CN118126185A

CN118126185A - Radionuclide-labeled anti-5T 4 nano antibody and preparation method and application thereof

Info

Publication number: CN118126185A
Application number: CN202311793516.1A
Authority: CN
Inventors: 康杰; 张顺杰; 汪笑盈; 萨日娜; 谭轶滔
Original assignee: Shanghai Huahe Pharmaceutical Co ltd
Current assignee: Shanghai Huahe Pharmaceutical Co ltd
Priority date: 2023-12-25
Filing date: 2023-12-25
Publication date: 2024-06-04

Abstract

The invention discloses an application of a radionuclide marked anti-5T 4 nano antibody in prognosis and diagnosis of cancer. Specifically, the invention discloses an immunoconjugate for detecting 5T4 molecules, which comprises a VHH chain of a specific anti-5T 4 nanobody and a radionuclide, and can be used for non-invasive detection of 5T4 expression of a subject to be detected. The immunoconjugate of the invention has high stability and high specificity, is suitable for the whole body detection of the primary and metastatic tumors targeted simultaneously, and has high accuracy and small radiation dose.

Description

Radionuclide-labeled anti-5T 4 nano antibody and preparation method and application thereof

Technical Field

The invention relates to the biotechnology field and the nuclear medicine field, in particular to a radionuclide-labeled anti-5T 4 nano antibody, and a preparation method and application thereof.

Background

Tumors are important diseases affecting human health, and tumor treatment mainly comprises traditional operation treatment, radiation treatment, chemotherapy and novel treatment modes such as rapid development targeting treatment, immunotherapy and the like in recent years. Targeted drugs have been a hotspot in the development of antitumor drugs. Tumor targeted drugs can be broadly divided into two broad classes, monoclonal antibodies and small molecule compounds.

5T4 is a protein encoded by the TPBG gene on the surface of highly glycosylated cells. 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 expressed in only a limited number of epithelial cells. But is highly expressed in many cancer cells. This makes 5T 4a very attractive target in tumor immunotherapy.

Therefore, the development of 5T 4-targeting agents or drugs is of great importance for the diagnosis and treatment of tumors.

Disclosure of Invention

The invention aims to provide a 5T4 protein-targeted nanobody conjugate, and a preparation method and application thereof.

In a first aspect of the invention there is provided an immunoconjugate comprising:

(a) An antibody moiety selected from a VHH chain of an anti-5T 4 nanobody, or an anti-5T 4 nanobody having said VHH chain, wherein said VHH chain comprises Complementarity Determining Regions (CDRs) as shown below:

CDR1 with the amino acid sequence shown as SEQ ID NO. 2, CDR2 with the amino acid sequence shown as SEQ ID NO. 3 and CDR3 with the amino acid sequence shown as SEQ ID NO. 4; and

(B) A coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a nucleic acid, or a combination thereof.

In another preferred embodiment, the detectable label includes (but is not limited to): radionuclides, luciferin, enzymes, and colloidal gold.

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

In another preferred embodiment, the radionuclide comprises:

(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 combinations thereof; and/or

(Ii) A therapeutic isotope selected from the group consisting of group 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.

In another preferred embodiment, the radionuclide is selected from: ga-68, F-18, I-131.

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%, e.g.96%, 97%, 98%, 99%) sequence identity thereto.

In a second aspect of the present invention, there is provided a radioactive nanoprobe comprising:

(B) A radionuclide coupled to (a).

In another preferred embodiment, the radionuclide comprises:

(Ii) A therapeutic isotope selected from the group consisting of group 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.

In a third aspect of the invention there is provided a 5T4 protein detection reagent comprising an immunoconjugate according to the first aspect of the invention or a radioactive nanoprobe according to the second aspect of the invention, and a detectably acceptable carrier.

In another preferred embodiment, the coupling moiety of the immunoconjugate is a diagnostic isotope.

In another preferred embodiment, the conjugated moiety of the radioactive nanoprobe is a diagnostic isotope.

In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.

In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of: isotope tracer, contrast agent, flow detection reagent, cell immunofluorescence detection reagent, nano magnetic particle and imaging agent.

In another preferred embodiment, the detection reagent is a contrast agent, and the contrast agent further comprises other agents for contrast.

In another preferred embodiment, the contrast agent is a contrast agent for MRI (magnetic resonance imaging) or CT (electronic computed tomography).

