A radionuclide-labeled anti-5T4 nanoantibody and its preparation method and application

Abstract

The present invention discloses an application of a radionuclide-labeled anti-5T4 nano antibody in the prognosis and diagnosis of cancer. Specifically, the present invention discloses an immunoconjugate for detecting 5T4 molecules, wherein the immunoconjugate comprises a specific VHH chain of an anti-5T4 nano antibody and a radionuclide, and can be used for non-invasive detection of 5T4 expression in a subject to be tested. The immunoconjugate of the present invention has high stability and high specificity, is suitable for systemic detection of simultaneously targeting primary and metastatic tumors, has high accuracy, and has a low radiation dose.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and nuclear medicine, and in particular to a radionuclide-labeled anti-5T4 nano antibody and a preparation method and application thereof. Background Art

[0002] Tumor is an important disease that affects human health. Tumor treatment mainly includes traditional surgical treatment, radiotherapy, chemotherapy and new treatment methods such as targeted therapy and immunotherapy that have developed rapidly in recent years. Targeted drugs have always been a hot spot in the research and development of anti-tumor drugs. Tumor targeted drugs can be roughly divided into two categories: monoclonal antibodies and small molecule compounds.

[0003] 5T4 is a highly glycosylated cell surface protein encoded by the TPBG gene. The molecular weight of human TPBG protein is about 72kDa. 5T4 is widely expressed in various trophoblast cells during embryonic development. For normal adult tissues, 5T4 is only expressed in a limited number of epithelial cells. But it is highly expressed in many cancer cells. This makes 5T4 a very attractive target in tumor immunotherapy.

[0004] Therefore, the development of reagents or drugs targeting 5T4 is of great significance for the diagnosis and treatment of tumors. Summary of the invention

[0005] The object of the present invention is to provide a nano antibody conjugate targeting 5T4 protein, and a preparation method and application thereof.

[0006] The first aspect of the present invention provides an immunoconjugate, wherein the immunoconjugate comprises:

[0007] (a) an antibody portion selected from the VHH chain of an anti-5T4 nanobody, or an anti-5T4 nanobody having said VHH chain, wherein said VHH chain comprises a complementary determining region (CDR) as shown below:

[0008] The amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; and

[0009] (b) a conjugation moiety selected from the group consisting of a detectable label, a drug, a toxin, a nucleic acid, or a combination thereof.

[0010] In another preferred embodiment, the detectable marker includes (but is not limited to): radionuclide, fluorescein, enzyme, and colloidal gold.

[0011] In another preferred embodiment, the coupling moiety is a radionuclide.

[0012] In another preferred embodiment, the radioactive nuclides include:

[0013] (i) a diagnostic isotope selected from the group consisting of Ga-68, F-18, I-123, Ga-67, Cu-64, Zr-89, C-11, Re-188, or a combination thereof; and / or

[0014] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of I-131, I-124, I-125, Lu-177, Tc-99m, In-111, 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, 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.

[0015] In another preferred embodiment, the radioactive nuclide is selected from: Ga-68, F-18, and I-131.

[0016] In another preferred embodiment, the amino acid sequence of the VHH chain is as shown in SEQ ID NO: 1 or has at least 80% (preferably 85%, more preferably 90%, most preferably 95%, for example 96%, 97%, 98%, 99%) sequence identity thereto.

[0017] A second aspect of the present invention provides a radioactive nanoprobe, wherein the radioactive nanoprobe comprises:

[0018] (a) an antibody portion selected from the VHH chain of an anti-5T4 nanobody, or an anti-5T4 nanobody having said VHH chain, wherein said VHH chain comprises a complementary determining region (CDR) as shown below:

[0019] The amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; and

[0020] (b) A radionuclide coupled to (a).

[0021] In another preferred embodiment, the radioactive nuclides include:

[0022] (i) a diagnostic isotope selected from the group consisting of Ga-68, F-18, I-123, Ga-67, Cu-64, Zr-89, C-11, Re-188, or a combination thereof; and / or

[0023] (ii) therapeutic isotopes, wherein the therapeutic isotopes are selected from the group consisting of I-125, I-131, I-124, Lu-177, Tc-99m, In-111, 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, 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.

[0024] In another preferred embodiment, the radioactive nuclide is selected from: Ga-68, F-18, and I-131.

