specific antigen on the surface of tumor cells, and precisely delivers and releases an anti-tumor drug (such as a small molecule chemotherapeutic drug, etc.) to the target tumor cells, thereby achieving the goal of precisely killing tumors. ADCs are currently already used for targeted administration to tumor tissues, but due to limitations such as large molecular weight and weak penetrability within tumors, there are significant differences in the administration effects within tumors, and their application to solid tumors is not desirable. Moreover, because ADCs have a long terminal-phase half-life in the body, it leads to unnecessary toxicity of the carried toxins to normal tissues and cells. On the other hand, a nanobody-drug conjugate (NDC) produced by a nanobody retains the advantages of conventional ADCs, while having high vascular permeability, strong tumor penetrability, a fast rate of reaching target cells, being able to improve the accumulation of drugs in tumors, and at the same time being able to appropriately control the plasma exposure amount and half-life of the drug, which is advantageous for further improving the therapeutic effect on solid tumors and the overall therapeutic area, and is expected to be the most promising innovative anti-tumor drug.

[0006] As described above, due to the important role of CD73 in mechanisms such as tumor progression, recurrence, and drug resistance, the development of Nb and NDC drugs targeting CD73 can provide new treatment strategies for the treatment of tumor patients with abnormal CD73 expression, either alone or in combination, in clinical practice.

Summary of the Invention

[0007] The object of the present invention is to provide a nanobody and a nanobody-drug conjugate targeting CD73, a method for producing the same, and their use.

[0008] In a first aspect of the present invention, there is provided a nanobody targeting CD73, wherein the complementarity-determining region CDR of the VHH chain is one or more selected from the following group: (1) CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, and CDR3 represented by SEQ ID NO: 3; or (2) CDR1 represented by SEQ ID NO: 5, CDR2 represented by SEQ ID NO: 6, and CDR3 represented by SEQ ID NO: 7; or (3) CDR1 represented by SEQ ID NO: 9, CDR2 represented by SEQ ID NO: 10, and CDR3 represented by SEQ ID NO: 11; or (4) CDR1 represented by SEQ ID NO: 13, CDR2 represented by SEQ ID NO: 14, and CDR3 represented by SEQ ID NO: 15; or (5) CDR1 represented by SEQ ID NO: 17, CDR2 represented by SEQ ID NO: 18, and CDR3 represented by SEQ ID NO: 19; or (6) CDR1 represented by SEQ ID NO: 21, CDR2 represented by SEQ ID NO: 22, and CDR3 represented by SEQ ID NO: 23; or (7) CDR1 represented by SEQ ID NO: 25, CDR2 represented by SEQ ID NO: 26, and CDR3 represented by SEQ ID NO: 27; or (8) CDR1 represented by SEQ ID NO: 29, CDR2 represented by SEQ ID NO: 30, and CDR3 represented by SEQ ID NO: 31; or (9) CDR1 represented by SEQ ID NO: 33, CDR2 represented by SEQ ID NO: 34, and CDR3 represented by SEQ ID NO: 35; or (10) CDR1 represented by SEQ ID NO: 5, CDR2 represented by SEQ ID NO: 54, and The CDR3 shown by SEQ ID NO: 7; or (11) CDR1 shown by SEQ ID NO: 5, CDR2 shown by SEQ ID NO: 55, and the CDR3 shown by SEQ ID NO: 7; or (12) CDR1 shown by SEQ ID NO: 5, CDR2 shown by SEQ ID NO: 56, and the CDR3 shown by SEQ ID NO: 7.

[0009] In another preferred example, any one of the amino acid sequences among the above amino acid sequences has further optionally undergone addition, deletion, modification and / or substitution of at least one amino acid, and includes a derived sequence that retains the CD73 binding affinity.

[0010] In another preferred example, the CDR of the nanobody targeting the above CD73 has one amino acid substituted and includes a derived sequence that retains the CD73 binding affinity. Preferably, it is a substitution of N in CDR2 shown by SEQ ID NO: 6 with an amino acid selected from the group consisting of A, G, or Q. In another preferred example, the CDR region of the VHH chain of the above nanobody includes an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence similarity to any one of SEQ ID NOs: 1 to 36.

[0011] In another preferred example, the amino acid sequence of the CDR region of the VHH chain of the above nanobody includes one or more amino acid substitutions, preferably conservative amino acid substitutions, compared to any one of SEQ ID NOs: 1 to 36.

[0012] In another preferred example, the VHH chain includes CDR1, CDR2, and CDR3 selected from the following combinations: (1) Complementary determining regions CDR1, CDR2, CDR3 shown by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 (corresponding to the CDRs of nanobody 3-D7); or (2) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 (corresponding to the CDRs of nanobody 5-E11); or (3) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 (corresponding to the CDRs of nanobody 4-D04); or (4) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 (corresponding to the CDRs of nanobody 4-B02); or (5) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 (corresponding to the CDRs of nanobody 1-E7); or (6) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23 (corresponding to the CDRs of nanobody 2-B10); or (7) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27 (corresponding to the CDRs of nanobody 2-G9); or (8) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31 (corresponding to the CDRs of nanobody 3-A3); or (9) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 (corresponding to the CDRs of nanobody 3-F1); or (10) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 5, SEQ ID NO: 54, and SEQ ID NO: 7 (corresponding to the CDRs of nanobody 5-E11 AS or 5-E11 ASCS); or (11) Complementary determining regions CDR1, CDR2, and CDR3 shown by SEQ ID NO: 5, SEQ ID NO: 55, and SEQ ID NO: 7 (corresponding to the CDRs of nanobody 5-E11 GS); or (12) Complementary determining regions CDR1, CDR2, and CDR3 (corresponding to the CDRs of nanobody 5-E11 QS) represented by Accession No. 5, Accession No. 56, and Accession No. 7. In another preferred example, the CDR1, CDR2, and CDR3 are separated by the framework regions FR1, FR2, FR3, and FR4 of the VHH chain.

[0013] In another preferred example, the VHH chain of the nanobody further comprises a framework region (FR). In another preferred example, the framework region is of human origin, murine origin, rabbit origin, or camel origin. In another preferred example, the framework region comprises a human-derived FR region, a murine- or camel-derived FR region.

[0014] In another preferred example, the VHH chain of the nanobody targeting CD73 has an amino acid sequence represented by Accession No. 4, Accession No. 8, Accession No. 12, Accession No. 16, Accession No. 20, Accession No. 24, Accession No. 28, Accession No. 32, or Accession No. 36. In another preferred example, the VHH chain of the nanobody targeting CD73 has an amino acid sequence represented by Accession No. 46, Accession No. 47, Accession No. 48, or Accession No. 49 (all of which are point mutation sequences of Accession No. 8).

[0015] In another preferred example, the nanobody targeting CD73 is a humanized nanobody, and the humanized nanobody has an amino acid sequence represented by Accession No. 50, Accession No. 51, or Accession No. 52. In another preferred example, the VHH chain of the nanobody targeting CD73 has an amino acid sequence represented by Accession No. 50, Accession No. 51, or Accession No. 52 of the 3-D7 humanized nanobody.

[0016] In a second aspect of the present invention, there is provided an antibody targeting CD73, which comprises the VHH chain of one or more of the nanobodies targeting CD73 described in the first aspect of the present invention.

[0017] In another preferred example, the VHH chain of the nanobody targeting CD73 has an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 or SEQ ID NO: 49.

[0018] In another preferred example, the antibody is a monomer, a bivalent antibody, and / or a multivalent antibody. In another preferred example, the antibody is an animal-derived antibody, a humanized antibody, a chimeric antibody or a chimeric antigen receptor (CAR) antibody. In another preferred example, the antibody is a humanized antibody, and the VHH chain of the nanobody targeting CD73 has an amino acid sequence represented by SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO: 52.

[0019] In another preferred example, the CDR region of the humanized antibody contains 1, 2, or 3 amino acid changes. In another preferred example, the animal is a non-human mammal, preferably a mouse, a sheep, a rabbit, or a camel. In another preferred example, the antibody is a double-stranded antibody or a single-stranded antibody. In another preferred example, the antibody is a monoclonal antibody. In another preferred example, the antibody is a partially or fully humanized monoclonal antibody. In another preferred example, the number of added, deleted, modified and / or substituted amino acids is 40% or less, preferably 20% or less, more preferably 10% or less of the total number of amino acids in the original amino acid sequence.

[0020] In another preferred example, the number of added, deleted, modified and / or substituted amino acids is 1 to 7, preferably 1 to 3, more preferably 1. In another preferred example, the sequence with addition, deletion, modification and / or substitution of said at least 1 amino acid is an amino acid sequence with at least 80% homology.

[0021] In another preferred example, the derivative sequence with addition, deletion, modification and / or substitution of said at least 1 amino acid has a function of suppressing the enzymatic catalysis of the CD73 protein on the cell surface or the recombinant CD73 protein. In another preferred example, said antibody is in the form of a drug conjugate. In another preferred example, the ELISA binding affinity EC of said nanobody for the extracellular region of human CD73 protein (CD73-ECD) 50 is 2.67 ng / mL to 13.48 ng / mL, the humanized antibody is 2.1 ng / mL to 2.5 ng / mL, or the binding constant KD value measured by ForteBio surface plasmon resonance (SPR) is 0.795 to 1.997 nM, and the humanized antibody is 0.472 nM to 1.297 nM.

[0022] In another preferred example, said antibody has one or more characteristics selected from the following group: (a) Suppressing the activity of catalyzing the hydrolysis of adenosine monophosphate (AMP) of CD73 to generate adenosine; (b) Specifically binding to CD73 of tumor cells and / or immune / stromal cells in the tumor microenvironment; (c) Suppressing the activity of catalyzing the hydrolysis of AMP of CD73 in the tumor / tumor microenvironment; (d) Suppressing the migration or metastasis of tumor cells; (e) Suppressing tumor growth and improving the anti-tumor therapeutic effect when used in combination; (f) Improving the effect of tumor immunity by promoting the proliferation, survival and function of immune cells; (g) After binding to tumor cells, being taken up into lysosomes in the cells; (h) Having the characteristic of passing through the blood-brain barrier and being distributed in the brain. (i) Having a good therapeutic effect on an intracranial tumor model; (j) Having a good therapeutic effect on many solid tumors.

[0023] In a third aspect of the present invention, there is provided a multispecific antibody comprising a nanobody targeting CD73 described in the first aspect of the present invention or an antibody targeting CD73 described in the second aspect of the present invention.

[0024] In another preferred example, the multispecific antibody further comprises a second antigen-binding region targeting one selected from the group consisting of EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or combinations thereof.

[0025] In another preferred example, the second antigen-binding region is a nanobody. In another preferred example, the multispecific antibody comprises one or more second antigen-binding regions. In another preferred example, the multispecific antibody further comprises an Fc segment of an antibody.

[0026] In another preferred example, the antigen-binding region is an antibody or an antibody fragment, and the antibody fragment includes (i) a Fab fragment, (ii) an F(ab’)2 fragment, (iii) an Fd fragment, (iv) an Fv fragment, (v) a single-chain Fv (scFv) molecule, (vi) a dAb fragment.

[0027] In a fourth aspect of the present invention, there is provided a recombinant protein having the following: (i) A nanobody targeting CD73 described in the first aspect of the present invention, or an anti-CD73 antibody described in the second aspect of the present invention; and (ii) A polypeptide molecule or fragment having any therapeutic function; and / or (iii) A functional region that improves the physical and chemical properties or drug-likeness of any protein. In another preferred example, improving the physical and chemical properties or drug-likeness includes extending the half-life of a nanobody targeting CD73.

[0028] In another preferred example, the recombinant protein further includes (iv) a tag sequence that aids in any expression and / or purification. In another preferred example, the tag sequence is selected from the group consisting of a 6His tag, a GGGS sequence, and a FLAG tag. In another preferred example, the recombinant protein is a monomer, dimer, or multimer.

[0029] In another preferred example, the polypeptide molecule or fragment having the therapeutic function includes, but is not limited to, polypeptide molecules or fragments targeting EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7.

[0030] In another preferred example, the polypeptide molecule or fragment having the therapeutic function includes, but is not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines. In another preferred example, the recombinant protein (or polypeptide) includes a fusion protein. In another preferred example, the fusion protein includes a multispecific antibody and a chimeric antibody. In another preferred example, the functional regions for improving the physical and chemical properties or drug-likeness of the protein include an Fc segment, an anti-albumin nanobody (HLE), and an albumin-binding domain (ABD).

[0031] In another preferred example, the fusion protein has the following elements from the N-terminus to the C-terminus. A-B (However, the A element is a nanobody targeting CD73, and the B element is an Fc segment, an albumin-binding domain (ABD), or an anti-albumin nanobody (HLE). "-" represents a peptide bond or a linker. )

[0032] In another preferred example, the VHH chain of the nanobody targeting CD73 is selected from the group consisting of amino acid sequences represented by SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO: 49. In another preferred example, the linker is (G4S)n, where n is a positive integer from 1 to 4, and may further include modification of one or more cysteine residues. In another preferred example, the Fc segment is a human IgG Fc segment. In another preferred example, the B element is an Fc segment, and the amino acid sequence of the fusion protein is selected from the group consisting of amino acid sequences represented by SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42. In another preferred example, the fusion protein is selected from the group consisting of 3-D7, 5-E11, 4-D04, 4-B02, 1-E7, 2-B10, 2-G9, 3-A3, 3-F1, 3-D7-FC1, 3-D7-FC2, 3-D7-FC3, 5-E11-FC1, 5-E11-FC2, 5-E11-FC3, 3-D7-HLE, 3-D7-ABD, 5-E11-ABD, 5-E11ASCS-FC1, 3D7-HM7-FC1, 3D7-HM8-FC1, 3D7-HM9-FC1, 3D7-HM9-ABD.

[0033] In a fifth aspect of the present invention, there is provided a CAR construct in which the antigen-binding region is the VHH chain of the nanobody described in the first aspect of the present invention.

[0034] In a sixth aspect of the present invention, there is provided a recombinant immune cell that expresses the exogenous CAR construct described in the fifth aspect of the present invention.

[0035] In another preferred example, the immune cell is selected from the group consisting of NK cells and T cells. In another preferred example, the immune cell is derived from a human or a non-human mammal (e.g., a mouse).

[0036] In a seventh aspect of the present invention, there is provided an immune complex containing the following: (a) an antibody moiety that is a nanobody targeting CD73 described in the first aspect of the present invention or an antibody targeting CD73 described in the second aspect of the present invention; and (b) a complex moiety complexed with the nanobody moiety, selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, an enzyme, a proteolytic agent, an oligonucleotide, or a combination thereof.

[0037] In another preferred example, the immune complex is a nanobody-drug conjugate. In another preferred example, the nanobody moiety and the complex moiety are complexed via a chemical bond or a linker. In another preferred example, the composite part is a chemical marker or a biomarker. In another preferred example, the chemical marker is an isotope, an immunotoxin and / or a chemical drug. In another preferred example, the biomarker is biotin, avidin or an enzyme marker. In another preferred example, the composite part is a drug or a toxin. In another preferred example, the drug is a cytotoxic drug.

[0038] In another preferred example, the cytotoxic drug is selected from the group consisting of an anti-tubulin drug, a DNA minor groove binding reagent, a DNA replication inhibitor, an alkylating reagent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemotherapy sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

[0039] Examples of particularly useful cytotoxic drug systems include, for example, DNA minor groove binding reagents, DNA alkylating reagents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatin, camptothecin, duocarmycin, etoposide, maytansine and maytansinoid (such as DM1 and DM4), taxane, benzodiazepine or benzodiazepine-containing drug (such as pyrrolo[1,4]benzodiazepine (PBD), indolinobenzodiazepine and oxazolidinobenzodiazepine) and vinca alkaloid, or a combination thereof.