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

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

In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as water agent, injection, lyophilized powder, tablet, buccal agent, and aerosol).

In a fourth aspect of the invention there is provided a pharmaceutical composition comprising an immunoconjugate according to the first aspect of the invention or a radioactive nanoprobe according to the second aspect of the invention, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the conjugate moiety of the immunoconjugate is a drug, toxin, and/or therapeutic isotope.

In another preferred embodiment, the conjugated moiety of the radioactive nanoprobe is a therapeutic isotope.

In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating tumors, such as cytotoxic drugs.

In another preferred embodiment, the pharmaceutical composition is used for the treatment or prevention of tumors that express 5T4 protein (i.e., 5T4 positive).

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

in another preferred embodiment, the pharmaceutical composition is in the form of an injection.

In a fifth aspect of the invention, there is provided a kit for detecting a 5T4 protein, the kit comprising a 5T4 protein detection reagent according to the third aspect of the invention and instructions.

In another preferred embodiment, the instructions describe that the kit is for non-invasively detecting 5T4 expression in a subject.

In another preferred embodiment, the subject comprises a human or non-human mammal (e.g., mouse, rat, rabbit, monkey, etc.).

In another preferred embodiment, the kit is used to detect tumors that express 5T4 protein (i.e., 5T4 positive).

In a sixth aspect of the invention, there is provided the use of an immunoconjugate according to the first aspect of the invention or a radioactive nanoprobe according to the second aspect of the invention for the preparation of (1) a detection reagent, a detection kit or a detection plate for detecting 5T4 protein; (2) A pharmaceutical composition for treating or preventing a tumor that expresses a 5T4 protein (i.e., 5T4 positive).

In another preferred embodiment, the immunoconjugate or radioactive nanoprobe is used for preparing a detection reagent, a detection kit or a detection plate for in vivo detection of 5T4 protein.

In a seventh aspect of the invention there is provided a method of preparing a radioactive nanoprobe according to the second aspect of the invention, the method comprising the steps of:

(S1) providing a VHH chain of an anti-5T 4 nanobody or an anti-5T 4 nanobody having said VHH chain;

(S2) adding a radionuclide chelator capable of binding to nanobodies, thereby forming a label precursor;

(S3) charging the radionuclide into the labeled precursor formed in step (S2), thereby forming a radionuclide-labeled nanobody from the radionuclide and the labeled precursor;

(S4) separating the radionuclide-labeled nanobody obtained in the step (S3), thereby obtaining the radioactive nano-probe.

In another preferred embodiment, the method further comprises: and evaluating the prepared radioactive nano probe.

In another preferred embodiment, the radionuclide chelator comprises a bifunctional chelator, a macrocyclic chelator, or a combination thereof.

In another preferred embodiment, the radionuclide chelator is selected from the group consisting of: NOTA, DFO, NODA, TCO, ±h ₃ RESCA-THP, or a combination thereof.

In another preferred embodiment, the VHH chain of the anti-5T 4 nanobody in step (S1) or the anti-5T 4 nanobody having said VHH chain is obtained by post-expression purification of the host cell;

Wherein the host cell comprises a eukaryotic cell and a prokaryotic cell;

Preferably, the host cell is pichia pastoris.

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

(S1) contacting a 5T4 protein of a subject with an immunoconjugate according to the first aspect of the invention, or a radioactive nanoprobe according to the second aspect of the invention;

(S2) detecting the formation of antigen-antibody complex, and if the antigen-antibody complex exists, proving that 5T4 protein exists in the object to be detected.

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

In another preferred embodiment, the method is an in vivo method for non-invasively detecting 5T4 expression in a subject.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the results of an on-the-fly thin layer chromatography assay of radionuclide ⁶⁸ Ga-labeled 5T4 antigen-targeted radioactive probes and in-vivo efficacy assays.

FIG. 2 shows the results of an on-the-fly thin layer chromatography assay of radionuclide ¹⁸ F-labeled 5T4 antigen-targeted radioactive probes and in vivo efficacy assays.

FIG. 3 shows the results of an on-the-fly thin layer chromatography assay of radionuclide ¹³¹ I-labeled 5T4 antigen-targeted radioactive probes and in vivo efficacy assays.