[0025] In another preferred embodiment, the amino acid sequence of the VHH chain is as shown in SEQ ID NO: 1 or has at least 80% (preferably 85%, more preferably 90%, most preferably 95%, for example 96%, 97%, 98%, 99%) sequence identity thereto.

[0026] The third aspect of the present invention provides a 5T4 protein detection reagent, which comprises the immunoconjugate as described in the first aspect of the present invention or the radioactive nanoprobe as described in the second aspect of the present invention, and a carrier acceptable in detection.

[0027] In another preferred embodiment, the conjugated portion of the immunoconjugate is a diagnostic isotope.

[0028] In another preferred embodiment, the coupling portion of the radioactive nanoprobe is a diagnostic isotope.

[0029] In another preferred embodiment, the test-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0030] In another preferred embodiment, the detection reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

[0031] In another preferred embodiment, the detection agent is a contrast agent, and the contrast agent also includes other agents used for contrast.

[0032] In another preferred embodiment, the contrast agent is a contrast agent used for MRI (magnetic resonance imaging) or CT (computer tomography).

[0033] In another preferred embodiment, the imaging agent chelates two or more signals simultaneously, such as Ga-68 and Gd, which are used for PET / CT and MRI simultaneously; or Tc-99m and a fluorescent agent, which are used for SPECT / CT and fluorescence detection simultaneously.

[0034] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0035] In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal preparation, inhaler).

[0036] The fourth aspect of the present invention provides a pharmaceutical composition, which comprises the immunoconjugate as described in the first aspect of the present invention or the radioactive nanoprobe as described in the second aspect of the present invention, and a pharmaceutically acceptable carrier.

[0037] In another preferred embodiment, the conjugated portion of the immunoconjugate is a drug, a toxin, and / or a therapeutic isotope.

[0038] In another preferred embodiment, the coupling portion of the radioactive nanoprobe is a therapeutic isotope.

[0039] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating tumors, such as cytotoxic drugs.

[0040] In another preferred embodiment, the pharmaceutical composition is used to treat or prevent tumors expressing 5T4 protein (ie, 5T4 positive).

[0041] In another preferred embodiment, the tumor includes (but is not limited to):

[0042] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0043] The fifth aspect of the present invention provides a kit for detecting 5T4 protein, wherein the kit comprises the 5T4 protein detection reagent as described in the third aspect of the present invention and instructions.

[0044] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting the expression of 5T4 in a subject to be tested.

[0045] In another preferred embodiment, the subject to be tested includes humans or non-human mammals (eg, mice, rats, rabbits, monkeys, etc.).

[0046] In another preferred embodiment, the kit is used to detect tumors expressing 5T4 protein (ie, 5T4 positive).

[0047] In the sixth aspect of the present invention, there is provided a use of the immunoconjugate as described in the first aspect of the present invention or the radioactive nanoprobe as described in the second aspect of the present invention for preparing (1) a detection reagent, a detection kit or a detection plate for detecting 5T4 protein; (2) a pharmaceutical composition for treating or preventing tumors expressing 5T4 protein (i.e., 5T4 positive).

[0048] In another preferred embodiment, the immunoconjugate or radioactive nanoprobe is used to prepare a detection reagent, a detection kit or a detection plate for detecting 5T4 protein in vivo.

[0049] The seventh aspect of the present invention provides a method for preparing the radioactive nanoprobe according to the second aspect of the present invention, the method comprising the steps of:

[0050] (S1) providing a VHH chain of an anti-5T4 nanobody or an anti-5T4 nanobody having said VHH chain;

[0051] (S2) adding a radionuclide chelator that can bind to the nanobody to form a labeled precursor;

[0052] (S3) adding a radionuclide to the labeled precursor formed in step (S2), so that the radionuclide and the labeled precursor form a radionuclide-labeled Nanobody;

[0053] (S4) separating the radionuclide-labeled nanobody obtained in step (S3) to obtain the radioactive nanoprobe.

[0054] In another preferred embodiment, the method further comprises: evaluating the prepared radioactive nanoprobe.

[0055] In another preferred embodiment, the radionuclide chelator includes a bifunctional chelator, a macrocyclic chelator, or a combination thereof.

[0056] In another preferred embodiment, the radionuclide chelator is selected from the following group: NOTA, DFO, NODA, TCO, ±H3RESCA-THP, or a combination thereof.

