Nuclide-labeled Trop2 single-domain antibody

Abstract

The invention relates to the field of biology, in particular to a nuclide labeled anti-Trop2 single-domain antibody, which has good targeting property, high uptake in tumor tissues, long tumor residence time and higher tumor / normal tissue ratio, and is expected to be used for early diagnosis, risk stratification and prognosis evaluation or treatment of Trop2 expression positive tumors.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to a prior application with patent application number 2023114971043 filed with the State Intellectual Property Office of China on November 10, 2022, entitled “Nuclear-labeled anti-Trop2 single-domain antibody”. The entire text of the prior application is incorporated herein by reference. Technical Field

[0003] The present invention relates to the biological field, in particular to a radionuclide-labeled anti-Trop2 single-domain antibody. Background Art

[0004] Trop2 is a cell surface glycoprotein encoded and expressed by the TACSTD2 (Tumor-associated calcium signal transducer 2) gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), gastrointestinal tumor-associated antigen (GA733-1), surface marker 1 (M1S1). It is mainly lowly expressed in epithelial cells in normal tissues, but is overexpressed in a variety of epithelial malignant tumor cell tissues, such as urothelial carcinoma, cervical cancer, triple-negative breast cancer, lung cancer, papillary thyroid carcinoma, endometrial cancer, prostate cancer, colorectal cancer, etc. Trop2 protein is a 36kDa single transmembrane protein composed of 323 amino acids, which is structurally divided into a hydrophobic signal peptide (1-26), an extracellular domain (27-274), a transmembrane region (275-297) and an intracellular tail (298-323). Trop2 protein belongs to the GA733 protein family and has a high structural sequence similarity with epithelial cell adhesion molecule (EpCAM, also known as Trop1, TACSTD1), with a sequence identity of 49%. In addition, Trop2 can cause strong endocytosis after binding to antibodies, and cell endocytosis may increase the tumor killing effect. Because the mechanism of action of Trop2 is still unclear, and it is highly expressed in a variety of tumor tissues, constitutively endocytic, and transferred to lysosomes, Trop2 has become a hot target after Her2.

[0005] The present invention performs radionuclide labeling on the VHH-Fc Trop2 heavy chain antibody obtained by the previous screening, and tests its tumor imaging and biodistribution effects, thereby completing the present invention. Summary of the invention

[0006] The present invention first provides a radionuclide-labeled anti-Trop2 single domain antibody, wherein the complementary determining region CDR of the anti-Trop2 single domain antibody includes CDR1 to CDR3 with the amino acid sequences shown below: CDR1 with an amino acid sequence as shown in one of SEQ ID NOs. 1 to 7, CDR2 with an amino acid sequence as shown in one of SEQ ID NOs. 8 to 14, and CDR3 with an amino acid sequence as shown in one of SEQ ID NOs. 15 to 21.

[0007] In some embodiments of the present invention, the complementary determining region CDR of the anti-Trop2 single domain antibody includes CDR1 to CDR3 with the amino acid sequences shown below:

[0008] (1) the amino acid sequence of CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of CDR2 is as shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.15; or

[0009] (2) the amino acid sequence of CDR1 is as shown in SEQ ID NO.2, the amino acid sequence of CDR2 is as shown in SEQ ID NO.9, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.16; or

[0010] (3) the amino acid sequence of CDR1 is as shown in SEQ ID NO.3, the amino acid sequence of CDR2 is as shown in SEQ ID NO.10, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.17; or

[0011] (4) the amino acid sequence of CDR1 is as shown in SEQ ID NO.4, the amino acid sequence of CDR2 is as shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.18; or

[0012] (5) the amino acid sequence of CDR1 is as shown in SEQ ID NO.5, the amino acid sequence of CDR2 is as shown in SEQ ID NO.12, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.19; or

[0013] (6) the amino acid sequence of CDR1 is as shown in SEQ ID NO.6, the amino acid sequence of CDR2 is as shown in SEQ ID NO.13, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.20; or

[0014] (7) The amino acid sequence of CDR1 is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.14, and the amino acid sequence of CDR3 is shown in SEQ ID NO.21.

[0015] The anti-Trop2 single domain antibody provided by the present invention may include a framework region FR, wherein the framework region FR includes FR1 to FR4 with the amino acid sequences shown below:

[0016] (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.22, the amino acid sequence of FR2 is as shown in SEQ ID NO.23, the amino acid sequence of FR3 is as shown in SEQ ID NO.24, and the amino acid sequence of FR4 is as shown in SEQ ID NO.25; or

[0017] (2) the amino acid sequence of FR1 is as shown in SEQ ID NO.26, the amino acid sequence of FR2 is as shown in SEQ ID NO.27, the amino acid sequence of FR3 is as shown in SEQ ID NO.28, and the amino acid sequence of FR4 is as shown in SEQ ID NO.29; or

[0018] (3) an amino acid sequence of FR1 as shown in SEQ ID NO.30, an amino acid sequence of FR2 as shown in SEQ ID NO.31, an amino acid sequence of FR3 as shown in SEQ ID NO.32, or an amino acid sequence of FR4 as shown in SEQ ID NO.33; or

[0019] (4) an amino acid sequence of FR1 as shown in SEQ ID NO.34, an amino acid sequence of FR2 as shown in SEQ ID NO.35, an amino acid sequence of FR3 as shown in SEQ ID NO.36, or an amino acid sequence of FR4 as shown in SEQ ID NO.25; or

[0020] (5) an amino acid sequence of FR1 as shown in SEQ ID NO.37, an amino acid sequence of FR2 as shown in SEQ ID NO.38, an amino acid sequence of FR3 as shown in SEQ ID NO.39, or an amino acid sequence of FR4 as shown in SEQ ID NO.40; or

[0021] (6) an amino acid sequence of FR1 as shown in SEQ ID NO.30, an amino acid sequence of FR2 as shown in SEQ ID NO.41, an amino acid sequence of FR3 as shown in SEQ ID NO.42, and an amino acid sequence of FR4 as shown in SEQ ID NO.43; or

[0022] (7) FR1 with an amino acid sequence as shown in SEQ ID NO.44, FR2 with an amino acid sequence as shown in SEQ ID NO.45, FR3 with an amino acid sequence as shown in SEQ ID NO.46, and FR4 with an amino acid sequence as shown in SEQ ID NO.25.

[0023] In a specific embodiment of the present invention, the amino acid sequence of the single domain antibody may include: a) an amino acid sequence as shown in one of SEQ ID NOs. 47 to 53, preferably as shown in SEQ ID NOs. 48-49; or, b) an amino acid sequence having a sequence identity of more than 80% with one of SEQ ID NOs. 47 to 53, and having the function of the amino acid sequence defined in a); specifically, the amino acid sequence in b) specifically refers to: an amino acid sequence as shown in one of SEQ ID NOs. 47 to 53 obtained by substitution, deletion or addition of one or more (specifically 1-20, 1-10, 1-5, or 1-3) amino acids, or an amino acid sequence as shown in one of SEQ ID NOs. 47 to 53 obtained by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and having the function of a polypeptide fragment as shown in one of SEQ ID NOs. 47 to 53, for example, the specific binding ability to Trop2. The amino acid sequence in b) may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more identity with one of SEQ ID NO. 47 to 53. The anti-Trop2 single domain antibody provided by the present invention can specifically bind to cells expressing Trop2. The above-mentioned "substitution, deletion or addition" is outside the CDR region, and the calculation of sequence identity does not include the CDR region.

[0024] In some embodiments of the present invention, the anti-Trop2 single domain antibody is a humanized antibody. Preferably, the amino acid sequence of the humanized anti-Trop2 single domain antibody is shown in one of SEQ ID NOs. 54 to 65; most preferably, SEQ ID NO. 57 or 59.

[0025] In some embodiments of the present invention, the anti-Trop2 single domain antibody further comprises a second domain for prolonging the in vivo half-life and / or having a binding effect on effector cells.

[0026] In some embodiments of the present invention, the second domain comprises a combination of one or more of a serum albumin fragment, a polyethylene glycol fragment, and a single domain antibody that binds to serum albumin.