Detailed Description

The present inventors have made extensive and intensive studies to provide, for the first time, a radionuclide-labeled anti-5T 4 nanobody conjugate. The inventor obtains a nanobody probe targeting 5T4 antigen by using a novel bifunctional chelating agent or other chelating agents to couple with the nanobody and then labeling the nanobody with ¹³¹I、⁶⁴Cu、⁶⁸Ga、¹⁸ F and other nuclides. The antigen targeted by the nanobody probe is 5T4, which is glycoprotein highly expressed in embryo trophoblast cells, and the glycoprotein is found by research to be highly expressed in various tumor cells, but rarely expressed in normal tissues. By coupling radionuclides to the nanobody, probes directed against a variety of tumor cells can be prepared, thereby applying them to the accurate diagnosis and treatment of tumors. The nano antibody probe can be used for diagnosing and treating the high-expression 5T4 tumor, and has good clinical application prospect.

On this basis, the present invention has been completed.

Terminology

As used herein, the terms "nanobody of the invention", "anti-5T 4 nanobody of the invention", "nanobody targeting 5T4 antigen" are used interchangeably and refer to a nanobody that specifically recognizes and binds to 5T4 protein. A preferred nanobody VHH chain comprises a CDR1 with an amino acid sequence shown as SEQ ID NO. 2, a CDR2 with an amino acid sequence shown as SEQ ID NO. 3 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 4; particularly preferred are nanobodies of the amino acid sequence of the VHH chain as shown in SEQ ID NO. 1. The nano antibody is expressed from pichia pastoris and has a structure of VHH-GSC.

SEQ ID NO:1

EVQLLESGGGLVQPGGSLRLSCAASGLTYCDSLMYWYRQGPGKGLEFVSFIDRAGRTSYADSVQGRFTISQDNAKNTVYLQMNNLKPEDTAMYYCKITCYNSGYQQWGQGTLVTVSS

Wherein the underlined parts are CDR1, CDR2 and CDR3 in this order, and the sequences separated by CDR regions are FR1, FR2, FR3 and FR4.

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; the constant region of the light chain is opposite the first constant region of the heavy chain and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the terms "single domain antibody", "nanobody" and "nanobody" have the same meaning, and refer to the variable region of a cloned antibody heavy chain, and a single domain antibody (VHH) consisting of only one heavy chain variable region is constructed, which is the smallest antigen-binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and together with the CDRs of the other chain form the antigen binding site of the antibody (see Kabat et al, NIH publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of radionuclides, drugs, toxins, cytokines (cytokines), enzymes and other diagnostic or therapeutic molecules with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the anti-5T 4 nanobody or fragment thereof.

As used herein, the term "heavy chain variable region" is used interchangeably with "V _H".

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (complementarity determining region, CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the coding sequence of the antibody and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which are capable of absorbing DNA, can be obtained after an exponential growth phase and treated by the CaCl ₂ method using procedures well known in the art. Another approach is to use MgCl ₂. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, 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.

Immunoconjugates

The invention provides an immunoconjugate, which comprises:

(a) An antibody moiety selected from a VHH chain of an anti-5T 4 nanobody, or an anti-5T 4 nanobody having said VHH chain, wherein the amino acid sequence of said VHH chain is shown in SEQ ID NO. 1; and

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

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

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

In another preferred embodiment, the conjugate is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticle, etc.

The immunoconjugate of the invention can be used for non-invasively detecting the 5T4 expression level of an object to be detected, and has the advantages of small size, high specificity, low immunogenicity, high accuracy and small radiation dose.

Radioactive nano probe

The invention provides a radioactive nano probe, which comprises (a) an antibody part, which is selected from a VHH chain of an anti-5T 4 nano antibody or an anti-5T 4 nano antibody with the VHH chain, wherein the amino acid sequence of the VHH chain is shown as SEQ ID NO. 1; and (b) a radionuclide coupled to (a).

In one embodiment of the present invention, a ⁶⁸ Ga-labeled radioactive nanoprobe is provided, which is prepared by coupling radionuclide ⁶⁸ Ga to the aforementioned anti-5T 4 nanobody by a chelator of radionuclides (e.g., bifunctional chelator NOTA).

In one embodiment of the present invention, a ¹⁸ F-labeled radioactive nanoprobe is provided, which is prepared by coupling a radionuclide ¹⁸ F to the aforementioned anti-5T 4 nanobody by a chelator of radionuclides (e.g., TCO or.+ -. H ₃ RESCA-THP).

In one embodiment of the present invention, a ¹³¹ I-labeled radioactive nanoprobe is provided, which is prepared by SGMIB conjugated with radionuclide ¹³¹ I and conjugated to the anti-5T 4 nanobody described above.

