WO2023164956A1 - 抗体偶联近红外ii区荧光探针、构建方法和应用 - Google Patents

抗体偶联近红外ii区荧光探针、构建方法和应用 Download PDF

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WO2023164956A1
WO2023164956A1 PCT/CN2022/079704 CN2022079704W WO2023164956A1 WO 2023164956 A1 WO2023164956 A1 WO 2023164956A1 CN 2022079704 W CN2022079704 W CN 2022079704W WO 2023164956 A1 WO2023164956 A1 WO 2023164956A1
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casr
parathyroid
antibody
targeting
fluorescent
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French (fr)
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郑伟慧
郭鹏
陈帆
程向东
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浙江省肿瘤医院
中国科学院基础医学与肿瘤研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones

Definitions

  • the invention belongs to the field of thyroid and parathyroid surgery, and relates to an antibody-coupled near-infrared region II fluorescent probe, a construction method and application thereof, in particular to an antibody-coupled near-infrared region II fluorescent probe and its construction method and application
  • the present invention implements surgical navigation and targeted identification of parathyroid glands by constructing specific CaSR molecular probes.
  • the parathyroid gland is an essential gland for life, and its main function is to regulate blood calcium in the human body.
  • Parathyroid glands have important functions and are difficult to identify. Surgery can easily cause hypoparathyroidism, and the missection rate is high. Therefore, accurate identification of parathyroid glands during surgery has important practical significance and is an important issue related to human health. Animal experiments have proved that if the gland is completely removed, the blood calcium content will drop sharply, and the muscles will spasm violently, leading to death. Clinically, the serious complications brought about by impaired parathyroid function even exceed the harm of thyroid cancer. Numbness of the hands and feet, general convulsions, neurasthenia, and spasm of the diaphragm. People with permanent hypoparathyroidism must rely on calcium supplements for life, which will cause a series of sequelae of calcium disorders such as kidney stones, gallstones, soft tissue calcification, cataracts, and central nervous system damage in the long run.
  • the parathyroid gland is the smallest organ in the human body, like a soybean. Each gland is 3-8mm long, 2-5mm wide, and 0.5-2mm thick. The total weight is less than 0.3g.
  • the position of the parathyroid gland is not fixed. The inferior parathyroid gland descends together with the thymus during embryonic development, and can appear anywhere from the mandibular angle to the pericardium, or even be buried in the thyroid gland.
  • the number of parathyroid glands is not fixed, usually 4, but there are also 3, or even 7.
  • the color of the parathyroid glands changes with age.
  • the surrounding tissues of the parathyroid glands are similar in color to the thyroid gland, and similar in shape to lymph nodes and fat. Therefore, it has been a medical challenge to accurately and quickly identify all parathyroid glands with the naked eye of a surgeon.
  • Impaired parathyroid function is one of the most common complications after thyroid surgery. Due to the huge amount of thyroid surgery, the parathyroid glands are at risk during surgery. In 2020, there will be 586,278 new cases of thyroid cancer worldwide. The incidence of the main thyroid cancer subtypes is increasing in 25 countries around the world.
  • the current parathyroid identification technology cannot meet this medical need.
  • the first type of negative imaging is to highlight the parathyroid glands by changing the color of the thyroid gland and lymph nodes because the parathyroid glands are small and their color is close to that of the thyroid gland.
  • the main applications are nano-carbon and mitoxantrone.
  • Nano-carbon is a carbon nano-material. Because carbon cannot be degraded in the human body, DNA will be damaged during cell replication, which has safety problems. Nano-carbon is easy to leak and easily pollutes the operation area.
  • Mitoxantrone is an anthracycline chemotherapeutic drug, which is a non-specific drug for the cell cycle and is toxic to normal cells.
  • the second category is the positive development of parathyroid glands, mainly including 5-aminolevulinic acid, indocyanine green, and methylene blue.
  • the positive contrast agent is mainly administered through systemic intravenous injection, but it is not targeted, and the dosage is large and the side effects are serious.
  • the third type is autofluorescence imaging of parathyroid glands. Autofluorescence has certain advantages, but due to weak autofluorescence, the tissue penetration depth is limited, and the surface of parathyroid glands is often covered with adipose tissue, so it cannot penetrate the fluorescence well. In addition, due to the common blood stains in the surgical area, metastatic lymph nodes, brown fat, collagen nodules, etc., it is easy to misjudgment and have certain false positives under autofluorescence imaging, so it has not been widely used clinically.
  • the present invention firstly determines the specifically highly expressed CaSR target on the surface of the parathyroid principal cell membrane, and then connects CaSR to different near-infrared region II fluorescent molecules (or near-infrared II region fluorescent molecules, near-infrared region II fluorescent molecules) to form antibody fluorescent conjugates. Lianwu, through animal experiments, finally screened the fluorescent molecular probes in the near-infrared II region with the best imaging effect, and used them for the development of parathyroid glands during surgery.
  • the purpose of the present invention is to reduce the missection rate of parathyroid glands during operation and reduce postoperative complications of hypoparathyroidism.
  • One of the objectives of the present invention is to provide the application of CaSR-targeting substances in the preparation of imaging agents.
  • the contrast agent is a parathyroid positive contrast agent; preferably, the contrast agent is used for intraoperative navigation of thyroid gland, parathyroid gland and/or lymph node tissue.
  • the substance targeting CaSR is an antibody, and the antibody is obtained by immunizing animals with at least one polypeptide antigen shown in SEQ ID NO.4-5;
  • the substance targeting CaSR is a monoclonal antibody, and the monoclonal antibody is obtained by immunizing animals with any one of the polypeptide antigens shown in SEQ ID NO.4-5; preferably, the SEQ ID NO.4 The C-terminus of ⁇ 5 is connected to cysteine;
  • the substance targeting CaSR is a monoclonal antibody, and the monoclonal antibody is obtained by immunizing animals with any one of the polypeptide antigens shown in SEQ ID NO.4-5; preferably, the SEQ ID NO.4
  • the expression region of ⁇ 5 is shown in SEQ ID NO.6;
  • the CaSR-targeting substance is selected from double-stranded RNA, shRNA or small molecule compounds.
  • the CaSR-targeting substance is coupled with a fluorescent molecule, which is an antibody-coupled fluorescent molecular probe;
  • the CaSR-targeting substance is coupled with a fluorescent molecule, which is an antibody-coupled fluorescent molecular probe, and the CaSR-targeting substance is directly coupled to the fluorescent molecule;
  • the CaSR-targeting substance is coupled with a fluorescent molecule, which is an antibody-coupled fluorescent molecular probe, and the CaSR-targeting substance is connected to the fluorescent molecule through a linker; preferably, the linker is selected from At least one of non-cleavable, reductively cleavable and protease cleavable, as shown in FIG. 16 .
  • the fluorescent molecule is a fluorescent molecule in the near-infrared II region; preferably, the fluorescent molecule in the near-infrared II region is an IRDye800 fluorescent molecule, and its structure is shown in Formula 1:
  • the CaSR-targeting substance when the CaSR-targeting substance is a monoclonal antibody, and when the antibody is directly coupled to a fluorescent molecule, it is expressed as CaSR-IRDye800 antibody-coupled fluorescent molecular probe, wherein the lysine in the antibody is The amino group is coupled to the n-hydroxysuccinimide ester on the fluorescent molecule IRDYe800.
  • one or more fluorescent molecules are coupled to the CaSR-targeting substance.
  • the fluorescence of the fluorescent molecules is obtained in the near-infrared II region.
  • Another object of the present invention is to provide a complex, which comprises a CaSR-targeted substance; preferably, the complex is selected from one or more of imaging agents, diagnostic tracers, and pharmaceutical compositions. kind.
  • the developing agent is a positive developing agent for parathyroid glands, and the positive developing agent for parathyroid glands is the above-mentioned antibody-coupled fluorescent molecular probe.
  • Another object of the present invention is to provide a method for screening effective drugs for the treatment of thyroid and/or lymph nodes, characterized in that the CaSR-targeted substance and drug molecule are constructed on the carrier, and the thyroid gland is stimulated by the CaSR-targeted substance. Positive imaging of paraglandular glands, evaluation of the effectiveness of drug molecules, and screening of drugs that are effective in the treatment of thyroid and/or lymph nodes.