[0040] In another preferred example, the toxin is selected from the following group: Auristatins (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlorotetracycline, maytansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A-chain, modeccin A-chain, alpha-streptococcus, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, croticin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, or a combination thereof.

[0041] In another preferred example, the composite part is a detectable marker. In another preferred example, the detectable marker contains a radionuclide, and the radionuclide includes the following: (i) Diagnostic isotopes selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or (ii) Therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0042] In another preferred example, the proteolytic agent is a degrading agent for tumor-related proteins, and the tumor-related proteins are selected from the group consisting of EGFR, NF-κB, RIPK2, BCR-ABL, HER2, c-Met, TBK1, CDK, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, TRK, Fak, BRD, AR, ER, MetAp-2, BCL-XL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, EZH2, IRAK4, STAT3FRS2, RAS (such as KRAS, HRAS and NRAS).

[0043] In another preferred example, the KRAS includes KRAS-G12C, KRAS-G12D, KRAS-G12V, etc. In another preferred example, the proteolytic agent includes PROTAC (proteolysis-inducing chimeric compound). In another preferred example, the PROTAC targets EGFR, KRAS (including KRAS-G12C, KRAS-G12D, KRAS-G12V, etc.). In another preferred example, the oligonucleotide includes antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA) or RNA aptamer.

[0044] In another preferred example, the complex is selected from a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme that generates a detectable product, a radionuclide, a biotoxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, gold nanoparticles / nanorods, virus particles, liposomes, magnetic nanoparticles, a prodrug-activating enzyme (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (such as cisplatin), or any form of nanoparticles, etc.

[0045] In another preferred example, the immune complex contains a nanobody targeting CD73 described in the first aspect of the present invention that is multivalent (for example, bivalent) or an antibody targeting CD73 described in the second aspect of the present invention. In another preferred example, the multivalent means that the amino acid sequence of the immune complex contains a plurality of repeated nanobodies targeting CD73 described in the first aspect of the present invention or antibodies targeting CD73 described in the second aspect of the present invention. In another preferred example, the detection is in vivo detection or in vitro detection. In another preferred example, the immune complex is used for the diagnosis and / or treatment of tumors expressing CD73 protein.

[0046] In another preferred example, the antibody-drug conjugate ADC is represented by the following molecular formula.

Chemical formula

[0047] In another preferred example, LU is maleimidocaproyl (MC), maleimide (MAL), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) linker linked to the antibody part, and valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), polyethylene glycol (PEG), and is one or more linking groups selected from those containing the same.

[0048] In another preferred example, the antibody is covalently bound to the linker by reacting with a moiety selected from the following group: maleimidocaproyl (MC), maleimide (MAL), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), and the like.

[0049] In another preferred example, D is a compound having antitumor activity selected from the following group: (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF); (ii) Toxins acting on DNA, such as duocarmycin, pyrrolobenzodiazepine (PBD); (iii) Topoisomerase inhibitors, such as camptothecin, SN38, etoposide, Dxd.

[0050] In another preferred example, the LU-D compound is selected from the following group:

Chemical formula

[0051] In the eighth aspect of the present invention, there is provided a pharmaceutical composition comprising: (i) A nanobody targeting CD73 described in the first aspect of the present invention, an antibody targeting CD73 described in the second aspect of the present invention, a multispecific antibody described in the third aspect of the present invention, a recombinant protein described in the fourth aspect of the present invention, a recombinant immune cell described in the sixth aspect of the present invention, or an immune complex described in the seventh aspect of the present invention; (ii) A pharmaceutically acceptable carrier.

[0052] In another preferred example, the pharmaceutical composition includes a single drug, a combined drug, or a synergistic drug. In another preferred example, the pharmaceutical composition further includes other bioactive substances, such as drugs for treating tumors. In another preferred example, the administration mode of the drug composition is selected from the group consisting of subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, micro-needle injection, oral administration, or nasal and oral spray and aerosol inhalation. In another preferred example, the dosage form of the drug composition is selected from the group consisting of liquid, solid, or gel-like. In another preferred example, the drug composition is a liquid preparation. In another preferred example, the drug composition is an injection.

[0053] In the ninth aspect of the present invention, there is provided the use of an active ingredient, wherein the active ingredient is selected from the group consisting of a nanobody targeting CD73 described in the first aspect of the present invention, an antibody targeting CD73 described in the second aspect of the present invention, a multispecific antibody described in the third aspect of the present invention, a recombinant protein described in the fourth aspect of the present invention, a recombinant immune cell described in the sixth aspect of the present invention, an immune complex described in the seventh aspect of the present invention, or a combination thereof, for (a) the manufacture of a detection reagent, detection plate or kit, and / or (b) the manufacture of a drug for preventing and / or treating CD73-related diseases.

[0054] In another preferred example, the detection reagent, detection plate or kit is used for the following: (1) Detection of CD73 protein in a sample; and / or (2) Detection of endogenous CD73 protein in tumor cells; and / or (3) Detection of tumor cells expressing CD73 protein.

[0055] In another preferred example, the types of detection include, but are not limited to, detection by flow cytometry, detection by cellular immunofluorescence, detection by enzyme-linked immunosorbent assay, detection by immunoblot, etc. In another preferred example, the detection reagent, detection plate or kit is used for the diagnosis of CD73-related diseases. In another preferred example, the drug is used for the treatment or prevention of tumors with high CD73 expression, tumor metastasis, or tumor drug resistance. In another preferred example, the tumor drug resistance includes drug resistance to tumor immunotherapeutic agents, drug resistance to tumor targeted therapeutic agents, drug resistance to conventional tumor chemotherapy, and insensitivity to radiotherapy.

[0056] In another preferred example, the drug is used for being selected from the following group: (a) Suppress the activity of catalyzing the hydrolysis of adenosine monophosphate (AMP) of CD73 to generate adenosine; (b) Specifically bind to CD73 of tumor cells and / or immune / stromal cells in the tumor microenvironment; (c) Suppress the activity of catalyzing the hydrolysis of AMP of CD73 in the tumor / tumor microenvironment; (d) Suppress the migration or metastasis of tumor cells; (e) Suppress tumor growth and improve the anti-tumor therapeutic effect when used in combination; (f) Improve the effect of tumor immunity by promoting the proliferation, survival, and function of immune cells.

[0057] In another preferred example, the CD73-related disease is selected from the group consisting of cancer, autoimmune disease, metabolism-related disease, infectious disease, or a combination thereof. In another preferred example, the CD73-related disease includes the occurrence, growth, and / or metastasis of tumors. In another preferred example, the cancer includes solid tumors and blood cancers. In another preferred example, the cancer is a tumor with high CD73 expression. In another preferred example, the tumors with high CD73 expression are selected from the group consisting of breast cancer, lung cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, rectal cancer, glioblastoma, melanoma, leukemia, lymphoma, or a combination thereof. In another preferred example, the cancer is a drug-resistant tumor. In another preferred example, the CD73-highly expressed tumor is characterized in that the ratio of the level L1 of CD73 transcript and / or protein in tumor tissue to the level L0 of transcript and / or protein in normal tissue is L1 / L0 ≧ 2, preferably ≧ 3.

[0058] In a tenth aspect of the present invention, there is provided a polynucleotide encoding a polypeptide selected from the following group: (1) A nanobody targeting CD73 according to the first aspect of the present invention, an antibody targeting CD73 according to the second aspect of the present invention, or a multispecific antibody according to the third aspect of the present invention; (2) A recombinant protein according to the fourth aspect of the present invention; or (3) A CAR construct according to the fifth aspect of the present invention.

[0059] In another preferred example, the polynucleotide contains RNA, DNA or cDNA.

[0060] In an eleventh aspect of the present invention, there is provided a vector comprising the polynucleotide according to the tenth aspect of the present invention.

[0061] In another preferred example, the vector includes bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0062] In a twelfth aspect of the present invention, there is provided a genetically engineered host cell, which comprises the vector according to the eleventh aspect of the present invention or in which the polynucleotide according to the tenth aspect of the present invention is integrated into the genome.

[0063] In a thirteenth aspect of the present invention, there is provided a method for detecting (including diagnostic or non-diagnostic) CD73 in a sample in vitro, the method comprising the following steps: (1) In vitro, contacting the sample with a nanobody targeting CD73 as described in the first aspect of the present invention, an antibody targeting CD73 as described in the second aspect of the present invention, or a multispecific antibody as described in the seventh aspect of the present invention; (2) Detecting whether an antigen-antibody complex is formed, where the formation of the complex means the presence of CD73 in the sample.

[0064] In another preferred example, the detection includes diagnostic or non-diagnostic ones.

[0065] In the fourteenth aspect of the present invention, a detection plate is provided, which includes a base sheet (support plate) and a measurement bar containing a nanobody targeting CD73 as described in the first aspect of the present invention, an antibody targeting CD73 as described in the second aspect of the present invention, or an immune complex as described in the seventh aspect of the present invention.

[0066] In the fifteenth aspect of the present invention, a kit is provided, which includes: (1) A first container containing a nanobody targeting CD73 as described in the first aspect of the present invention or an antibody targeting CD73 as described in the second aspect of the present invention; and / or (2) A second container containing a secondary antibody of a nanobody targeting CD73 as described in the first aspect of the present invention or an antibody targeting CD73 as described in the second aspect of the present invention; Or, the detection plate as described in the fourteenth aspect of the present invention.

[0067] In the sixteenth aspect of the present invention, a method for producing a recombinant polypeptide is provided, which is characterized by including the following steps: (a) Culturing the host cell as described in the twelfth aspect of the present invention under conditions suitable for expression; (b) Isolate the recombinant polypeptide from the culture, wherein the recombinant polypeptide is a nanobody targeting CD73 described in the first aspect of the present invention, an antibody targeting CD73 described in the second aspect of the present invention, a multispecific antibody described in the third aspect of the present invention, or a recombinant protein described in the fourth aspect of the present invention.

[0068] In the seventeenth aspect of the present invention, there is provided a method for treating a CD73-related disease, comprising administering to a subject in need thereof a nanobody targeting CD73 described in the first aspect of the present invention, an antibody targeting CD73 described in the second aspect of the present invention, a multispecific antibody described in the third aspect of the present invention, a recombinant protein described in the fourth aspect of the present invention, a recombinant immune cell described in the sixth aspect of the present invention, an immune complex described in the seventh aspect of the present invention, a pharmaceutical composition described in the eighth aspect of the present invention, or a combination thereof.

[0069] In another preferred example, the method further comprises co-administering to a subject in need thereof another drug or treatment method for treatment. In another preferred example, the other drug or treatment method includes an anti-tumor immunotherapeutic agent, a tumor-targeted therapeutic agent, tumor chemotherapy, or tumor radiotherapy. In another preferred example, the anti-tumor immunotherapeutic agent includes a PD-1 or PD-L1 monoclonal antibody.

[0070] In the eighteenth aspect of the present invention, there is provided a method for producing a chimeric antibody, comprising the following steps: Clone the nucleotide sequence of the VHH sequence derived from alpaca of the nanobody targeting CD73 described in the first aspect of the present invention into an expression vector containing the nucleotide sequence of the constant region of a human antibody, and then transfect animal cells to express a human-alpaca chimeric antibody.

[0071] In the nineteenth aspect of the present invention, there is provided a method for producing a humanized antibody, comprising the following steps: The nucleotide sequence of the CDR region of the VHH chain of the nanobody targeting CD73 described in the first aspect of the present invention is introduced into a template of a nucleotide sequence containing the FR region of a human-derived antibody, and further cloned into an expression vector containing the constant region of a human antibody, and then transfected into animal cells to express a humanized antibody.

[0072] In the twentieth aspect of the present invention, there is provided a method for suppressing the growth and metastasis of tumor cells, which comprises administering to a subject in need thereof the nanobody targeting CD73 described in the first aspect of the present invention, the antibody targeting CD73 described in the second aspect of the present invention, or the nanobody-drug conjugate described in the present invention, or the CAR-T cells of the nanobody, or a combination thereof.

[0073] In the twenty-first aspect of the present invention, there is provided a method for suppressing the growth of tumors in a model animal, which comprises administering to a subject in need thereof the nanobody targeting CD73 described in the first aspect of the present invention, the antibody targeting CD73 described in the second aspect of the present invention, the nanobody-drug conjugate described in the present invention, or the CAR-T cells of the nanobody.

[0074] In another preferred example, the drug may be administered alone or in combination with means including tumor immunotherapy, tumor-targeted drugs, cytotoxic drugs, and radiotherapy.

[0075] In the twenty-second aspect of the present invention, there is provided a method for producing the nanobody-drug conjugate described in the seventh aspect of the present invention, which comprises the following steps: (1) Reacting the antibody with a reducing reagent in a buffer to obtain a reduced antibody; (2) Crosslinking (coupling) the DL linker-drug conjugate with the reduced antibody obtained in step (1) in a mixture of a buffer and an organic solvent to obtain antibody-drug conjugates 1a, 1b, 1c, or 1d having different DAR values.

[0076] In another preferred example, the crosslinking reaction of the production method is as shown in FIG. 46. In another preferred example, in the step (1), the antibody is reduced with a reducing reagent, so that the inter-chain disulfide bond of the antibody is reduced and a thiol group is generated. In another preferred example, in the step (1), the reducing reagent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), β-mercaptoethanol, β-mercaptoethylamine hydrochloride, or dithiothreitol (DTT).

[0077] In another preferred example, the buffer solution is selected from the group consisting of potassium dihydrogen phosphate-sodium hydroxide (KH2PO4-NaOH) / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA) buffer solution, disodium hydrogen phosphate-citric acid / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA), boric acid-borax / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA), histidine-sodium hydroxide / sodium chloride (NaCl) / diethylenetriaminepentaacetic acid (DTPA), and PBS / diethylenetriaminepentaacetic acid (DTPA).

[0078] In another preferred example, in the step (2), the volume ratio of the organic solvent in the reaction solution is 15% or less. In another preferred example, the organic solvent in the step (2) is selected from the group consisting of acetonitrile (ACN), dimethylformamide (DMF), dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO).

[0079] Furthermore, a reagent for the linking group (previously dissolved in acetonitrile (ACN), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or diethylacetamide (DMA) at 10 mg / ml) is added so that the volume ratio of the organic solvent in the reaction solution becomes 15% or less, and a coupling reaction is carried out with stirring at 0-37°C for 2-4 hours. When reducing with TCEP, the substitution maleimide-based compound can be added and coupled directly without removing the remaining TCEP.

[0080] The coupling reaction mixture is purified by filtration through a desalting column with sodium succinate / NaCl buffer or histidine-acetic acid / sucrose gel, and the peak samples are collected based on the UV280 absorbance value. Or perform ultrafiltration several times. Then, sterilize by filtration and store the obtained product at a low temperature.

[0081] The drug-antibody ratio (DAR) of the obtained antibody-drug conjugate is relatively uniform. For NDCs with some variation in DAR, when a sample with better uniformity is required, further separation and purification can be carried out by hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), ion exchange chromatography (IEC), but it is not limited to these methods.

[0082] Of course, within the scope of the present invention, it is understood that each of the above technical features of the present invention and each of the specifically described technical features below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited number of pages, it will not be explained one by one here.

Brief Description of the Drawings

[0083]

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Mode for Carrying Out the Invention

[0084] The inventor of the present invention has conducted extensive and in-depth research, immunized alpacas and constructed libraries, screened a large number of phage display libraries and yeast display libraries, and obtained many nanobodies such as 3-D7, 5-E11, 4-D04, and 4-B02. The said antibody can bind specifically to human CD73 protein. As measured by ELISA, its EC 50They are 13.48 ng / mL, 8.72 ng / mL, 2.67 ng / mL, and 3.03 ng / mL respectively. The antibody has efficient inhibitory activity against both recombinantly expressed or cell-derived human CD73 enzymes. It can effectively reverse the growth inhibitory effect of AMP on human T cells. In addition, the nanobody-drug conjugate (CD73-NDC) designed based on 3-D7, 5-E11, and 4-D04 can specifically kill tumors with high CD73 expression while maintaining the same effect as the above antibody, showing good tumor inhibitory activity and potential ability to regulate the immune microenvironment in many aspects. Based on this, the present invention was completed.