[0027] In some embodiments of the present invention, the second domain comprises an immunoglobulin Fc region, preferably selected from a human immunoglobulin Fc region.

[0028] In some embodiments of the present invention, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is selected from a combination of one or more of IgG1, IgG2, IgG3, or IgG4 subtypes.

[0029] In some embodiments of the present invention, the immunoglobulin Fc region is the Fc of human natural IgG1, and its amino acid sequence is shown in SEQ ID NO.66.

[0030] In some embodiments of the present invention, the anti-Trop2 single domain antibody comprising the second domain is as shown in SEQ ID NOs. 67-85; further preferably as shown in SEQ ID NOs. 68, 69, 76-79.

[0031] In some embodiments of the present invention, the anti-Trop2 single domain antibody further comprises a second domain for recognizing and binding to another tumor or cancer marker;

[0032] Preferably, the second domain is a single-chain antibody. Preferably, the other tumor or cancer marker is ER2, CD20, PTK7, HSG or PSMA.

[0033] In a specific embodiment of the present invention, the structure of the radionuclide-labeled antibody is formula (I):

[0034] VHH-R1-L-R2-N(I), where:

[0035] VHH is an anti-Trop2 single-domain antibody;

[0036] R1 is the linking group between L and the antibody;

[0037] L is the linker arm or does not exist;

[0038] R2 is a connecting group or a direct bond between N and L;

[0039] N is a radionuclide labeled group or nanoaggregate;

[0040] Wherein R1-L-R2-N can be one or more, which means that each VHH antibody can be coupled with one or more R1-L-R2-N, and when the same antibody is coupled with multiple R1-L-R2-N, they can be the same or different. In a specific embodiment of the present invention, R1 is an amide bond, which is modified on one or more lysine side chain amino groups or N-terminal amino groups of VHH.

[0041] In a specific embodiment of the present invention, R1 is preferably a sulfhydryl reactive group, an amino reactive group, a carboxyl reactive group, a dithiol bridging group, etc.; for antibodies introduced with non-natural amino acids, it can also be selected from click chemistry reactive groups such as ketones, hydrazines or hydrazides, azides, alkynes, cyclopropenes or dienes; the antibody connection site includes any applicable amino acid residue or N297 sugar chain coupling of the CH2 domain, such as fucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (SA) introduced by sugar engineering; the connection reaction includes a chemical reaction or an enzymatic reaction, such as using transglutaminase (MTGase) to transfer the amine-containing linker L or the radionuclide-labeled group or nanoaggregate N to the deglycosylated antibody.

[0042] Preferably, R1 is an amino reactive group, such as an amide bond (formed by the reaction of an NHS active ester with a lysine side chain amino group or an N-terminal amino group).

[0043] In a specific embodiment of the present invention, R2 is a direct bond, and L is a part of N or does not exist. A person skilled in the art may encounter such a situation when labeling an antibody using a commercially available chelating agent. For example, when labeling an antibody using a chelating agent Hynic-NHS (chemical structure a) or DOTA-NHS (chemical structure b), R2 is a direct bond, L does not exist, and R1 is an amide bond.

[0044]

[0045]

[0046] For another example, when the antibody is labeled with the chelating agent NOTA-C6-NHS, R2 is a direct bond, L is a C6 alkyl group, and R1 is an amide bond.

[0047] In a specific embodiment of the present invention, the terms "radionuclide" and "nuclide" have the same meaning, and the terms "nuclide labeling" and "radioactive labeling" have the same meaning, which means that the antibody is modified by a radioactive isotope in a covalent or non-covalent manner. The radionuclide labeling can be a radionuclide labeled compound or nanoaggregate, preferably a chelating agent for chelating a radionuclide, and the radionuclide is selected from non-metallic isotopes including 11 C. 14 C. 13 N. 18 F. 131 I. 123 I. 124 I and 125 I; or selected from metal isotopes including: 86 Y. 90 Y. 211 At 153 Sm,186 Re, 188 Re, 67 Cu, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 111 In, 62 Cu, 64 Cu, 89 Zr, 177 Lu, 99m Tc, 67 Ga, 68 Ga and 153 Gd; the above radioactive isotopes are used for gamma-ray imaging or positron emission imaging, preferably for PET / CT or SPETCT imaging, or for radiotherapy of tumors.

[0048] Preferably, in the case of heavy metal ions or radionuclide ions, a chelating agent or a multidentate ligand is used for complexation to form a chelate (especially with a radioactive metal isotope), and optionally contains a co-ligand (such as tricine and TPPTS). Non-limiting examples of such chelating agents are hydrazinonicotinic acid HYNIC (also known as: hydrazinonicotinamide); 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 2,2',2"-(1,4,7-triazacyclononane-1,4,7-triyl) triacetic acid (NOTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA), 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (H P-DO3A); 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine (AAZTA) and its derivatives; preferred chelating agents include one or more of HYNIC, EDTA, HP-DO3A, DTPA, DOTA, NOTA, TRAP, NODAGA, HBED-CC, DOTAGA, DOTA(GA)2 and their derivatives; when the radioactive isotope is a non-metallic ion, it can be chelated by forming a complex with a metal ion such as aluminum, for example, Al 18 F.

[0049] Preferably, the chelating agent is hydrazinonicotinic acid HYNIC, and the radioactive isotope is 99m Tc, the cooperative ligands are trihydroxymethylglycine (tricine) and triphenylphosphine trisulfonate (TPPTS).

[0050] In certain embodiments, L has about 1-100 linking atoms and optionally includes vinyloxy ((CH2CH2O)) moieties, amine, ester, amide, ketone, urea, carbamate, and carbonate functional groups;

[0051] Preferably, L is a straight or branched chain alkyl group of 2 to 20 carbon atoms;

[0052] Preferably, L is a polyethylene glycol group of 2 to 10 ethyleneoxy ((CH2CH2O)) units;

[0053] Preferably, L is absent.

[0054] In a preferred embodiment of the present invention, R2 is a direct bond, L is absent, R1 is an amide bond or a thioether bond (such as formed by the reaction of maleimide and a sulfhydryl group), and the chelating agent is selected from one or more of HYNIC, EDTA, HP-DO3A, DTPA, DOTA, NOTA, TRAP, NODAGA, HBED-CC, DOTAGA or DOTA(GA)2.

[0055] The second aspect of the present invention provides a tumor imaging agent or therapeutic agent comprising the above-mentioned radionuclide-labeled anti-Trop2 single-domain antibody as an active ingredient.

[0056] The present invention also provides a method for preparing the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody, comprising the following steps:

[0057] 1) Modification of anti-Trop2 single domain antibodies using chelators;

[0058] 2) Chelated nuclides.

[0059] The present invention also provides a synthetic kit for preparing the above-mentioned radionuclide-labeled anti-Trop2 single-domain antibody, comprising:

[0060] 1) anti-Trop2 single domain antibodies, chelators and radionuclide reagents; or

[0061] 2) Chelator-modified anti-Trop2 single domain antibodies and nuclide reagents.

[0062] The present invention also provides a stable pharmaceutical composition comprising:

[0063] (1) the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody and (2) an anti-radiation degradation stabilizer such as ascorbic acid and / or its salt; preferably, the composition is in the form of an injection.

[0064] The present invention also provides a drug combination or a drug administration combination, comprising:

[0065] (1) the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody, or the above-mentioned imaging agent or the above-mentioned pharmaceutical composition; and (2) another imaging agent or diagnostic agent such as a NIR contrast agent, or a component or agent having cancer therapeutic activity.

[0066] The present invention also provides uses of the radionuclide-labeled anti-Trop2 single domain antibody, the tumor imaging agent or therapeutic agent, the pharmaceutical composition, the drug combination or the drug administration combination, including:

[0067] 1) Used for preparing tumor detection or treatment reagents or kits;

[0068] 2) Used for preparing tumor diagnosis or treatment drugs or compositions;

[0069] 3) Used to prepare molecular probes targeting tumor tissues or cells.

[0070] The tumor tissue or cells overexpress Trop2 antigen (Trop2-expressing cell-associated tumor).