As used herein, the terms "radioactive nanoprobe", "radioactive probe", "anti-5T 4 nanobody radioactive probe" are used interchangeably and refer to the above probe molecules prepared according to the present invention.

Detection reagent and kit

The invention provides a 5T4 protein detection reagent, which comprises the immunoconjugate or the radioactive nano-probe and a carrier acceptable in detection. In one embodiment of the invention, the immunoconjugate or the conjugated portion of the radioactive nanoprobe is a diagnostic isotope.

Preferably, the detection reagent of the present invention is a reagent for in vivo detection, such as a contrast agent.

The invention also provides a kit containing the immunoconjugate or the radioactive nano-probe, and in a preferred embodiment of the invention, the kit further comprises a container and instructions for use.

Preferably, the kit of the invention is an in vivo diagnostic kit for non-invasively detecting the 5T4 expression level of a test subject.

Pharmaceutical composition

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an immunoconjugate or a radioactive nanoprobe as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical compositions of the invention can be used directly to bind PD-L1 protein molecules and thus can be used to treat tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the immunoconjugate or radioactive nanoprobe of the invention described above, together with a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the conjugates of the invention may also be used with other therapeutic agents.

In using the pharmaceutical composition, a safe and effective amount of the immunoconjugate or the radioactive nanoprobe is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight and in most cases no more than about 50 milligrams/kg body weight, preferably the dose is about 10 micrograms/kg body weight to about 10 milligrams/kg body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The method of the invention

The present invention provides a method for preparing a radioactive nano-probe, in particular, a method for preparing ⁶⁸Ga、¹⁸ F or ¹³¹ I labeled anti-5T 4 nano-antibody radioactive probe.

In one embodiment of the present invention, there is provided a method for preparing ⁶⁸ Ga-labeled anti-5T 4 nanobody radioactive probe, comprising the specific steps of:

(1) Mixing a bifunctional chelating agent NOTA and a nano antibody according to an adaptive proportion, and obtaining a labeled precursor Nb-NOTA under specific conditions;

(2) Antibodies were labeled with ⁶⁸ Ga: leaching ⁶⁸ Ga solution by using 2mL of 0.1M HCl solution, regulating pH value of ⁶⁸ Ga solution to about 4-4.4 by using saturated sodium acetate, uniformly mixing with labeled precursor Nb-NOTA, and reacting for 10-15min at room temperature;

(3) Purifying the reaction solution of the step (2) by using a PD10 column, thereby obtaining the radioactive probe.

In another embodiment of the present invention, there is provided a method for preparing ¹⁸ F-labeled anti-5T 4 nanobody radioactive probe, comprising the specific steps of:

(1) Mixing a chelating agent TCO with a nano antibody according to an adaptive proportion, and obtaining a protein labeling precursor TCO-Nb under specific conditions;

(2) Obtaining ¹⁸ F marked small molecule marked precursor by adopting a click chemistry method, and separating the small molecule marked precursor by using HPLC;

(3) Purifying the collected ¹⁸ F marked small molecule marked precursor, adding the protein marked precursor TCO-Nb in the step (1), and reacting for 10-15min under the adaptive reaction condition;

(4) Purifying the reaction solution of the step (3) by using a PD-10 column, thereby obtaining the radioactive probe.

In yet another embodiment of the present invention, there is provided a method for preparing ¹⁸ F-labeled anti-5T 4 nanobody radioactive probe, comprising the specific steps of:

(1) Mixing a chelating agent+/-H ₃ RESCA-THP with the nano antibody according to an adaptive proportion, reacting for 2-3H at room temperature, and purifying the chelating reaction liquid by using PD-10 to obtain a chelating precursor+/-H ₃ RESCA-THP-Nb;

(2) The ¹⁸ F aqueous solution produced by the cyclotron was passed through an activated QMA column and washed with 0.9% sodium chloride solution to give Na ¹⁸ F solution.