[0085] Term As used herein, the terms "nanobody of the present invention", "anti-CD73 nanobody of the present invention", and "CD73 nanobody of the present invention" can be used interchangeably, and all refer to nanobodies that specifically recognize and bind to CD73 (including human CD73). Particularly preferred are nanobodies in which the amino acid sequence of the VHH chain is represented by SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 46, 47, 48, or 49.

[0086] As used herein, the term "antibody" or "immunoglobulin" is a glycoprotein heterotetramer of about 150,000 daltons with similar structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is bound to the heavy chain by one covalent disulfide bond, but the number of disulfide bonds between the heavy chains depends on the immunoglobulin isotype. Each heavy chain and light chain also have intra-chain disulfide bonds at regular intervals. Each heavy chain has a variable region (VH) at one end and a number of constant regions beyond that. Each light chain has a variable region (VL) at one end and a constant region at the other end. The constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light and heavy chains.

[0087] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning and refer to the variable region of the heavy chain of a monoclonal antibody, but construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment having complete function. Usually, first an antibody lacking the natural light chain and the constant region 1 (CH1) of the heavy chain is obtained, and then the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0088] As used herein, the term "variable" means that in an antibody, a part of the variable region has different sequences, whereby the binding and specificity of each specific antibody to its specific antigen are constituted. However, the variability is not uniformly distributed throughout the variable region of the antibody. It is concentrated in three fragments called complementarity-determining regions (CDRs) or hypervariable regions in the variable regions of the light and heavy chains. In the variable region, the relatively conserved part is called the framework region (FR). The variable regions of the natural heavy and light chains are basically in a β-sheet structure, respectively, and are linked by three CDRs that form connecting loops, and include four FR regions that are sometimes in a partial β-sheet structure. The CDRs in each chain are close in the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits different effector functions such as participating in the antibody-dependent cytotoxicity of the antibody.

[0089] As is known to those skilled in the art, immune complexes and fusion expression products include complexes formed by the binding of a drug, toxin, cytokine, radionuclide, enzyme and other diagnostic or therapeutic molecules to the antibody or fragment thereof of the present invention. Further, the present invention includes a cell surface marker or antigen bound to the anti-CD73 protein antibody or fragment thereof.

[0090] As used herein, the terms "heavy chain variable region" and "V H " can be used interchangeably. As used herein, the terms "variable region" and "complementarity determining region (CDR)" can be used interchangeably.

[0091] In one preferred embodiment of the present invention, it includes three complementarity determining regions, namely CDR1, CDR2, and CDR3 of the heavy chain variable region of the antibody. In one preferred embodiment of the present invention, the heavy chain of the antibody includes the above heavy chain variable region and heavy chain constant region.

[0092] In the present invention, the terms "antibody of the present invention", "protein of the present invention", or "polypeptide of the present invention" can be used interchangeably, and all refer to a polypeptide that specifically binds to the CD73 protein, for example, a protein or polypeptide having a heavy chain variable region. These may or may not contain the starting methionine.

[0093] In addition, the present invention provides other proteins or fusion expression products of the antibody of the present invention. Specifically, the present invention includes any protein or protein complex having a heavy chain containing this variable region and a fusion expression product (i.e., an immune complex and a fusion expression product) as long as the variable region is the same as or has at least 90%, preferably at least 95% homology with the heavy chain variable region of the antibody of the present invention.

[0094] Generally, the antigen-binding properties of an antibody are characterized by three specific regions located in the heavy-chain variable region, which are called the variable regions (CDRs), and are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not participate directly in the binding reaction. These CDRs form a loop structure and approach each other in terms of spatial structure by the β-sheets formed by the FRs therein. The CDRs in the heavy chain and the corresponding CDRs in the light chain constitute the antigen-binding site of the antibody. By comparing the amino acid sequences of homologous antibodies, it is possible to confirm which amino acids constitute the FR or CDR regions. The variable region of the heavy chain of the antibody of the present invention is particularly noted because at least a part thereof is involved in antigen binding. Therefore, a molecule having a heavy-chain variable region chain of a CDR-containing monoclonal antibody is included in the present invention if the CDRs thereof have a homology of 90% or more (preferably 95% or more, most preferably 98% or more) with the CDRs identified herein.

[0095] Anti-CD73 nanobody The present invention provides highly specific and high-affinity nanobodies that target some CD73, contain only the heavy chain, and the heavy chain contains the amino acid sequence (shown in Table-1 and Table-2) of the heavy-chain variable region (VH).

[0096] Preferably, the amino acid sequence of the heavy-chain variable region (VL) has CDR1, CDR2, and CDR3 of the following polypeptide sequences: a1) CDR1 is SEQ ID NO: 1: GFLLDYYV, SEQ ID NO: 5: GFTLDYYN, SEQ ID NO: 9: GFTLDYYN, SEQ ID NO: 13: GFPLDYYS, SEQ ID NO: 17: GFTLDYYN, SEQ ID NO: 21: GFTLDDNA, SEQ ID NO: 25: GFLLDYYT, SEQ ID NO: 29: GFPLDYYT, SEQ ID NO: 33: GFTLDYYT, a2) The CDR2 is SEQ ID NO: 2: ISGSDRST, SEQ ID NO: 6: SSNSGGST (or SEQ ID NO: 54: SSASGGST, SEQ ID NO: 55: SSGSGGST, or SEQ ID NO: 56: SSQSGGST), SEQ ID NO: 10: ISSSEGST, SEQ ID NO: 14: IGSSDGST, SEQ ID NO: 18: ISNSDGST, SEQ ID NO: 22: MRTSDGDT, SEQ ID NO: 26: ISSSDSSP, SEQ ID NO: 30: ISSSDSSP, SEQ ID NO: 34; IRSSDSSP, a3) The CDR3 is SEQ ID NO: 3: AADPSPVTVQAMCLPRFPVPY, SEQ ID NO: 7: AAYRGWHPSLDARVYDY, SEQ ID NO: 11: AADRYYYCSLHVSQYDY, SEQ ID NO: 15: AALKYYYCSGHEAIYDY, SEQ ID NO: 19: AAYRGWHPSLDARVYDY, SEQ ID NO: 23: AAALGTYYSGFYYLAGDGMDY, SEQ ID NO: 27: ALRANVYDY, SEQ ID NO: 31: SLRANVYDY, SEQ ID NO: 35: SLTANVYDY, a4) A sequence having CD73 binding affinity, wherein any one of the amino acid sequences undergoes at least one amino acid addition, deletion, modification, and / or substitution.

[0097] In another preferred example, the sequence that has undergone the addition, deletion, modification, and / or substitution of the at least 1 amino acid is preferably an amino acid sequence with at least 80% homology, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%. Preferably, the antibody has the function of suppressing the enzymatic catalysis of cell surface CD73 protein and recombinant CD73 protein, and can be rapidly taken up by cells and enter lysosomes.

[0098] The antibody of the present invention may be a double-stranded or single-stranded antibody, and may be selected from animal-derived antibodies, chimeric antibodies, human-animal chimeric antibodies, preferably humanized antibodies, and more preferably fully humanized antibodies.

[0099] Derivatives of the antibody according to the present invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab’, (Fab’)2 or other known antibody derivatives in the art, and may also be any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies. Here, the animal is preferably a mammal, such as a mouse.

[0100] The antibody of the present invention may also be a chimeric antibody, a humanized antibody, a CDR grafted and / or modified antibody targeting human CD73.

[0101] In one preferred embodiment of the present invention, any one or more of the sequences of SEQ ID NO: 1-3, SEQ ID NO: 5-7, SEQ ID NO: 9-11, SEQ ID NO: 13-15, SEQ ID NO: 17-19, SEQ ID NO: 21-23, SEQ ID NO: 25-27, SEQ ID NO: 29-31, SEQ ID NO: 33-35, SEQ ID NO: 54-56, or sequences having CD73 binding affinity after undergoing at least one amino acid addition, deletion, modification and / or substitution thereof are located in the CDR region of the variable region (VH).

[0102] In the above content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably 40% or less of the total number of amino acids in the original amino acid sequence, more preferably 35% or less, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, more preferably 15-20%.

[0103] In the above content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids may be 1-7, more preferably 1-5, more preferably 1-3, more preferably 1-2.

[0104] In another preferred example, the original antibody is human-alpaca chimeric antibody 3-D7, 5-E11, 4-D04, 4-B02, 1-E7, 2-B10, 2-G9, 3-A3, 3-F1. In another preferred example, the numbers of the amino acid sequences of the heavy chain variable regions (VH) of the chimeric antibody are shown in Table 1. In another preferred example, the numbers of the amino acid sequences of the complementarity-determining regions (CDR) of the antibody are shown in Table 2.

[0105] The antibody of the present invention may be used in combination, and may also be used for the construction of CAR constructs, recombinant immune cells containing CAR constructs, antibody-drug conjugates, etc., and may be used for (a) the production of detection reagents, detection plates or kits, and / or (b) the production of drugs for preventing and / or treating CD73-related diseases. [Table 1]

[0106] [Table 2]

[0107] Recombinant protein (or fusion protein) The present invention further includes a recombinant protein (or fusion protein) containing the CD73 nanobody of the present invention. One preferred fusion protein is a multispecific antibody, and the multispecific antibody further includes a second antigen-binding region targeting one selected from the group consisting of EGFR, TGFβ, BCMA, B7H6, GUCY2C, DLL3, CD38, CD123, CD19, CD20, CD22, B7-H3, GPC3, HER2, PMSA, CD28, 4-1BB, OX40, CD40, CD27, CD3, CTLA4, PD1, PDL1, BCMA, GLP-1, Trop2, TIGIT, LAG-3, FGL1, TLR7, or combinations thereof.

[0108] Preferably, the multispecific antibody includes one or more second antigen-binding regions or further includes a third antigen-binding region.

[0109] In addition, the recombinant protein (or fusion protein) of the present invention may further contain a tag sequence (e.g., 6His tag, GGGS sequence, FLAG tag) that aids in any expression and / or purification, in addition to the CD73 nanobody of the present invention, or a polypeptide molecule or fragment having any therapeutic function, or any protein functional region that physically, chemically, or pharmaceutically aids.

[0110] The present invention includes not only complete antibodies but also antibody fragments having immunological activity or fusion proteins composed of antibodies and other sequences. Therefore, the present invention further includes fragments, derivatives, and analogs of the said antibodies.

[0111] As used herein, the terms "fragment", "derivative", and "analog" basically refer to polypeptides that maintain the same biological function or activity as the antibodies of the present invention. Fragments, derivatives, and analogs of the polypeptides of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having substituents on one or more amino acid residues, or (iii) polypeptides fused with another compound (e.g., a compound that extends the half-life of the polypeptide such as polyethylene glycol), or (iv) polypeptides with an additional amino acid sequence fused to this polypeptide (e.g., a leader sequence or a secretion sequence or a sequence for purifying this polypeptide or a protein precursor sequence, or a fusion protein formed with a 6His tag). Based on the disclosure herein, these fragments, derivatives, and analogs are within the scope known to those skilled in the art. The antibody of the present invention refers to a polypeptide containing the above CDR region and having an activity of binding to the CD73 protein. The term further includes forms of mutations of the polypeptide containing the above CDR region that have the same function as the antibody of the present invention. These forms of mutations include deletions, insertions and / or substitutions of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or more (usually within 20, preferably within 10, more preferably within 5) amino acids to the C-terminus and / or N-terminus, but are not limited thereto. For example, in this field, when substituting with amino acids having similar or close functions, usually the function of the protein is not changed. Also, addition of one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term further includes active fragments and active derivatives of the antibody of the present invention.

[0112] The forms of mutations of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing with the coding DNA of the antibody of the present invention under conditions of high or low stringency, and polypeptides or proteins obtained with antiserum against the antibody of the present invention.

[0113] The present invention further includes fusion proteins of other polypeptides, such as nanobodies or fragments thereof. In addition to almost full-length polypeptides, the present invention further includes fragments of the nanobody of the present invention. Usually, the fragment has at least about 50 consecutive amino acids, 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 of the antibody of the present invention.

[0114] In the present invention, the "conservative variant of the antibody of the present invention" refers to a polypeptide in which, when compared with the amino acid sequence of the antibody of the present invention, 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids are substituted with amino acids having similar or close properties. These polypeptides with conservative mutations are preferably generated by substituting amino acids as shown in Table A.

Table A

[0115] Furthermore, the present invention provides a polynucleotide molecule encoding the above antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0116] The polynucleotide encoding the mature polypeptide of the present invention includes a coding sequence encoding only the mature polypeptide, the coding sequence of the mature polypeptide and various additional coding sequences, and the coding sequence of the mature polypeptide (and any additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" may be a polynucleotide encoding this polypeptide, or may be a polynucleotide further including additional coding and / or non-coding sequences.

[0117] The present invention further relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides according to the present invention under stringent conditions. In the present invention, "stringent conditions" refer to (1) low ionic strength and high temperature, for example, hybridization and elution at 0.2×SSC, 0.1% SDS, 60°C, or (2) addition of a denaturing agent during hybridization, for example, adding 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll at 42°C, or (3) hybridizing only when the identity between the two sequences is at least 90% or more, preferably 95% or more. And the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0118] The full-length nucleotide sequence or fragment of the antibody of the present invention is usually obtained by PCR amplification method, recombination method or artificial synthesis method. As an applicable method, especially when the length of the fragment is short, the related sequence is synthesized by the artificial synthesis method. Usually, first a number of small fragments are synthesized and then ligated to obtain a long fragment of the sequence. Also, the coding sequence of the heavy chain may be fused integrally with an expression tag (for example, 6His) to form a fusion protein.

[0119] Production of Antibodies The sequence of the DNA molecule of the antibody or its fragment of the present invention can be obtained by ordinary techniques, for example, by methods such as PCR amplification or genomic library screening. Also, the coding sequences of the light chain and the heavy chain may be fused integrally to form a single-chain antibody.

[0120] Once the related sequence is obtained, a large amount of the related sequence can be obtained by the recombination method. In this case, usually, after cloning the sequence into a vector, it is introduced into cells, and then the related sequence is isolated from the host cells grown by ordinary methods. Also, especially when the length of the fragment is short, the related sequences may be synthesized by artificial synthesis methods. Usually, first a number of small fragments are synthesized and then ligated to obtain a long fragment of the sequence.

[0121] Currently, it is already possible to obtain the DNA sequence encoding the antibody (or its fragment, or its derivative) of the present invention entirely by chemical synthesis. Furthermore, this DNA sequence may be introduced into various known DNA molecules (or vectors, etc.) and cells well-known in this field. Also, mutations can be introduced into the protein sequence of the present invention by chemical synthesis.

[0122] Furthermore, the present invention relates to vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences. These vectors can be used for the transformation of appropriate host cells so as to express proteins.

[0123] The host cell may be a prokaryotic cell, for example, a bacterial cell, or a lower eukaryotic cell, for example, a yeast cell, or a higher eukaryotic cell, for example, a mammalian cell. Suitable animal cells include, but are not limited to, CHO-S and HEK-293 cells.

[0124] Usually, the host cells obtained by transformation are cultured under conditions appropriate for the expression of the antibody of the present invention. Then, it is purified by ordinary separation and purification means well-known to those skilled in the art, such as the ordinary purification steps of globulin, for example, Protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and affinity chromatography, to obtain the antibody of the present invention.