[0071] The above reagents or kits, drugs or compositions or molecular probes are expected to be used for diagnosing, treating or preventing tumors associated with cells expressing Trop2.

[0072] The tumor associated with the cells expressing Trop2 is selected from one or more combinations of urothelial carcinoma, cervical cancer, triple-negative breast cancer, gastric cancer, pancreatic cancer, lung cancer, papillary thyroid carcinoma, endometrial cancer, prostate cancer, colorectal cancer, and head and neck squamous cell carcinoma.

[0073] The present invention also provides a chelator-modified anti-Trop2 single domain antibody, wherein the anti-Trop2 single domain antibody and the chelator are as described in the first aspect of the present invention;

[0074] Preferably, the chelator-modified anti-Trop2 single domain antibody is as shown in (Formula II):

[0075] VHH-R1-L-R2-Ch(II), wherein:

[0076] VHH is an anti-Trop2 single-domain antibody;

[0077] R1 is the linking group between L and the antibody;

[0078] L is the linker arm or does not exist;

[0079] R2 is a connecting group or a direct bond between Ch and L;

[0080] Ch is a chelating agent;

[0081] R1-L-R2-Ch can be one or more; when the same antibody is coupled with multiple R1-L-R2-Ch, they can be the same or different;

[0082] In a specific embodiment of the present invention, VHH, R1, L or R2 are as described in the first aspect of the present invention;

[0083] In a specific embodiment of the present invention, the chelating agent is as described in the first aspect of the present invention.

[0084] Beneficial technical effects

[0085] The radionuclide-labeled anti-Trop2 single-domain antibody provided by the present invention has excellent targeting, high uptake in tumor tissue, long tumor retention time, and a high tumor / normal tissue ratio (i.e., target / non-target ratio), and is expected to be used for early diagnosis, treatment, risk stratification or prognosis evaluation of Trop2-expressing positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 WB verified the expression of TROP2 in cell lines (upper: ACTIN; lower: TROP2);

[0087] Figure 2 99m Determination of labeling rate of Tc-labeled Hynic-C10;

[0088] Figure 3 99m Radiochemical purity determination of Tc-labeled Hynic-C10;

[0089] Figure 4 177 Determination of labeling rate of Lu-labeled DOTA-B9;

[0090] Figure 5 177 Radiochemical purity determination of Lu-labeled DOTA-B9;

[0091] Figure 6 99m Tc-labeled antibody biodistribution results;

[0092] Figure 7 99m Representative MIP images of Tc-labeled antibodies (a) and temporal changes of tumor uptake (b);

[0093] Figure 8 177 Lu-labeled antibody imaging results;

[0094] Fig. 9 177 Lu-labeled antibody biodistribution. DETAILED DESCRIPTION

[0095] The present inventors completed the present invention by performing nuclide modification on the basis of the single domain antibody specifically binding to Trop2 antigen discovered in the early stage.

[0096] The terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to the variable region of the heavy chain of an antibody, which is a single domain antibody (VHH) consisting of only one heavy chain variable region, and is the smallest antigen-binding fragment with complete functions. Usually, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained from alpaca immune serum, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region. A single domain antibody (VHH) is composed of four "framework regions" respectively referred to in the art and hereinafter as "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", wherein the framework region is separated by three "complementarity determining regions" or "CDRs" respectively referred to in the art and hereinafter as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". Therefore, the general structure or sequence of a single domain antibody (VHH) can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody (VHH) has an antigen binding site, which confers specificity to an antigen.

[0097] The terms "single domain antibody", "heavy chain single domain antibody", "VHH domain", "VHH", "VHH antibody fragment", "VHH antibody" and "nanobody" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably.

[0098] The term "single-chain antibody (scFv)" generally refers to an antibody formed by connecting the heavy chain variable region and the light chain variable region of an antibody via a connecting peptide. In order, the C-terminus of the heavy chain variable region can be connected to the N-terminus of the light chain variable region via a connecting peptide, or the C-terminus of the light chain variable region can be connected to the N-terminus of the heavy chain variable region via a connecting peptide. The connecting peptide is preferably selected from a flexible polypeptide chain composed of alanine and / or serine and / or glycine, and the length of the connecting peptide can be 3 to 40 amino acids.

[0099] The term "IMGT numbering system" is an integrated information system specifically for immunoglobulins (IG), T cell receptors (TCR) and major histocompatibility complex (MHC) in humans and other vertebrates, namely THE INTERNATIONAL IMMUNOGENETICS INFORMATION (Lafranc et al., 2003, Dev. Comp. Immunol. 27(1):55-77). Log in to IMGT (http: / / www.imgt.org / IMGT_vquest) and analyze the antibody light and heavy chain genes to determine the framework regions (FR) and complementarity determining regions (CDR) of the variable region. The "position" of CDR within the structure of the immunoglobulin variable domain is conserved between species and exists in a structure called a loop, so it is easy to identify CDR and framework residues by using a numbering system that aligns the variable domain sequence according to structural features. This information can be used to transplant and replace CDR residues from an immunoglobulin from one species into an acceptor framework that is usually from a human antibody. Unless otherwise indicated, in this specification, claims and drawings, anti-Trop2 single domain antibodies are numbered according to the IMGT numbering method to determine CDR regions and FR regions.

[0100] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance, and immunofluorescence.

[0101] Herein, the term "humanization" refers to a molecule having an antigen binding site that is substantially derived from an immunoglobulin of a non-human species, wherein the rest of the immunoglobulin structure of the molecule is based on the structure and / or sequence of a human immunoglobulin. The antigen binding site may comprise a complete variable domain fused to a constant domain, or may comprise only a complementarity determining region (CDR) transplanted to an appropriate framework region in a variable domain. The antigen binding site may be wild type, or modified by one or more amino acid substitutions, such as modification to be closer to a human immunoglobulin. Certain forms of humanized antibodies retain all CDR sequences (e.g., humanized single domain antibodies containing all three CDRs from alpacas). Other forms have one or more CDRs that have been altered relative to the original antibody.

[0102] The term "effector function" when referring to an antibody refers to those biological activities attributable to the Fc region of the antibody and which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0103] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins (or antibodies in general) bound to certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) that have Fc receptors (FcRs) enable these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. NK cells, as the main cells mediating the ADCC effect, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In order to evaluate the ADCC activity of the target molecule, an in vitro ADCC activity assay can be performed (see, e.g., U.S. Pat. Nos. 5,500,362 and 5,821,337). Effector cells suitable for the assay include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0104] The term "complement-dependent cytotoxicity" or "CDC" is another method of cell killing mediated by antibodies. For complement activation, IgM is the most effective isotype. IgG1 and IgG3 are also very effective in mediating CDC through the classical complement activation pathway. Preferably, in this cascade, the formation of an antigen-antibody complex results in the exposure of multiple C1q binding sites (C1q is one of the three subcomponents of complement C1) that are adjacent to the CH2 domain of the participating antibody molecule (e.g., IgG molecule). Preferably, the exposed C1q binding site converts the previously low-affinity C1q-IgG interaction into a high-affinity interaction, which triggers a cascade involving a series of other complement proteins and causes the proteolytic release of effector cell chemotactic / activators C3a and C5a. Preferably, the complement cascade ultimately forms a membrane attack complex, which creates holes in the cell membrane, facilitating the free passage of water and solutes into and out of the cell.

[0105] The term "T cell-dependent cytotoxicity (T cell-dependent cell-mediated cytotoxicity)" or "TDCC activity" is also called T cell-mediated tumor cell killing. The main participants are CD8+T cells. Anti-Trop2 / CD3 bispecific antibodies cross-link with target cells and T cells expressing Trop2 antigens to produce the TDCC effect and thus kill target cells.

[0106] In the present invention, the term "endocytosis" or "endocytosis" or "endocytosis ability" means that after the Anti-Trop2 antibody binds to the Trop2 antigen on the cell surface, it enters the tumor cell through Trop2 antigen-mediated endocytosis and is engulfed by the intracellular lysosome. The endocytosis of Trop2 has been confirmed to be clathrin-mediated endocytosis (CME).

[0107] "Sequence identity" or "sequence identity" between different polypeptide sequences refers to the percentage of identical amino acids between the sequences. "Sequence similarity" refers to the percentage of identical or conservative amino acids in different sequences. Methods for evaluating sequence identity or sequence similarity between amino acid sequences are known to those skilled in the art. For example, the BLAST program of the NCBI database can be used to determine identity.

[0108] The term "antigen" is a target to which an antibody can selectively bind. The target antigen can be a polypeptide, protein, nucleic acid, cell, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments herein, the target antigen is a cell expressing Trop2, more preferably, a portion of a Trop2 molecule.

[0109] The present invention first provides a radionuclide-labeled anti-Trop2 single-domain antibody:

[0110] VHH-R1-L-R2-N(I)

[0111] Wherein VHH is the anti-Trop2 single domain antibody provided by the present invention;

[0112] R1 is the linking group between L and the antibody;

[0113] L is the linker arm or does not exist;

[0114] R2 is a connector or direct bond between N and L; R2 is a direct bond, which means that L is a part of N or does not exist;

[0115] N is a radionuclide labeled group or nanoaggregate.

[0116] Wherein R1-L-R2-N can be one or more, which means that each VHH antibody can be coupled with one or more R1-L-R2-N, and when the same antibody is coupled with multiple R1-L-R2-N, they can be the same or different. In a specific embodiment of the present invention, R1 is an amide bond, which is modified on one or more lysine side chain amino groups or N-terminal amino groups of VHH.

[0117] In a specific embodiment of the present invention, R1 is preferably a sulfhydryl reactive group, an amino reactive group, a carboxyl reactive group, a dithiol bridging group, etc.; for antibodies introduced with non-natural amino acids, it can also be selected from click chemistry reactive groups such as ketones, hydrazines or hydrazides, azides, alkynes, cyclopropenes or dienes; the antibody connection site includes any applicable amino acid residue or N297 sugar chain coupling of the CH2 domain, such as fucose, galactose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acid (SA) introduced by sugar engineering; the connection reaction includes a chemical reaction or an enzymatic reaction, such as using transglutaminase (MTGase) to transfer the amine-containing linker L or the radionuclide-labeled group or nanoaggregate N to the deglycosylated antibody.

[0118] Preferably R1 is an amino reactive group, such as NHS active ester, which reacts with the side chain amino group of lysine to form an amide bond.

[0119] In a specific embodiment of the present invention, the term "nuclide labeling" or "radioactive labeling" has the same meaning, which is to modify the antibody with a radioactive isotope in a covalent or non-covalent manner. The radioactive isotope can be a radioactive isotope labeled compound or nanoaggregate, preferably a chelating agent that chelates different metal ions. The radioactive isotope is selected from non-metallic isotopes including 11 C. 14 C. 13 N. 18 F. 131 I. 123 I. 124 I and 125 I; or selected from 86 Y. 90 Y. 211 At 153 Sm, 186 Re, 188 Re, 67 Cu, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 111 In, 62 Cu, 64 Cu, 89 Zr, 177 Lu, 99m Tc, 67 Ga, 68 Ga and 153 Gd; preferably 177 Lu or 99m Tc; the above radioactive isotopes are used for gamma-ray imaging or positron emission imaging, preferably for PET / CT or SPETCT imaging.

[0120] Preferably, the complexation is carried out using a chelating agent or a polydentate ligand to form a chelate (especially with a radioactive metal), and optionally includes co-ligands (such as tricine and TPPTS). Non-limiting examples of such chelating agents are hydrazinonicotinic acid HYNIC; 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 2,2',2"-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA), 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A); 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); methyl)]amino-6-methylperhydro-1,4-diazepine (AAZTA) and its derivatives; preferred chelating agents include one or more of HYNIC, EDTA, HP-DO3A, DTPA, DOTA, NOTA, TRAP, NODAGA, HBED-CC, DOTAGA, DOTA(GA)2 and their derivatives (see CN114364405A); when the radioactive isotope is a non-metallic ion, it can form a complex with a metal ion such as aluminum to achieve chelation, and the representative structure is reported in CN102066974B, and the typical structure is 18 F-Al-NOTA.

[0121] In the present invention, linker or spacer has the same meaning, which is any divalent chemical group; a representative linker can have about 1 to about 100 linking atoms, and any ethyleneoxy moiety, amine, ester, amide, ketone, urea, carbamate and carbonate functional groups. Other linkers used in the method of the present invention can have about 1 to about 50 linking atoms, or about 1 to about 10 linking atoms, or about 5 to about 10 linking atoms.

[0122] Preferably, L is a straight or branched chain alkyl group of 2 to 20 carbon atoms;

[0123] Preferably, L is a polyethylene glycol group having 2 to 10 ethyleneoxy ((CH2CH2O)) units.

[0124] Preferably, L is absent to reduce the effect of the nuclide on the antibody affinity.

[0125] The present invention also uses the method of nuclide-chelating group-anti-Trop2 single domain antibody or anti-Trop2 single domain antibody-chelating group-nuclide to describe the above-mentioned nuclide-labeled antibody, wherein the anti-Trop2 single domain antibody is any of the antibodies described above in the present invention, the chelating group is any of the chelating groups described above in the present invention, and the nuclide is any of the isotope nuclides described above in the present invention. For example, VHH-DOTA- 177 The meaning of Lu is 177 Lu modifies the anti-Trop2 single domain antibody of the present invention by DOTA chelation; 99m The meaning of Tc-Hynic-B9 is 99m Tc modified the B9 antibody (aTrop2-1B9huV3-FC) by Hynic chelation. The above statement only indicates that the modified antibody contains the above three components, and does not exclude that the labeled antibody also includes other chemical components or modifications, such as connecting arms, connecting groups and co-ligands, nor does it limit the number of modified antibody nuclides. Preferred labeled antibodies of the present invention include:

[0126] VHH-DOTA-nuclide, VHH-NOTA-nuclide, VHH-DTPA-nuclide, VHH-Hynic-nuclide, VHH-EDTA-nuclide;

[0127] or

[0128] VHH-chelating group- 68 Ga, VHH-chelate group- 177 Lu, VHH-chelate group- 99m Tc;

[0129] or

[0130] VHH-DOTA- 68 Ga、VHH-NOTA- 68 Ga、VHH-DTPA- 68 Ga, VHH-DOTA- 177 Lu, VHH-NOTA- 177 Lu, VHH-DTPA- 177 Lu, VHH-Hynic- 99m Tc.

[0131] Preferably, (aTrop2-1B9huV3-FC)-DOTA- 68 Ga, (aTrop2-1B9huV3-FC)-NOTA- 68 Ga, (aTrop2-1B9huV3-FC)-DTPA- 68 Ga, (aTrop2-1B9huV3-FC)-DOTA-177 Lu, (aTrop2-1B9huV3-FC)-NOTA- 177 Lu, (aTrop2-1B9huV3-FC)-DTPA- 177 Lu, (aTrop2-1B9huV3-FC)-Hynic- 99m Tc;

[0132] or preferably, (aTrop2-1C10huV3-FC)-DOTA- 68 Ga, (aTrop2-1C10huV3-FC)-NOTA- 68 Ga, (aTrop2-1C10huV3-FC)-DTPA- 68 Ga, (aTrop2-1C10huV3-FC)-DOTA- 177 Lu, (aTrop2-1C10huV3-FC)-NOTA- 177 Lu, (aTrop2-1C10huV3-FC)-DTPA- 177 Lu, (aTrop2-1C10huV3-FC)-Hynic- 99m Tc.

[0133] In a specific embodiment of the present invention, the radionuclide-labeled anti-Trop2 single domain antibody further comprises a second modification, such as a fluorescent imaging group, a chemiluminescent group, a photoacoustic response imaging group, an MRI enhancement group or an X-ray response group.