[0057] In another preferred embodiment, the VHH chain of the anti-5T4 nanobody or the anti-5T4 nanobody having the VHH chain in step (S1) is obtained by expressing in a host cell and then purifying;

[0058] Wherein, the host cells include eukaryotic cells and prokaryotic cells;

[0059] Preferably, the host cell is Pichia pastoris.

[0060] In an eighth aspect of the present invention, a method for detecting 5T4 protein is provided, comprising the steps of:

[0061] (S1) contacting the immunoconjugate described in the first aspect of the present invention or the radioactive nanoprobe described in the second aspect of the present invention with the 5T4 protein of the subject to be tested;

[0062] (S2) Detecting the formation of an antigen-antibody complex. If the antigen-antibody complex exists, it indicates that the 5T4 protein exists in the object to be tested.

[0063] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0064] In another preferred embodiment, the method is an in vivo method for non-invasively detecting the expression of 5T4 in a subject to be tested.

[0065] In another preferred embodiment, the subject to be tested includes humans or non-human mammals (eg, mice, rats, rabbits, monkeys, etc.).

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 Radionuclides are shown 68 Real-time thin-layer chromatography detection results of Ga-labeled radioactive probe targeting 5T4 antigen and in vivo effect detection diagram.

[0068] Figure 2 Radionuclides are shown 18 Real-time thin-layer chromatography detection results of F-labeled radioactive probe targeting 5T4 antigen and in vivo effect detection diagram.

[0069] Figure 3 Radionuclides are shown 131 Real-time thin-layer chromatography detection results of I-labeled radioactive probe targeting 5T4 antigen and in vivo effect detection diagram. DETAILED DESCRIPTION

[0070] After extensive and in-depth research, the inventors have provided a radionuclide-labeled anti-5T4 nanobody conjugate for the first time. The inventors have used a novel bifunctional chelator or other chelators to couple with the nanobody to conduct 131 I. 64 Cu, 68 Ga, 18F and other nuclides are used to label the nanoantibody probes, thereby obtaining a nanoantibody probe targeting the 5T4 antigen. The antigen targeted by the nanoantibody probe of the present invention is 5T4, which is a glycoprotein highly expressed in embryonic trophoblast cells. The protein has been found to be highly expressed in a variety of tumor cells, but rarely expressed in normal tissues. By coupling radionuclides to the nanoantibody, probes targeting a variety of tumor cells can be prepared, thereby applying them to the precise diagnosis and treatment of tumors. The nanoantibody probe of the present invention can be used for the diagnosis and treatment of tumors with high expression of 5T4, and has good clinical application prospects.

[0071] On this basis, the present invention has been completed.

[0072] the term

[0073] As used herein, the terms "nanoantibodies of the present invention", "anti-5T4 nanoantibodies of the present invention", and "nanoantibodies targeting 5T4 antigens" are used interchangeably and all refer to nanoantibodies that specifically recognize and bind to 5T4 proteins. A preferred nanoantibody VHH chain comprises a CDR1 with an amino acid sequence as shown in SEQ ID NO:2, a CDR2 with an amino acid sequence as shown in SEQ ID NO:3, and a CDR3 with an amino acid sequence as shown in SEQ ID NO:4; a nanoantibody with an amino acid sequence as shown in SEQ ID NO.:1 is particularly preferred. The nanoantibody in the present invention is derived from Pichia pastoris expression and has a structure of VHH-GSC.

[0074] SEQ ID NO:1

[0075] EVQLLESGGGLVQPGGSLRLSCAAS GLTYCDSLMY WYRQGPGKGLEFVSF ID R AGR SYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYC KITCYNSGYQQ WGQGTLVTVSS

[0076] Among them, the underlined parts are CDR1, CDR2 and CDR3, and the parts separated by CDR are FR1, FR2, FR3 and FR4.

[0077] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0078] As used herein, the terms "single domain antibody" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0079] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of light and heavy chains. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region does not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0080] As known to those skilled in the art, immunoconjugates and fusion expression products include: radionuclides, drugs, toxins, cytokines, enzymes and other diagnostic or therapeutic molecules combined with antibodies of the present invention or fragments thereof to form conjugates. The present invention also includes cell surface markers or antigens combined with the anti-5T4 nanobody or fragments thereof.

[0081] As used herein, the term "heavy chain variable region" is synonymous with "V H” can be used interchangeably.