[0125] The obtained monoclonal antibody can be identified by ordinary means. For example, the binding specificity of the monoclonal antibody can be measured by immunoprecipitation or an in vitro binding assay (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of the monoclonal antibody can be measured, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0126] The antibody of the present invention can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, recombinant proteins can be isolated and purified by various isolation methods using their physical and chemical properties 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 regeneration treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques, and combinations of these methods.

[0127] Nanobody-drug conjugate (NDC) The present invention also provides an immune complex based on the antibody of the present invention, preferably a nanobody-drug conjugate (NDC).

[0128] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, and the antibody and the effector molecule are coupled, preferably chemically coupled. Here, the effector molecule is preferably a drug having therapeutic activity. The effector molecule may also be one or more of a toxin protein, a chemotherapeutic agent, a small molecule drug or a radionuclide.

[0129] The antibody of the present invention and the effector molecule may be coupled by a coupling agent. Examples of the coupling agent may be any one or more of a non-selective coupling agent, a coupling agent using a carboxy group, a peptide chain, and a coupling agent using a disulfide bond. The non-selective coupling agent is a compound that conjugates and links an effector molecule and an antibody, such as glutaraldehyde. The coupling agent using a carboxy group may be any one or more of a cis-aconitic anhydride-based coupling agent (for example, cis-aconitic anhydride) and an acylhydrazone-based coupling agent (the coupling site is acylhydrazone).

[0130] Some residues in the antibody (such as Cys and Lys) are used for the linkage with many functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody may be coupled to a functional agent to form an antibody-functional agent complex. The functional agent (such as a drug, a detection reagent, a stabilizer) is coupled (conjugated) to the antibody. The functional agent may be directly or indirectly linked to the antibody via a linker.

[0131] Nanobodies can be made into antibody-drug conjugates (ADCs) by conjugating drugs. Typically, an ADC contains a linker positioned between the drug and the antibody. The linker may be a cleavable linker or a non-cleavable linker. A cleavable linker typically degrades readily in the intracellular environment, for example, by cleaving at the site of interest, releasing the drug from the antibody. Suitable cleavable linkers include, for example, linkers that are degraded by enzymes that can degrade peptidyl-containing linkers, such as proteases (e.g., lysosomal proteases or endosomal proteases) in the cell, or sugar linkers, such as glucuronic acid-containing linkers that can be degraded by glucuronidase. Peptidyl linkers may contain, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable cleavable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze when the pH is less than 5.5, such as hydrazone linkers) and linkers that can be degraded under reducing conditions (disulfide bond linkers). Non-cleavable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0132] Before conjugating to the antibody, the linker has a reactive group that can react with certain amino acid residues, and the conjugation is achieved via the reactive group. A reactive group specific for thiol groups is preferred, and includes, for example, maleimide-based compounds, halogenated amides (e.g., iodinated, brominated, or chlorinated), halogenated esters (e.g., iodinated, brominated, or chlorinated), halogenated methyl ketones (e.g., iodinated, brominated, or chlorinated), halogenated benzyls (e.g., iodinated, brominated, or chlorinated), vinyl sulfones, pyridyl disulfides, mercury derivatives, such as 3,6-di(mercury methyl) dioxane (the counterion is acetate ion, chloride ion, or nitrate ion), and polymethylene dimethyl sulfide thiosulfonate. The linker may contain, for example, maleimide conjugated to the antibody via thiosuccinimide.

[0133] The drug may be any cytotoxic drug, drug that inhibits cell growth, or immunosuppressive drug. In an embodiment, the linker connects the antibody and the drug, and the drug has a functional group capable of binding to the linker. For example, the drug may have an amino group, a carboxy group, a thiol group, a hydroxy group, or a ketone group capable of binding to the linker. When the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0134] Useful drug types include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drug systems include, for example, DNA minor groove binding reagents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatin, camptothecin, duocarmycin, etoposide, maytansine and maytansinoid (such as DM1 and DM4), taxane, benzodiazepine or benzodiazepine-containing drug (such as pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloid.

[0135] In the present invention, the drug-linker can be used for the formation of NDC in a single simple step. In another embodiment, the bifunctional linker compound can be used for the formation of NDC in a two-step or multi-step method. For example, a cysteine residue reacts with the reactive part of the linker in the first step, and in a subsequent step, the functional group in the linker reacts with the drug to form NDC.

[0136] Generally, the functional groups in the linker are selected so as to be prone to react specifically with appropriate reactive groups in the drug moiety. As a non-limiting example, a moiety based on an azide compound can be used for reaction with a reactive alkynyl group in a specific drug moiety. The drug conjugates to the linker by addition via a 1,3-dipole between the azide group and the alkynyl group. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azide groups), isocyanates and isothiocyanates (suitable for reaction with amines and alcohols), and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other ligation means are well known to those skilled in the art, for example, as described in "Bioconjugation Techniques", 2nd Edition (Elsevier). For those skilled in the art, for the selective reaction of the drug moiety and the linker, when selecting complementary pairs of reactive functional groups, either of the complementary pairs can be used for both the linker and the drug.

[0137] Preferably, the conjugate moiety conjugated to the nanobody moiety in the nanobody-drug conjugate NDC of the present invention contains a proteolytic agent or an oligonucleotide drug. Preferably, the oligonucleotide drug is a nucleic acid drug targeting small molecules, including antisense nucleic acid (ASO), small interfering RNA (siRNA), microRNA (miRNA), small activating RNA (saRNA), messenger RNA (mRNA), aptamer, ribozyme, antibody-nucleic acid drug conjugate (ARC), etc.

[0138] As used herein, the proteolytic agent is a degrading agent for tumor-related proteins, and the tumor-related proteins are selected from the following group: (1) Kinases, such as RIPK2, BCR-ABL, EGFR, HER2, c-Met, TBK1, CDK2 / 4 / 6 / 9, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, Fak, etc.; (2) BET proteins, such as BRD2 / 4 / 6 / 9; (3) Nuclear receptors, such as AR, ER, etc.; (4) Other proteins, such as MetAp-2, Bcl-xL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, FRS2, RAS (KRAS, HRAS, and NRAS), etc.

[0139] Preferably, the proteolytic agent includes a PROTAC (proteolysis-targeting chimera compound), and more preferably, the PROTAC targets EGFR, KRAS (e.g., KRAS-G12C, KRAS-G12D).

[0140] The present invention also provides a method for manufacturing an NDC, which may further include a step of binding an antibody to a drug-linker compound under conditions sufficient for the formation of an antibody conjugate (NDC). In some embodiments, the method of the present invention includes a step of binding an antibody to a bifunctional linker compound under conditions sufficient for the formation of an antibody-linker complex. In these embodiments, the method of the present invention further includes a step of binding the antibody-linker complex to a drug moiety under conditions sufficient for the drug moiety to conjugate to the antibody via the linker.

[0141] In some embodiments, the nanobody-drug conjugate NDC is represented by the following molecular formula.

Chemical formula

[0142] Drug As used herein, "drug" broadly refers to any compound having any desired biological activity and having reactive functional groups such that the conjugate according to the present invention can be easily produced. The desired biological activity includes the diagnosis, cure, alleviation, treatment, and prevention of diseases in humans or other animals. Thus, any compound having the necessary reactive functional groups is included in the term "drug" and includes drugs identified in official national pharmacopoeias and, for example, official similar therapeutic-pharmacopoeias in the United States, official national formularies, or any supplements thereof. Typical drugs are shown in the Physician's Desk Reference (PDR) and the Orange Book of the US Food and Drug Administration (FDA). Of course, as new drugs are discovered and developed, these drugs are also incorporated into the "drugs" in the drug conjugates according to the present invention.

[0143] Drugs that can be used in the construction of the NDC of the present invention include, but are not limited to, cytotoxic agents (e.g., cytotoxic small molecule drugs).

[0144] The term "cytotoxic agent" refers to a substance that suppresses or inhibits the expression activity or function of cells and / or destroys cells. The term includes radioisotopes, chemotherapeutic agents and toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants or animals, and fragments and / or variants thereof. Examples of cytotoxic agents include auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlorotetracycline, mitansinoids, ricin, ricin A-chain, combretastatin, duocarmycin, drostatins, adriamycin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A-chain, modeccin A-chain, alpha-streptococcus, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotonin, calicheamicin, Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents, and radioisotopes, such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioisotopes of Lu including Lu177, but are not limited thereto. Antibodies may be coupled with an anti-cancer prodrug activating enzyme that can convert a prodrug into its active form.

[0145] One preferred drug of the present invention is maytansine or a maytansinoid. Maytansine compounds inhibit cell proliferation by suppressing the formation of microtubules by tubulin. Maytansinoids are derivatives of maytansine. Both maytansine and maytansinoids have efficient cytotoxicity, but are greatly limited in clinical use for cancer treatment, mainly due to the low selectivity of such molecules for tumors. However, due to such high cytotoxicity, they become the first candidates for the drug part of antibody-drug conjugates. Below, the structure of deacetylmaytansine is shown. [Chemical formula]

[0146] Another preferred drug of the present invention is an auristatin-based drug. Auristatin-based drugs are analogs of dolastatin 10, and the latter is a bioactive polypeptide isolated from the body of the marine mollusk Amathia. Dolastatin 10 inhibits the polymerization of tubulin by binding to tubulin (the same binding region as vincristine). Dolastatin 10, auristatin PE, and auristatin E are all linear polypeptides and contain four amino acids (three of which are unique to dolastatin-based compounds) and a C-terminal amide group. Two representative auristatin-based compounds, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), are both first candidates for the drug of antibody-drug conjugates. [Chemical formula]

[0147] Another preferred drug of the present invention is Tubulysin. Tubulysin was first isolated from a myxobacterium culture by a research team and is a very effective cell growth inhibitor that acts by inducing apoptosis by suppressing the polymerization of tubulin. Among Tubulysins, Tubulysin D is the most effective and has an activity 10 to 100 times stronger than many other tubulin regulators (including epothilone, vinblastine, and taxol). Taxol and vinblastine are currently used in the treatment of many cancers, while epothilone derivatives are being evaluated for activity in clinical trials. Synthetic derivatives of Tubulysin D provide the necessary information related to inhibition and major binding interactions and have excellent properties as anticancer agents, which are either isolated entities or chemical warheads in target antibodies or ligands. Tubulysin D is a complex tetrapeptide, divided into four regions of Mep (D-N-methylpiperidinecarboxylic acid), Ile (isoleucine), Tuv (tubuvaline), and Tup (tubuphenylalanine), and is represented by the following formula. [Chemical formula]

[0148] Another preferred drug of the present invention is a microorganism-derived cryptophycin derivative that can suppress the polymerization of tubulin. Cryptophycin is a novel antitumor active substance isolated from a cyanobacterium culture that can suppress the production of tubulin and has many major activities. Cryptophycin is a lipophilic compound containing two peptide bonds and two ester bonds and has five optically active centers and one epoxy group. The structures of cryptophycin derivatives CP1 and CP2 are represented by the following formula. [Chemical formula]

[0149] Another preferred drug of the present invention is taltobulin (HTI-286, SPA-110), a novel antitubulin agent. Taltobulin suppresses the polymerization of nascent tubulin, interferes with tubulin organization in cells, and induces mitotic arrest and apoptosis. Taltobulin is a potent inhibitor of cell proliferation, with an average IC50 of 2.5 nM against 18 human tumor cell lines. Compared with currently used antitubulin agents, taltobulin is not a suitable substrate for p-glycoprotein. The structure of taltobulin is shown in the following figure.

Chemical formula

[0150] In one aspect, the drug is the camptothecin-based drug derivative SN-38. SN-38 is the bioactive metabolite of irinotecan hydrochloride (CPT-11) and is a topoisomerase inhibitor. The inhibition of DNA topoisomerase I by SN-38 is the strongest, suppressing DNA synthesis in a dose-dependent and time-dependent manner and leading to frequent single-strand breaks in DNA. The structure of SN-38 is shown in the following figure.

Chemical formula

[0151] In one aspect, the drug is the camptothecin-based drug derivative exatecan. It is a synthetic homolog of the topoisomerase I inhibitor camptothecin, with stronger activity than SN-38. The inhibition of DNA topoisomerase I by it is the strongest, suppressing DNA synthesis in a dose-dependent and time-dependent manner and leading to frequent single-strand breaks in DNA. The structure of exatecan is shown by the following formula.

Chemical formula

[0152] Another preferred drug of the present invention is an amatoxin drug (α-Amanitin), and its structure is shown in the following figure. α-Amanitin is a mycotoxin derived from Amanita phalloides, a bicyclic octapeptide, and can inhibit the transcription of eukaryotic RNA polymerase II and RNA polymerase III. [Chemical formula]

[0153] Another preferred drug of the present invention is benzodipyrrole antibiotics (such as duocarmycin, CC-1065, etc.) and other cyclopropapyrroloind-4-one (CPI) derivatives. Such compounds are effective DNA minor groove binding-alkylating reagents. Cyclopropabenzindol-4-one (CBI) homologs have a more stable chemical structure, higher biological activity, and are easier to synthesize compared to their parent compounds containing natural CPI alkylating subunits. One representative CBI derivative is a phenolic hydroxy-protected derivative CBI, which has attenuated prodrug toxicity and enhanced water solubility (the general formula of the CBI-seco structure is shown in the following figure). [Chemical formula]

[0154] Another suitable drug of the present invention is pyrrolobenzodiazepines (pyrrolo[2,1-c][1,4]benzodiazepines, PBDs) or PBD dimers. PBDs are natural products produced by Streptomyces bacteria, and their unique property is to form non-rotating conjugate adducts in the DNA minor groove, precisely in the purine-guanine-purine sequence. The targeting of PBDs to DNA sequences as partial small molecules and their use as novel anti-cancer and antibacterial drugs have attracted attention. Dimers obtained by linking the C8 / C8’ hydroxy groups of two PBD units with a flexible carbon chain have enhanced biological activity. PBD dimers are said to lead to their biological activity by causing sequence-selective DNA damage, for example, by cross-linking between reverse 5’-Pu-GATC-Py-3’ strands. These compounds have been proven to be efficient cytotoxic drugs and are useful as drug candidates for antibody-drug conjugates.

Chemical formula

[0155] Another suitable drug of the present invention is a PNU-159682 derivative, and PNU-159682 is the main active metabolite of nemorubicin in human liver microsomes, with an activity 3000 times higher than that of MMDX and adriamycin.

Chemical formula

[0156] On the other hand, the drugs are not limited to the types mentioned above, but include all drugs that can be used in antibody-drug conjugates. And in particular, cytotoxins that coordinate by having, for example, an alkaline amine group (primary amine or secondary amine) through an amide bond with a linker, such as the structures of cytotoxins D1-D12 shown above.

[0157] Linking group Based on the mechanism of drug release inside cells, the "linker" or "linker of antibody-drug conjugate" is divided into two types: non-cleavable linker and cleavable linker. In the antibody-drug conjugate containing a non-cleavable linker, the mechanism of drug release is that after the conjugate binds to the antigen and is taken up by the cell, the antibody is enzymatically degraded in the lysosome, releasing the active molecule composed of the small molecule drug, the linker, and the amino acid residues of the antibody. The resulting change in the molecular structure of the drug does not reduce its cytotoxicity, but since the active molecule has a charge (amino acid residues), it cannot penetrate nearby cells. Therefore, such an active drug cannot kill tumor cells (antigen-negative cells) that do not express the nearby target antigen (bystander effect).

[0158] The cleavable linker can literally cleave inside the target cell to release the active drug (the small molecule drug itself). Cleavable linkers are divided into two main types: chemically labile linkers and enzymatically labile linkers. Chemically labile linkers can be selectively cleaved by the properties of plasma and cytoplasm. Such properties include the pH value, glutathione concentration, etc. Linkers sensitive to the pH value are usually also called acid-cleavable linkers. Such linkers are relatively stable in the neutral environment of the blood (pH 7.3 - 7.5), but are hydrolyzed in weakly acidic endosomes (pH 5.0 - 6.5) and lysosomes (pH 4.5 - 5.0). Many of the first-generation antibody-drug conjugates use such linkers, for example, hydrazone, carbonate, acetal, ketal systems. Due to the limited plasma stability of acid-cleavable linkers, antibody-drug conjugates based on such linkers usually have a relatively short half-life (2 - 3 days). Such a short half-life restricts to some extent the use of pH-sensitive linkers in the new generation of antibody-drug conjugates.