  • the present invention starts from reducing the missection rate of parathyroid glands, and draws on the design principle of antibody-drug conjugate (ADC) through highly expressed targets of parathyroid glands and fluorescent molecular probes.
  • ADC antibody-drug conjugate
  • Molecular targeting of specific parathyroid gland tissues enables precise intraoperative identification and real-time navigation.
  • the application of this concept will provide a new research idea for the field of surgery and intraoperative imaging of important normal organs.
  • FIG. 1 Positive imaging of the parathyroid glands.
  • the parathyroid glands are located dorsally to the thyroid gland and are targeted by near-infrared II fluorescence for intraoperative real-time navigation.
  • Calcium-sensing receptors recognize targets on the membrane surface of parathyroid principal cells through linker-coupled near-infrared II fluorescent molecules.
  • C Schematic diagram of the chemical reaction of calcium-sensing receptor coupled with IRDye800 fluorescent molecular probe.
  • Figure 3 Calcium sensitive receptor monoclonal antibody sequence analysis and protein detection.
  • Figure 3A Sequence analysis based on human sequences (human, mouse, rat);
  • Figure 3B conserveed domains;
  • Figure 3C Homologous sequences are 96.8% homologous to rabbit sequences in the UniProt database;
  • Figure 3D Antigen preparation, preparation of antigen expression and purification of recombinant protein by synthesizing polypeptides and polypeptide coupling, the following are: M 20812, CHE 20801, E1, E2, 20854, 20847, 20846, 20845.
  • Figure 4 is a post-translational modification map; wherein, amino acids at positions 90, 130, 261, 287, 386, 400, 446, 468, 488, 541, and 594 have GlcNAc glycosylation modifications, and amino acids at positions 920 and 1061 There are phosphorylation modifications. In addition, disulfide bonds were formed between amino acids 60 and 101, 236 and 561, 358 and 395, 437 and 449, 542 and 562, 546 and 565, 568 and 582, and 585 and 598.
  • FIG. 5 Diagram showing the transmembrane domain analysis.
  • the extracellular regions are: 20-612aa, 671-681aa, 746-769aa, 829-836aa.
  • the transmembrane regions are: 613-635aa, 650-670aa, 682-700aa, 725-745aa, 770-792aaa, 806-828aa, 837-862aa.
  • Intracellular region 636-649aa, 701-724aa, 793-805aa, 863-1078aa.
  • Figure 6 is a signal peptide analysis diagram; the predicted signal peptide is 1-19aa.
  • FIG. 1 Schematic diagram of antibody preparation process.
  • FIG. 8 Immunohistochemical results of expression of calcium-sensing receptor targets in human parathyroid glands.
  • A HE staining and corresponding calcium-sensing receptor protein (CaSR) of parathyroid gland (Parathyroid Gland, PG), lymph node (Lymph Node, LN), thyroid gland (Thyroid Gland, TG) in the same tissue section of the same patient Immunohistochemical picture (2X).
  • B Immunohistochemical images of CaSR in the parathyroid glands of 3 different patients, multiples (10X, 20X, 40X). CaSR staining intensity > 95%, strong positive (+++).
  • FIG. 9 Localization of CaSR content in human parathyroid gland tissue and expression of CaSR protein in rats.
  • CaSR is highly expressed in human hyperplastic parathyroid tissue, and there are three peaks, namely PBS, IgG and CaSR.
  • B Human hyperplastic parathyroid tissue was made into a single cell suspension, and CaSR was expressed on the surface of the cell membrane.
  • B Rat parathyroid tissue highly expresses CaSR, lymph nodes and thyroid are negative.
  • FIG. 10 Animal anatomical model establishment of rat parathyroid gland.
  • A Anatomical diagram of a live rat, the neck is exposed and depilated, the front of the neck is cut in the middle, the anterior neck muscle is stretched to both sides, the thyroid gland and the left and right parathyroid glands are identified with the naked eye, the size of the laryngeal tissue in vitro is about 6mm*10mm, and the thyroid gland is 3mm *4mm, parathyroid gland 1mm*2mm.
  • B Pathological HE-stained serial sections to find the parathyroid gland layer, and the section shows the tissues around the parathyroid gland: thyroid cartilage, trachea, esophagus, thyroid gland, and anterior cervical band muscle and other structures.
  • C Pathological HE staining confirms the position of the parathyroid gland in rats, which is close to the surface of the thyroid gland, with a complete outer membrane and a dense structure.
  • FIG. 11 New Zealand rabbit parathyroid animal anatomical model establishment.
  • A Anatomical map of New Zealand in vivo. Anterior median incision was made on the neck. The trachea was exposed after the anterior cervical muscle was separated. The thyroid gland and parathyroid gland were identified with the naked eye.
  • B Frontal view of New Zealand rabbit vivisection, showing thyroid cartilage, trachea, anterior cervical band muscle, thyroid, etc. The parathyroid glands are close to the muscles, red in color, smooth in surface, strip-shaped, bilaterally asymmetrical and scattered.
  • C Pathological HE staining confirmed the parathyroid gland tissue (blue) of New Zealand rabbit, and the surrounding adipose tissue (white) in the upper left corner.
  • D After zooming in on the local area, abundant chief cells and eosinophils can be seen in the parathyroid tissue (bar 50um).
  • CaSR/IgG-IRDYe800 compound characterizes imaging in the near-infrared I/II region.
  • A Fluorescence spectra of CaSR-IRDYe800 and different gradient concentrations of IRDYe800.
  • C Imaging images of CaSR-IRDYe8 and the control in the near-infrared I region.
  • FIG. 13 Near infrared region II imaging of CaSR-IRDYe800 compound after local injection in mice.
  • A Near-infrared II general fluorescence imaging of IRDYe800 after local injection of pectoralis major, right thyroid, and left parathyroid in rats.
  • B Pseudo-color HOT fluorescence imaging in the near-infrared II zone after local injection of IRDYe800.
  • C Fluorescence imaging of the parathyroid glands of mice locally injected with CaSR-IRDYe800 in the near-infrared region I.
  • D Near-infrared II region bright-field imaging of the parathyroid glands of mice locally injected with CaSR-IRDYe800.
  • E Fluorescence and pseudo-color imaging in the near-infrared II region of the parathyroid glands of mice injected locally with CaSR-IRDYe800 (different exposure times).
  • F Fluorescence and pseudocolor imaging of isolated laryngeal tissue after local injection of CaSR-IRDYe800 into mouse parathyroid glands.
  • FIG. 14 In vivo CaSR/IgG-IRDYe800 compound CaSR/IgG-IRDYe800 compound after tail vein injection for 24 hours and near-infrared region II imaging.
  • A Bright-field and fluorescence images of the near-infrared region II after tail vein injection of mice (normal saline, CaSR-IRDYe800, IgG-IRDYe800).
  • B Normal fluorescence and pseudo-color HOT images in the near-infrared II region of CaSR-IRDYe800 mice after tail vein injection for 24 hours.
  • C Normal fluorescence and pseudo-color HOT images in the near-infrared II region of CaSR-IRDYe800 mice after tail vein injection for 24 hours.
  • Fig. 15 Real-time navigation parathyroid identification diagram during operation in Example 2.
  • A Opto Medic imaging system, including host, fluorescent probe, and monitor.
  • B Frontal view of SD rats, before and after injection of the fluorescence patterns, it is difficult to identify the parathyroid glands before the injection, but after the injection, the bilateral parathyroid glands are clearly identifiable and the fluorescence signal is strong.
  • C The right side view of SD rats, the fluorescence patterns before and after injection, the right parathyroid gland was difficult to identify before injection, but the right parathyroid gland was clearly identifiable after injection, with strong fluorescence signal.
  • D The left side view of SD rats, the fluorescence patterns before and after injection, the parathyroid glands were difficult to identify before injection, but the left parathyroid glands were clearly identifiable after injection, with strong fluorescence signals.
  • Figure 16 Schematic diagram of the connection of antibodies and fluorescent molecules through three linkers.