[0082] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0083] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0084] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0085] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0086] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by synthesizing multiple small fragments first and then connecting them. In addition, the coding sequence of the antibody and the expression tag (such as 6His) can also be fused together to form a fusion protein.

[0087] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0088] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0089] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0090] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0091] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0092] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0093] Immunoconjugates

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

[0095] (a) an antibody portion selected from the VHH chain of an anti-5T4 nanobody, or an anti-5T4 nanobody having said VHH chain, wherein the amino acid sequence of said VHH chain is as shown in SEQ ID NO: 1; and

[0096] (b) a conjugation moiety selected from the group consisting of a detectable label, a drug, a toxin, a nucleic acid, or a combination thereof.

[0097] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0098] In another preferred embodiment, the coupling moiety is a detectable label.

[0099] In another preferred embodiment, the coupling moiety is a radionuclide.

[0100] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0101] The immunoconjugate of the present invention can be used for non-invasive detection of the 5T4 expression level of a test subject, has a small size and high specificity, low immunogenicity, high accuracy, and low radiation dose.

[0102] Radioactive Nanoprobes

[0103] The present invention provides a radioactive nanoprobe, which comprises (a) an antibody portion, which is selected from the VHH chain of an anti-5T4 nanoantibody, or an anti-5T4 nanoantibody having the VHH chain, wherein the amino acid sequence of the VHH chain is as shown in SEQ ID NO: 1; and (b) a radionuclide coupled to (a).

[0104] In one embodiment of the present invention, there is provided a 68 Ga-labeled radioactive nanoprobes are prepared by chelating radionuclides (e.g., bifunctional chelator NOTA) to 68 Ga was coupled to the aforementioned anti-5T4 nanobody to prepare it.

[0105] In one embodiment of the present invention, there is provided a 18F-labeled radioactive nanoprobes are prepared by chelating radionuclides (e.g., TCO or ±H3RESCA-THP) to 18 F was prepared by coupling to the aforementioned anti-5T4 nanobody.

[0106] In one embodiment of the present invention, there is provided a 131 I-labeled radioactive nanoprobes are prepared by SGMIB and radionuclides. 131 I is prepared by combining and coupling to the aforementioned anti-5T4 nanobody.

[0107] As used herein, the terms "radioactive nanoprobe", "radioactive probe", and "anti-5T4 nanoantibody radioactive probe" can be used interchangeably and all refer to the above-mentioned probe molecules prepared by the present invention.

[0108] Detection Reagents and Kits

[0109] The present invention provides a 5T4 protein detection reagent, which comprises the immunoconjugate or radioactive nanoprobe of the present invention and a carrier acceptable in detection. In one embodiment of the present invention, the coupling part of the immunoconjugate or radioactive nanoprobe is a diagnostic isotope.

[0110] Preferably, the detection agent of the present invention is an agent for in vivo detection, such as a contrast agent.

[0111] The present invention also provides a kit containing the immunoconjugate or radioactive nanoprobe of the present invention. In a preferred embodiment of the present invention, the kit further includes a container and instructions for use.

[0112] Preferably, the kit of the present invention is an in vivo diagnostic kit for non-invasively detecting the 5T4 expression level of a subject to be tested.

[0113] Pharmaceutical composition

[0114] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned immunoconjugate or radioactive nanoprobe, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0115] The pharmaceutical composition of the present invention can be directly used to bind to the PD-L1 protein molecule, and thus can be used to treat tumors. In addition, other therapeutic agents can also be used simultaneously.

[0116] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned immunoconjugate or radioactive nanoprobe of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the conjugate of the present invention can also be used with other therapeutic agents.

[0117] When using a pharmaceutical composition, a safe and effective amount of an immunoconjugate or a radioactive nanoprobe is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0118] Method of the present invention

[0119] The present invention provides a method for preparing a radioactive nanoprobe, specifically, providing a method for preparing 68 Ga, 18 F. or 131 I-labeled anti-5T4 nanobody radioactive probe method.

[0120] In one embodiment of the present invention, there is provided a method for preparing 68 The method for preparing a Ga-labeled anti-5T4 nanobody radioactive probe comprises the following specific steps:

[0121] (1) mixing the bifunctional chelator NOTA and the nanobody in an appropriate ratio to obtain the labeled precursor Nb-NOTA under specific conditions;

[0122] (2) Adoption 68 Ga-labeled antibodies: Elute with 2 mL of 0.1 M HCl solution 68 Ga solution, using saturated sodium acetate 68 The Ga solution was adjusted to pH 4-4.4 and then mixed evenly with the labeled precursor Nb-NOTA and reacted at room temperature for 10-15 minutes;

[0123] (3) Purifying the reaction solution of step (2) using a PD10 column to obtain the radioactive probe.

[0124] In another embodiment of the present invention, there is provided a preparation 18 The method for preparing a radioactive probe for an anti-5T4 nanobody labeled with F comprises the following specific steps:

[0125] (1) using a chelating agent TCO and a nanobody to mix in an appropriate ratio, and obtaining a protein-labeled precursor TCO-Nb under specific conditions;

[0126] (2) Obtained by click chemistry 18 F-labeled small molecule labeling precursor, and separated the small molecule labeling precursor using HPLC;

[0127] (3) Collect the 18 After the F-labeled small molecule labeling precursor is purified, the protein labeling precursor TCO-Nb in step (1) is added and reacted for 10-15 minutes under the appropriate reaction conditions;

[0128] (4) Purifying the reaction solution of step (3) using a PD-10 column to obtain the radioactive probe.

[0129] In another embodiment of the present invention, there is provided a preparation 18 The method for preparing a radioactive probe for an anti-5T4 nanobody labeled with F comprises the following specific steps:

[0130] (1) using a chelating agent ±H3RESCA-THP and a nanobody to mix in an appropriate ratio, reacting at room temperature for 2-3 hours, and using PD-10 to purify the chelating reaction solution to obtain a chelating precursor ±H3RESCA-THP-Nb;

[0131] (2) Produced by cyclotron 18 F aqueous solution, passed through the activated QMA column, and washed with 0.9% sodium chloride solution to obtain Na 18 Solution F.

[0132] (3) Add a specific amount of AlCl3 solution into Na 18 F solution, react for 5 min, then put the chelating precursor ±H3RESCA-THP-Nb into the above solution and react for 10 min;

[0133] (4) Purifying the reaction solution of step (3) using a PD-10 column to obtain the radioactive probe.

[0134] In another embodiment of the present invention, there is provided a method for preparing 131 The method for preparing an anti-5T4 nanobody radioactive probe labeled with I comprises the following specific steps:

[0135] (1) SGMTB and radioisotopes 131 I was reacted at room temperature for 15 minutes to obtain a small molecule labeling precursor, which was purified by HPLC using 0.1% TFA in water as phase A and acetonitrile as phase B, and the ratio of phases A and B was adjusted to obtain a labeling precursor SGMIB with a radiochemical purity greater than 95%;

[0136] (2) The labeled precursor SGMIB was reacted with the antibody Nb-H006 at 37°C for 30 min.

[0137] (3) Purifying the reaction solution of step (2) using a PD-10 column to obtain the radioactive probe.

[0138] The main advantages of the present invention are:

[0139] (1) The antibody involved in the immunoconjugate or radioactive nanoprobe of the present invention is a nanobody expressed by the Pichia pastoris system, and has a structure of VHH. The antibody has a highly specific binding to the 5T4 antigen.

[0140] (2) The immunoconjugate or radioactive nanoprobe prepared by the present invention has the advantages of high stability, low immunogenicity, and low production cost.

[0141] Example 1 Radionuclides 68 Preparation of Ga-labeled radioactive probe targeting 5T4 antigen

[0142] This embodiment provides 68 Ga-labeled nanobody Nb and its preparation method, the specific preparation method is as follows: (1)

[0144]

[0145] To 0.5 mg of nanobody Nb-H006 (PBS as solvent), add 12 times the molar amount of P-SCN-Bn-NOTA (dissolved in DMSO) of the nanobody dropwise, mix the two evenly, adjust the pH to 8.5-8.7 with 0.05 M sodium bicarbonate / sodium carbonate mixture, and incubate at room temperature for 2 h with shaking. Use 1N hydrochloric acid to adjust the pH to 7.0-7.4 to terminate the reaction.

[0146] Use 5*5mL PBS to pre-equilibrate the PD-10 desalting column, discard the effluent, put the above reaction solution into the desalting column, discard the first 2.5mL effluent, use 3.5mL PBS to elute the labeled precursor, and use a 10KDa ultrafiltration centrifuge tube to concentrate the eluate to obtain the labeled precursor. (2)

[0148]

[0149] Use 0.1M hydrochloric acid to 68 Ga is eluted from the generator and the pH is adjusted to 4.4-4.6 using saturated sodium acetate solution. 68 Ga solution, mixed evenly, reacted at room temperature for 5-10 minutes, and the labeling yield was detected by iTLC. When the labeling rate reached about 90%, the product was purified by PD MiniTrap G-25, wherein PD MiniTrap G-25 was pre-activated with 3*2.5mL PBS. After the sample entered the packed bed, 1mL PBS was used for elution, and the eluate was collected to obtain the labeled product. Radioactive instant thin layer chromatography (iTLC) detected that the radiochemical purity was >95%.

[0150] The obtained labeled product was used to perform in vivo effect detection, and the method was as follows: 100uL (about 100uCi) of the labeled product was injected into the tail vein of tumor-bearing mice, and small animal PET / CT imaging was performed at specific time points.

[0151] The real-time thin layer chromatography and in vivo effects of the product are as follows Figure 1 Radioactive real-time chromatography showed that the radiochemical purity of the labeled product was > 95%.

[0152] The results showed that 30 minutes after tail vein injection, small animal PET / CT scanning revealed obvious accumulation of the labeled product in the tumor site at this time point.

[0153] Example 2: Click chemistry for radionuclides 18 Preparation of F-labeled radioactive probe targeting 5T4 antigen

[0154] This embodiment provides 18 F-labeled nanobody Nb and its preparation method, the specific preparation method is as follows:

[0155] (1) The protein labeling precursor TCO-Nb was prepared as follows:

[0156] ±H3RESCA-THP chelator dissolved in DMSO was added dropwise to 0.5 mg of nanoantibody Nb-H006 (PBS as solvent), the ratio of chelator to antibody was about 10:1, and after mixing evenly, the reaction solution was placed on a shaker at room temperature for 2-3 hours.

[0157] The MiniTrap G-25 desalting column was pre-equilibrated with 3*2.5 mL of sodium acetate (pH 4.4), and the effluent was discarded. The above reaction solution was added to the desalting column, the first 0.5 mL of the effluent was discarded, and the protein-labeled precursor was eluted with 1 mL of sodium acetate (pH 4.4) to obtain the protein-labeled precursor TCO-Nb.

[0158] (2) Preparation of [F-18]FBA-OSu:

[0159]

[0160] [F-18]FBA-OSu is obtained by the above chemical method, and the specific implementation method is as follows: the aqueous solution containing 18F- produced by the cyclotron is passed through light QMA column, enrich 18F-, and then use KOTf solution (10mg / 1mL H2O) (100, 200uL) to 18 F was released into the labeled tube. 1 mL of MeCN was added and concentrated under nitrogen flow at 100 °C, and repeated 3 to 5 times. K was obtained. 18 F. After cooling, the pre-reaction solution was added and the reaction was carried out at 100°C for 5 min. The separated product was purified by HPLC. The labeled precursor [F-18]FBA-OSu was eluted with ethanol and then dried by nitrogen flow.

[0161] (3) The protein labeling precursor TCO-Nb and [F-18]FBA-OSu were mixed evenly, reacted at room temperature for 10-15 minutes, and purified by PD-10 to obtain a labeled product. The radiochemical purity was > 95% by radioactive instant thin layer chromatography.

[0162] The obtained labeled product is used to perform in vivo effect detection, and the specific method is the same as that in Example 1.

[0163] The instant thin layer chromatography and in vivo effects of the labeled product are as follows Figure 2 Radioactive real-time chromatography showed that the radiochemical purity of the labeled product was > 95%.

[0164] The results showed that 30 minutes after tail vein injection, small animal PET / CT scanning revealed obvious accumulation of the labeled product in the tumor site at this time point.

[0165] Example 3 Radionuclides 131 Preparation of I-labeled radioactive probe targeting 5T4 antigen

[0166] This embodiment provides 131 I labeled nano antibody Nb and its preparation method, the specific method is as follows: (1)

[0168]

[0169] Will Na 131 I (5mci, about 50μL) was added with 3% acetic acid methanol solution to adjust the pH to acidic, 1mg SGMTB was dissolved in methanol, 10μL (50μg) was taken and added to the labeling bottle, reacted at room temperature for 15min, and methanol was evaporated with nitrogen. The reaction product was co-evaporated twice with acetonitrile to remove impurities, and then dissolved with an appropriate amount of acetonitrile, separated by HPLC to obtain the labeling precursor.