[0134] The fluorescent imaging group is a fluorescent compound or nanoaggregate that is excited by one radiation wavelength and detected by a second, different radiation wavelength, including visible and near-infrared fluorescent molecules; preferably, it includes fluorescein molecules, coumarin molecules, rhodamine molecules, cyanine dye molecules, preferably cyanine dye (Cy dye) or indocyanine green dye (ICG dye), BODIPY molecules, square acid molecules, phosphorescent molecules, semiconductor molecules, carbon quantum dots, silicon quantum dots, sulfur quantum dots, One or more of selenium quantum dots, tellurium quantum dots, phosphorus quantum dots, perovskite quantum dots, upconversion rare earth nanomaterials, long afterglow nanomaterials and chelating agents, preferably Cy5.5, Cy3, Cy5, Alexa 680, DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindolyl dicarbocyanine perchlorate) and DiR (1,1'-dioctadecyl-3,3,3',3'-tetramethylindolyl tricarbocyanine iodide).

[0135] The chemiluminescent group refers to a luminescent group in the system that releases energy through a redox reaction to transition from a ground state to an excited state, and then returns to the ground state to release energy in the form of radiant luminescence (ultraviolet light, visible light or near-infrared light), such as luminol and its derivatives, luciferin analogs, peroxyoxalate and dioxetanes.

[0136] The photoacoustic response imaging group is a compound or nano-aggregate that can generate targeted photoacoustic imaging signals in solution, cells and in vivo after being excited; such as ICG compounds.

[0137] The MRI enhancing group is a compound or nano-aggregate that enhances magnetic resonance imaging; for example, ferromagnetic metals, paramagnetic small molecule probes (such as complexes of Gd(III), Dy(III), Fe(III) and Mn(II)) and superparamagnetic nanoparticles (such as iron oxide nanoparticles).

[0138] The X-ray responsive group is a compound or nanoaggregate that enhances CT imaging; such as a compound or nanoaggregate with X-ray absorption ability, for example, metal nanoparticles such as Au, Pt, Bi, Ta, Yb, etc.; or Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au, Yb, Dy, Cu, Rh, Ag, Ir and I.

[0139] Method and kit for preparing radionuclide labeled antibodies

[0140] Those skilled in the art can use optional technical means in the field of bioconjugation to label the antibodies of the present invention, preferably using a chelating agent, which is well known to those skilled in the art and is easily commercially available, for example: HYNIC-NHS, DTPA-NHS, p-NH2-Bn-DTPA, p-SCN-Bn-DTPT, DOTA-NHS, Bn-SCN-DOTA, N3-DOTA-tBu, DOTA(tBu)-COOH, NOTA-NHS, etc.

[0141] There is no limitation on the method for preparing the radionuclide-labeled antibody. For example, the antibody is first modified with a chelating agent (referred to as a radionuclide-labeled precursor in the present invention) and then the radionuclide is chelated.

[0142] Therefore, the present invention also provides a synthesis kit for the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody, comprising:

[0143] 1) Anti-Trop2 single domain antibodies, chelators and radionuclide reagents;

[0144] or

[0145] 2) Chelator-modified anti-Trop2 single domain antibodies and nuclide reagents.

[0146] Uses and compositions of radionuclide labeled antibodies

[0147] The present invention also provides a stable pharmaceutical composition comprising:

[0148] (1) the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody and (2) an anti-radiation degradation stabilizer such as ascorbic acid and / or its salt; preferably, the composition is in the form of an injection.

[0149] The present invention also provides a drug combination or a drug administration combination, comprising:

[0150] (1) the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody, or the above-mentioned imaging agent or the above-mentioned pharmaceutical composition; and (2) another imaging agent or diagnostic agent such as a NIR contrast agent, or a component or agent having cancer therapeutic activity.

[0151] The present invention also provides uses of the radionuclide-labeled anti-Trop2 single domain antibody, the tumor imaging agent or therapeutic agent, the pharmaceutical composition, the drug combination or the drug administration combination, including:

[0152] 1) Used for preparing tumor detection or treatment reagents or kits;

[0153] 2) Used for preparing tumor diagnosis or treatment drugs or compositions;

[0154] 3) Used to prepare molecular probes targeting tumor cells.

[0155] The tumor tissue or cells overexpress Trop2 antigen (Trop2-expressing cell-associated tumor).

[0156] The above reagents or kits, drugs or compositions or molecular probes are expected to be used for diagnosing, treating or preventing tumors associated with cells expressing Trop2.

[0157] The tumor associated with cells expressing Trop2 is selected from one or more combinations of urothelial carcinoma, cervical cancer, triple-negative breast cancer, gastric cancer, pancreatic cancer, lung cancer, papillary thyroid carcinoma, endometrial cancer, prostate cancer, colorectal cancer, and head and neck squamous cell carcinoma.

[0158] In the present invention, the term "pharmaceutical composition" refers to a preparation in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain other components that are unacceptably toxic to the subject receiving the pharmaceutical composition.

[0159] In the present invention, the term "pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives; the composition of the present invention preferably includes an anti-radiation degradation stabilizer such as ascorbic acid and / or its salts.

[0160] The present invention also provides a method for diagnosing or treating tumors, comprising administering the above-mentioned radionuclide-labeled anti-Trop2 single domain antibody, the above-mentioned tumor imaging agent or therapeutic agent, the above-mentioned pharmaceutical composition, the above-mentioned drug combination or administration combination to a subject.

[0161] The term "subject" refers to any member of the animal kingdom, typically mammals. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sport or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is a human.

[0162] Materials and methods

[0163] 1. Antibody information

[0164] The antibody sequences used in the present invention are shown in the sequence table, and the affinity of representative antibodies to Trop2 is shown in Table 1:

[0165] Table 1: Affinity of humanized Anti-Trop2 VHH-Fc fusion protein for Trop2

[0166] Sample name <![CDATA[EC 50 (nM)]]> aTrop2-1A11huV1-Fc 0.092 aTrop2-1A11huV3-Fc 0.115 aTrop2-1B9huV1-Fc 0.069 aTrop2-1B9huV3-Fc 0.061 aTrop2-1C10huV1-Fc 0.112 aTrop2-1C10huV3-Fc 0.090 aTrop2-1C11huV1-Fc 0.187 aTrop2-1C11huV3-Fc 0.220 aTrop2-2B7huV3-Fc 0.140 aTrop2-2E3huV3-Fc 0.089

[0167] Antibody affinity determination method:

[0168] Use coating solution to dilute Trop2-His fusion protein to 1μg / mL, add 100μL to each well of the ELISA plate, and coat overnight at 4℃. Wash three times with PBST, add 5% skim milk powder-PBST to block at 37℃ for 2 hours. Dilute Anti-Trop2 VHH-Fc fusion protein with 1% BSA-PBST gradient, add to the blocked ELISA plate, and incubate at 37℃ for 1 hour. After washing with PBST 5 times, add 100μL of 1:10000 diluted HRP-Goat anti human IgG Fc antibody (ThermoScientific) to each well and incubate at 37℃ for 1 hour. After washing with PBST 6 times, add TMB substrate and incubate at 37℃. After incubation for 5 minutes to develop color, add 1M sulfuric acid to terminate the reaction, and at OD 450nm The absorbance was measured. GraphPad Prism v5.0 software was used for data processing and graphing analysis to obtain the EC value of Anti-Trop2 VHH-Fc for Trop2-His. 50 The value reflects the affinity of the antibody for Trop2.

[0169] Table 1 shows that the affinity of the humanized Trop2 antibodies of the present applicant to Trop2 can reach the nanomolar level, and has a strong affinity for Trop2.

[0170] The following examples of the present invention use aTrop2-1B9huV3-FC antibody (abbreviated as B9) and aTrop2-1C10huV3-FC antibody (abbreviated as C10), and hRS7 (anti-Trop2 mAb) as a positive control (according to patent US9770517B).

[0171] 2. Experimental Animals and Cell Lines

[0172] 1. Basic information of experimental animals:

[0173] Species and strains: BALB / c Nude mice

[0174] Sex / Number: 15 females, 15 males

[0175] Weight / Age: 6-8 weeks, 18-22g

[0176] Grade: SPF grade

[0177] (II) Tumor cell lines

[0178] Trop2 high-expressing cell line: human prostate cancer PC-3, culture medium conditions are RPMI 1640 medium, containing 10% fetal bovine serum and 1% penicillin-streptomycin. After cell expansion, inoculate in the right armpit of nude mice (male), the injection dose for each mouse is 1×10 7 indivual.

[0179] Trop2 low-expressing cell line: human breast cancer cell line MDA-MB-231, culture medium conditions are DMEM medium, containing 10% fetal bovine serum and 1% penicillin-streptomycin. After cell expansion, inoculate in the right armpit of nude mice (female), the injection dose of each mouse is 5×10 6 indivual.

[0180] (III) WB verification of TROP2 expression in cell lines

[0181] (1) Protein extraction:

[0182] a) Discard the culture medium, wash the cells (PC3, MDA-MB-231) with pre-cooled PBS, add total protein extract, pipette thoroughly, and then place on ice for 10-20 minutes, then aspirate the homogenate and place it in a 1.5 ml centrifuge tube;

[0183] b) Centrifuge at 10,000 rpm and 4°C for 10 min, take an appropriate amount of supernatant and place it in a new 1.5 ml centrifuge tube and freeze it at -80°C.

[0184] (2) Protein concentration determination:

[0185] a) Measure the concentration using the BCA method:

[0186] Drawing of standard curve: Take an ELISA plate and add reagents according to Table 2:

[0187] Table 2: BCA standard curve gradient

[0188] Hole No. 0 1 2 3 4 5 6 7 Protein solution (μL) 0 1 2 4 8 12 16 20 Deionized water (μL) 20 19 18 16 12 8 4 0 Corresponding protein content (μg) 0 0.5 1 2 4 6 8 10

[0189] b) Sample concentration determination: 1 μl of the protein to be tested and 19 μl of 0.9% saline;

[0190] c) According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly;

[0191] d) Add 200 μL of BCA working solution to each well;

[0192] e) Place the ELISA plate at 37°C for 30 minutes, and then perform colorimetric analysis at 562 nm. Draw a standard curve with protein content (μg) as the ordinate and absorbance as the abscissa;

[0193] f) According to the absorbance value of the measured sample, the corresponding protein content can be found on the standard curve.

[0194] (3) SDS-PAGE electrophoresis;

[0195] (4) Immunoblotting - membrane transfer;

[0196] (5) Immunoassay;

[0197] (6) Chemiluminescence;

[0198] (7) WB results (electrophoresis sequence: MDA-MB-231, PC3):

[0199] Table 3: WB results of TROP2 cell lines

[0200] Figure 1 superior ACTIN 10% sds-page Figure 1 Down TROP2 10% sds-page

[0201] (8) Grayscale analysis

[0202] Table 4: Grayscale analysis results

[0203] sample MDA-MB-231 PC3 ACTIN 180.995 187.6 TROP2 39.636 87.86

[0204] from Figure 1 As can be verified from Table 4, human prostate cancer PC-3 is a Trop2 high-expressing cell line, and human breast cancer cell line MDA-MB-231 is a Trop2 low-expressing cell line.

[0205] 3. Main instruments

[0206] Table 5: Main instruments

[0207]

[0208] Example

[0209] Example 1 99m Tc-labeled VHH-Fc Trop2 heavy chain antibody imaging experiment (taking C10 as an example)

[0210] (I) Purification of precursor (C10):

[0211] (1) Remove the bottom plug and top cap of the Zeba column, place it in a 1.5 mL EP tube, and centrifuge at 1500 g for 1 min at 4 °C to remove the storage solution;

[0212] (2) Mark the bevel of the Zeba column and keep the higher side of the bevel facing outward during subsequent centrifugation.

[0213] (3) Prepare 1 mL of 1× modification buffer;

[0214] (4) Add 300 μL of 1× modification buffer to the Zeba column and centrifuge three times at 1500 g for 2 min at 4°C;

[0215] (5) Loading: Place the pretreated Zeba column in a new 1.5 mL EP tube, add 2 nmol of the precursor (C19) to the center of the Zeba column resin, and centrifuge at 1500 g for 2 min at 4°C to obtain the purified precursor (C10), about 100 μL.

[0216] (II) Coupling of precursor (C10) with Hynic-NHS and purification

[0217] (1) Weigh 0.2 mg of Hynic-NHS and dissolve it in 40 μL of DMSO;

[0218] (2) Add 2 nmol purified C10 (about 100 μL) and 40 nmol Hynic-NHS (about 2.5 μL) at an equivalent ratio of precursor: chelating agent = 1:20;

[0219] (3) 4°C, 400 rpm metal bath shaking reaction overnight (about 10 h);

[0220] (4) Take a new Zeba column and purify Hynic-C10 using the same method as the precursor purification to obtain about 100 μL of purified sample.

[0221] (three) 99m Tc-labeled Hynic-C10

[0222] (1) Preparation of Tricine solution: weigh 100 mg of Tricine powder and dissolve it in 1 mL of ultrapure water to make a 100 mg / ml solution;

[0223] (2) Preparation of SnCl2 solution: Dissolve large particles of SnCl2 in UP water to make a 7 mg / ml solution;

[0224] (3) 99m Tc labeling: Take the purified and concentrated antibody Hynic-C10 (about 100 μL), add 100uTricine, 4uLSnCl2, 500uL 99m Tc solution (about 15.8 mCi) and mix thoroughly. Shake and react for 30 minutes at room temperature and in the dark. 99m Tc-Hynic-C10);

[0225] (4) Determination of labeling rate ( Figure 2 ): Take 2uL of the mixed solution after the reaction and measure it by iTLC, using PBS as the developing solvent.

[0226] (5) Determination of radiochemical purity ( Figure 3 ): The mixed solution after the reaction was passed through a PD-10 column (same as above), and 8 tubes of products were collected. The radioactivity of each tube was measured in turn. The third tube with the highest activity was taken for iTLC measurement, and PBS was used as the developing solvent.

[0227] Table 6: 99m Determination of radiochemical purity of Tc-Hynic-C10

[0228] Serial number 1 2 3 4 5 6 7 8 PD-10 column Activity (μCi) 93 1580 4180 3230 890 470 510 570 7430 .

[0229] (IV) Animal Imaging

[0230] (1) Collect the third tube product with the highest activity for subsequent imaging and biodistribution studies;

[0231] (2) Method of administration:

[0232] a) Dosage frequency: Single dose

[0233] b) Dosage: 1mCi / rat

[0234] c) Dosing volume: 100 μL / mouse

[0235] d) Administration route: Tail vein injection

[0236] e) Dosing container: Use disposable sterile insulin syringe and needle, specification 1ml, model U-40, manufacturer: Becton Dickinson Medical Devices (Shanghai) Co., Ltd., complete the injection within 30 seconds, and press the needle hole with a cotton ball.

[0237] (3) Anesthesia method: Use the Portable Anesthesia System for small animals and isoflurane gas anesthesia. Isoflurane, specification: 100 ml, storage condition: room temperature, manufacturer: Reward Life Science Technology Co., Ltd. Before imaging, place the animal in an anesthesia induction box. After anesthesia, place the animal in the PET / CT animal cabin and keep it in a prone position. Continuous anesthesia during PET / CT scanning and imaging lasts about 10-20 minutes. The isoflurane flow rate is set to 1.5 LPM (liters / minute).

[0238] (4) SPECT / CT imaging was performed 2 h, 6 h, 12 h, 24 h, and 36 h after administration, and a biodistribution study was performed after the imaging was completed at the 36 h time point.

[0239] (V) The labeling, imaging and biodistribution study methods of precursors (B9, hRS7) are the same as above.

[0240] Table 7: 99m Determination of radiochemical purity of Tc-Hynic-B9

[0241] Serial number 1 2 3 4 5 6 7 PD-10 column Activity (μCi) 28 897 2230 1060 162 61 57 9550

[0242] Table 8: 99m Determination of radiochemical purity of Tc-Hynic-hRS7

[0243] Serial number 1 2 3 4 5 6 7 PD-10 column Activity (μCi) 95 3130 5640 1390 155 77 65 2210 .