  • ADC antibody drug conjugate
  • G protein-coupled receptors sense changes in extracellular calcium ion concentrations and play a key role in maintaining calcium homeostasis. Senses fluctuations in circulating calcium concentrations and regulates parathyroid hormone (PTH) production in the parathyroid glands (by similarity). The activity of this receptor is mediated by G proteins that activate the phosphatidylinositol-calcium second messenger system.
  • PTH parathyroid hormone
  • CaSR calcium-sensitive receptor protein
  • CaSR is a member of the G protein-coupled receptor family. It is mainly expressed specifically in the parathyroid gland and senses changes in the concentration of extracellular blood calcium. When the blood calcium decreases, CaSR regulates the parathyroid cells to secrete parathyroid hormone and mobilizes bone calcium into the body. Blood, maintain normal physiological concentration of blood calcium (2.25 ⁇ 2.75mmol/L). CaSR plays a key role in accurately maintaining calcium homeostasis in the human body. It is through CaSR that the parathyroid glands complete the physiological function of regulating blood calcium levels in the body. In order to achieve positive development of parathyroid glands, targeting CaSR is an ideal target: First, CaSR is located in the cell membrane and can be targeted and recognized without entering the cell. Secondly, the expression of CaSR in other human tissues is relatively small and weak, and the target specificity is strong. Finally, the CaSR protein sequence is conserved, with high homology among different species, and is widely used.
  • the inventor has completed the design and preparation of the CaSR antibody in the previous research, and the antigen design is aimed at the CaSR protein of three species of human, rat, and mouse (CaSR protein size (aa): 1078; the sequences are respectively as SEQ ID NO.1 ⁇ 3), the homology comparison analysis is shown in Figure 2 (the homology of the three is 92.9%), and the protein sequence is analyzed with human sequence ( Figure 3A-B). The homology of the three is 92.9%, compared with the rabbit sequence in the UniProt database, the homology is 96.8% (Fig. 3C).
  • the monoclonal antibody of the calcium-sensing receptor satisfies the follow-up animal experiments in terms of species and quantity, and can be used in clinical research even after the antibody is humanized.
  • amino acids at positions 90, 130, 261, 287, 386, 400, 446, 468, 488, 541, and 594 have GlcNAc glycosylation modifications, and amino acids at positions 920 and 1061 exist Phosphorylation modification.
  • disulfide bonds were formed between amino acids 60 and 101, 236 and 561, 358 and 395, 437 and 449, 542 and 562, 546 and 565, 568 and 582, and 585 and 598.
  • transmembrane domain analysis is shown in Figure 5, the extracellular region is: 20-612aa, 671-681aa, 746-769aa, 829-836aa.
  • the transmembrane regions are: 613-635aa, 650-670aa, 682-700aa, 725-745aa, 770-792aaa, 806-828aa, 837-862aa.
  • Intracellular region 636-649aa, 701-724aa, 793-805aa, 863-1078aa.
  • the signal peptide analysis is shown in Figure 6, and the predicted signal peptide is 1-19aa.
  • the peptide technical route was 20-32aa, the sequence was YGPDQRAQKKGDI (SEQ ID NO.4), which was located in the extracellular region of the CaSR protein, and the other polypeptide sequence was 212 -225aa, the sequence is IAADDDYGRPGIEK (SEQ ID NO.5), which is located in the extracellular region of the CaSR protein.
  • polypeptide antigen For the polypeptide antigen, it needs to be coupled with the carrier protein KLH for animal immunization. Therefore, cysteine will be added to the C-terminal of the polypeptide antigen sequence for the coupling of the carrier protein.
  • the principle of selection analyze the antigenicity of itself, the hydrophilicity of the sequence, and the exposure of the epitope, and select the final appropriate segment as the antigenic sequence.
  • the preparation process is shown in Figure 7.
  • Cell line screening, ascites preparation, and monoclonal antibody purification are prepared by general recombinant methods. At this stage, the whole gene has been synthesized to protein expression and purification, and the expression region is 448-610aa. The sequence of this fragment is as follows:
  • this project can express and purify the expected target protein, which can meet the immune requirements.
  • Quality control standard Use indirect ELISA to analyze the titer and production level of antibodies in mouse serum.
  • the titer level is 1:72900 diluted OD450nm value is greater than 0.3, and a small serum sample is sent to the customer for verification.
  • Indirect ELISA was used to analyze the titer of the antibody in the ascites, and at the same time to analyze the subtype.
  • the actual antibody subtype choose an appropriate purification method to purify the antibody, and obtain two purified monoclonal antibodies, that is, antibodies targeting CaSR.
  • the present inventors detected the specific high expression of CaSR in human parathyroid tissue sections by immunohistochemical method, and proved that the expression of CaSR was on the cell membrane surface of parathyroid principal cells ( FIG. 8 ).
  • a total of 24 patients with thyroid cancer were extracted, and a small amount of parathyroid gland tissue was occasionally found in the postoperative pathology, and the tissue of the central lymph node dissection was white.
  • CaSR protein was selectively expressed on the surface of parathyroid gland cells, but negatively expressed in thyroid and lymph nodes, which suggested that CaSR protein was a specific expression target of parathyroid glands.
  • the expression intensity of 1 patient was 60%
  • the expression intensity of 2 patients was 80%
  • the expression intensity of the remaining 21 patients were all >95% (+++).
  • FIG. 9A-B Through flow cytometry experiments, it was confirmed that the CaSR protein content in the fresh tissue single cell suspension of human diseased parathyroid glands was high ( FIG. 9A-B ).
  • Patients with clinically suspected thyroid cancer and parathyroid hyperplasia signed informed consent.
  • the hyperplastic parathyroid gland and the surrounding thyroid tissue were taken, cut into pieces, and lysed to make a single-cell suspension.
  • CaSR fluorescent antibody was incubated for 45 minutes, and after washing, the main cell population was collected by flow cytometry (CytoFLEX) at around 10 3 .
  • the expression level of CaSR in human hyperplastic parathyroid gland was 11188, the control IgG antibody was 4567, and the control PBS solution was 111, the peaks were independent and separated (Fig. 9A).
  • laser confocal microscopy ZEISS was used to observe the parathyroid staining ( FIG. 9B ).
  • the inventor has obtained the animal experiment qualification certificate (Certificate No. 22-09016), and through the anatomical study of rats and New Zealand rabbits, combined with the experience of human neck surgery, the establishment of rat and New Zealand rabbit thyroid Anatomical model of paraglandular glands (Fig. 10, 11).
  • the ethics approval document (ethics batch number 2021-05-002) was obtained from the animal ethics committee.
  • potassium chloride was administered intravenously to kill.
  • the larynx (thyroid and parathyroid glands are too small) of SD rats were excised in vitro, including thyroid cartilage, epiglottis, thyroid, parathyroid gland, trachea, esophagus and other structures, which were immersed in formaldehyde solution. Entirely excised parathyroid glands of New Zealand rabbits were immersed in formaldehyde.
  • HE staining Hematoxylin-eosin staining
  • the parathyroid glands of SD rats are small, and it is difficult to slice them separately.
  • a total of 16 white slices were cut, and each layer was less than 1mm.
  • the bilateral parathyroid glands were cut in the same slice (Fig. 10B).
  • New Zealand rabbit parathyroid tissue was fixed separately.
  • HE staining baked slices for 5 minutes, xylene dewaxing for 3 times for 5 minutes each, gradient alcohol for 4 times for 1 minute each, water washing, hematoxylin staining for 3 minutes and 30 seconds, water washing, eosin for 40 seconds, gradient alcohol for 5 times for 1 minute each , xylene for 30 seconds, and finally sealed with a sealing machine.
  • the results of HE staining were diagnosed by two senior experts from the Clinical Pathology Department of the Cancer Hospital. Microscopically, parathyroid tissue was found to be rich in chief cells and eosinophils (Fig. 10C, Fig. 11C-D).
  • the schematic diagram of the synthesis principle is shown ( FIG. 1C ).
  • Three groups were set up, namely CaSR-IRDye800, IgG-IRDye800 and pure IRDye800.
  • the molar masses of antibodies and fluorescent molecules are configured in a ratio of 1:10. Configure it into a 500ul system, shake it overnight for 12 hours, and keep it away from light. Unbound antibody dye was separated by ultrafiltration (30 kD cut-off). Through dialysis bags, purified under the action of a magnetic stirrer.