[0170] (2) After dissolving the nanobody Nb-H006 in a borate solution at pH 8.5, the nanobody was added to the labeled precursor and reacted for 30 minutes to obtain a labeled product, which was purified using a PD-10 column. The radiochemical purity was > 95% using radioactive real-time thin layer chromatography.

[0171] The obtained labeled product is used to perform in vivo effect detection, and the specific method is the same as that in Example 1.

[0172] The instant thin layer chromatography and in vivo effects of the labeled product are as follows Figure 3 Radioactive real-time chromatography showed that the radiochemical purity of the labeled product was > 95%.

[0173] 60 minutes after tail vein injection, small animal SPECT / CT scanning revealed obvious accumulation of labeled products in the tumor site at this time point.

[0174] In summary, the nanoantibody Nb targeting human 5T4 antigen provided by the present invention has high specificity for 5T4 antigen and can be used to prepare a variety of antibody conjugates, among which the radionuclide provided by the present invention 68 Ga, 18 The preparation method of the F-labeled radioactive probe has high labeling rate and radiochemical purity, good in vitro and in vivo stability, high binding affinity to 5T4 antigen, good imaging effect, and can be used for accurate diagnosis and treatment of 5T4-positive tumor lesions. The radionuclide-labeled nanoantibody is expected to become a radioactive probe with good application prospects.

[0175] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A radioactive nanoprobe, characterized in that: The radioactive nanoprobe comprises: (a) an antibody portion selected from the VHH chain of an anti-5T4 nanobody, wherein the VHH chain comprises a complementary determining region (CDR) as shown below: The amino acid sequence of CDR1 is shown in SEQ ID NO:2, the amino acid sequence of CDR2 is shown in SEQ ID NO:3, and the amino acid sequence of CDR3 is shown in SEQ ID NO:4; and (b) a radionuclide coupled to (a), wherein the radionuclide is selected from Ga-68 or F-18.

2. The radioactive nanoprobe according to claim 1, characterized in that: The amino acid sequence of the VHH chain is as shown in SEQ ID NO: 1 or has at least 80% sequence identity thereto.

3. A 5T4 protein detection reagent, comprising the radioactive nanoprobe as claimed in claim 1, and a carrier acceptable in detection.

4. The 5T4 protein detection reagent according to claim 3, characterized in that The detection reagent is a contrast agent.

5. The 5T4 protein detection reagent according to claim 4, characterized in that The contrast agent is a contrast agent used for MRI (magnetic resonance imaging) or CT (computer tomography).

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the radioactive nanoprobe as claimed in claim 1 and a pharmaceutically acceptable carrier.

7. A kit for detecting 5T4 protein, characterized in that: The kit contains the 5T4 protein detection reagent as described in claim 3 and instructions.

8. The kit according to claim 7, characterized in that The instructions state that the kit is used for non-invasively detecting the expression of 5T4 in a subject to be tested.

9. The kit according to claim 8, characterized in that The kit is used for detecting tumors expressing 5T4 protein.

10. Use of the radioactive nanoprobe according to claim 1 for preparing a detection reagent, a detection kit or a detection plate for detecting 5T4 protein.

11. A method for preparing the radioactive nanoprobe according to claim 1, characterized in that: The method comprises the steps of: (S1) providing the VHH chain of the anti-5T4 nanobody; (S2) adding a radionuclide chelator that can bind to the nanobody to form a labeled precursor; (S3) adding a radionuclide to the labeled precursor formed in step (S2), so that the radionuclide and the labeled precursor form a radionuclide-labeled Nanobody; (S4) separating the radionuclide-labeled nanobody obtained in step (S3) to obtain the radioactive nanoprobe.

12. The method according to claim 11, characterized in that The radionuclide chelator includes a bifunctional chelator, a macrocyclic chelator, or a combination thereof.

13. The method according to claim 12, characterized in that The radionuclide chelator is selected from the group consisting of NOTA, DFO, NODA, TCO, ±H3RESCA-THP, or a combination thereof.

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