[0159] The glutathione-sensitive linking group is also called a disulfide bond linking group. The drug release is due to the difference between the high concentration of glutathione (in the millimolar range) in cells and the relatively low concentration of glutathione (in the micromolar range) in blood. Tumor cells, in particular, have a low oxygen content, which leads to an enhanced activity of reductase and thus a higher glutathione concentration. Since the disulfide bond has thermodynamic stability, it has good stability in plasma. Enzymatically labile linking groups, such as peptide linking groups, can better control drug release. Peptide linking groups are effectively cleaved by proteases in lysosomes, such as Cathepsin B or plasmin (the content of such enzymes increases in some tumor tissues). Such peptide linkages are said to be very stable in plasma circulation because proteases usually do not have activity due to the inappropriate extracellular pH value and serum protease inhibitors. Due to high plasma stability and good intracellular cleavage selectivity and effectiveness, enzymatically labile linking groups are widely used as cleavable linking groups for antibody-drug conjugates. Typical enzymatically labile linking groups include Val-Cit (VC), Val-Ala (VA), Gly-Gly-Phe-Gly (GGFG), etc.

[0160] The self-releasing linking group is generally sandwiched between the cleavable linking group and the active drug or is part of the cleavable linking group itself. The mechanism of action of the self-releasing linking group is that when the cleavable linking group cleaves under appropriate conditions, the self-releasing linking group spontaneously rearranges and further releases the active drug linked to it. Commonly seen suicide linking groups include p-aminobenzyl alcohol-based (PAB) and β-glucuronide-based (β-Glucuronide), etc.

[0161] Applications The present invention provides uses of the present invention, such as uses in the manufacture of diagnostic agents or in the manufacture of drugs for preventing and / or treating CD73-related diseases. The CD73-related diseases include the occurrence, growth and / or metastasis of tumors, tumor drug resistance diseases, inflammation, metabolism-related diseases, etc.

[0162] The uses of the antibody, NDC, CAR-T, etc. of the present invention include, but are not limited to, the following. (i) To diagnose, prevent, and / or treat the occurrence, growth, and / or metastasis of tumors, particularly tumors with high CD73 expression. The tumors include, but are not limited to, breast cancer (e.g., triple-negative breast cancer), lung cancer (e.g., non-small cell lung cancer), pancreatic adenocarcinoma, malignant glioma, gastric cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, leukemia, myeloma, angiosarcoma, etc., particularly triple-negative breast cancer, non-small cell lung cancer, pancreatic adenocarcinoma, malignant glioma, more preferably triple-negative breast cancer and / or non-small cell lung cancer. (ii) To diagnose, prevent, and / or treat autoimmune diseases. The autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, type I diabetes, psoriasis, multiple sclerosis. (iii) To diagnose, prevent, and / or treat inflammation. The inflammation includes, but is not limited to, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, Reiter's syndrome, psoriatic arthritis, infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, glomerulonephritis, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, acute lung injury, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis. (iv) To diagnose, prevent, and / or treat metabolism-related diseases. The metabolism-related diseases include, but are not limited to, diabetes, diet-induced obesity, and adipose inflammation.

[0163] Drug composition The present invention also provides a composition. In a preferred example, the composition is a pharmaceutical composition containing the above-mentioned antibody or its active fragment, or its fusion protein, or its NDC, or the corresponding CAR-T cells, and a pharmaceutically acceptable vehicle. Usually, these substances are formulated with a non-toxic, inert, pharmaceutically acceptable aqueous carrier. The pH value depends on the nature of the substances being formulated and the disease to be treated, but is usually about 5 - 8, preferably about 6 - 8. The formulated pharmaceutical composition can be administered by conventional routes, including but not limited to intratumoral, intraperitoneal, intravenous, or topical administration.

[0164] The antibody according to the present invention can be expressed intracellularly by a nucleotide sequence and used for cell therapy. For example, the antibody can be used in chimeric antigen receptor T cell immunotherapy (CAR-T), etc.

[0165] Since the pharmaceutical composition of the present invention can be directly used for binding to the CD73 protein molecule, it is useful for the prevention and treatment of diseases such as tumors. It may also be used in combination with other therapeutic agents.

[0166] The pharmaceutical composition of the present invention contains 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-mentioned monoclonal antibody (or its complex) of the present invention and a pharmaceutically acceptable vehicle or excipient. Such vehicles include, but are not limited to, saline, buffer solutions, glucose, water, glycerin, ethanol, and combinations thereof. The formulation of the drug corresponds to the dosage form. The pharmaceutical composition of the present invention may be an injection, for example, it can be prepared by a conventional method with an aqueous solution containing physiological saline or glucose and other adjuvants. The pharmaceutical composition is manufactured under aseptic conditions in the case of an injection. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. Also, the polypeptide of the present invention can be used in combination with other therapeutic agents.

[0167] When using the pharmaceutical composition, a safe and effective amount of the immune conjugate is administered to a mammal, and the safe and effective amount is usually at least about 10 μg / kg body weight and often about 50 mg / kg body weight or less, preferably the dosage is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dosage should further consider factors such as the mode of administration and the health status of the patient, and all are within the skill range of a skilled doctor.

[0168] Regarding the NDC, the nanobody-drug conjugate provided by the present invention targets specific cell populations and can release the drug in an active form into cells by binding to specific proteins (antigens) on the cell surface and through the uptake of the conjugate or the penetration of the drug. Therefore, the nanobody-drug conjugate of the present invention can be used for the treatment of target diseases, and the above-mentioned antibody-drug conjugate can be administered to a subject (such as a human) in a therapeutically effective amount via an appropriate route. The subject in need of treatment may be a patient at risk of or suspected of suffering from a disease related to the activity or expression level of a specific antigen. Such patients can be identified by routine health examinations.

[0169] When treating with the nanobody-drug conjugate of the present invention, it can be delivered by conventional methods in this field. For example, it can be introduced into cells by liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or vector can be delivered directly at the site by injection or an infusion pump.

[0170] The main advantages of the present invention are as follows. 1. The nanobody of the present invention has excellent biological activity and specificity, and has a high affinity (its EC 50 measured by ELISA is 2.67 to 13.48 ng / mL), and has the inhibitory activity of the CD73 enzyme function (its IC 50 measured by enzyme activity is 4.66 to 38.18 ng / mL). In addition, it has a good binding affinity for CD73 of tumor cells (its EC 50has a concentration of 0.037 - 0.227 μg / mL) and inhibits the function of tumor cell CD73 enzyme (IC 50 value is 0.083 - 0.48 μg / mL), and is useful as a therapeutic antibody targeting CD73. 2. The nanobody and nanobody-drug conjugate (NDC) of the present invention have significant anti-tumor activity, but have no obvious toxicity or side effects on normal cells. 3. The nanobody of the present invention has a significant growth protection effect on human T cells, can effectively reverse the growth inhibition of adenosine monophosphate (AMP) on T cells, and can promote the expression and secretion of INFγ, and its EC 50 is 0.0008 - 0.0247 μg / mL. 4. According to the designs of FIGS. 45a, b, c of the present invention, CD73-nanobody-drug conjugates with the required DAR value can be conveniently produced, and their cross-linking reaction formulas are shown in FIGS. 46 1a, 1b, 1c or 1d respectively. 5. The nanobody-drug conjugate (NDC) described in the present invention has excellent CD73-dependent anti-tumor activity, that is, it has no obvious toxicity or side effects on CD73 normal or low-expression cells, but has very high killing activity on CD73 medium- and high-expression tumor cells, and its IC 50 measured in the cell growth inhibition test is 0.0025 - 0.0658 μg / mL. 6. In the intermittent administration model, NDC shows a stronger anti-tumor killing effect than the normal antibody-drug conjugate (ADC) of the same target. 7. The NDC described in the present invention has no obvious toxicity or side effects on the growth of normal human T cells, and its IC 50 measured in the cell growth inhibition test is >15 μg / mL. 8. The NDC of the present invention has a significant growth protection effect on human T cells, can effectively reverse the growth inhibition of adenosine monophosphate (AMP) on T cells and promote the expression and secretion of INFγ, and its EC 50 is 0.0046 - 0.0379 μg / mL. 9. The NDC of the present invention has extremely excellent in vivo antitumor activity, and can completely suppress tumor growth or regress tumors in many tumor models after administration by single intravenous injection. 10. The nanobody and NDC of the present invention can pass through the blood-brain barrier, have the property of distributing in the brain, and exhibit excellent therapeutic effects on intracranial tumors. 11. The humanized antibody and NDC of the present invention similarly exhibit the above or more advantages.

[0171] Hereinafter, specific examples are combined to further describe the present invention. It is understood that these examples are only used to explain the present invention and do not limit the scope of the present invention. Experimental methods for which detailed conditions are not shown in the following examples are usually according to normal conditions such as those described in, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York, Cold Spring Harbor Laboratory Press, 1989), or according to the recommended conditions of the manufacturer. Unless otherwise specified, % and parts are calculated by weight.

[0172] Example 1 Discovery and Production of Nanobody Targeting Human CD73 First, the extracellular region of human CD73 protein (CD73-ECD) was produced as an antigen. Referring to the 27th to 547th amino acids of NCBI:NP_002517.1, an antigen with a C-terminus polyhistidine-tag was obtained by gene cloning technology and a mammalian vector expression system. WELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKAHHHHHHHHHH

[0173] Using the protein of the extracellular region of CD73 produced as described above, alpacas were immunized with an immunization interval of 21 days. Ten days after the last immunization, some peripheral blood was collected (PBMC). After isolation of PBMC and cloning of VHH antibody fragments, a phage display library and a yeast display library were constructed. Through processes such as screening, single clone ELISA, and FACS screening, four nanobodies (3-D7, 5-E11, 4-D04, 4-B02) with high activity and independent sequences were obtained from the phage display library, and five active antibodies (1-E7, 2-B10, 2-G9, 3-A3, 3-F1) with independent sequences were obtained from the yeast display library.

[0174] Example 2 Measurement of the Affinity of CD73 Nanobodies for Human CD73 Antigen by SPR Surface Plasmon Resonance (SPR) measurement: Antibodies were immobilized with Protein A sensors, and the height of immobilization was about 1.5 nM. The buffer was PBST (PBS + 0.02% Tween 20), and samples of CD73-ECD protein were diluted to 500, 250, 125, 62.5, 31.3, 0 nM. Affinity detection: Kinetic characterization analysis was performed at a detection temperature of 25 °C for 60 s of equilibration, 180 s of binding, and 180 s of dissociation using the ForteBio OCTET R2 system. As shown in Figure 1, the detection results indicated that 3-D7, 5-E11, 4-D04, and 4-B02 had strong affinity for CD73-ECD, with binding constants (KD) of 1.997 nM, 0.826 nM, 0.919 nM, and 0.795 nM, respectively.

[0175] Example 3 Measurement of the Affinity of CD73 Nanobodies for Human CD73 Antigen by ELISA The extracellular region of CD73 protein (CD73-ECD) was diluted to 1 μg / mL with the coating solution, and the ELISA plate was coated at 100 μL / well at 4 °C overnight. The remaining antigen was washed away, blocked with 1% BSA at room temperature for 2 h, and then each monoclonal antibody diluted in a 5-fold gradient was added at 100 μL / well and incubated at room temperature for 1 h. Unbound antibodies were washed away, and Staphylococcus protein A labeled with horseradish peroxidase at an appropriate concentration was added at 100 μL / well and incubated at room temperature for 0.5 h. Unbound secondary antibodies were washed away, TMB chromogenic solution was added and reacted for about 15 min, 1 M HCl was added at 50 μL / well to stop the chromogenic reaction, and then the absorbance at 450 nm was measured and the data were analyzed.

[0176] As shown in Figure 2, the detection results indicated that 3-D7, 5-E11, 4-D04, and 4-B02 had strong affinity for CD73-ECD, and the EC 50 were 13.48 ng / mL, 8.72 ng / mL, 2.67 ng / mL, and 3.03 ng / mL, respectively. For five yeast-derived antibodies, transient transfection and expression were performed in HEK293F cells. Subsequently, after culturing for 2 days, the supernatant was collected, and ELISA measurements were performed using 100 μL, 10 μL, and 1 μL. As shown in Figure 11, the detection results indicated that all of the nanobodies 1-E7, 2-B10, 2-G9, 3-A3, and 3-F1 had strong CD73-ECD binding affinities. As described above, the CD73 nanobodies of this example have good binding activity with the human CD73 antigen.

[0177] Example 4 Measurement of the inhibitory activity of the CD73 nanobody on the catalytic function of recombinant human CD73 enzyme The human recombinant CD73 enzyme (CD73 extracellular region) was diluted to 0.1 μg / mL with the antigen diluent and uniformly spread on a 96-well low-adsorption culture plate at 25 μL / well. 50 μL of the CD73 antibody diluted from 300 ng / mL to 0.137 ng / mL in a 3-fold gradient was added to the culture plate and uniformly mixed (the final concentration was 1350 ng / mL to 0.069 ng / mL). After incubation at 37 °C for 1 h, 25 μL of the mixture containing 1.2 mM AMP and 0.4 mM ATP was added, and incubation was carried out at 37 °C for 1 h. 50 μL of the above reaction solution was taken out and placed in another 96-well plate. 50 μL of the CellTiter-Glo reagent was added to each well, uniformly mixed, and reacted for 3 - 5 min avoiding light. The fluorescence signal intensity was detected using a microplate reader.

[0178] As shown in Figure 3, the detection results indicated that all of 3-D7, 5-E11, 4-D04, and 4-B02 had the activity to significantly inhibit the hydrolysis of AMP by recombinant CD73 protease, and their IC 50 values were 38.18 ng / mL, 9.47 ng / mL, 5.63 ng / mL, and 4.66 ng / mL, respectively. As described above, the CD73 monoclonal antibody of this example has significant inhibitory activity on the catalytic function of the recombinant human CD73 enzyme.

[0179] Example 5 Measurement of the binding affinity of CD73 nanobody to CD73 on the surface of tumor cells Using triple-negative breast cancer cells MDA-MB-231, non-small cell lung cancer cells NCI-H1299, PC9, NCI-H1975, SW1573, NCI-H1373, HCC44 with medium to high CD73 expression as target cells, the test antibody diluted from 10 μg / mL to 0.016 μg / mL in a 5-fold gradient of 100 μL was used as the primary antibody, and 1×10 5 cells suspended in 100 μL of RPMI-1640 serum-free medium were uniformly mixed, diluted to a final concentration of 5 μg / mL to 0.008 μg / mL, incubated at 4°C for 1 h, the cells were washed twice with PBS to remove unbound primary antibody, and then the target cells were incubated with 200 μL of secondary antibody labeled with 2 μg / mL of PE at 4°C for 30 min, the cells were washed twice with PBS to remove unbound secondary antibody, and finally the cells were resuspended in 200 μL of PBS, and the binding affinity of the test antibody to CD73 on the corresponding cell surface was measured by a flow cytometer.

[0180] As shown in the detection results in Figure 4A, 3-D7, 5-E11, 4-D04, and 4-B02 had excellent binding affinity for MDA-MB-231, and the EC 50 were 0.12 μg / mL, 0.037 μg / mL, 0.059 μg / mL, and 0.037 μg / mL, respectively. As shown in the detection results in Figure 4B, 3-D7, 5-E11, 4-D04, and 4-B02 had excellent binding affinity for NCI-H1299, and the EC 50 were 0.222 μg / mL, 0.068 μg / mL, 0.080 μg / mL, and 0.078 μg / mL, respectively.

[0181] As shown in the detection results in Figure 5A, 3-D7, 5-E11, 4-D04, and 4-B02 had excellent binding affinity for PC9, and the EC 50 were 0.133 μg / mL, 0.041 μg / mL, 0.057 μg / mL, and 0.044 μg / mL, respectively. As shown in Figure 5B, the detection results indicate that 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinities for NCI-H1975, and the EC 50 values were 0.106 μg / mL, 0.057 μg / mL, 0.068 μg / mL, and 0.055 μg / mL, respectively.

[0182] As shown in Figure 6A, the detection results indicate that 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinities for SW1573, and the EC 50 values were 0.116 μg / mL, 0.062 μg / mL, 0.067 μg / mL, and 0.061 μg / mL, respectively. As shown in Figure 6B, the detection results indicate that 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinities for NCI-H1373, and the EC 50 values were 0.227 μg / mL, 0.135 μg / mL, 0.163 μg / mL, and 0.112 μg / mL, respectively.

[0183] As shown in Figure 7, the detection results indicate that 3-D7, 5-E11, 4-D04, and 4-B02 have excellent binding affinities for HCC44, and the EC 50 values were 0.234 μg / mL, 0.096 μg / mL, 0.114 μg / mL, and 0.105 μg / mL, respectively.

[0184] For six yeast-derived antibodies, transient transfection and expression were performed in HEK293F cells. Subsequently, after culturing for 2 days, the supernatant was collected and incubated with MDA-MB-231 cells at 100 μL, 10 μL, and 1 μL, followed by flow cytometry measurement to determine their binding activities. As shown in Figure 12, the detection results indicate that all of the nanobodies 1-E7, 2-B10, 2-G9, 3-A3, and 3-F1 had strong CD73-ECD binding affinities.

[0185] As shown in the above results, the CD73 nanobodies of this example can target CD73 of human-derived tumor cells and have good binding activities.

[0186] Example 6: Influence of CD73 nanobody on the catalytic function of CD73 enzyme on the surface of tumor cells Triple-negative breast cancer cells MDA-MB-231 with high CD73 expression were used as target cells. An appropriate number of tumor cells (confirmed by preliminary experiments) were seeded in 96-well plates and cultured at 37 °C for 16 hours. After that, the cells were washed three times with serum-free RPMI-1640 medium, and 50 μL of the test antibody diluted from 10 μg / mL to 0.0045 μg / mL in a 3-fold gradient was added to the 96-well plates. After incubating at 37 °C for 30 min, 25 μL of 0.9 mM AMP was added, and the cells were cultured at 37 °C and 5% CO2 for 3 h. Then, 25 μL of the above culture supernatant was taken out and put into another 96-well plate, and 25 μL of 0.1 mM ATP was added and mixed uniformly. 50 μL of CellTiter-Glo reagent was added to each well, mixed uniformly, and reacted for 3 - 5 min in the dark, and the fluorescence signal intensity was detected by a microplate reader.

[0187] As shown in the detection results in Figure 8, 3-D7, 5-E11, 4-D04, and 4-B02 can all significantly inhibit the function of catalyzing AMP hydrolysis of CD73 on the surface of MDA-MB-231 cells, and the IC 50 was 0.483 μg / mL, 0.088 μg / mL, 0.093 μg / mL, and 0.083 μg / mL, respectively.

[0188] Example 7: Protective effect of CD73 nanobody on the proliferation of T cells and the expression of IFN-γ Regeneration, amplification, and screening of PBMC: First, PBMC were regenerated and cultured in a medium containing 500 ng / mL of CD3 / CD28 antibody and 100 IU / mL of IL-2 for 3 - 4 days, and then PBMC were screened by a screening kit (Stemcell, Cat#1795) to obtain CD3-positive T cells.

[0189] T cell proliferation assay: The T cells obtained from the above screening were fluorescently labeled, and the pre-prepared CFSE (carboxyfluorescein succinimidyl ester) was added to the cell suspension (final concentration 2.5 μM). After labeling at 37 °C for 5 min, it was washed three times with PBS. Then, the T cells labeled with CFSE were plated in a 96-well plate (2×10 4 cells / well), 50 μL of serially diluted nanobody (final concentration 15 μg / mL - 0.00019 μg / mL, n = 4) was added to each well, and then 50 μL of adenosine monophosphate (AMP, final concentration 0.25 mM) was added, mixed uniformly, cultured for 4 - 5 days, then the culture supernatant was collected, read by a fluorescence-activated cell sorter (FACS) to count the number of cells in a predetermined volume, and a cell growth curve was drawn by Flowjo software to calculate the EC 50 value. 50 μL / well of the culture supernatant of T cells was taken for the detection of IFN-γ protein concentration, an ELISA kit was used, and the techniques and operation procedures provided by the kit were referred to.

[0190] As shown in Figure 9, the CD73 nanobodies 3-D7, 5-E11, 4-D04, and 4-B02 have a significant growth protection effect on human T cells, and can effectively reverse the growth inhibition of AMP on T cells. Their EC 50 values were 0.0247 ± 0.0052 μg / mL, 0.0008 ± 0.0002 μg / mL, 0.0025 ± 0.0007 μg / mL, and 0.0012 ± 0.0004 μg / mL, respectively.

[0191] As shown in Figure 10, the CD73 nanobodies 3-D7, 5-E11, 4-D04, and 4-B02 were able to effectively reverse the inhibitory effect of AMP on the expression / secretion of INF-γ in T cells. As shown in the above results, the CD73 nanobody of this example not only inhibits the activity of the CD73 enzyme on the surface of tumor cells, but also inhibits the active function of the CD73 enzyme on the surface of T cells, reverses the influence of AMP on the proliferation and function of T cells, and can protect T cells.

[0192] Example 8 Uptake into Lysosomes in Cells by Binding of CD73 Antibody to Tumor Cells MDA-MB-231 cells were seeded in a 26-well plate at a density of 50% and cultured at 37°C for 6 h. Then, 5 μg / mL of the CD73 nanobody was added, and the mixture was incubated at 37°C for 4 h or at 4°C for 1 h, respectively. The cells were washed three times with PBS to remove the unbound antibody, and then fixed with 4% paraformaldehyde at room temperature for 30 min. After washing three times with PBS, the cells were permeabilized with 0.4% Triton X-100 for 10 min. After washing three times with PBS, the cells were incubated with the Lamp-2 (rabbit anti-human) antibody at 37°C for 1 h to label the position of cell lysosomes. The unbound antibody was washed away with PBS, and then incubated with the sheep anti-human secondary antibody labeled with R-PE and the donkey anti-rabbit secondary antibody labeled with Alexa Fluor 488 at 37°C for 30 min. The unbound secondary antibody was washed away, and the cells were stained with DAPI for 10 min to label the position of the cell nucleus. Then, the uptake of the antibody was observed under a fluorescence microscope (200×).

[0193] As shown in Fig. 13, all of 3-D7, 5-E11, 4-D04, and 4-B02 were rapidly and significantly taken up into lysosomes by MDA-MB-231 cells. From these results, it was shown that the antibody of the present invention is suitable for the production of NDC, and CD73-NDC has good NDC drug properties, suggesting its potential as a therapeutic agent that targets CD73-positive tumors in a broad and highly specific manner.

[0194] Example 9 Production of 3-D7-vc-MMAE, 5-E11-vc-MMAE, and 4-D01-vc-MMAE (I) Production of 3-D7-vc-MMAE Conjugate The 3-D07 protein stock solution was diluted to 5 mg / mL with a reaction buffer of 50 mM potassium dihydrogen phosphate - sodium hydroxide (KH2PO4 - NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.4. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) with a molar ratio of 3 - 4-fold excess was added, and the reaction solution was stirred at 25 °C for 2.5 hours.

[0195] The above reaction solution was cooled to 0 - 10 °C, an appropriate amount of diethylacetamide (DMA) was added without generation, and mc-vc-MMAE (pre-dissolved in DMA at 10 mg / ml) with a molar ratio of 6-fold excess was added so that the volume ratio of DMA in the reaction system was 10% or less. The coupling was carried out with stirring at 10 - 25 °C for 2 hours. The coupling reaction mixture was purified by filtration through a desalting column with a histidine - acetic acid / sucrose gel at pH 6.0, and the peak samples were collected according to the UV280 ultraviolet absorption value. Then, it was sterilized with a filtration device with a pore size of 0.22 μm and stored at -80 °C. When calculating the difference between the mass number and the theoretical molecular weight of the antibody conjugate 3-D7-vc-MMAE by mass spectrometry, the accurate drug loading amount was about 2 (Figure 21).

[0196] (II) Preparation of 5-E11-vc-MMAE conjugate The 5-E11 protein stock solution was diluted to 5 mg / mL with a reaction buffer of 50 mM potassium dihydrogen phosphate - sodium hydroxide (KH2PO4 - NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.4. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) with a molar ratio of 3 - 4-fold excess was added, and the reaction solution was stirred at 25 °C for 2.5 hours.

[0197] The above reaction solution was cooled to 0 - 10 °C, an appropriate amount of diethylacetamide (DMA) was added without generation, and a 6-fold excess molar ratio of mc-vc-MMAE (pre-dissolved in DMA at 10 mg / ml) was added so that the volume ratio of DMA in the reaction system was 10% or less, and the mixture was coupled with stirring at 10 - 25 °C for 2 hours. The coupling reaction mixture was purified by filtration through a desalting column with histidine - acetic acid / sucrose gel at pH 6.0, and the peak samples were collected according to the UV280 ultraviolet absorption value. Then, it was sterilized by a filtration device with a pore size of 0.22 μm and stored at -80 °C. When calculating the difference between the mass number and the theoretical molecular weight of the antibody conjugate 5-E11-vc-MMAE by mass spectrometry, the accurate drug loading amount was about 2 (Figure 22).

[0198] (III) Preparation of 4-D04-vc-MMAE conjugate The stock solution of 4-D04 protein was diluted to 5 mg / mL with a reaction buffer of 50 mM potassium dihydrogen phosphate - sodium hydroxide (KH2PO4-NaOH) / 150 mM sodium chloride (NaCl) / 1 mM diethylenetriaminepentaacetic acid (DTPA), pH 7.4, and a 3 - 4-fold excess molar ratio of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was added, and the reaction solution was stirred at 25 °C for 2.5 hours.

[0199] The above reaction solution was cooled to 0 - 10 °C, an appropriate amount of diethylacetamide (DMA) was added without generation, and a 6-fold excess molar ratio of mc-vc-MMAE (pre-dissolved in DMA at 10 mg / ml) was added so that the volume ratio of DMA in the reaction system was 10% or less, and the mixture was coupled with stirring at 10 - 25 °C for 2 hours. The coupling reaction mixture was purified by filtration through a desalting column with histidine - acetic acid / sucrose gel at pH 6.0, and the peak samples were collected according to the UV280 ultraviolet absorption value. Then, it was sterilized by a filtration device with a pore size of 0.22 μm and stored at -80 °C. When calculating the difference between the mass number and the theoretical molecular weight of the antibody conjugate 4-D04-vc-MMAE by mass spectrometry, the accurate drug loading amount was about 2 (Figure 23).

[0200] Example 10 In vitro antitumor activity of CD73-NDC against CD73-highly expressing tumor cells The cell lines used in the examples were purchased from the American Type Culture Collection (ATCC), the Cell Library of the Chinese Academy of Sciences, and Nanjing Kebai Biotechnology Co., Ltd., cultured according to the corresponding instructions, and included MDA-MB-453, MDA-MB-231, PC9, NCI-H1975, NCI-H441, and SW1573. The above cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate at a density of 200-2000 cells / well (depending on the cell growth rate) at 150 μL / well, cultured at 37°C and 5% CO2 for about 5 h, and then different concentrations of CD73-NDC (15 μg / mL to 0.00019 μg / mL) were added. For each drug concentration, 2-4 duplicate wells, as well as wells for the corresponding solvent control and blank control, were set up. After acting for 5-9 days (depending on the cell growth rate, ensuring sufficient cell division), the culture medium was discarded, MTS reaction solution (purchased from Promega, cat# G3581) was added at 100 μL / well, and the reaction was carried out at 37°C until a predetermined color depth was reached. The cell viability of each group was measured (OD490 nm), and the cell survival rate was calculated using the formula survival rate = (OD administration - OD blank) / (OD control - OD blank) × 100%. The above data were analyzed by GraphPad Prism 8 software, and the IC 50 values of the above CD73 antibody-drug conjugate in different cell lines were calculated.

[0201] The results of the in vitro antitumor activities of two preferred CD73-NDCs: 3-D7-MMAE, 5-E11-MMAE and CD73-ADC Hu001-MMAE as a control are shown in Figures 14 to 16 respectively.

[0202] As shown in Fig. 14A, CD73-NDC did not show an obvious growth inhibitory effect on MDA-MB-453 cells with low CD73 expression, but showed a strong cell growth inhibitory effect on all of MDA-MB-231 with medium / high CD73 expression (Fig. 14B), PC9 (Fig. 15A), NCI-H1975 (Fig. 15B), NCI-H441 (Fig. 16A), and SW1573 (Fig. 16B). Overall, from the cytotoxicity (IC50 value) of CD73-NDC, it was shown that the cytotoxic activity of CD73-NDC was directly related to the CD73 expression level of the test cells, so it was determined to be specific cytotoxicity targeting CD73. The IC50 values of some cell growth inhibition tests are summarized in Table-3.

Table 3

[0203] Example 11 Protective effect of CD73-NDC on the proliferation of T cells and the expression of IFN-γ All human peripheral blood mononuclear cell (PBMC) cryopreservation tubes were provided by Jiangsu Xidier Biological Technology Co., Ltd. First, PBMC were cultured in medium containing 500 ng / mL of CD3 / CD28 antibody and 100 IU / mL of IL-2 for 3 to 4 days of regeneration culture, and then screened by a screening kit (provider: Stemcell, Cat#1795) to obtain CD3-positive T cells, which were further fluorescently labeled. The pre-prepared fluorescent dye CFSE (carboxyfluorescein succinimidyl ester) was added to the cell suspension (final concentration 2.5 μM), incubated at 37 °C for 5 min, washed 3 times with PBS, and then used for the test.

[0204] First, the effect of different concentrations of CD73-NDC on the T cell growth curve was observed. T cells labeled with CFSE were plated in a 96-well plate (2×10 4(cells / well), CD73-NDC diluted in gradient (final concentration 15 μg / mL to 0.000192 μg / mL, n = 2) or the control solvent was added to each well. After culturing for 5 days, the number of live cells was read by FACS, and a dose-response curve was made to calculate the IC50 value. As shown in Figure 17A, 3-D7-MMAE and 5-E11-MMAE did not show significant toxicity or side effects in the tested concentration range (IC 50 > 15 μg / mL).

[0205] In another set of tests, T cells labeled with CFSE were plated in a 96-well plate (2 × 10 4 (cells / well), 50 μL of NDC diluted in gradient (final concentration 3 μg / mL to 0.00019 μg / mL, n = 2) was added to each well, and then 50 μL of AMP (final concentration 0.25 mM) was added, mixed uniformly, and after culturing for 4 - 5 days, the culture supernatant was collected, read by a fluorescence-activated cell sorter (FACS) to count the number of cells in a predetermined volume, and a cell growth curve was drawn by Flowjo software to calculate the EC50 value. The results, as shown in Figure 17B, 3-D7-MMAE and 5-E11-MMAE had a significant growth-protective effect on human T cells and could effectively reverse the growth inhibition of AMP on T cells, and their EC 50 were 0.0379 ± 0.0066 μg / mL and 0.0046 ± 0.0012 μg / mL respectively.

[0206] 50 μL / well of the T cell culture supernatant was taken for detecting the IFN-γ protein concentration. An ELISA kit (Sinobiological, Cat#KIT11725A) was used, and the techniques and operating procedures provided by the kit were referred to. The results, as shown in Figure 18, 3-D7-MMAE and 5-E11-MMAE could effectively reverse the inhibitory effect of AMP on the expression / secretion of INF-γ in T cells. From the results of this example, it was shown that CD73-NDC had no toxic effect on T cells and could effectively reverse the inhibitory effect of AMP / Ado on the proliferation of T cells.

[0207] Example 12 Expression production and purification of mass production of CD73 nanobody and fusion protein The variable region sequences of 3-D7 and 5-E11 nanobodies were cloned into an hIgG1 expression vector, Fc fragments with different designs (3-D7-FC1, 3-D7-FC2, 3-D7-FC3, 5-E11-FC1, 5-E11-FC2, 5-E11-FC3), or an expression vector for recombinant / fusion protein (3-D7-HLE, 3-D7-ABD, 5-E11-ABD), transiently transfected into HEK293, and then cultured in suspension for 6 days in serum-free medium. The culture supernatant was collected for purification, protein quantification, and SDS-PAGE detection. As shown in FIGS. 19 and 20, chimeric nanobodies and recombinant fusion proteins derived from 3-D7 and 5-E11 could all be efficiently expressed, produced, and purified.

[0208] Example 13 Production and activity detection of CD73 nanobody 5-E11-derived antibodies Since there was an undesirable amino acid sequence (hot spot) in CDR2 of the original sequence of 5-E11 VH (SEQ ID NO: 8), after mutation and modification, 5-E11-derived antibodies 5-E11AS, 5-E11GS, 5-E11QS, and 5-E11ASCS were obtained, and the corresponding sequences are SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, and SEQ ID NO: 49, respectively.

[0209] As a result of detecting the CD73 antigen ELISA binding affinity according to the method of Example 3, as shown in FIG. 24, 5-E11AS, 5-E11GS, and 5-E11QS have the same ELISA binding affinity as the original antibody 5-E11.

[0210] Based on the above mutants, 5-E11ASCS further designed and manufactured was subjected to CD73 antigen ELISA binding affinity detection according to the method of Example 3. As a result, as shown in FIG. 25, 5-E11ASCS has the same ELISA binding affinity as the original antibody 5-E11, and the EC 50 was 0.018 μg / mL and 0.05 μg / mL, respectively. When the binding affinity of the antibody for CD73 on the surface of SW1573 cells was measured according to the method of Example 5, as shown in Fig. 26, 5-E11ASCS had the same cell FACS binding affinity as the original antibody 5-E11, and the EC 50 were 0.015 μg / mL and 0.013 μg / mL, respectively.

[0211] Example 14 Humanization and Activity Detection of Nanobody 3-D7 From the sequence information of the original antibody, a homologous model of the antibody was obtained by model construction, and the amino acids in the framework within the 5 Å range of the CDR were analyzed. These amino acid sites usually affect the conformation of the CDR or the antigen-binding activity. After obtaining the human germline by IMGT analysis and ligating the framework of the selected human germline to the CDR of the antibody, the sequences of the framework regions of the designed humanized antibody and the original antibody were compared, and by analyzing the results of the homologous model construction of the parental antibody, the amino acids similar to the surface residues of the human antibody were used for replacement on the premise of reducing the heterology while maintaining the activity of the antibody, and humanized antibodies 3D7-HM7, 3D7-HM8, and 3D7HM9 were obtained, and the corresponding sequences are SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52.

[0212] According to the surface plasmon resonance (SPR) measurement method of Example 2, the affinity of the humanized antibody for the antigen was detected. As shown in Fig. 27, the results showed that 3D7-HM7, 3D7-HM8, and 3D7-HM9 had very close antigen-binding affinities to the original antibody 3-D7, and the binding constants were 1.297 nM, 1.173 nM, 0.638 nM, and 0.472 nM, respectively.

[0213] According to the ELISA measurement method of Example 3, the affinities of the humanized antibodies for human and cynomolgus monkey CD73-ECD were detected respectively. As shown in Fig. 28, the results showed that 3D7-HM9-FC1 and 3D7-HM9-FCWT had ELISA binding EC for human CD73-ECD 50It was 2.5 ng / mL and 2.09 ng / mL. As shown in Figure 29, the results show that 3D7-HM9-FC1 and 3D7-HM9-FCWT have ELISA binding ECs against cynomolgus CD73-ECD 50 It was 1.88 ng / mL and 2.06 ng / mL.

[0214] According to the method for measuring the catalytic functional activity of the recombinant human CD73 enzyme of Example 4, the inhibitory activity of the humanized antibody was detected. As shown in Figure 30, the results show that 3D7-HM7, 3D7-HM8, and 3D7-HM9 have a functional inhibitory activity of the CD73 enzyme that is very close to that of the original antibody 3-D7, and the IC 50 was 45.65 ng / mL, 55.55 ng / mL, 18.74 ng / mL, and 36.3 ng / mL, respectively.

[0215] According to the method for measuring the binding affinity of the tumor cell surface CD73 of Example 5, the binding activity of the humanized antibody to NCI-H1299 cells was detected. As shown in Figure 31, the results show that 3-D7-HM7, 3-D7-HM8, and 3-D7-HM9 have a tumor cell binding affinity that is very close to that of the original antibody 3-D7, and the EC 50 was 0.02 μg / mL, 0.043 μg / mL, 0.02 μg / mL, and 0.019 μg / mL, respectively.

[0216] According to the method for measuring the catalytic function of the CD73 enzyme on the tumor cell surface of Example 6, the inhibitory activity of the humanized antibody was detected. As shown in Figure 32, the results show that 3D7-HM9-FC1, 3D7-HM9-ABD, and 3D7-HM9-FCWT have an inhibitory activity that is very close to that of the original antibody 3-D7, and the IC 50 was 0.098 μg / mL, 0.123 μg / mL, and 0.126 μg / mL, respectively.

[0217] According to the measurement method for the proliferation of T cells in Example 7, CFSE-labeled T cells were plated in a 96-well plate (2×10 4(cells / well), 50 μL of gradient-diluted nanobody (final concentration 15 μg / mL to 0.00019 μg / mL, n = 4) was added to each well, and then 50 μL of adenosine monophosphate (AMP, final concentration 0.25 mM) was added, mixed uniformly, cultured for 4 - 5 days, and then read by a fluorescence-activated cell sorter (FACS) to count the number of cells in a predetermined volume. As shown in Fig. 33, 3D7-HM9-FC1 and 3D7-HM9-ABD can effectively reverse the inhibitory effect of AMP on T cell proliferation, and the EC 50 was 0.024 μg / mL and 0.0467 μg / mL respectively.

[0218] According to Example 11, the effect of humanized NDC on T cell proliferation was detected. As shown in Fig. 34, 3D7-HM9-FC1 and 3D7-HM9-ABD showed no obvious toxicity or side effects on T cell proliferation, and the IC 50 was > 10 μg / mL. In another set of detections, 0.25 mM AMP was added to the T cell proliferation incubation culture medium. As shown in Fig. 35, 3D7-HM9-FC1 can effectively reverse the inhibitory effect of AMP on T cell proliferation, and the EC 50 was 0.0197 μg / mL.

[0219] According to Example 9, vc-MMAE conjugates of humanized antibody fusion proteins 3D7-HM9-FC1 and 3D7-HM9-ABD were prepared. For the antibody alone and the MMAE conjugate, the purity and conjugation degree were detected by methods such as hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC). It was expected that the antibody FC1 fusion protein would form a dimer conjugate with a target DAR of 4, and the antibody-ABD structure would be a monomer conjugate with a target DAR of 1. As shown in Fig. 36, when the 3D7-HM9-FC1-MMAE conjugate was detected and analyzed by HIC, a single conjugate tumor peak was shown, and the expected DAR value was 4. As shown in Figure 37, when the 3D7-HM9-ABD-MMAE conjugate was detected and analyzed by HIC, a single conjugate tumor peak was shown and the expected DAR value was 1.

[0220] The in vitro antitumor activity of the humanized NDC with high CD73 expression against tumor cells was detected according to Example 10. The detected cells included lung cancer cell lines HCC44, PC9, H1975, SW1573, NCI-H441, Calu-1, triple-negative breast cancer cell line MDA-MB-231, pancreatic adenocarcinoma cell line BXPC3, HPAF-II, and glioblastoma cell line U87MG. The above cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate at a density of 200-2000 cells / well (depending on the cell growth rate) at 150 μL / well, and after treatment with drugs at 37°C for 5-9 days (depending on the cell growth rate to ensure sufficient cell division), the culture medium was discarded and the cell viability (OD490nm) of each group was measured.

[0221] Table 4 summarizes the excellent in vitro antitumor activities of the humanized NDC 3D7-HM9-FC1-MMAE, 3D7-HM9-FCWT-MMAE, and 3D7-HM9-ABD-MMAE against the above tumor cell lines, which were consistent with the predictions.

Table 4

[0222] Example 15 Killing effect of intermittent administration of CD73-NDC on tumor cells H1975 and SW1573 cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate at 1000 cells per well and cultured at 37 °C and 5% CO2 in 150 μL / well for about 16 h. 5 μg / mL of hIgG1-MMAE, 3D7-FC1-MMAE, 5E11-FC1-MMAE and the ADC Hu001-MMAE with the same target were added, and the cells were treated for 24 h. The original medium was discarded, and the cells were gently washed once with PBS. Fresh complete medium was added again, and the cells were cultured for an additional 4 days under drug-free conditions. The culture medium was discarded, 100 μL / well of MTS reaction solution was added, and the reaction was allowed to proceed at 37 °C until the expected color intensity was reached. The cell viability (OD490nm) of each group was read using a microplate reader. As shown in Figure 38, CD73-NDC showed a stronger killing effect on tumor cells on day 1 of intermittent administration compared to the ADC with the same target.

[0223] Example 16 In Vivo Anti-Tumor Effect of CD73 Nanobody (Subcutaneous Tumor Model) 4-5-week-old nude mice were purchased and adaptively bred in an SPF-class animal laboratory for 1 week. PC9 cells in the logarithmic growth phase were digested, cooled to 4 °C, and centrifuged at 1000 rpm for 5 min using a pre-cooled centrifuge. The supernatant was discarded, and the cells were resuspended in PBS according to the cell count to adjust the cell density to 25×10 6 cells / mL. 200 μL of the cell suspension was inoculated subcutaneously into the flanks of nude mice at 5×10 6 cells / tumor. The growth status of the tumors was observed. When the tumors grew to about 200 mm 3 , the mice were grouped according to the tumor volume and body weight. In the PC9 transplanted tumor experiment (n = 8), on the 15th day after tumor inoculation, 3 mg / kg of hIgG1-MMAE, 3D7-FC1-MMAE, and 5E11-FC1-MMAE were administered by a single injection via the tail vein. The growth status of the tumors and the condition of the mice were observed regularly. Data on the length and width of the tumors were collected twice a week, and the tumor volume (mm 3) The volume of the tumor was calculated using the formula of length × width × width ÷ 2. The growth curve of the tumor and the body weight curve of the mice were plotted. After the experiment ended, the mice were euthanized with carbon dioxide, the tumors were collected, the weights of the tumors were measured, and the tumor inhibition rate was calculated. The tumors were quickly frozen with liquid nitrogen and then stored in a -80°C refrigerator. As shown in Figure 39, after a single administration of 3 mg / kg, both 3D7-FC1-MMAE and 5E11-FC1-MMAE showed excellent anti-tumor efficacy, leading to significant tumor regression.

[0224] Example 17 In vivo anti-tumor effect of CD73 nanobody (orthotopic lung tumor model) 4- to 5-week-old nude mice were purchased and adaptively bred in an SPF-class animal laboratory for 1 week. HCC44-luc cells in the logarithmic growth phase were digested, cooled in a centrifuge at 4°C, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended with PBS according to the cell number, and adjusted to a cell density of 40×10 6 cells / mL. An equal volume of high-concentration Matrigel was added, that is, cell suspension:Matrigel = 1:1. After the mice were anesthetized with abectin (400 μL / 20 g), an incision was made at a position near the lower part of the right flank of the mice (the skin and muscle are a total of 3 layers), and the pink lungs could be seen. When making the incision, be careful to avoid the Y-shaped blood vessels on the skin, and it may be inserted at the middle position of the Y shape. A line was drawn at the 3-mm position of the insulin needle, that is, the depth at which the needle was inserted, and the cells were slowly inserted, 50 μL was inoculated into each mouse, a total of 1×10 6 cells / mouse. After the injection was completed, it was stopped for 5-10 s, and the needle was withdrawn while rotating, and the wound was sutured. In one set of experiments, on the 13th day after model construction, imaging was performed with a bioluminescence imaging device, and the mice were divided according to the signal value. On the 13th day, 3 mg / kg of hIgG1-MMAE, 3D7-FC1-MMAE, and 5-E11-MMAE were injected from the tail vein (n = 6), and a total of 1 administration was given. On the 13th and 20th days, 3 mg / kg of 3D7-ABD-MMAE was injected from the tail vein (n = 4), and a total of 2 administrations were given. On the 20th, 27th, and 34th days, imaging was performed with a bioluminescence imaging device to detect and record the fluorescence value.

[0225] As shown in Figure 40, after a single administration of 3D7-FC1-MMAE and 5E11-FC1-MMAE at 3 mg / kg, both showed excellent antitumor efficacy, leading to tumor regression. After two administrations of 3D7-ABD-MMAE, it showed excellent antitumor efficacy, leading to tumor regression. In another set of experiments, on the 20th day after model establishment, 3 mg / kg of hIgG1-MMAE and 3D7-HM9-FCWT-MMAE were injected via the tail vein, and the drug efficacy was observed in the above steps. As shown in Figure 41, after a single administration of 3D7-HM9-FCWT-MMAE at 3 mg / kg, it showed excellent antitumor efficacy, leading to regression of most tumors.

[0226] Example 18 Isotope I 125 Detection of the entry of labeled antibodies into the brain First, H1975-luc cells were inoculated into the brain. Approximately 1-2 weeks after inoculation, the signal value of the luciferase substrate was detected to confirm that tumor cells had formed tumors in the brain. 3D7-FC1, 3D7-ABD, 5E11-ABD labeled with radioactive isotope I 125 and the corresponding IgG antibody Hu001 were injected into the mouse body via the tail vein. The final dose for each mouse was 1 mg / kg, and the radioactivity was 300 μCi. After administration, radiation detection was performed using a small animal SPECT / CT bioimaging system at 1 hour and 3 hours respectively, and the results were statistically analyzed. As shown in Figure 42, at 1 hour and 3 hours after administration, there were very obvious radioactive signals in the brains of mice for 3D7-FC1, 3D7-ABD, and 5E11-ABD. When the mice were observed 360° with imaging software, it was confirmed that the signal values were at the tumor inoculation site in the brain. On the other hand, the signal value of the corresponding Hu001 in the brain was low.

[0227] Example 19 In vivo antitumor effect of CD73 nanobody (intracranial model) In a set of experiments, human lung cancer cells H1975-luc were inoculated into the brain. On the 12th day, tumor fluorescence signal values were obtained by small animal in vivo imaging. According to the fluorescence signal values, on the 13th day, 5 mg / kg of hIgG1-MMAE and 3D7-FC1-MMAE were respectively administered via the tail vein (n = 9). Tumor fluorescence signal values were detected by in vivo imaging every 7 days and curves were plotted. As shown in Fig. 43, after a single intravenous administration of 5 mg / kg 3D7-FC1-MMAE, compared with hIgG1-MMAE, the growth of mouse lung cancer brain transplanted tumors was completely inhibited, and in some cases, the tumor signal values completely disappeared.

[0228] In another set of experiments, after H1975-luc was inoculated into the brain and in vivo imaged on the 14th day, grouping was performed. On the 15th day, 5 mg / kg of hIgG1-MMAE and 3D7-HM9-FCWT-MMAE were respectively administered via the tail vein (n = 5). Tumor fluorescence signal values were detected by in vivo imaging every 7 days and curves were plotted. As shown in Fig. 44, after a single intravenous administration of 5 mg / kg 3D7-HM9-FCWT-MMAE, compared with hIgG1-MMAE, the growth of brain transplanted tumors was completely inhibited in 4 / 5 mice. In 1 mouse, the tumor disappeared and then reappeared.

[0229] As described above, in the research of the examples regarding CD73-NDC, the following was revealed. 1. Both 3-D7-MMAE and 5-E11-MMAE, which are CD73-NDC, have good CD73-specific tumor cell killing activity, that is, they have a very strong inhibitory effect on the proliferation of CD73 medium / high expression tumor cells, but have no significant toxicity to the proliferation of CD73 low expression cells. 2. 3-D7-MMAE and 5-E11-MMAE, which are CD73-NDC, have no significant toxicity to the cell proliferation of normal human T cells. 3. 3-D7-MMAE and 5-E11-MMAE, which are CD73-NDCs, have no toxic effect on T cells and can effectively reverse the inhibitory effect of AMP / Ado on T cell proliferation. 4. In the intermittent dosing model, NDC shows a stronger antitumor killing effect than the conventional antibody-drug conjugate (ADC) of the same target. 5. The NDC of the present invention has extremely excellent in vivo antitumor activity and can completely inhibit tumor growth or regress tumors in many tumor models after administration by single intravenous injection. 6. The nanobody and NDC of the present invention can pass through the blood-brain barrier, have the property of distributing in the brain, and show excellent therapeutic effects on intracranial tumors. 7. The humanized antibody and NDC of the present invention similarly show many of the above advantages.

[0230] The sequence of the nanobody of the present invention SEQ ID NO: 1: 3-D7 CDR1 GFLLDYYV SEQ ID NO: 2: 3-D7 CDR2 ISGSDRST SEQ ID NO: 3: 3-D7 CDR3 AADPSPVTVQAMCLPRFPVPY SEQ ID NO: 4: 3-D7 VHQRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAADPSPVTVQAMCLPRFPVPYWGQGTQVTVST SEQ ID NO: 5: 5-E11 CDR1 GFTLDYYN SEQ ID NO: 6: 5-E11 CDR2 SSNSGGST SEQ ID NO: 7: 5-E11 CDR3 AAYRGWHPSLDARVYDY SEQ ID NO: 8: 5-E11 VH ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSNSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSS Sequence number 9: 4-D04 CDR1 GFTLDYYN Sequence number 10: 4-D04 CDR2 ISSSEGST Sequence number 11: 4-D04 CDR3 AADRYYYCSLHVSQYDY Sequence number 12: 4-D04 VH QLQLVESGGGLVQPGGSLRLSCAASGFTLDYYNIGWFRQAPGKEREGVSCISSSEGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADRYYYCSLHVSQYDYWGQGTLVTVSS Sequence number 13: 4-B02 CDR1 GFPLDYYS Sequence number 14: 4-B02 CDR2 IGSSDGST Sequence number 15: 4-B02 CDR3 AALKYYYCSGHEAIYDY Sequence number 16: 4-B02 VH EVQLVESGGGLVQPGGSLRLSCAASGFPLDYYSIGWFRQAPGKEREGVSCIGSSDGSTYYADSVKGRFTISRDNAENTVWLQMNSLKPEDTAVYYCAALKYYYCSGHEAIYDYWGQGTQVTVSG Sequence number 17: 1-E7 CDR1 GFTLDYYN Sequence number 18: 1-E7 CDR2 ISNSDGST Sequence number 19: 1-E7 CDR3 AAYRGWHPSLDARVYDY Sequence number 20: 1-E7 VH QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYNIGWFRQAPGKEREGVSCISNSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYRGWHPSLDARVYDYWGQGTQVTVSS Sequence number 21: 2-B10 CDR1 GFTLDDNA Sequence number 22: 2-B10 CDR2 MRTSDGDT Sequence number 23: 2-B10 CDR3 AAALGTYYSGFYYLAGDGMDY Sequence number 24: 2-B10 VH QLQLVESGGGLVQPGGSLRLSCAASGFTLDDNAIGWFRQAPGKEREGVACMRTSDGDTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAALGTYYSGFYYLAGDGMDYWGKGTLVTVSS Sequence number 25: 2-G9 CDR1 GFLLDYYT Sequence number 26: 2-G9 CDR2 ISSSDSSP Sequence number 27: 2-G9 CDR3 ALRANVYDY Sequence number 28: 2-G9 VH QLQLVESGGGLVQPGGSLRLSCAASGFLLDYYTIGWFRQAPGKELEGVSCISSSDSSPYDADSVKGRFTMSRDNANNTVYLQMNSLKPEDTAVYYCAAQRYYYCALRANVYDYWGQGTQVTVST Sequence number 29: 3-A3 CDR1 GFPLDYYT Sequence number 30: 3-A3 CDR2 ISSSDSSP Sequence number 31: 3-A3 CDR3 SLRANVYDY Sequence number 32: 3-A3 VH QVQLVESGGGSVQPGGSLRLSCAASGFPLDYYTIGWFRQAPGKELEGVSCISSSDSSPYDADSVKGRFTMSRDNANNTVYLQMNSLKPEDTAVYYCAAQRYYYCSLRANVYDYWGQGTQVTVSS Sequence number 33: 3-F1 CDR1 GFTLDYYT Sequence number 34: 3-F1 CDR2 IRSSDSSP Sequence number 35: 3-F1 CDR3 SLTANVYDY Sequence number 36: 3-F1 VH QVQLVESGVSLVQPGGSLRLSCAASGFTLDYYTIGWFRQAPGKELEGISCIRSSDSSPYDADSVKDRFTMSRDNANNTVYLQMNSLKPEDTADYYCAAQRYYYCSLTANVYDYWGQGTQVTVSS Sequence number 37: 3-D7-FC1 QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAADPSPVTVQAMCLPRFPVPYWGQGTQVTVSTEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 38: 3-D7-FC2 QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAADPSPVTVQAMCLPRFPVPYWGQGTQVTVSTGGGGSGGGGSGGGGSPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC Accession number 39: 3-D7-FC3 QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAADPSPVTVQAMCLPRFPVPYWGQGTQVTVSTEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC Accession number 40: 5E11-FC1 ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSNSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSSEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Accession number 41: 5E11-FC2 ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSNSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSSGGGGSGGGGSGGGGSPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC Sequence number 42: 5E11-FC3 ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSNSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSSEPKSSDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC Sequence number 43: 3-D7-HLE QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAADPSPVTVQAMCLPRFPVPYWGQGTQVTVSTGGGGSGGGGCGGGGSEVQLVESGGGVVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKERERVVSISRGGGYTYYADSVKGRFTISRDNSENTVYLQMNSLRPEDTALYYCAAARYWATGSEYEFDYWGQGTLVTVSSAHHHHHHC Sequence number 44: 3-D7-ABD QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAADPSPVTVQAMCLPRFPVPYWGQGTQVTVSTGGGGSGGGGCGGGGSQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPAHHHHHHC Sequence number 45: 5-E11-ABD ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSNSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSSGGGGSGGGGCGGGGSQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPAHHHHHHC Sequence number 46: 5-E11AS VH ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSASGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSS Accession number 47: 5-E11GS VH ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSGSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSS Accession number 48: 5-E11QS VH ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSCSSQSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSS Accession number 49: 5-E11-ASCS VH ALQLVESGGGLVQPGGSLRLSCEVSGFTLDYYNIGWFRQAPGKEREGVSSSSASGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCAAYRGWHPSLDARVYDYWGQGTEVTVSS Accession number 50: 3D7-HM7 VH QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNSKNTVYLQMGSLRAEDTAVYYCAADPSPVTVQAMCLPRFPVPYWGQGTLVTVSS Accession number 51: 3D7-HM8 VH QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNSKNTVYLQMSSLRAEDTAVYYCAADPSPVTVQAMCLPRFPVPYWGQGTLVTVSS Accession number 52: 3D7-HM9 VH QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAADPSPVTVQAMCLPRFPVPYWGKGTLVTVSS SEQ ID NO: 53: 3D7-HM9-ABD QRQLVESGGGLVQPGGSLRLSCAASGFLLDYYVIGWFRQAPGKEREGVSCISGSDRSTYYADSVKGRFTISRDNAKNTVYLQMSSLRAEDTAVYYCAADPSPVTVQAMCLPRFPVPYWGKGTLVTVSSGGGGSGGGGSGGGGSQHDEAVDANSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPAAACAAAHHHHHH SEQ ID NO: 545-E11 AS CDR2 or 5-E11 ASCS CDR2: SSASGGST SEQ ID NO: 555-E11 GS CDR2 SSGSGGST SEQ ID NO: 565-E11 QS CDR2 SSQSGGST

[0231] All documents related to the present invention are cited herein as references as if each were cited separately. After reading the above content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and it should be understood that those equivalent forms are included in the claims of the present invention.

Claims

1. A nanobody targeting CD73, wherein the complementarity-determining region CDR of the VHH chain of the nanobody is one or more selected from the following group: (1) CDR1 represented by SEQ ID NO: 1, CDR2 represented by SEQ ID NO: 2, and CDR3 represented by SEQ ID NO: 3; Or, (2) CDR1 represented by SEQ ID NO: 5, CDR2 represented by SEQ ID NO: 6, and CDR3 represented by SEQ ID NO: 7; Or, (3) CDR1 represented by SEQ ID NO: 9, CDR2 represented by SEQ ID NO: 10, and CDR3 represented by SEQ ID NO: 11, Or, (4) CDR1 represented by SEQ ID NO: 13, CDR2 represented by SEQ ID NO: 14, and CDR3 represented by SEQ ID NO: 15; Or, (5) CDR1 represented by SEQ ID NO: 17, CDR2 represented by SEQ ID NO: 18, and CDR3 represented by SEQ ID NO: 19, Or, (6) CDR1 represented by SEQ ID NO: 21, CDR2 represented by SEQ ID NO: 22, and CDR3 represented by SEQ ID NO: 23; Or, (7) CDR1 represented by SEQ ID NO: 25, CDR2 represented by SEQ ID NO: 26, and CDR3 represented by SEQ ID NO: 27; Or, (8) CDR1 represented by SEQ ID NO: 29, CDR2 represented by SEQ ID NO: 30, and CDR3 represented by SEQ ID NO: 31; Or, (9) CDR1 represented by SEQ ID NO: 33, CDR2 represented by SEQ ID NO: 34, and CDR3 represented by SEQ ID NO: 35; Or (10) CDR1 represented by SEQ ID NO: 5, CDR2 represented by SEQ ID NO: 54, and CDR3 represented by SEQ ID NO: 7; Or (11) CDR1 represented by SEQ ID NO: 5, CDR2 represented by SEQ ID NO: 55, and CDR3 represented by SEQ ID NO: 7; Or (12) CDR1 represented by SEQ ID NO: 5, CDR2 represented by SEQ ID NO: 56, and CDR3 represented by SEQ ID NO:

7.

2. The nanobody according to claim 1, wherein the VHH chain of the nanobody further comprises a framework region (FR).

3. An antibody targeting CD73, comprising the VHH chain of a nanobody targeting CD73 according to one or more of claims 2.

4. A multispecific antibody comprising the nanobody targeting CD73 according to claim 1 or the antibody targeting CD73 according to claim 3.

5. A recombinant protein, characterized by having the following: (i) A nanobody targeting CD73 according to claim 1, or an antibody targeting CD73 according to claim 3; and (ii) A polypeptide molecule or fragment having any therapeutic function; and / or (iii) A functional region that improves the physical and chemical properties or drug-likeness of any protein.

6. The recombinant protein according to claim 5, characterized by having the following elements from the N-terminus to the C-terminus. A - B (However, the A element is a nanobody targeting CD73, and the B element is an Fc segment, an albumin-binding domain (ABD), or an anti-albumin nanobody (HLE). "-" represents a peptide bond or a linker.)

7. The recombinant protein according to claim 5, characterized in that the VHH chain of the nanobody targeting CD73 is selected from the group consisting of amino acid sequences represented by SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, or SEQ ID NO:

49.

8. A CAR construct, characterized in that the antigen-binding region is the VHH chain of the nanobody according to claim 1.

9. A recombinant immune cell, characterized by expressing the exogenous CAR construct according to claim 8.

10. An immune complex, characterized by containing the following: (a) An antibody moiety, which is a nanobody targeting CD73 according to claim 1 or an antibody targeting CD73 according to claim 3; and (b) A complex moiety complexed with the nanobody moiety, selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, an enzyme, a proteolytic agent, an oligonucleotide, or a combination thereof.

11. The proteolytic agent is a degrader of tumor-related proteins, and the tumor-related proteins are selected from the group consisting of EGFR, NF-κB, RIPK2, BCR-ABL, HER2, c-Met, TBK1, CDK, ALK, Akt, CK2, ERK1 / 2, FLT3, PI3K, BTK, TRK, Fak, BRD, AR, ER, MetAp-2, BCL-XL, Sirt2, HDAC6, Pirin, SMAD3, ARNT, PCAF / GCN5, Tau, EZH2, IRAK4, STAT3FRS2, RAS (such as KRAS, HRAS, and NRAS). The immunocomplex according to claim 10, characterized in that it is so selected.

12. The immunocomplex according to claim 10, characterized in that it is an antibody-drug conjugate ADC represented by the following molecular formula. 【Chemical Formula 15】 (However, nAb is a nanobody targeting CD73. LU is a linker (also called a linking group). D is a drug. And the subscript p is a value selected from 1-8.)

13. The immunocomplex according to claim 10, characterized in that LU is one or more linking groups selected from those in which maleimidocaproyl (MC), maleimide (MAL), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC) linkers are linked to the antibody part, and valine-citrulline (VC), valine-alanine (VA), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine (AAA), p-aminobenzyloxycarbonyl (PAB), polyethylene glycol (PEG) are included.

14. The immunocomplex according to claim 10, characterized in that D is a compound having antitumor activity selected from the following groups: (i) Tubulin inhibitors, such as maytansine derivatives (DM1, DM4), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF); (ii) Toxins acting on DNA, such as duocarmycin, pyrrolobenzodiazepine (PBD); (iii) Topoisomerase inhibitors, such as camptothecin, SN38, etoposide, Dxd.

15. A pharmaceutical composition, characterized by containing the following: (i)The nanobody targeting CD73 according to claim 1, the antibody targeting CD73 according to claim 3, the multispecific antibody according to claim 4, the recombinant protein according to any one of claims 5 to 7, the recombinant immune cell according to claim 9 or the immune complex according to any one of claims 10 to 14; and (ii)A pharmaceutically acceptable carrier.

16. Use of an active ingredient, wherein the active ingredient is selected from the group consisting of the nanobody targeting CD73 according to claim 1, the antibody targeting CD73 according to claim 3, the multispecific antibody according to claim 4, the recombinant protein according to any one of claims 5 to 7, the recombinant immune cell according to claim 9 or the immune complex according to any one of claims 10 to 14, or a combination thereof, for (a) the manufacture of a detection reagent, detection plate or kit, and / or (b) the manufacture of a drug for preventing and / or treating CD73-related diseases.

17. A method for detecting CD73 in a sample in vitro (including diagnostic or non-diagnostic ones), characterized by comprising the following steps: (1)In vitro, contacting the sample with the nanobody targeting CD73 according to claim 1, the antibody targeting CD73 according to claim 3 or the immune complex according to any one of claims 10 to 14; (2)Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex means the presence of CD73 in the sample.

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