  • the antibody concentration was determined by the BCA method (Enhanced BCA protein Assay kit), and a standard concentration gradient was configured to draw a standard curve. The 96-well plate was incubated for 30 minutes, and the absorbance value at 562nm was detected on the machine, which was brought into the standard curve to obtain the antibody concentration. According to molecular weight, obtain molar mass 3.185nmol.
  • the synthesized CaSR-IRDYe800 conjugate was imaged under a near-infrared I region instrument, and the fluorescence intensity of the conjugate was seen to be around 10 10 ( FIG. 12C ).
  • 200ul of the synthesized conjugate was covered with 1mm, 3mm, and 5mm thick chicken breasts and imaged. It was found that the fluorescence intensity of 1mm and 3mm was about 109 , and almost no fluorescence could be seen at 5mm thickness (Fig. 12D).
  • a negative control IgG-IRDYe800 was also synthesized at the same time (FIG. 12G). The remaining synthetic compounds were stored lyophilized.
  • the present inventors synthesized CaSR-IRDYe800 conjugates, and observed the preliminary imaging effect through two different ways of injection into the local anatomical area of mice and tail vein injection (Figure 13).
  • IRDYe800 was injected into the local anatomical area of the rat, including the pectoralis major, right thyroid gland, and left parathyroid gland, and it was found that the fluorescence intensity was high in the near-infrared II region ( Figure 13).
  • Figure 13 We locally injected the synthesized CaSR-IRDYe800 conjugate into bilateral mouse parathyroid glands. Under the near-infrared I region, we can see that there are weak fluorescent signals in both thyroid regions, but the signals are not focused, and the parathyroid glands cannot be distinguished. Gland (Fig. 13C). Then, observed under near-infrared II region, bright field (Fig.
  • Physiological saline, CaSR-IRDYe800 and IgG-IRDYe800 were injected into the tail vein respectively, and imaged in the near-infrared II region 24 hours later ( Figure 14).
  • Fig. 14A 3 mice were observed under bright field and normal fluorescence (Fig. 14A), and the IgG-IRDYe800 conjugate was found to be fluorescently deposited in the liver, but no obvious fluorescent signal was seen.
  • Fig. 14B, C we observed the injected CaSR-IRDYe800 and CaSR-IRDYe800 under ordinary fluorescence and pseudo-color HOT respectively (Fig. 14B, C). Bilateral symmetrical fluorescence signals were found in the neck of CaSR-IRDYe800, but no neck signal in IgG-IRDYe800 (Fig. 14C).
  • the system is Opto Medic imaging research equipment provided by Hangzhou Weiyuan Company ( Figure 15A).
  • the fluorescence excitation light wavelength currently used is 805nm, and the near-infrared zone II navigation will be carried out in the future, and real-time navigation imaging will be carried out after systemic intravenous injection at the same time.
  • the surgical preparation of SD rats take SD female rats, weighing 300g. Isoflurane inhalation anesthesia, hair removal, neck supine position, occipital padded, the same as the clinical conventional thyroid surgery position. Disinfection, draping, incision in the middle of the neck, and operation were performed as described above for the establishment of the rat parathyroid animal model. The bilateral anterior cervical muscle retractors are pulled to the outside to fully expose the thyroid gland, trachea, larynx and other organ tissues.

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Abstract

在甲状腺及甲状旁腺外科领域，涉及一种抗体偶联近红外II区荧光探针、构建方法和应用，应用具体为靶向CaSR的物质在制备显影剂尤其是甲状旁腺正显影剂中的应用。靶向CaSR的物质为抗体，其通过连接子共价偶联有荧光分子，可以靶向特定甲状旁腺组织，能实现术中精准辨识，实时导航，减少甲状旁腺误切率，应用前景广泛。

Description

抗体偶联近红外II区荧光探针、构建方法和应用 技术领域
本发明属于甲状腺及甲状旁腺外科领域，涉及一种抗体偶联近红外II区荧光探针、构建方法和应用，具体涉及一种抗体偶联近红外II区荧光探针及其构建方法和用于甲状旁腺显影的应用，本发明通过构建特异性CaSR分子探针，实现手术导航，靶向识别甲状旁腺的应用。
背景技术
甲状旁腺是生命必需腺体，主要功能是调节人体血钙。甲状旁腺功能重要，不易识别，手术易引起甲状旁腺功能减退，误切率高，因此，术中准确识别甲状旁腺具有重要现实意义，是关乎人类健康的重要问题。动物实验证明，如将此腺完全切除，则致血钙含量急骤下降，肌肉强烈痉挛而导致死亡。临床上，甲状旁腺功能受损所带来的严重并发症，甚至超过甲状腺癌的危害。手足麻木、全身抽搐、神经衰弱，膈肌痉挛。永久性甲状旁腺功能低下者，须终身依赖钙剂，远期将引起肾结石、胆结石、软组织钙化、白内障及中枢神经受损等一系列钙紊乱的后遗症。
甲状旁腺的胚胎发育、大体解剖、形态位置等诸多因素，造成了甲状旁腺识别的困难。其不易识别的原因主要有以下几个方面：第一，甲状旁腺是人体内最小的器官，似黄豆，每个腺体长3～8mm，宽2～5mm，厚0.5～2mm。总重量不到0.3g。第二，甲状旁腺位置不固定，下极甲状旁腺在胚胎发育过程同胸腺一起下降，可出现在下颌角至心包的任何区域内，甚至埋在甲状腺内。第三，甲状旁腺的数量不固定，一般4枚，但也有3枚，甚至7枚。第四，甲状旁腺的颜色，随着年龄的变化而变化。最后，甲状旁腺的周围组织，颜色与甲状腺接近，形态与淋巴结、脂肪相近。因此，凭借外科医师的肉眼，准确而快速的识别所有甲状旁腺一直是医学挑战。
甲状旁腺功能受损是甲状腺手术后最常见的并发症之一。由于甲状腺手术量巨大，术中甲状旁腺面临风险的人群庞大。2020年全球新发甲状腺癌586278例。全球25个国家，主要甲状腺癌亚型的发病率还在增加。
在进行大量甲状腺手术过程中，甲状旁腺误切的情况不容忽视。2021年， Barrios在Surgery总结了1114例甲状腺切除术，甲状旁腺平均误切率22.4％(范围16.9％～43.6％)，不同手术量外科医师，在淋巴结清扫过程中的甲状旁腺误切率(图1)。有经验的外科医师最低也有7.7％误切率。同样，在
Figure PCTCN2022079704-appb-000001
和Philips的报道中甲状旁腺的误切率分别高达21.8％和13.6％。甲状旁腺误切是导致术后一过性和永久性甲状旁腺功能减退的独立危险因素。
当前甲状旁腺识别技术无法满足该医学需求。目前临床术中甲状旁腺显影主要有三类方式，第一类负向显影，由于甲状旁腺微小，颜色跟甲状腺接近，通过改变甲状腺和淋巴结的颜色来凸显甲状旁腺。主要应用有纳米碳和米托蒽醌。纳米碳是碳纳米材料，由于碳元素无法在人体降解，细胞复制过程中会损伤DNA，存在安全问题，且纳米碳易于渗漏，对术区易造成污染。米托蒽醌是蒽环类的化疗药物，为细胞周期非特异性药物，对正常细胞的具有毒性，这些特性使米托蒽醌不是理想的负显影剂。第二类是正向显影甲状旁腺，主要包括5-氨基乙酰丙酸、吲哚菁绿、亚甲蓝。正显影剂主要通过全身静脉注射的方式进行给药，但不具有靶向性，使用剂量大，副反应严重。第三类是甲状旁腺自体荧光显影，自体荧光有一定优势，但由于自体荧光弱，组织穿透深度有限，且甲状旁腺表面常覆盖脂肪组织，因此无法很好透过荧光。此外，由于常见的术区血迹、转移淋巴结、棕色脂肪、胶原结节等的存在，在采用自体荧光显影下容易存在误判，存在一定的假阳性，因此临床上尚未广泛应用。
本发明人在梳理目前国内外术中甲状旁腺显影领域主要的研究时，发现虽然显影方式和显影剂种类较多，但都存在不足之处。这从另一个层面说明，甲状旁腺的识别是一个尚未被解决的临床重要问题，手术引起的甲状旁腺误切或功能减退仍困扰着当今的外科医生。
发明内容
本发明首先确定甲状旁腺主细胞膜表面特异性高表达CaSR靶点，然后将CaSR连接不同近红外区II荧光分子(或近红外II区荧光分子、近红外二区荧光分子)，形成抗体荧光偶联物，通过动物体内实验，最后筛选得到最优成像效果的近红外II区荧光分子探针，用于手术中甲状旁腺的显影。本发明的目的在于降低术中甲状旁腺误切率，减少术后甲状旁腺功能减退并发症。
本发明的目的之一是提供靶向CaSR的物质在制备显影剂中的应用。
其中，所述显影剂为甲状旁腺正显影剂；优选地，所述显影剂用于甲状腺、甲状旁腺和/或淋巴结组织术中导航。
作为优选，所述靶向CaSR的物质为抗体，所述抗体通过SEQ ID NO.4～5所示的至少一种多肽抗原免疫动物获得；
作为优选，所述靶向CaSR的物质为单克隆抗体，所述单克隆抗体通过SEQ ID NO.4～5所示的任一项多肽抗原免疫动物获得；优选地，所述SEQ ID NO.4～5的C端连接半胱氨酸；
作为优选，所述靶向CaSR的物质为单克隆抗体，所述单克隆抗体通过SEQ ID NO.4～5所示的任一项多肽抗原免疫动物获得；优选地，所述SEQ ID NO.4～5的表达区域如SEQ ID NO.6所示；
作为优选，所述靶向CaSR的物质选自双链RNA、shRNA或小分子化合物。
作为优选，所述靶向CaSR的物质上偶联有荧光分子，为抗体偶联荧光分子探针；
作为优选，所述靶向CaSR的物质上偶联有荧光分子，为抗体偶联荧光分子探针，所述靶向CaSR的物质与荧光分子直接偶联；
作为优选，所述靶向CaSR的物质上偶联有荧光分子，为抗体偶联荧光分子探针，所述靶向CaSR的物质与荧光分子通过连接子连接；优选地，所述连接子选自不可裂解、还原裂解和蛋白酶裂解中的至少一种，如图16所示。
作为优选，所述荧光分子为近红外II区荧光分子；优选地，所述近红外II区荧光分子为IRDye800荧光分子，其结构如式1所示：
Figure PCTCN2022079704-appb-000002
作为优选，当所述靶向CaSR的物质为单克隆抗体，所述抗体与荧光分子直接偶联时，表示为CaSR-IRDye800抗体偶联荧光分子探针，其中，所述抗体赖氨酸中的氨基与荧光分子IRDYe800上的n羟基琥珀酰亚胺酯偶联。
作为优选，所述靶向CaSR的物质上偶联的荧光分子为一个或一个以上。
作为优选，在近红外II区下获得所述荧光分子的荧光。
本发明的另一目的是提供一种复合物，所述复合物包含靶向CaSR的物质；优选地，所述复合物选自显影剂，诊断示踪剂，药物组合物中的一种或几种。
所述显影剂为甲状旁腺正显影剂，所述甲状旁腺正显影剂为如上所述的抗体偶联荧光分子探针。
本发明的另一目的是提供一种筛选对甲状腺和/或淋巴结治疗有效的药物的方法，其特征在于，将靶向CaSR的物质与药物分子构建到载体上，通过靶向CaSR的物质对甲状旁腺的正显影，对药物分子的有效性进行评价，筛选对甲状腺和/或淋巴结治疗有效的药物。
本发明的有益效果：
(1)新思路：本发明从减少甲状旁腺误切率出发，通过甲状旁腺高表达靶点与荧光分子探针借鉴抗体偶联药物(antibody-drug conjugate,ADC)的设计原理，利用荧光分子靶向特定甲状旁腺组织，实现术中精准辨识，实时导航。应用该理念，将为外科领域，术中显影重要正常器官，提供崭新的研究思路。
(2)新前景：目前针对甲状旁腺的显像尚缺乏特异性的正显影技术，本项目 利用甲状旁腺的主细胞特异性表达CaSR靶点，满足外科医生术中实时显像甲状旁腺的迫切需求，具有明确临床应用转化前景。
附图说明
图1.甲状旁腺正显影图。(A)甲状旁腺位于甲状腺背侧，通过近红外II荧光靶向，实现术中实时导航。(B)钙敏感受体通过连接子偶联近红外II荧光分子识别甲状旁腺主细胞膜表面的靶标。(C)钙敏感受体偶联IRDye800荧光分子探针化学反应示意图。
图2.人、大鼠、小鼠三种物种的CaSR蛋白同源性性比对分析。
图3.钙敏感受体单克隆抗体序列分析及蛋白检测。图3A：以人的序列为基础序列分析(人、小鼠、大鼠)；图3B：保守结构域；图3C:同源序列与UniProt数据库中兔的序列，96.8％同源；图3D：抗原制备，通过合成多肽，多肽偶联制备抗原表达纯化重组蛋白，其中下方依次为：M 20812，测20801，E1，E2，20854，20847，20846，20845。
图4为翻译后修饰图；其中，第90、130、261、287、386、400、446、468、488、541、594位置的氨基酸存在GlcNAc的糖基化修饰，第920、1061位的氨基酸存在磷酸化修饰。此外，60与101，236与561，358与395，437与449，542与562，546与565，568与582，585与598氨基酸之间会形成而二硫键。
图5.为跨膜结构域分析图。其中，胞外区为：20-612aa，671-681aa，746-769aa，829-836aa。跨膜区为：613-635aa，650-670aa，682-700aa，725-745aa，770-792aaa，806-828aa，837-862aa。胞内区：636-649aa，701-724aa，793-805aa，863-1078aa。
图6为信号肽分析图；预测信号肽为1-19aa。
图7.抗体制备流程示意图。
图8.人甲状旁腺表达钙敏感受体靶点的免疫组化结果。(A)同一患者同一张组织切片中的甲状旁腺(Parathyroid Gland,PG)、淋巴结(Lymph Node,LN)、甲状腺(Thyroid Gland,TG)的HE染色和相应的钙敏感受体蛋白(CaSR)免疫组化图(2X)。(B)3例不同患者甲状旁腺CaSR免疫组化图，倍数(10X、20X、40X)。CaSR染色强度＞95％，强阳性(+++)。
图9.人甲状旁腺组织CaSR含量定位及大鼠CaSR蛋白表达。(A)人增生甲状旁腺组织中CaSR高表达，三个峰，分别为PBS、IgG和CaSR。(B)人增生甲状旁 腺组织制成单细胞悬液，可见CaSR表达于细胞膜表面。(B)大鼠甲状旁腺组织高表达CaSR，淋巴结和甲状腺阴性。
图10.大鼠甲状旁腺动物解剖模型建立。(A)活体大鼠解剖图，颈部暴露脱毛，颈前正中切开，颈前肌牵拉至两侧，肉眼辨识甲状腺及左右甲状旁腺，喉组织离体大小约6mm*10mm，甲状腺3mm*4mm，甲状旁腺1mm*2mm。(B)病理HE染色连续切片寻找甲状旁腺层面，断层显示甲状旁腺周围组织：甲状软骨、气管、食管、甲状腺、颈前带状肌等结构。(C)病理HE染色确诊大鼠甲状旁腺的位置，紧贴甲状腺表面，外膜完整，结构致密。
图11.新西兰兔甲状旁腺动物解剖模型建立。(A)活体新西兰解剖图，颈前正中切开，分离颈前肌后显露气管，肉眼辨识甲状腺及甲状旁腺后离体，一侧甲状腺大小约15mm*10mm，甲状旁腺3mm*5mm。(B)新西兰兔活体解剖正面观，显示甲状软骨、气管、颈前带状肌、甲状腺等。甲状旁腺紧贴肌肉，色红，表面光滑，长条状，双侧不对称，散在。(C)病理HE染色确诊新西兰兔甲状旁腺组织(蓝)，周围左上角脂肪组织(白)。(D)放大局部后可见甲状旁腺组织内丰富的主细胞和嗜酸细胞(标尺50um)。
图12.CaSR/IgG-IRDYe800化合物表征近红外I/II区的成像。(A)CaSR-IRDYe800和不同梯度浓度的IRDYe800的荧光光谱图。(B)不同浓度梯度IRDYe800的标准斜率曲线(R2＝0.970)，其中，横坐标从左至右依次为0、0.2、0.4、0.6、0.8、1、1.2；纵坐标从下至上依次为0.00E+00、5.00E+05、1.00E+06、1.50E+06、2.00E+06、2.50E+06。(C)CaSR-IRDYe8及对照在近红外I区下的成像图。(D)CaSR-IRDYe800在96孔板表面覆盖不同厚度鸡胸肉的近红外I区成像图。(E)CaSR-IRDYe800在近红外II区的成像图。(F)CaSR-IRDYe800在96孔板表面覆盖不同厚度鸡胸肉的近红外II区成像图。(G)IgG-IRDYe800化合物在近红外II区成像图。
图13.CaSR-IRDYe800化合物在鼠局部注射后近红外II区成像。(A)IRDYe800在大鼠胸大肌、右侧甲状腺、左侧甲状旁腺局部注射后的近红外II区普通荧光成像。(B)IRDYe800局部注射后近红外II区伪彩HOT荧光成像。(C)CaSR-IRDYe800局部注射小鼠甲状旁腺在近红外I区荧光成像。(D)CaSR-IRDYe800局部注射小鼠甲状旁腺在近红外II区明场成像。(E) CaSR-IRDYe800局部注射小鼠甲状旁腺在近红外II区荧光及伪彩成像(不同曝光时间)。(F)CaSR-IRDYe800局部注射小鼠甲状旁腺后离体喉组织的荧光及伪彩成像。
图14.体内CaSR/IgG-IRDYe800化合物尾静脉注射24小时后近红外II区成像。(A)小鼠尾静脉注射后近红外II区明场及荧光图像(生理盐水、CaSR-IRDYe800、IgG-IRDYe800)。(B)CaSR-IRDYe800小鼠尾静脉注射24小时后近红外II区普通荧光及伪彩HOT图像。(C)CaSR-IRDYe800小鼠尾静脉注射24小时后近红外II区普通荧光及伪彩HOT图像。(D)小鼠离体后心、肝、脾、肺、肾的近红外II区普通荧光图。(E)CaSR-IRDYe800小鼠喉离体后的明场、普通荧光及伪彩HOT图。
图15.实施例2术中实时导航甲状旁腺识别图。(A)Opto Medic显像系统，包括主机、荧光探头、显示器。(B)SD大鼠正面观，注射前和注射后的荧光模式图，注射前甲状旁腺辨识困难，注射后双侧甲状旁腺清晰可辨，荧光信号强。(C)SD大鼠右面观，注射前和注射后的荧光模式图，注射前右侧甲状旁腺辨识困难，注射后右侧甲状旁腺清晰可辨，荧光信号强。(D)SD大鼠左面观，注射前和注射后的荧光模式图，注射前甲状旁腺辨识困难，注射后左侧甲状旁腺清晰可辨，荧光信号强。
图16.抗体与荧光分子通过三种连接子连接的示意图。
具体实施方式
为解决术中导航甲状旁腺的问题，本发明人根据自身临床和研究经验，提出抗体偶联药物(antibody drug conjugate，ADC)的结构设计。它的设计原理是通过靶向甲状旁腺上特定靶标的单克隆抗体(mAb)与荧光分子(warhead)通过化学连接子(Linker)进行共价偶联，形成复合型大分子靶向荧光探针。开发具有甲状旁腺特异靶向性的荧光分子正显影剂，用于解决术中甲状旁腺识别的关键问题。
G蛋白偶联受体可感知细胞外钙离子浓度的变化，并在维持钙稳态中起关键作用。感知循环钙浓度的波动并调节甲状旁腺中甲状旁腺激素(PTH)的产生(通过相似性)。该受体的活性由激活磷脂酰肌醇-钙第二信使系统的G蛋白介导。针对甲状旁腺正显影缺乏有效分子靶点的问题，本发明人在前期研究中发现钙敏感受体蛋白(calcium-sensitive receptor,CaSR)在人体甲状旁腺主细胞膜表 面特异性的表达，在周围甲状腺和其他细胞表面上不表达，是一种甲状旁腺靶向荧光探针的有效分子靶点，因此，我们设计和开发了CaSR单克隆抗体，用做ADC的靶向分子(图1)。
基于ADC药物设计理念，为实现甲状旁腺正显影，首先我们从甲状旁腺细胞表面特异性高表达靶点入手，通过文献及书籍调研，锁定甲状旁腺主细胞表面特异性高表达的CaSR作为我们的靶点。甲状旁腺构成相对简单，周围一层薄结缔组织被膜，实质有主细胞和嗜酸细胞构成，嗜酸细胞有主细胞转化而来，因此聚焦主细胞的表面膜蛋白找到合适靶点是正确的途径。
CaSR是G蛋白偶联受体家族成员，主要特异性表达在甲状旁腺，感知细胞外血钙的浓度变化，当血钙降低时，CaSR调节甲状旁腺细胞分泌甲状旁腺素，动员骨钙入血，维持血钙正常生理浓度(2.25～2.75mmol/L)。CaSR在精准维持人体钙稳态中发挥关键作用，甲状旁腺正是通过CaSR，完成调节体内血钙水平的生理功能。为实现甲状旁腺的正显影，靶向CaSR是理想靶点：首先，CaSR位于细胞膜，不需进入细胞，即可被靶向识别。其次，CaSR在人体其它组织中表达相对少且弱，靶点专一性强。最后，CaSR蛋白序列保守，不同物种之间同源性高，应用广泛。
具体方案：
1.设计和制备钙敏感受体单克隆抗体
本发明人在前期研究中已经完成CaSR抗体的设计和制备，抗原设计针对人、大鼠、小鼠三种物种的CaSR蛋白(CaSR蛋白大小(aa)：1078；序列分别如SEQ ID NO.1～3所示)，同源性比对分析如图2所示(三者的同源性为92.9％)，蛋白序列以人的序列进行分析(图3A-B)。三者的同源性92.9％，比对UniProt数据库中兔的序列，同源性96.8％(图3C)。该钙敏感受体的单克隆抗体在物种和数量上满足后续动物实验，甚至人源化抗体后，可供临床研究使用。