(3) Adding a specific amount of AlCl ₃ solution into Na ¹⁸ F solution, reacting for 5min, adding chelation precursor + -H ₃ RESCA-THP-Nb into the solution, and reacting for 10min;

In yet another embodiment of the present invention, there is provided a method of preparing ¹³¹ I-labeled anti-5T 4 nanobody radioactive probe, comprising the specific steps of:

(1) SGMTB and radioisotope ¹³¹ I are reacted for 15min at room temperature to obtain a small molecular marker precursor, water with 0.1% TFA is used as an A phase and acetonitrile is used as a B phase by HPLC, and the ratio of the two phases of A, B is adjusted to obtain the marker precursor SGMIB with the radiochemical purity of more than 95%;

(2) The labeled precursor SGMIB was reacted with antibody Nb-H006 at 37℃for 30min.

(3) Purifying the reaction solution of the step (2) by using a PD-10 column, thereby obtaining the radioactive probe.

The invention has the main advantages that:

(1) The antibody related to the immunoconjugate or the radioactive nano-probe is a nano-antibody expressed by a Pichia pastoris system, the structure of the antibody is VHH, and the antibody has high specific binding to a 5T4 antigen.

(2) The immunoconjugate or the radioactive nano-probe prepared by the invention has the advantages of high stability, low immunogenicity, low production cost and the like.

EXAMPLE 1 preparation of radionuclide ⁶⁸ Ga-labeled 5T4 antigen-targeting radiolabel

The embodiment provides ⁶⁸ Ga-labeled nanobody Nb and a preparation method thereof, concrete preparation the method comprises the following steps:

(1)

To 0.5mg nanobody Nb-H006 (PBS as solvent) was added dropwise 12-fold molar amount of P-SCN-Bn-NOTA (dissolved in DMSO) of nanobody, the two were mixed well, pH was adjusted to 8.5-8.7 using 0.05M sodium bicarbonate/sodium carbonate mixture, and incubated with shaking at room temperature for 2H. The reaction was stopped by adjusting the pH to 7.0-7.4 using 1N hydrochloric acid.

The PD-10 desalting column was pre-equilibrated with 5X 5mL of PBS, the effluent was discarded, the reaction solution was put on the desalting column, the first 2.5mL of effluent was discarded, the labeled precursor was eluted with 3.5mL of PBS, and the eluate was concentrated using a 10kDa ultrafiltration centrifuge tube. A labeled precursor is obtained.

(2)

⁶⁸ Ga was eluted from the generator using 0.1M hydrochloric acid and the pH was adjusted to 4.4-4.6 using saturated sodium acetate solution. The labeling precursor is put into a ⁶⁸ Ga solution which is regulated, uniformly mixed, reacted for 5-10min at room temperature, the labeling yield is detected by using iTLC, and when the labeling yield reaches about 90%, the product is purified by using PD MINITRAP G-25, wherein PD MINITRAP G-25 is activated by using 3 x 2.5mL PBS in advance. After the sample entered the packed bed, elution was performed using 1mL PBS and the eluate was collected to give the labeled product. The radiochemical purity is more than 95% by radioactive Instant Thin Layer Chromatography (iTLC).

The obtained labeled product is used for detecting the in vivo effect, and the method comprises the following steps: tumor bearing mice were injected with 100uL (about 100 uCi) of the labeled product intravenously and imaged using small animal PET/CT at specific time points.

The in-vivo effect of the product by instant thin layer chromatography is shown in figure 1. Radioactive instant chromatography showed that the labeled product was > 95% radiochemical purity.

The results showed that at this time point, after 30min of tail vein injection, significant accumulation of the labeled product at the tumor site was observed using PET/CT scan of the small animals.

Example 2 preparation of radionuclide ¹⁸ F-labeled 5T4 antigen-targeting radiolabeled Probe Using click chemistry

The ¹⁸ F labeled nanobody Nb and the preparation method thereof provided in the embodiment are as follows:

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

To 0.5mg nanobody Nb-H006 (PBS as solvent) was added dropwise ± H ₃ RESCA-THP chelator dissolved in DMSO, at a chelator to antibody ratio of about 10:1, after being uniformly mixed, the reaction liquid is placed on a shaking table to react for 2-3 hours at room temperature.

The reaction solution was put on a desalting column, the effluent was discarded, 3X 2.5mL of sodium acetate (pH 4.4) was pre-equilibrated, MINITRAPG-25 desalting column was pre-equilibrated, and the first 0.5mL of effluent was discarded, and the protein-labeled precursor was eluted with 1mL of sodium acetate (pH 4.4) to obtain TCO-Nb protein-labeled precursor.