[0244] Example 2 177 Lu-labeled VHH-Fc Trop2 heavy chain antibody imaging experiment (taking B9 as an example)

[0245] (I) Purification of precursor (B9):

[0246] Table 9: Purification of precursor (B9)

[0247] Serial number 1 2 3 4 5 Concentration (mg / mL) 0.026 0.435 0.599 0.0935 0.077

[0248] (1) Purification using PD-10 column;

[0249] (2) The second and third tubes with the highest concentration were ultrafiltrated and concentrated. After centrifugation at 13,000 g and 4°C for 25 min, they were inverted into new 2 mL EP tubes and centrifuged at 1,000 g and 4°C for 2 min to collect the purified precursor, about 210 μL, 3.5 nmol.

[0250] (II) Coupling of precursor (B9) with DOTA-NHS and purification

[0251] (1) Weigh 4.3 mg of DOTA-NHS and dissolve it in 30 μL of DMSO;

[0252] (2) 3.5 nmol of purified B9 (about 210 μL) and 40 nmol of DOTA-NHS (about 3 μL) were added at an equivalent ratio of precursor to chelating agent = 1:30, and then 0.1 M Na2CO3 buffer was added to adjust the pH of the system to 9.0;

[0253] (3) 4 °C, 400 rpm metal bath shaking reaction for 4 h;

[0254] (4) Take a new PD-10 column and purify DOTA-B9 using the same precursor purification method. Take the second and third tubes with the highest concentrations for ultrafiltration and concentration, and collect the purified DOTA-B9, about 157 μL, 2.1 nmol.

[0255] Table 10: Purification of DOTA-B9

[0256] Serial number 1 2 3 4 5 Concentration (mg / mL) 0.0845 0.155 0.2225 0.053 0.065

[0257] (three) 177 Lu Mark DOTA-B9

[0258] (1) 177 Lu labeling: Take the purified and concentrated precursor DOTA-B9, add 300μL NH4OAc buffer to adjust the system pH to 5.0, and rinse repeatedly with NH4OAc buffer 177 After the LuCl3 tank wall, add 3mCi 177LuCl3 (about 10μL). Shake and react for 1h at 37℃ in the dark;

[0259] (2) Determination of labeling rate ( Figure 4 ): Take 2uL of the mixed solution after the reaction and measure it by iTLC, using 0.5M EDTA as the developing solvent;

[0260] (3) Determination of radiochemical purity ( Figure 5 ): The mixed solution after the reaction was passed through a PD-10 column (same as above), and 8 tubes of products were collected. The radioactivity of each tube was measured in turn. The second tube with the highest activity was taken for iTLC measurement, and PBS was used as the developing solvent.

[0261] Table 11: 177 Radiochemical Purity Determination of Lu-DOTA-B9

[0262] Serial number 1 2 3 4 5 6 7 PD-10 column Activity (μCi) 41 355 310 49 12 33 92 1500

[0263] (IV) Animal Imaging

[0264] (1) Collect the products from the second and third tubes with the highest activity for subsequent imaging and biodistribution studies;

[0265] (2) Method of administration:

[0266] a) Dosage frequency: Single dose

[0267] b) Dosage: 120μCi / rat

[0268] c) Dosing volume: 200 μL / mouse

[0269] d) Administration route: Tail vein injection

[0270] e) Dosing container: Use disposable sterile insulin syringe and needle, specification 1ml, model U-40, manufacturer: Becton Dickinson Medical Devices (Shanghai) Co., Ltd., complete the injection within 30 seconds, and press the needle hole with a cotton ball.

[0271] (3) Anesthesia method: Use the Portable Anesthesia System for small animals and isoflurane gas anesthesia. Isoflurane, specification: 100 ml, storage condition: room temperature, manufacturer: Reward Life Science Technology Co., Ltd. Before imaging, place the animal in the anesthesia induction box. After anesthesia, place the animal in the PET / CT animal cabin and keep it in a prone position. Continuous anesthesia during PET / CT scanning and imaging lasts about 10-20 minutes. The isoflurane flow rate is set to 1.5 LPM (liters per minute).

[0272] (4) SPECT / CT imaging was performed 6 h, 24 h, 48 h, 72 h, and 120 h after administration, and a biodistribution study was performed after the imaging was completed at the 120 h time point.

[0273] Example 3 Results Analysis

[0274] 99m Analysis of Tc-labeled antibody results

[0275] Figure 6 Shows 99m Biodistribution of the three Tc-labeled antibodies (PC3 tumor-bearing mice, 36 h (n=3)). All three antibodies have good tumor aggregation ability, and the target organ / non-target organ uptake ratio T / M (tumor-to-muscle ratio) is high; 99m Tc-Hynic-B9 and 99m Tc-Hynic-C10 has a similar tumor-to-blood ratio (T / B) as the positive control, and the lower T / B value is consistent with the longer blood circulation time of the antibody.

[0276] Figure 7 shows99m Representative MIP images of Tc-labeled antibodies ( Figure 7a ) and the temporal changes in tumor uptake ( Figure 7b ), 99m Tc-Hynic-B9 and 99m Tc-Hynic-C10 has good uptake and long retention time in the tumor site. The tumor site is still visible at the 36h imaging point. 99m Tc-Hynic-B9 and 99m The higher uptake of Tc-Hynic-C10 and the reduced background suggest that the 99m Tc imaging time to obtain a higher target-to-species ratio.

[0277] 177 Analysis of Lu-labeled antibody results

[0278] Figure 8 Shows 177 In Lu labeling experiments, 177 Representative MIP images of Lu-DOTA-B9 antibody (PC3 tumor-bearing mice and MDA-MB-231 tumor-bearing mice, n=3), Fig. 9 Shows 177 The biodistribution of Lu-DOTA-B9 antibody (120h) shows good tumor uptake and long tumor retention time, indicating therapeutic potential. From the biodistribution results, the tumor uptake value of the Trop2 high-expression tumor-bearing mouse (PC3) model is higher than that of the Trop2 low-expression tumor-bearing mouse (MDA-MB-231) model. Sequence information:

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

Claims

1. A radionuclide-labeled anti-Trop2 single domain antibody, wherein the complementary determining region (CDR) of the anti-Trop2 single domain antibody comprises CDR1 to CDR3 with the amino acid sequences shown below: CDR1 with an amino acid sequence as shown in one of SEQ ID NOs. 1 to 7, CDR2 with an amino acid sequence as shown in one of SEQ ID NOs. 8 to 14, and CDR3 with an amino acid sequence as shown in one of SEQ ID NOs. 15 to 21.

2. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 1, wherein the complementarity determining region (CDR) of the anti-Trop2 single domain antibody comprises CDR1 to CDR3 whose amino acid sequences are as follows: (1) the amino acid sequence of CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of CDR2 is as shown in SEQ ID NO.8, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.15; or (2) the amino acid sequence of CDR1 is as shown in SEQ ID NO.2, the amino acid sequence of CDR2 is as shown in SEQ ID NO.9, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.16; or (3) the amino acid sequence of CDR1 is as shown in SEQ ID NO.3, the amino acid sequence of CDR2 is as shown in SEQ ID NO.10, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.17; or (4) the amino acid sequence of CDR1 is as shown in SEQ ID NO.4, the amino acid sequence of CDR2 is as shown in SEQ ID NO.11, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.18; or (5) the amino acid sequence of CDR1 is as shown in SEQ ID NO.5, the amino acid sequence of CDR2 is as shown in SEQ ID NO.12, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.19; or (6) the amino acid sequence of CDR1 is as shown in SEQ ID NO.6, the amino acid sequence of CDR2 is as shown in SEQ ID NO.13, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.20; or (7) The amino acid sequence of CDR1 is shown in SEQ ID NO.7, the amino acid sequence of CDR2 is shown in SEQ ID NO.14, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

21.

3. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 2, wherein the anti-Trop2 single domain antibody comprises a framework region FR, wherein the framework region FR comprises FR1 to FR4 whose amino acid sequences are as follows: (1) the amino acid sequence of FR1 is as shown in SEQ ID NO.22, the amino acid sequence of FR2 is as shown in SEQ ID NO.23, the amino acid sequence of FR3 is as shown in SEQ ID NO.24, and the amino acid sequence of FR4 is as shown in SEQ ID NO.25; or (2) the amino acid sequence of FR1 is as shown in SEQ ID NO.26, the amino acid sequence of FR2 is as shown in SEQ ID NO.27, the amino acid sequence of FR3 is as shown in SEQ ID NO.28, and the amino acid sequence of FR4 is as shown in SEQ ID NO.29; or (3) an amino acid sequence of FR1 as shown in SEQ ID NO.30, an amino acid sequence of FR2 as shown in SEQ ID NO.31, an amino acid sequence of FR3 as shown in SEQ ID NO.32, or an amino acid sequence of FR4 as shown in SEQ ID NO.33; or (4) an amino acid sequence of FR1 as shown in SEQ ID NO.34, an amino acid sequence of FR2 as shown in SEQ ID NO.35, an amino acid sequence of FR3 as shown in SEQ ID NO.36, or an amino acid sequence of FR4 as shown in SEQ ID NO.25; or (5) an amino acid sequence of FR1 as shown in SEQ ID NO.37, an amino acid sequence of FR2 as shown in SEQ ID NO.38, an amino acid sequence of FR3 as shown in SEQ ID NO.39, or an amino acid sequence of FR4 as shown in SEQ ID NO.40; or (6) an amino acid sequence of FR1 as shown in SEQ ID NO.30, an amino acid sequence of FR2 as shown in SEQ ID NO.41, an amino acid sequence of FR3 as shown in SEQ ID NO.42, and an amino acid sequence of FR4 as shown in SEQ ID NO.43; or (7) FR1 with an amino acid sequence as shown in SEQ ID NO.44, FR2 with an amino acid sequence as shown in SEQ ID NO.45, FR3 with an amino acid sequence as shown in SEQ ID NO.46, and FR4 with an amino acid sequence as shown in SEQ ID NO.

25.

4. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 1, wherein the anti-Trop2 single domain antibody comprises: a) an amino acid sequence shown in one of SEQ ID NOs. 47 to 53; or, b) an amino acid sequence having a sequence identity of more than 80% with one of SEQ ID NOs. 47 to 53, and having the function of the amino acid sequence defined in a); Preferably, the anti-Trop2 single domain antibody is a humanized antibody, and the amino acid sequence of the humanized antibody is shown in one of SEQ ID NOs. 54 to 65.

5. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 1, further comprising a second domain for prolonging the half-life in vivo and / or having a binding effect on effector cells; Preferably, the second domain comprises a combination of one or more of a serum albumin fragment, a polyethylene glycol fragment, and a single domain antibody that binds to serum albumin; Preferably, the second domain comprises an immunoglobulin Fc region, and the immunoglobulin Fc region is the Fc of human natural IgG1, and its amino acid sequence is shown in SEQ ID NO.66; Preferably, the anti-Trop2 single-domain antibody comprising the second domain is as shown in SEQ ID NOs.67-85; further preferably, as shown in SEQ ID NOs.68, 69, 76-79. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 1 , further comprising a second domain for recognizing and binding to another tumor or cancer marker.

7. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 1, wherein the radionuclide is selected from a non-metallic isotope or a metal isotope: the non-metallic isotope is selected from 11 C. 14 C. 13 N. 18 F. 131 I. 123 I. 124 I. 125 I at least one; the metal isotope is selected from 86 Y. 90 Y. 211 At 153 Sm, 186 Re, 188 Re, 67 Cu, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 111 In, 62 Cu, 64 Cu, 89 Zr, 177 Lu, 99m Tc, 67 Ga, 68 Ga, 153 At least one of Gd.

8. According to the radionuclide-labeled anti-Trop2 single-domain antibody of claim 7, the metal isotope is labeled with the antibody through a chelator; the chelator is selected from at least one of HYNIC, EDTA, HP-DO3A, DTPA, DOTA, NOTA, TRAP, NODAGA, HBED-CC, DOTAGA, and DOTA(GA)2.

9. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 8, wherein the radionuclide-labeled anti-Trop2 single domain antibody comprises: VHH-DOTA-Nuclide, VHH-NOTA-Nuclide, VHH-DTPA-Nuclide, VHH-Hynic-Nuclide or VHH-EDTA-Nuclide; wherein VHH is an anti-Trop2 single domain antibody as defined in any one of claims 1-6.

10. The radionuclide-labeled anti-Trop2 single domain antibody according to any one of claims 1 to 6, wherein the structure is: VHH-R1-L-R2-N(I), where: VHH is an anti-Trop2 single-domain antibody; R1 is the linking group between L and the antibody; L is the linker arm or does not exist; R2 is a connecting group or a direct bond between N and L; N is a nuclide-labeled group or nanoaggregate.

11. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 10, wherein R1 is selected from a thiol reactive group, an amino reactive group, a carboxyl reactive group, and a dithiol bridging group; Preferably, R1 is an amide bond, R2 is a direct bond, and L is a part of N or does not exist.

12. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 10, wherein: The nuclide is selected from 11 C. 14 C. 13 N. 18 F. 131 I. 123 I. 124 I. 125 At least one of I; or selected from 86 Y. 90 Y. 211 At 153 Sm, 186 Re, 188 Re, 67 Cu, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 111 In, 62 Cu, 64 Cu, 89 Zr, 177 Lu, 99m Tc, 67 Ga, 68 Ga, 153 At least one of Gd; The group or nanoaggregate is a chelating agent, and the chelating agent is selected from at least one of HYNIC, EDTA, HP-DO3A, DTPA, DOTA, NOTA, TRAP, NODAGA, HBED-CC, DOTAGA, DOTA(GA)2 and derivatives thereof.

13. The radionuclide-labeled anti-Trop2 single domain antibody according to claim 10, wherein: L has about 1-100 linking atoms and optionally includes vinyloxy, amine, ester, amide, ketone, urea, carbamate, and carbonate functional groups; or, L is a straight or branched alkyl group with 2 to 20 carbon atoms; or L does not exist.

14. A tumor imaging agent or therapeutic agent comprising the radionuclide-labeled anti-Trop2 single-domain antibody according to any one of claims 1 to 13 as an active ingredient.

15. A method for preparing the radionuclide-labeled anti-Trop2 single domain antibody according to any one of claims 1 to 13, comprising the following steps: 1) Modification of anti-Trop2 single domain antibodies using chelators; 2) Chelated nuclides.

16. A synthetic kit for preparing the radionuclide-labeled anti-Trop2 single domain antibody according to any one of claims 1 to 13, comprising: 1) an anti-Trop2 single domain antibody, a chelating agent, and a reagent for providing a nuclide; or, 2) Chelating agent-modified anti-Trop2 single domain antibody and reagent for providing nucleoside.

17. A stable pharmaceutical composition comprising: The radionuclide-labeled anti-Trop2 single domain antibody and the anti-radiation degradation stabilizer according to any one of claims 1 to 13.

18. A drug combination or administration combination comprising: (1) the radionuclide-labeled anti-Trop2 single domain antibody of any one of claims 1 to 13, the imaging agent or therapeutic agent of claim 14, or the pharmaceutical composition of claim 17; and (2) another imaging agent or diagnostic agent, or a component or agent having cancer therapeutic activity.

19. Use of the radionuclide-labeled anti-Trop2 single domain antibody according to any one of claims 1 to 13, the imaging agent or therapeutic agent according to claim 14, the pharmaceutical composition according to claim 17, or the pharmaceutical combination or administration combination according to claim 18, comprising: 1) Used for preparing tumor detection or treatment reagents or kits; 2) Used for preparing tumor diagnosis or treatment drugs or compositions; 3) Used to prepare molecular probes targeting tumor tissues or cells; Preferably, the tumor is a tumor associated with cells expressing Trop2.

20. A chelator-modified anti-Trop2 single domain antibody, the anti-Trop2 single domain antibody being as defined in any one of claims 1 to 6; Preferably, the chelator-modified anti-Trop2 single domain antibody is as shown in (Formula II): VHH-R1-L-R2-Ch(II), wherein: VHH is an anti-Trop2 single-domain antibody; R1 is the linking group between L and the antibody; L is the linker arm or does not exist; R2 is a connecting group or a direct bond between Ch and L; Ch is a chelating agent.

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