翻译后修饰如图4所示，第90、130、261、287、386、400、446、468、488、541、594位置的氨基酸存在GlcNAc的糖基化修饰，第920、1061位的氨基酸存在磷酸化修饰。此外，60与101，236与561，358与395，437与449，542与562，546与565，568与582，585与598氨基酸之间会形成而二硫键。
跨膜结构域分析如图5所示，胞外区为：20-612aa，671-681aa，746-769aa， 829-836aa。
跨膜区为：613-635aa，650-670aa，682-700aa，725-745aa，770-792aaa，806-828aa，837-862aa。
胞内区：636-649aa，701-724aa，793-805aa，863-1078aa。
信号肽分析如图6所示，预测信号肽为1-19aa。
1.1抗原设计
1.1.1序列信息
>sp|P41180|CASR_HUMAN Extracellular calcium-sensing receptor OS＝Homo sapiens OX＝9606 GN＝CASR PE＝1 SV＝3(SEQ ID NO.1)
Figure PCTCN2022079704-appb-000003
>sp|P48442|CASR_RAT Extracellular calcium-sensing receptor OS＝Rattus norvegicus OX＝10116 GN＝Casr PE＝1 SV＝1(SEQ ID NO.2)
Figure PCTCN2022079704-appb-000004
Figure PCTCN2022079704-appb-000005
>sp|Q9QY96|CASR_MOUSE Extracellular calcium-sensing receptor OS＝Mus musculus OX＝10090 GN＝Casr PE＝1 SV＝2(SEQ ID NO.3)
Figure PCTCN2022079704-appb-000006
1.1.2抗原设计
Figure PCTCN2022079704-appb-000007
Figure PCTCN2022079704-appb-000008
在抗原分析的时候，设计两种抗原技术路线，多肽技术路线选择的是20-32aa，序列为YGPDQRAQKKGDI(SEQ ID NO.4)，该序列位于CaSR蛋白的胞外区，另一段多肽序列为212-225aa，序列为IAADDDYGRPGIEK(SEQ ID NO.5)，该序列位于CaSR蛋白的胞外区。
针对多肽抗原的话，后续需要偶联载体蛋白KLH后，用于动物的免疫，因此会在多肽抗原序列的C端加半胱氨酸用于载体蛋白的偶联。
选择的原则：从本身抗原性，序列的亲水性，表位的暴露性进行分析，选择最终合适的区段作为抗原序列。
1.2.抗体制备实验流程
1.2.1整体实验方案
制备流程如图7所示，通过抗原制备，免疫小鼠，并监控小鼠血清效价，待合格后取效价最高的小鼠进入细胞融合、阳性细胞单克隆化、分泌钙敏感受体抗体的细胞株筛选、腹水制备、单克隆抗体纯化的通用重组方法制备。现阶段，已全基因合成到蛋白表达纯化，表达区域448-610aa。该片段的序列如下：
Figure PCTCN2022079704-appb-000009
1.2.2实验流程及技术指标
1.2.2.1抗原制备
通过合成多肽，将多肽偶联KLH制备抗原/原核表达纯化重组蛋白。
如图3D所示，该项目能表达纯化出预期的目的蛋白，可以满足免疫要求。
质控标准：纯度>85％。
1.2.2.2免疫及血清评价
按照免疫流程，免疫5只6周龄Balb/c小鼠。
质控标准：采用间接ELISA分析小鼠血清中抗体的效价及产生水平，效价 水平为1:72900稀释OD450nm值大于0.3，同时寄送血清小样给客户进行验证。
1.2.2.3细胞融合
根据1.2.2.2的免疫血清效价结果和目标抗体的产生情况，选择最优的小鼠安排进入融合。
1.2.2.4阳性细胞单克隆化及具有分泌目的应用抗体的细胞株筛选
融合之后，利用间接Elisa筛选细胞上清中阳性抗体，利用特异性筛选，筛选特异性单抗，再通过两步有限稀释法获得单克隆抗体细胞。
1.2.2.5腹水制备
选择2个具有高亲和力的单抗细胞株，制备腹水。
检测：采用间接ELISA分析腹水中抗体的效价，同时进行亚型的分析。
1.2.2.6单克隆抗体的纯化
根据实际的抗体亚型，选择合适的纯化方式进行抗体的纯化，获得2个纯化单抗，即靶向CaSR的抗体。
检测：1.2.2.4相同的方法进行抗体功能的验证。
2.证明CaSR在甲状旁腺中特异性强表达(人、大鼠)
本发明人在前期实验中，通过免疫组化法检测CaSR在人甲状旁腺组织切片中特异性高表达，并证明CaSR的表达位置在甲状旁腺主细胞的细胞膜表面(图8)。共提取24例甲状腺癌患者，术后病理偶见少量甲状旁腺组织，中央区淋巴结清扫的组织白片。我们发现CaSR蛋白选择性表达在甲状旁腺细胞表面，而在甲状腺、淋巴结中均阴性表达，这提示CaSR蛋白是甲状旁腺特异性的表达靶点。在检测的24例患者中，1例表达强度60％，2例表达强度80％，其余21例表达强度均＞95％(+++)。
通过流式细胞实验，证实人病变甲状旁腺的新鲜组织单细胞悬液中CaSR蛋白含量高(图9A-B)。患者为临床怀疑甲状腺癌且伴有甲状旁腺增生者，签署知情同意。术中取增生甲状旁腺及周围部分甲状腺组织，剪碎，裂解，制成单细胞悬液。孵育CaSR荧光抗体45分钟，洗后，流式细胞仪(CytoFLEX)收集主细胞群在10 3附近。人增生甲状旁腺中CaSR表达量11188，对照IgG抗体4567， 对照PBS溶液111，峰值独立，分开(图9A)。在人的甲状旁腺细胞中，应用激光共聚焦显微镜(ZEISS)观察旁腺染色情况(图9B)。
通过WB实验，在SD大鼠中切取甲状旁腺、甲状腺和淋巴结，证实甲状旁腺组织特异性高表达CaSR，而甲状腺和淋巴结不表达(图9C)。
3.建立大鼠、新西兰兔甲状旁腺的解剖动物模型
本发明人在前期实验中，已获得动物实验资格认可证(证字第22-09016号)，通过对大鼠和新西兰兔的解剖学习，结合人体颈部手术经验，建立大鼠和新西兰兔甲状旁腺的解剖模型(图10、11)。动物实验前，获得动物伦理委员的伦理批件(伦理批号2021-05-002)。
SD大鼠实验，大鼠甲状旁腺微小(1mm*2mm)，通过病理连续切片证实。称取6-8周，重量约250-300克，雌雄各半，SD大鼠6只。新西兰兔实验，兔甲状旁腺散在，根据临床外科解剖经验判断，通过病理HE染色证实。称取3-4月，重量约2.5-3千克，雌雄各半，新西兰兔6只。
解剖过程，SD大鼠异氟烷吸入麻醉维持，新西兰兔注射戊巴比妥静脉麻醉，颈仰卧位，垫枕(效仿人甲状腺切除手术体位)。颈前正中切开，分离胸骨甲状肌和舌骨甲状腺肌至两侧并固定，显露气管，环状软骨水平显露甲状腺上极。大鼠甲状旁腺一对，位于甲状腺上极，对称，色粉白，与甲状腺紧贴，仔细观察，有包膜(图10A)。新西兰兔的甲状旁腺，上极多位于腺体内，肉眼解剖未见。下极旁腺，远离甲状腺，散在分布，贴附胸骨甲状腺肌，色红，长条状，质地均一。根据甲状旁腺周围血管分布走向的末端，提示甲状旁腺的肉眼判断(图11A-B)。解剖辨识甲状旁腺后，氯化钾静脉注射致死。离体切除SD大鼠喉(甲状腺及甲状旁腺太小)，包括甲状软骨、会厌、甲状腺、甲状旁腺、气管、食管等结构浸入甲醛溶液。完整切除新西兰兔甲状旁腺浸入甲醛。
病理诊断，通过(Hematoxylin)-伊红(Eosin)染色(简称HE染色)，金标准证实。SD大鼠甲状旁腺微小，单独组织切片困难，通过连续切喉组织，共切16张白片，每层小于1mm，在一个层面，切得双侧甲状旁腺在同一片子(图10B)。新西兰兔甲状旁腺组织单独固定。HE染色：烤片5分钟，二甲苯脱蜡3遍各5分钟，梯度酒精4次各1分钟，水洗，苏木素染液3分30秒，水洗，伊红色40秒，梯度酒精5次各1分钟，二甲苯30秒，最后封片机封片。HE染色结果有肿 瘤医院临床病理科两位资深专家诊断。显微镜下可见甲状旁腺组织丰富的主细胞和嗜酸细胞(图10C、图11C-D)。
4.制备CaSR-IR800荧光分子探针
首先配置50mM，pH＝8.5的磷酸钾缓冲液作为反应体系。通过NHS酯化反应，碱性环境(PH＝8.5)下，合成CaSR抗体偶联荧光分子探针，合成原理示意图(图1C)。设置3组，分别为CaSR-IRDye800、IgG-IRDye800和纯IRDye800。抗体与荧光分子的摩尔质量按照1：10比例配置。配置成500ul的体系，摇床，过夜12h，注意避光。超滤分离未结合的抗体的染料(30kD截留量)。通过透析袋，在磁力搅拌器作用下纯化。
表征合成的CaSR-IRDye800的抗体浓度和荧光强度。抗体浓度应用BCA法测定(Enhanced BCA protein Assay kit)，配置标准浓度梯度绘制标准曲线，96孔板孵育30min，上机检测在562nm处的吸光度值，带入标准曲线求得抗体浓度。根据分子量，求得摩尔质量3.185nmol。
表征合成的CaSR-IRDye800的荧光强度。应用稳态/瞬态荧光光谱仪检测荧光强度。将合成的CaSR-IRDye800偶联物进行荧光光谱分析，激发光波长738nm，吸收光波长825nm。在浓度分别为1ug/ml、0.5ug/ml、0.25ug/ml、0.125ug/ml、0.0625ug/ml的IRDYe800做标准曲线(R 2＝0.970)，从而测得染料的浓度1.703ug/ml，根据分子量，求得染料的摩尔质量0.848nmol(图12A、B)。因此，抗体与染料的之比1：3.75。
将合成的CaSR-IRDYe800偶联物在近红外I区仪器下成像，可见偶联物荧光强度在10 10左右(图12C)。取200ul合成后的偶联物表面覆盖1mm、3mm、5mm厚的鸡胸肉后成像，发现1mm和3mm荧光强度10 9左右，5mm厚处几乎无荧光可透出(图12D)。我们用相同的样品近红外II区仪器下成像，发现在鸡胸肉覆盖5mm，仍有荧光可透出，这为后续动物实验成像做准备，优选近红外II区仪器(图12E-F)。同时也合成阴性对照IgG-IRDYe800(图12G)。剩余合成化合物冻干保存。
实施例1
动物实验(局部注射、全身尾静脉注射)
本发明人合成CaSR-IRDYe800偶联物，通过小鼠局部解剖区域注射和尾静 脉注射的两种不同方式，观察初步成像效果(图13)。
局部注射方法，在大鼠的局部解剖区域，包括胸大肌、右侧甲状腺和左侧甲状旁腺分别注射少量IRDYe800，发现在近红外II区下，荧光强度高(图13)。我们将合成的CaSR-IRDYe800偶联物，局部注射双侧小鼠甲状旁腺，在近红外I区下，可见双侧甲状腺区均有较弱的荧光信号，但信号不聚焦，无法分辨甲状旁腺(图13C)。然后，在近红外II区下观察，明场(图13D)，普通荧光和伪彩HOT下，在不同的曝光时间(20s、25s)可见甲状旁腺清晰的荧光信号(图13E)。最后将小鼠喉离体后成像，发现双侧甲状旁腺，荧光信号仍清晰(图13F)。此部分实验，说明合成的CaSR-IRDYe800偶联物可以在现有的成像系统中成功显像甲状旁腺。
尾静脉分别注射生理盐水、CaSR-IRDYe800和IgG-IRDYe800，24小时后在近红外II区成像(图14)。首先将3只小鼠置于明场和普通荧光下观察(图14A)，发现IgG-IRDYe800偶联物肝脏荧光沉积，余未见明显荧光信号。接着，我们将注射CaSR-IRDYe800和CaSR-IRDYe800分别于普通荧光和伪彩HOT下观察(图14B、C)。发现CaSR-IRDYe800颈部双侧对称荧光信号，而IgG-IRDYe800无颈部信号(图14C)。然后，离体观察3只小鼠正常重要器官中的荧光信号(图14D)。最后将IgG-IRDYe800，离体喉，在甲状腺、甲状旁腺及气管离体组织中观察到荧光信号(图14F)。因此，证实CaSR-IRDYe800可以靶向甲状旁腺，特异性显影甲状旁腺。
实施例2
为了验证甲状旁腺实时术中导航系统的可操作性，我们用SD大鼠设计如下实验。
第一，操作系统的准备，主要包括开放式荧光导航系统、荧光探头、荧光半定量分析软件工作站。系统由杭州潍源公司提供的Opto Medic影像科研设备(图15A)。目前采用的荧光激发光波长805nm，后续将进行近红外Ⅱ区导航，同时进行全身静脉注射后实时导航成像。
第二，SD大鼠的手术准备，取SD雌性大鼠，重300g。异氟烷吸入麻醉，脱毛，颈仰卧位，枕部垫高，同临床常规甲状腺手术体位。消毒，铺巾，颈部正中切口，手术操作如上述大鼠甲状旁腺动物模型的建立。双侧颈前肌拉钩牵引至 外侧，充分暴露甲状腺、气管、喉等器官组织。
第三，实时导航甲状旁腺，将荧光探头连接主机，多种显影模式可供选择，高清白光、标准荧光、多彩荧光。常规暴露术区，肉眼辨识甲状旁腺困难，凭建模时掌握的经验，判断甲状旁腺的位置。荧光探头进行白平衡，对焦。拍摄各个模式下甲状旁腺的左侧、右侧及正面观(图15B、C、D)。甲状旁腺处注射0.5μl的CaSR-IRDYe800，浓度10 -3μg/ml。同样的模式拍一组照。比较注射前和注射后甲状旁腺的显影情况，术中实时调节探头的角度和距离。
实验结果，注射后甲状旁腺处的荧光信号强，清晰可辨。该系统目前符合实验操作需求，为后续的动物实验及临床转化试验提供前期基础。
以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。

Claims (10)

  1. 靶向CaSR的物质在制备显影剂中的应用。
  2. 如权利要求1所述的应用，其特征在于，所述显影剂为甲状旁腺正显影剂；优选地，所述显影剂用于甲状腺、甲状旁腺和/或淋巴结组织术中导航。
  3. 如权利要求1所述的应用，其特征在于，选自以下至少任一项：
    1)所述靶向CaSR的物质为抗体，所述抗体通过SEQ ID NO.4～5所示的至少一种多肽抗原免疫动物获得；
    2)所述靶向CaSR的物质为单克隆抗体，所述单克隆抗体通过SEQ ID NO.4～5所示的任一项多肽抗原免疫动物获得；优选地，所述SEQ ID NO.4～5的C端连接半胱氨酸；
    3)所述靶向CaSR的物质为单克隆抗体，所述单克隆抗体通过SEQ ID NO.4～5所示的任一项多肽抗原免疫动物获得；优选地，所述SEQ ID NO.4～5的表达区域如SEQ ID NO.6所示；
    4)所述靶向CaSR的物质选自双链RNA、shRNA或小分子化合物。
  4. 如权利要求1所述的应用，其特征在于，选自以下至少任一项：
    1)所述靶向CaSR的物质上偶联有荧光分子，为抗体偶联荧光分子探针；
    2)所述靶向CaSR的物质上偶联有荧光分子，为抗体偶联荧光分子探针，所述靶向CaSR的物质与荧光分子直接偶联；
    3)所述靶向CaSR的物质上偶联有荧光分子，为抗体偶联荧光分子探针，所述靶向CaSR的物质与荧光分子通过连接子连接；优选地，所述连接子选自不可裂解、还原裂解和蛋白酶裂解中的至少一种。
  5. 如权利要求4所述的应用，其特征在于，所述荧光分子为近红外II区荧光分子；优选地，所述近红外II区荧光分子为IRDye800荧光分子，其结构如式1所示：
    Figure PCTCN2022079704-appb-100001
  6. 如权利要求5所述的应用，其特征在于，当所述靶向CaSR的物质为单克隆抗体，所述抗体与荧光分子直接偶联时，表示为CaSR-IRDye800抗体偶联荧光分子探针，其中，所述抗体赖氨酸中的氨基与荧光分子IRDYe800上的n羟基琥珀酰亚胺酯偶联。
  7. 如权利要求4～6任一项所述的应用，其特征在于，选自以下至少任一项：
    1)所述靶向CaSR的物质上偶联的荧光分子为一个或一个以上。
    2)在近红外II区下获得所述荧光分子的荧光。
  8. 一种复合物，其特征在于，所述复合物包含靶向CaSR的物质；优选地，所述复合物选自显影剂，诊断示踪剂，药物组合物中的一种或几种。
  9. 如权利要求8所述的复合物，其特征在于，所述显影剂为甲状旁腺正显影剂，所述甲状旁腺正显影剂为如权利要求4～6任一项所述的应用中所述的抗体偶联荧光分子探针。
  10. 一种筛选对甲状腺和/或淋巴结治疗有效的药物的方法，其特征在于，将靶向CaSR的物质与药物分子构建到载体上，通过靶向CaSR的物质对甲状旁腺的正显影，对药物分子的有效性进行评价，筛选对甲状腺和/或淋巴结治疗有效的药物。
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