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

[ F-18] FBA-OSu is obtained by the above chemical method, and the specific embodiments are as follows: passing the 18F-containing aqueous solution produced by the cyclotron through Light QMA column, 18F-enriched, followed by KOTf solution (10 mg/1mL H ₂ O) (100, 200 uL) was used to release ¹⁸ F on the QMA column into the labeling tube. 1mL of MeCN was added, and the mixture was concentrated under a nitrogen stream at 100℃and repeated 3 to 5 times. K ¹⁸ F was obtained. Cooling, adding the pre-reaction liquid, and reacting at 100 ℃ for 5min. Separation by HPLC. The resulting product was isolated and purified by C18 column, eluting the labeled precursor [ F-18] FBA-OSu with ethanol, and then blow-dried by nitrogen flow.

(3) Uniformly mixing a protein labeling precursor TCO-Nb and [ F-18] FBA-OSu, reacting for 10-15min at room temperature, and purifying by using PD-10 to obtain a labeling product. Radiochemical purity > 95% was detected using radioactive instant thin layer chromatography.

Using the obtained labeled product, in vivo efficacy was examined in the same manner as in example 1.

The in vivo effect of the labeled product on the instant thin layer chromatography is shown in FIG. 2. Radioactive instant chromatography showed that the labeled product was > 95% radiochemical purity.

Example 3 preparation of radionuclide ¹³¹ I-labeled 5T4 antigen-targeting radiolabeled probe

The ¹³¹ I labeled nanobody Nb and the preparation method thereof provided by the embodiment are as follows:

(1)

Na ¹³¹ I (5 mci, about 50. Mu.L) was added to a 3% methanol solution of acetic acid to adjust pH to acidity, 1: 1mg SGMTB was dissolved in methanol, 10. Mu.L (50. Mu.g) was added to a flask, and the mixture was reacted at room temperature for 15 minutes, and methanol was evaporated with nitrogen gas. The reaction product was co-evaporated twice with acetonitrile to remove impurities, and then dissolved with an appropriate amount of acetonitrile, and separated by HPLC to obtain a labeled precursor.

(2) Nanobody Nb-H006 was dissolved using borate solution at ph8.5, added to the labeling precursor, reacted for 30min to obtain the labeled product, and purified using PD-10 column. Radiochemical purity > 95% was detected using radioactive instant thin layer chromatography.

The in vivo effect of the labeled product on the instant thin layer chromatography is shown in FIG. 3. Radioactive instant chromatography showed that the labeled product was > 95% radiochemical purity.

After 60min tail vein injection, a clear accumulation of the labeled product at the tumor site was observed at this time point using small animal SPECT/CT scan.

In conclusion, the nanometer antibody Nb targeting the human 5T4 antigen has higher specificity on the 5T4 antigen, and can be used for preparing various antibody conjugates, wherein the preparation method of the radionuclide ⁶⁸Ga、¹⁸ F-labeled radioactive probe provided by the invention has high labeling rate, radiochemical purity, good in-vivo and in-vitro stability, high binding force on the 5T4 antigen and good imaging effect, and can be used for accurately diagnosing and treating 5T4 positive tumor focus. The radionuclide labeled nano antibody is expected to be a radioactive probe with good application prospect.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. An immunoconjugate, said immunoconjugate comprising:

2. The immunoconjugate of claim 1, wherein 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 radioactive nanoprobe, wherein the radioactive nanoprobe comprises:

(B) A radionuclide coupled to (a).

4. The radioactive nanoprobe of claim 3, wherein said radionuclide comprises:

5. The radioactive nanoprobe of claim 3, wherein the amino acid sequence of the VHH strand is as shown in SEQ ID No. 1 or has at least 80% sequence identity thereto.

6. A 5T4 protein detection reagent comprising the immunoconjugate of claim 1 or the radioactive nanoprobe of claim 3, and a detectably acceptable carrier.

7. A pharmaceutical composition comprising the immunoconjugate of claim 1 or the radioactive nanoprobe of claim 3, and a pharmaceutically acceptable carrier.

8. A kit for detecting a 5T4 protein, comprising the 5T4 protein detection reagent of claim 6 and instructions.

9. Use of the immunoconjugate of claim 1 or the radioactive nanoprobe of claim 3 for the preparation of (1) a detection reagent, a detection kit or a detection plate for detecting 5T4 protein; (2) A pharmaceutical composition for treating or preventing a tumor that expresses a 5T4 protein (i.e., 5T4 positive).

10. A method of preparing the radioactive nanoprobe of claim 3, said method comprising the steps of: