WO2024149232A1 - Nanoanticorps b12 ciblant ceacam5 et utilisation du nanoanticorps b12 - Google Patents

Nanoanticorps b12 ciblant ceacam5 et utilisation du nanoanticorps b12 Download PDF

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Publication number
WO2024149232A1
WO2024149232A1 PCT/CN2024/071306 CN2024071306W WO2024149232A1 WO 2024149232 A1 WO2024149232 A1 WO 2024149232A1 CN 2024071306 W CN2024071306 W CN 2024071306W WO 2024149232 A1 WO2024149232 A1 WO 2024149232A1
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nanobody
ceacam5
seq
nanoantibody
present application
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Chinese (zh)
Inventor
赵善超
李志杰
肖兆铭
王继刚
刘亚伟
石广为
张晓东
杨诚
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Third Affiliated Hospital Of Southern Medical University Academy Of Orthopedics Guangdong Province
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Third Affiliated Hospital Of Southern Medical University Academy Of Orthopedics Guangdong Province
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Priority to GB2410580.1A priority Critical patent/GB2629534A/en
Publication of WO2024149232A1 publication Critical patent/WO2024149232A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/005—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3007—Carcino-embryonic Antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of biotechnology, and in particular to a nano antibody B12 targeting CEACAM5 and its application.
  • PCa prostate cancer
  • CRPC castration-resistant prostate cancer
  • Androgen receptor signal amplification such as androgen receptor gene and promoter amplification and androgen receptor splice variant AR-V7 formation, is an important factor leading to castration-resistant prostate cancer.
  • second-line endocrine therapy that effectively blocks endogenous androgen synthesis (abiraterone) or androgen receptor nuclear entry (enzalutamide) is the current main treatment method.
  • abiraterone abiraterone
  • enzalutamide androgen receptor nuclear entry
  • the above treatment can prolong the survival period of CRPC patients, 25% of CRPC clinical cases still experience disease progression after treatment and rapidly develop distant tumor metastasis and invasion of surrounding tissues in a short period of time, resulting in a median survival period of only 7 months, so it is called aggressive variant prostate cancer.
  • invasive variant prostate cancer has great heterogeneity at the histopathological level.
  • invasive variant prostate cancer lacks the adenocarcinoma tissue characteristics of prostate adenocarcinoma tissue and presents many pathological phenotypes including neuroendocrine carcinoma, small cell carcinoma, squamous cell carcinoma, ductal carcinoma, etc.
  • invasive variant prostate cancer presents neuroendocrine carcinoma and small cell carcinoma phenotypes in most clinical cases.
  • immunohistochemistry confirms that invasive variant prostate cancer also does not express a series of tissue markers expressed by prostate adenocarcinoma, such as the common PSMA, ERG, AR and PSA.
  • Lineage plasticity refers to the process of reverse differentiation or transdifferentiation in which cells regulate cell development through epigenetic remodeling to obtain a survival advantage phenotype. At the same time, it is also an important mechanism leading to tumor resistance.
  • Current studies have revealed that prostate cancer acquires a series of survival advantage phenotypes, including neuroendocrine phenotypes, through lineage plasticity pathways, and causes the disease to progress to the invasive variant prostate cancer stage.
  • Epigenetic remodeling is the core factor driving the above process. In prostate cancer, multiple epigenetic factors related to androgen receptor signal transduction are significantly remodeled. This leads to the loss of androgen receptor signals and abnormal activation of signaling pathways related to survival advantage phenotypes such as stem cells and neural development.
  • invasive variant prostate cancer can re-express membrane antigens related to embryonic and organ development. Therefore, effectively identifying CRPC-NE-specific membrane proteins and developing related antibody-conjugated drugs (ADCs) have great potential significance for the diagnosis and treatment of such diseases.
  • ADCs antibody-conjugated drugs
  • CEACAM5 is a type of mammalian immunoglobulin-related glycoprotein that is highly expressed in digestive and respiratory tract tumors and is developed for intestinal tumor-related ADC treatment. Recent studies have found that CEACAM5 can be expressed on the surface of invasive variant prostate tissue membrane as a downstream target gene of the neural development-related transcription factor ASCL1. CEACAM5 on the surface of tumor cells can be shed into the blood to form serum CEA and is used for clinical diagnosis. Related studies have also confirmed that the expression product of the CEACAM5 gene, CEA, is elevated in the serum of patients with invasive variant prostate cancer and is closely related to tumor organ metastasis. The above studies suggest that CEACAM5 is a potential target for clinical diagnosis and treatment of patients with invasive variant prostate cancer.
  • the purpose of the present application is to provide a nano-antibody B12, which has high affinity activity and binding activity, and can effectively detect CEACAM5 and prevent and treat CEACAM5-related diseases.
  • the first aspect of the present application provides a nanoantibody B12 targeting CEACAM5, wherein the nanoantibody B12 comprises three complementary determining regions, namely CDR1, CDR2 and CDR3; wherein the amino acid sequence of the CDR1 is shown in SEQ ID NO.6, the amino acid sequence of the CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of the CDR3 is shown in SEQ ID NO.8.
  • the nanoantibody B12 also contains four framework regions, namely FR1, FR2, FR3 and FR4; the amino acid sequence of the FR1 is shown in SEQ ID NO.2, the amino acid sequence of the FR2 is shown in SEQ ID NO.3, the amino acid sequence of the FR3 is shown in SEQ ID NO.4, and the amino acid sequence of the FR4 is shown in SEQ ID NO.5.
  • amino acid sequence of the nanoantibody B12 is shown in SEQ ID NO.1.
  • the second aspect of the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody B12 described in the first aspect of the present application.
  • the nucleic acid molecule of the present application can be synthesized, for example, by standard chemical synthesis methods and/or recombinant methods, or semi-synthetically produced, for example, by combining chemical synthesis and recombinant methods.
  • the coding sequence and the transcriptional regulatory element and/or Or connection with other amino acid coding sequences can be carried out using established methods, such as restriction enzyme digestion, ligation and molecular cloning.
  • nucleotide sequence encoding the nanoantibody B12 is the nucleotide sequence shown in SEQ ID NO.9.
  • the third aspect of the present application provides a vector, which comprises the nucleic acid molecule described in the second aspect of the present application.
  • the fourth aspect of the present application provides a host cell, which comprises the nucleic acid molecule described in the second aspect of the present application, or comprises the vector described in the third aspect of the present application.
  • the fifth aspect of the present application provides a drug conjugate, wherein the drug conjugate contains:
  • the conjugated moiety is selected from the group consisting of a detectable label, a drug, a toxin, a cytokine or an enzyme.
  • the drug conjugate contains the nanobody B12 and a toxin
  • the toxin is PE38 toxin.
  • the sixth aspect of the present application provides a composition, which comprises the Nanobody B12 described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the vector described in the third aspect of the present application, the host cell described in the fourth aspect of the present application, or the drug conjugate described in the fifth aspect of the present application.
  • the seventh aspect of the present application provides the use of the nanoantibody B12 described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the vector described in the third aspect of the present application, the host cell described in the fourth aspect of the present application, or the drug conjugate described in the fifth aspect of the present application in the preparation of a product for detecting castration-resistant neuroendocrine stage prostate cancer.
  • the eighth aspect of the present application provides the use of the nanoantibody B12 described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the vector described in the third aspect of the present application, the host cell described in the fourth aspect of the present application, or the drug conjugate described in the fifth aspect of the present application in the preparation of a drug for treating or preventing castration-resistant neuroendocrine stage prostate cancer.
  • the drug includes at least one of a CAR T-related drug, a CAR NK-related drug, a bispecific antibody-related drug, and a nanomedicine.
  • the ninth aspect of the present application provides the use of the nanoantibody B12 described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the vector described in the third aspect of the present application, the host cell described in the fourth aspect of the present application, or the drug conjugate described in the fifth aspect of the present application in the preparation of a drug for imaging the neuroendocrine stage of castration-resistant prostate cancer.
  • the tenth aspect of the present application provides the use of the nanobody B12 described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the vector described in the third aspect of the present application, the host cell described in the fourth aspect of the present application, or the drug conjugate described in the fifth aspect of the present application in the preparation of a product for detecting CEACAM5.
  • the eleventh aspect of the present application provides the use of the nanobody B12 described in the first aspect of the present application, the nucleic acid molecule described in the second aspect of the present application, the vector described in the third aspect of the present application, the host cell described in the fourth aspect of the present application, or the drug conjugate described in the fifth aspect of the present application in the preparation of products for diagnosing, treating, and preventing CEACAM5-related diseases.
  • the present application provides a nano antibody B12 targeting CEACAM5, which has a high affinity and binding force with CEACAM5.
  • the nano antibody B12 targets the neuroendocrine prostate cancer-specific membrane marker CEACAM5, which is conducive to the development of nano antibody-related drugs for neuroendocrine prostate cancer-specific membrane marker CEACAM5, and achieves targeted treatment of castration-resistant neuroendocrine prostate cancer, which has a very large clinical application value; it is also conducive to the development of drugs for diseases related to CEACAM5, and realizes the diagnosis and treatment of diseases related to CEACAM5.
  • nano antibodies are conducive to wide application.
  • Figures 1 and 2 are experimental results showing that CEACAM5 is a membrane marker for invasive variant prostate cancer in Example 1 of the present application;
  • Figures 3A and 3B are graphs showing the results of nanobody screening in Example 2 of the present application.
  • Figure 3C and Figure 3D are the results of purification and expression of Nanobodies in Example 2 of the present application.
  • Figures 3E to 3G are the affinity validation results of Nanobody B12 in Example 2 of the present application.
  • Figures 3H to 3I are graphs showing the specificity validation results of Nanobody B12 in Example 2 of the present application.
  • Figure 4 is a diagram showing the in vivo imaging results of Nanobody B12 in Example 3 of the present application.
  • FIGS 5A to 5D are diagrams showing the preparation and identification results of immunotoxin B12-PE38 in Example 4 of the present application;
  • 5E and 5F are graphs showing the experimental results of PC3 phagocytosis and uptake of immunotoxin B12-P38 in vitro in Example 4 of the present application;
  • FIGS 5G to 5I are graphs showing the experimental results of the in vitro killing of PC3 cells by the immunotoxin B12-PE38 in Example 4 of the present application;
  • Figures 6 to 8 are in vivo experimental results of the treatment of CEACAM5 positive cell line transplants with immunotoxin B12-PE38 in Example 4 of the present application;
  • FIG9 is a diagram showing the in vivo experimental results of the immunotoxin B12-PE38 combined with docetaxel in the treatment of PC3 cell line transplants in Example 4 of the present application;
  • FIG. 10 is a graph showing the experimental results of the immunotoxin B12-PE38 in Example 4 of the present application for treating invasive prostate cancer cell line PC3 tibial orthotopic transplanted tumors.
  • nano antibody B12 has a high binding affinity with the neuroendocrine prostate cancer-specific membrane marker CEACAM5.
  • the antibody can target the neuroendocrine prostate cancer-specific membrane marker CEACAM5, which is conducive to the development of nano antibody-related drugs related to the neuroendocrine prostate cancer-specific membrane marker CEACAM5, and achieve targeted treatment of castration-resistant neuroendocrine prostate cancer, which has great clinical application value; it is also conducive to the development of Drugs for diseases related to CEACAM5 can be used to diagnose and treat diseases related to CEACAM5.
  • nano antibodies are suitable for wide application due to their small molecular weight.
  • affinity refers to the binding ability between a macromolecule and its bound antigen, in particular the binding ability between a nanobody and its bound antigen, such as the binding ability between the nanobody B12 of the present application and the pathological form of CEACAM5 protein as defined above.
  • the affinity of the Nanobody B12 of the present application can be measured in vitro by several methods, including surface plasmon resonance or by ELISA, as described in the Examples.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present application to form conjugates.
  • cytokines cytokines
  • the present application also includes cell surface markers or antigens combined with the nanobody B12 or its fragments.
  • the present application provides a nanoantibody B12 targeting CEACAM5, wherein the nanoantibody B12 comprises three complementary determining regions, namely CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8.
  • SEQ ID NO.6 is: GVTFSTYGMG
  • SEQ ID NO.7 is: GTYSDGST
  • SEQ ID NO.8 is: APKHEYGTNWYERTIYSNELDY.
  • CDR1, CDR2 and CDR3 are sufficient to define an antigen binding site. It is also known to those skilled in the art that two Nanobodies recognizing the same antigen compete for binding to the antigen.
  • the present application also provides the framework region of nanoantibody B12, which comprises four framework regions, namely FR1, FR2, FR3 and FR4; the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, and the amino acid sequence of FR4 is shown in SEQ ID NO.5.
  • SEQ ID NO.2 is: AVQLVESGGGLVQPGESLRLSCAAS;
  • SEQ ID NO.3 is: WARQVPGKGLEWVC;
  • SEQ ID NO.4 is:
  • SEQ ID NO.5 is: WGQGTQVTVSS.
  • framework region refers to the amino acid sequence inserted between CDRs.
  • SEQ ID NO.1 is:
  • the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody B12 of the present application.
  • nucleotide sequence encoding Nanobody B12 is the nucleotide sequence shown in SEQ ID NO.9.
  • SEQ ID NO.9 is:
  • the nucleic acid molecule is a DNA or RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, bacteriophage or viral vector.
  • vector refers to vehicles that can introduce DNA or RNA sequences into host cells in a manner that transforms the host and promotes the expression (eg, transcription and translation) of the introduced sequences.
  • the present application provides, in one aspect, a vector comprising the nucleic acid of the present application.
  • Non-limiting examples of plasmid vectors include pQE-12, pUC-series, pBluescript (Stratagene), pET-series expression vectors (Novagen) or pCRTOPO (Invitrogen), ⁇ gt11, pJOE, pBBR1-MCS series, pJB861, pBSMuL, pBC2, pUCPKS, pTACT1, pTRE, pCAL-n-EK, pESP-1, pOP13CAT, E-027pCAG Kosak-Cherry (L45a) vector system, pREP (Invitrogen), pCEP4 (Invitrogen), pMC1neo (Stra tagene), pXT1 (Stra tagene), pSG5 (Str a tagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVn
  • Non-limiting examples of plasmid vectors suitable for Pichia pastoris include, for example, plasmids pAO815, pPIC9K, and pPIC3.5K (all Invitrogen).
  • Another vector suitable for expressing proteins in Xenopus embryos, zebrafish embryos, and a variety of mammalian and avian cells is the multi-purpose expression vector pCS2+.
  • a vector may contain one or more origins of replication (ori) and genetic systems for cloning or expression, one or more markers for selection in a host (e.g., antibiotic resistance), and one or more expression cassettes.
  • the established method connects the coding sequence contained in the vector to transcriptional regulatory elements and/or to other amino acid coding sequences.
  • regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure transcriptional initiation, internal ribosome entry sites (IRES) and optional regulatory elements that ensure transcriptional termination and transcript stability.
  • Non-limiting examples of such regulatory elements that ensure transcriptional initiation include promoters, translation initiation codons, enhancers, insulators and/or regulatory elements that ensure transcriptional termination, which are included in the downstream of the nucleic acid molecule of the present application. Further examples include Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing, nucleotide sequences encoding secretion signals, or signal sequences depending on the expression system used, which can guide the expressed protein to a cell compartment or culture medium.
  • the vector may also contain additional expressible polynucleotides encoding one or more protein chaperones to promote correct protein folding.
  • viral vectors include adenovirus, retrovirus, herpes virus and AAV vectors.
  • recombinant viruses can be produced by techniques well known to those skilled in the art, such as by transfection of packaging cells or by transient transfection of helper plasmids or viruses.
  • Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.
  • the application provides a host cell transfected, transduced or transformed according to the nucleic acid and/or vector of the application on the other hand.
  • transformation refers to the introduction of an "external” (i.e., external or extracellular) gene or DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a substance of interest, typically a protein encoded by the gene or the introduced sequence.
  • a host cell that receives and expresses the introduced DNA or RNA has been "transformed".
  • the nucleic acid molecules and/or vectors of the present application can be designed to be introduced into cells by, for example, chemical-based methods (polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nuclear transfection), non-chemical methods (electroporation, sonoporation, phototransfection, gene electrotransfer, fluid delivery or transformation that occurs naturally when cells are in contact with the nucleic acid molecules of the present application), particle-based methods (gene guns, magnetic transfection, puncture transfection), phage vector-based methods, and viral methods.
  • chemical-based methods polyethyleneimine, calcium phosphate, liposomes, DEAE-dextran, nuclear transfection
  • non-chemical methods electroporation, sonoporation, phototransfection, gene electrotransfer, fluid delivery or transformation that occurs naturally when cells are in contact with the nucleic acid molecules of the present application
  • particle-based methods gene guns, magnetic transfection, puncture transfection
  • phage vector-based methods and viral methods.
  • expression vectors derived from, for example, retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, Semliki viruses, or bovine papilloma viruses can be used to deliver nucleic acid molecules to targeted cell populations.
  • the baculovirus system can also be used as a vector in the eukaryotic expression system of the nucleic acid molecules of the present application.
  • the nucleic acid according to the present application can be used to produce the Nanobody B12 of the present application in a suitable expression system.
  • expression system refers to host cells and vectors that are compatible under appropriate conditions, for example, for expressing proteins encoded by exogenous DNA carried by the vector and introduced into host cells.
  • Conventional expression systems include Escherichia coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors thereof.
  • Other embodiments of host cells include prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • Specific embodiments include, but are not limited to, Escherichia coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells and HEK293T cells, etc.) and primary or established mammalian cell cultures (e.g., derived from lymphoblasts, fibroblasts, epithelial cells, neural cells, adipocytes, etc.).
  • mammalian cell lines e.g., Vero cells, CHO cells, 3T3 cells, COS cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells and HEK293T cells, etc.
  • primary or established mammalian cell cultures e.g.,
  • any suitable host cell/vector system can be used for the expression of the DNA sequence encoding the antibody molecule of the present application.
  • Bacteria e.g., E. coli
  • eukaryotic e.g., mammalian host cell expression systems
  • the above-mentioned cells include (but are not limited to) mammalian cells, plant cells, insect cells, fungal cells, or cells of bacterial origin.
  • mammalian cells one selected from (but not limited to) the group consisting of CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, HEK293 cells, and HEK293T cells can be preferably used as a host cell. Any cell known to those skilled in the art that can be used as a mammalian host cell can be used in the art.
  • the present application also relates to Nanobody B12 bound to a detectable marker.
  • Nanobody B12 bound to a detectable marker means herein that the detectable marker is directly or indirectly bound to Nanobody B12, for example via a cleavable or non-cleavable linker peptide, or is incorporated into Nanobody B12.
  • the detectable marker may in particular be bound to Nanobody B12 by substitution (for example by replacing H with I at the level of tyrosine residues), by complexation or by chelation.
  • detectable marker herein means a compound that produces a detectable signal. When it is attached to a tracer, it can monitor what the tracer becomes in vivo.
  • Detectable markers can be MRI contrast agents, scintigraphy contrast agents, X-ray imaging contrast agents, ultrasound contrast agents, optical imaging contrast agents.
  • Examples of detectable markers include radioactive elements, fluorophores such as fluorescein, Alexa or cyanine; chemiluminescent compounds such as luminol; bioluminescent compounds such as luciferase or alkaline phosphatase; and contrast agents such as nanoparticles or gadolinium. The selection of suitable detectable markers depends on the detection system used and is within the capabilities of those skilled in the art.
  • the detectable marker when the detection system is MRI, the detectable marker is preferably iron oxide nanoparticles or gadolinium; when the detection system is fluorescence imaging, the detectable marker is preferably fluorescein, Alexa or cyanine; when the detection system is chemiluminescence imaging, the detectable marker is preferably luminol; when the detection system is bioluminescence imaging, the detectable marker is preferably luciferase or alkaline phosphatase; when the detection system is nuclear imaging, the detectable marker is preferably a radioactive element, such as gallium ( 68Ga ) for PET imaging, or technetium 99m ( 99mTc ) for SPECT imaging.
  • the detectable marker when the detection system is MRI, the detectable marker is preferably iron oxide nanoparticles or gadolinium; when the detection system is fluorescence imaging, the detectable marker is preferably fluorescein, Alexa or cyanine; when the detection system is chemiluminescence imaging, the detectable marker is
  • the detectable marker is a radioactive element.
  • radioactive elements used in nuclear imaging techniques include technetium 99m ( 99m Tc), iodine 123 ( 123 I), iodine 125 ( 125 I), fluorine 18 ( 18 F), gallium 68 ( 68 Ga) and any other radioactive element that can be used on humans. Therefore, preferably, the radioactive element is selected from 99m Tc, 123 I, 125 I, 18 F and 68 Ga. Most preferably, the radioactive element is 99m Tc or 68 Ga, more preferably 99m Tc.
  • the nanobody B12 targeting CEACAM5 described in the present application can also be conjugated to a therapeutic agent to form an immunoconjugate, such as an antibody-drug conjugate (ADC).
  • Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents.
  • the antibody and therapeutic agent are preferably conjugated via a cleavable linker (e.g., a peptidyl, disulfide or hydrazone linker).
  • the present application also provides other polypeptides, such as fusion proteins comprising nano antibody B12 or fragments thereof.
  • the present application also includes fragments of nano antibody B12 of the present application.
  • the fragment has at least about 50 consecutive amino acids of the antibody of the present application, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.
  • Nanobody B12 according to the present application can be administered to a patient using any administration method known to a person skilled in the art.
  • Nanobody B12 can be administered, for example, orally, by inhalation or parenterally (in particular by intravenous injection).
  • Nanobody B12 can be in the form of injectable solutions and suspensions, packaged in vials or bottles.
  • Parenteral administration forms are usually obtained by mixing Nanobody B12 according to the present application with buffers, stabilizers, preservatives, solubilizers, isotonic agents and suspending agents. According to known techniques, these mixtures can be sterilized and packaged in the form of intravenous injections.
  • buffers include buffers, stabilizers, preservatives, solubilizers, isotonic agents and suspending agents.
  • these mixtures can be sterilized and packaged in the form of intravenous injections.
  • Those skilled in the art can, for example, use phosphate-based buffers as buffers.
  • Embodiments of suspending agents include methylcellulose, gum arabic and sodium carboxymethylcellulose.
  • Embodiments of stabilizers include sodium sulfite and sodium metabisulfite
  • preservatives include sodium p-hydroxybenzoate, sorbic acid, cresol and chlor
  • Nanobody B12 administered naturally depends on the route of administration, the height and/or weight of the patient, and the detection technique used.
  • the term "patient” refers to a human being who presents symptoms associated with a CEACAM5-related disease.
  • the disease includes solid tumors or hematological malignancies, inflammatory diseases, and the like.
  • Non-limiting examples of cancers for treatment include castration-resistant neuroendocrine stage prostate cancer.
  • the methods described herein can also be used to treat metastatic cancer, unresectable cancer, refractory cancer, and/or recurrent cancer.
  • the present application relates to the use of the Nanobody B12 in a method for diagnosis or prognosis.
  • diagnosis method refers to a method by which it can be determined whether an individual suffers from a pathological condition.
  • prognostic method or “prognosis” herein means a method which makes it possible to determine whether an individual is at risk of developing a pathological condition.
  • Nanobody B12 as defined above is used for the diagnosis or prognosis of a CEACAM5-related disease.
  • the present application also relates to the use of Nanobody B12 in the preparation of a medicament, in particular a medicament for treating CEACAM5-related diseases.
  • a therapeutic method comprising administering a therapeutically effective amount of Nanobody B12 as defined above to a patient in need thereof is also part of the present application.
  • treatment of a CEACAM5-related disease is intended to mean “therapeutic treatment” (or curative treatment) of a CEACAM5-related disease, including slowing or inhibiting the progression of a CEACAM5-related disease. It is also intended to mean “preventive treatment” of a CEACAM5-related disease.
  • prevention is intended to mean preventing or delaying the onset of a CEACAM5-related disease. The occurrence or reduction in the intensity of clinical or biochemical manifestations associated with the disease.
  • CEACAM5-related disease A person skilled in the art knows by virtue of his general knowledge how to determine the clinical or biochemical manifestations associated with a given CEACAM5-related disease and which can be improved (treatment) or prevented, delayed or reduced in intensity (prevention).
  • the biological parameter of interest may be the presence and localization of pathological forms of CEACAM5.
  • the present application more specifically relates to Nanobody B12 as defined above, for use in the treatment and/or prevention of CEACAM5-related diseases, preferably for the treatment and/or prevention of castration-resistant neuroendocrine stage prostate cancer.
  • the present application also relates to the use of Nanobody B12 for the preparation of a medicament intended for the treatment of CEACAM5-related diseases and/or the prevention of CEACAM5-related diseases in patients who may develop CEACAM5-related diseases.
  • the present application also relates to a method for treating a CEACAM5-related disease and/or preventing a CEACAM5-related disease in a patient in need thereof, comprising administering to a patient in need thereof a therapeutically effective amount of Nanobody B12 as defined above.
  • Nanobody B12 can be administered in a suitable form, for example, orally, by inhalation, parenterally (particularly by intravenous injection).
  • Nanobody B12 can be in the form of an injectable solute and suspension packaged in a vial or bottle.
  • the form of parenteral administration is obtained by mixing Nanobody B12 with a buffer, a stabilizer, a preservative, a solubilizer, an isotonic agent, and a suspending agent. According to known techniques, these mixtures are subsequently sterilized and then packaged in the form of an intravenous injection.
  • a buffer a person skilled in the art can use an organophosphate-based buffer.
  • suspending agents examples include methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, gum arabic, and sodium carboxymethylcellulose.
  • stabilizers used according to the present application are sodium sulfite and sodium metabisulfite, while sodium p-hydroxybenzoate, sorbic acid, cresol, and chlorocresol can be mentioned as preservatives.
  • the present application also relates to a pharmaceutical composition comprising a combination of Nanobody B12 and a pharmaceutically acceptable carrier.
  • pharmaceutically or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic or otherwise troublesome reactions when administered to mammals, particularly humans.
  • the expression "pharmaceutically acceptable carrier” includes any solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic or absorption delaying agent, etc.
  • pharmaceutically acceptable carrier includes any solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic or absorption delaying agent, etc.
  • the use of these media and agents for pharmaceutically active substances is well known to those skilled in the art. Except for the case where conventional media or agents are incompatible with the active ingredient, their use in pharmaceutical compositions can be envisioned. Additional active ingredients can also be incorporated into the composition.
  • the present application also relates to the use of Nanobody B12 in detecting CEACAM5 in a sample in vitro.
  • sample means a portion of a larger element.
  • a sample is a substance of biological origin. It contains cells and/or other molecular entities to be characterized and/or identified based on, for example, physical, biochemical, chemical and/or physiological characteristics.
  • disease sample or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain cells and/or molecular entities to be characterized.
  • Samples include, but are not limited to, tissue samples (e.g., tumor tissue samples), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymphatic fluid, synovial fluid, Follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.
  • Loss of AR expression is an important pathological feature of invasive variant prostate cancer.
  • hormone-sensitive prostate adenocarcinoma cell line LNCAP lactaminopyruvate, hematoma-derived cells 22RV1 and C4-2, and invasive variant prostate cancer PC3 and DU145 with small cell carcinoma characteristics were selected.
  • the above cell lines were implanted into poly-lysine-coated Perkin Elmer ultra cell 96-well plates.
  • the specific steps are as follows: a) Place the cells on ice, wash three times with PBS, and fix with 0.25% paraformaldehyde for 10 minutes; b) Wash three times with PBS and block with 4% BSA for 1 hour; c) Dilute CEACAM5 antibody with blocking solution at 1:100 and incubate with cells at room temperature for 1 hour; d) Wash three times with pre-cooled PBS, dilute Alexa 594-conjugated secondary antibody with 1:1000 with blocking solution, and incubate with cells for 1 hour; e) Wash three times with pre-cooled PBS, perform nuclear staining with Hoechst 33342, and detect the expression of CEACAM5 protein under a fluorescent inverted microscope.
  • the cell lines used for immunofluorescence, the hormone-sensitive prostate adenocarcinoma cell line VCAP, and the invasive variant prostate cancer-derived cell line NCI-H660 with neuroendocrine phenotype characteristics were selected. After trypsin digestion, the cells were collected and washed with PBS. After 10 minutes of fixation with 4% PFA, they were washed again with PBS. The antibody used this time was a direct-labeled flow antibody (Biolegend, cat.392805). CEACAM5 antibody was diluted with 4% BSA solution at a ratio of 1:100 and incubated at room temperature for 1 hour and washed 3 times with PBS. The direct-labeled flow antibody was directly loaded on the machine.
  • Neuroendocrine carcinoma is the most common pathological type of invasive variant prostate cancer. Therefore, according to the recommendation of the International Prostate Cancer Foundation, using any one of the SYN, CHGA and CD56 markers as the standard, 4 clinical specimens were collected from the Third affiliated Hospital of Southern Medical University. The same method was used to collect 3 PDX model tissues. At the same time, the PC3 cell line CDX model (OCT embedded) collected in previous experiments was used. Normal human tissue, prostate cancer, benign prostatic hyperplasia and normal prostate tissue were derived from tissue chips (Zhongke Guanghua). Paraffin-embedded tissues were dewaxed and hydrated for antigen retrieval.
  • the tissue was embedded in OCT, and after sectioning, PBS was used to remove OCT and fixed with acetone/methanol solution (4:1) for 10 minutes. All tissues were stained with 3% The endogenous peroxidase activity was blocked with hydrogen peroxide solution. After washing with TBS 2-3 times, 4% donkey serum was used for blocking for 1 hour. Then, CEACAM5 antibody was diluted with blocking solution at 1:100 and incubated with the chip for 1 hour. Wash with TBS again for 2-3 times, biotin-labeled secondary antibody was diluted with blocking solution at 1:200, and incubated with the chip for 1 hour.
  • CEACAM5 antigen was analyzed in the whole cell lineage derived from prostate cancer by cell immunofluorescence analysis and flow cytometry membrane surface antigen detection. As shown in Figure 1A and Figure 1B, it can be found that CEACAM5 is only expressed in castration-resistant prostate cancer cell lines showing non-epithelial characteristics. Its positive rate in the NCI-H660 cell line with neuroendocrine characteristics is 100%, while the positive rate in PC3 cells showing small cell differentiation characteristics is 24%. As for prostate cancer cell lines with glandular epithelial differentiation, they are all negatively expressed. Among them, the above experiment selected the human colon cancer cell line Ht29 with positive expression of CEACAM5 as a positive reference.
  • CEACAM5 was expressed in clinical specimens, PDX models and PC3CDX models of invasive variant prostate cancer with neuroendocrine characteristics, but no significant expression was found in prostate adenocarcinoma tissue (grouped according to the International Society of Urological Pathology prostate cancer pathological grading system, ISUP grading), prostate hyperplasia and normal prostate tissue.
  • CEACAM5 is an important membrane protein marker of CRPC-NE and can be used as a potential target for antibody targeted therapy.
  • CEACAM5-FC protein was purchased from ACROBiosystems.
  • the natural alpaca-derived phage display nanoantibody library was screened using the immunotube method, and the selected phage display library had a capacity of 2x10 9 .
  • the screening steps are as follows: a) The target protein was coated on the immunotube at a concentration of 50 ⁇ g/mL, and three rounds of enrichment screening were performed; b) The third round of phage eluate was used for plating, and 192 monoclonal clones were randomly selected for ELISA verification. The third round of phage eluate was used for plating and monoclonal clones were selected for ELISA.
  • the ELISA reading of CEACAM5 was greater than 3 times the corresponding BSA reading, and the ELISA reading of the FC antigen was less than 2 times the corresponding BSA reading as the positive standard; c) The positive monoclonal clones identified by the secondary phage ELISA were sent to the public The sequence information is determined by sequencing; d) the screened nanoantibodies are designed and synthesized according to the sequencing information, and expressed and purified by Escherichia coli; e) the affinity of the nanoantibodies is preliminarily identified by ELISA affinity experiment.
  • the nanobody gene sequence was cloned into the PET-22B vector, and the hemagglutinin HA tag and HIS TAG were fused and expressed for subsequent detection.
  • the expression and purification steps are as follows: a), because it is a periplasmic expression system, 1.0mM concentration of ITPG was used for induction overnight at 30°C, 250rpm; b), the bacteria were collected by centrifugation, washed with PBS, and resuspended in a phosphate solution containing 10nM imidazole; c), polymyxin was added at a ratio of 500,000 units per 50ml of bacterial solution, and the bacteria were broken on a shaker at 37°C for 1 hour; d), 17000g, 4°C centrifugation for 30min, the supernatant was taken and incubated with Ni filler at 4°C for 1 hour; e), the bacterial protein was removed by washing with a gradient imidazole/phosphate solution (10mM and 20mM).
  • ELISA plates were coated with CEACAM5-FC protein.
  • FC antigen and BSA were coated as negative reference.
  • various concentrations of nanoantibodies were added and incubated at room temperature for 1 hour, rinsed with PBS 3 times, and incubated with anti-HA antibody at room temperature for 1 hour.
  • the signal was amplified by horseradish peroxidase-labeled anti-HA antibody and developed with TMB.
  • This experiment is used to verify the direct interaction between the purified nanobodies expressed in vitro and the purified antigen proteins in vitro, and to calculate the equilibrium constant between the two.
  • the purified antigen proteins are fixed on the chip, and nanobodies of different concentrations are added sequentially to analyze the affinity with the antigen proteins, and the reaction signals within 360 seconds are recorded, the kinetic curves are made, and the relevant parameters are calculated.
  • the nanobody and IR800-Mal were reacted at a feed ratio of 5:1 at 4°C overnight.
  • the purity was then analyzed by SDS-PAGE gel, and the labeling effect of the protein was detected in fluorescence mode.
  • HT29 and PC3 cells positive for CEACAM5 were fixed and planted in 96-well plates with 4% paraformaldehyde. 4% BSA was blocked for 1 hour.
  • the nanobody was diluted with blocking solution to a gradient working solution and incubated with the cells at room temperature for 1 hour. PBST was washed 3 times. The binding of nanoantibodies on the cell surface was detected by a fully automatic electrophoresis fluorescence immunoassay.
  • the lentivirus used was synthesized by Weigen Biotech, and its nucleotide sequence is as follows (SEQ ID NO.10):
  • 0.5*10 6 cells were seeded in a six-well plate. After 12 hours, OPTI-MEM medium was replaced and the MOI was 30. Lentivirus and polyamide (10 ⁇ g/ml) were added to promote viral infection. After 48 hours, OPTI-MEM medium was removed and complete medium containing 2 ⁇ g/ml puromycin was added to select infected cells of the test tube. The selection was continued for 4 days until no dead cells were floating. The complete medium was replaced and the culture was expanded. The efficiency of CEACAM5 knockdown was analyzed by qRT-PCR and immunoblotting.
  • Total cell protein was extracted using RIPA strong lysis buffer containing PMSF, protease and phosphatase inhibitors, and quantified by BCA. Take the nitrocellulose membrane and draw a 1cm2 grid with a pencil. Prepare 5 ⁇ L of the test solution according to the sample protein concentration of 20.0 ⁇ g and drop it into the grid. Dry at room temperature. After blocking with 10% skim milk powder for 1 hour, use 4% BSA to prepare 15ml of nanoantibody working solution at a concentration of 20 ⁇ g/ml. Place the nanoantibody working solution and nitrocellulose membrane in a 10cm dish and incubate overnight at 4°C. Wash 3 times with PBST, add anti-HA antibody coupled with horseradish peroxide at a ratio of 1:1000 and incubate at room temperature for 1 hour. Wash again with PBST. ECL development and exposure.
  • Negative control and CEACAM5 knockdown PC3 cells seeded in 96-well plates were fixed with 4% paraformaldehyde. Blocked with 4% BSA for 1 hour. Nanobodies were diluted to gradient working solutions with blocking solution and incubated with cells at room temperature for 2 hours. Washed 3 times with PBST. Anti-HA mouse monoclonal antibody diluted 1:100 was added and incubated at room temperature for 2 hours, and then washed 3 times with PBST. Finally, Alexa 488-conjugated anti-mouse secondary antibody diluted 1:200 was added and incubated at room temperature for 1 hour, and washed 3 times with PBST to remove unbound antibodies. The results were analyzed using a fluorescent immunoassay analyzer.
  • nanobody B12 (as shown in FIG3B ), which has a good affinity for CEACAM5 antigen.
  • the gene sequence of nanobody B12 obtained by sequencing is as follows:
  • the PET.22B vector was introduced and induced for expression in Escherichia coli.
  • the Ni column captured the B12 nanobody with the His-tagged protein.
  • the bacterial protein was removed by washing with a gradient imidazole/phosphate solution (10mM and 20mM). High-purity nanobody B12 was obtained.
  • the HIS and HA tags of the nanobody were verified by using Coomassie blue staining and protein immunoblotting, and it was determined that the purified product was nanobody B12.
  • FIG3E is a graph showing the ELISA test results of the nanoantibody in the affinity verification test of the nanoantibody B12
  • FIG3F is a graph showing the surface plasmon resonance test results of the nanoantibody B12 in the affinity verification test
  • FIG3G is a graph showing the Cell ELISA test results of the nanoantibody B12 in the affinity verification test.
  • the nanoantibody B12 has a high degree of specificity and affinity in the process of recognizing the CEACAM5 antigen, and the nanoantibody B12 can effectively recognize the CEACAM5 antigen.
  • This example confirms the efficacy of the nanoantibody B12 in targeting the CEACAM5 molecule and the positive cell line by ELISA, SPR and Cell ELISA.
  • FIG3H is a diagram showing the results of the construction of the PC3 cell line with CEACAM5 knockdown in the specificity validation test of the nanoantibody B12
  • FIG3I is a diagram showing the results of the Cell ELISA experiment in the specificity validation test of the nanoantibody B12
  • FIG3J is a diagram showing the results of the dot blot experiment in the specificity validation test of the nanoantibody B12.
  • the invasive variant prostate cancer cell line PC3 with stable downregulation of CEACAM5 antigen was constructed by lentiviral transfection, and the knockdown efficiency was confirmed by qRT-PCR and immunoblotting, and the protein level could be reduced to 75% of the control group.
  • FIG3I and FIG3J respectively, using Cell ELISA and Dot blot experiments to confirm that CEACAM5 knockout can reduce the nanoantibody B12 signal bound to the PC3 cell line membrane surface and targeting its cell lysate by 30%.
  • the above results are consistent with the immunoblot characterization of the knockdown cell line, confirming that the nanoantibody B12 has good specificity in targeting the CEACAM5 antigen-positive prostate cancer cell line.
  • Nanobody B12 and IR800-Mal were added at a ratio of 2:1, and control nanobody C9 and IR800-Mal were added at a ratio of 2:1, and reacted overnight at 4°C.
  • the invasion-variant prostate cancer cell line PC3 was expanded in vitro and a cell suspension of 5*10 7 /ml was prepared. 100 ⁇ l of the cell suspension was subcutaneously injected into the right limb of 5-6 week-old male nude mice. In vivo imaging was performed when the tumor grew to 300-500 mm 3 .
  • the fluorescent molecule-labeled nanobody was injected into the circulation of nude mice via tail vein injection. Imaging was performed at 2, 4, 8, 12 and 24 hours. The nude mice were then killed, and their vital organs were collected and imaged again to confirm the imaging ability of nanobody B12 in vivo.
  • Nanobody B12 can effectively target and recognize the prostate cancer cell line PC3 expressing CEACAM5 antigen in vivo.
  • Fluorescent dye IR800 was labeled with nanobody B12 and non-targeted nanobody C and injected into the circulation of male nude mice with PC3 xenografts constructed through the tail vein. Referring to Figure 4A, the results showed that after 24 hours, IR800-labeled nanobody was significantly enriched in PC3 xenografts.
  • the fluorescence signal detection analysis of the excised nude mouse organs and PC3 xenografts confirmed that the nanobody B12 was significantly enriched in the CEACAM5-positive PC3 tumor.
  • tissue nanobody detection analysis was performed, see Figure 4C, by targeting VHH antibodies to detect nanobodies and using immunofluorescence as a technical means for analysis, it was found that there was a significant fluorescent signal in the PC3 xenografts that received B12 injection.
  • nanobody B12 can effectively recognize the grafts constructed from the invasive variant prostate cancer cell line PC3 in vivo.
  • the immunotoxin B12-PE38 gene sequence (SEQ ID NO.11) was cloned into the PET-15B vector, and the hemagglutinin HA tag and HIS TAG were fused and expressed for subsequent detection.
  • the expression and purification steps are as follows: a) Induce overnight with 1.0mM ITPG at 30°C, 220rpm; b) Collect the bacteria by centrifugation, wash with PBS, resuspend the bacteria with phosphate solution containing 10nM imidazole, and use ultrasonic lysis; c) Centrifuge at 17000g, 4°C for 30min, take the supernatant and incubate with Ni filler at 4°C for 1 hour; d) Use gradient imidazole/phosphate solution (10mM and 20mM) to wash and remove bacterial proteins.
  • the B12-PE38 gene sequence is the B12-PE38 amino acid sequence (SEQ ID NO.12) generated by conversion of the EMBOSS Backtranseq (https://www.ebi.ac.uk/) online database, and the generated nucleotide sequence corresponds to the species (Escherichia coli).
  • immunotoxin B12-PE38 was labeled with fluorescent molecule Cy5 (ratio 2:1) and confirmed by gel electrophoresis.
  • the labeled immunotoxin molecules were sterilized by filtration using a 0.22 ⁇ m filter and added to PC3 culture medium (with different concentration/time gradients and treatment conditions).
  • PC3 cells were seeded in poly-lysine-coated Perkin Elmer ultra cell 96-well plates and washed three times with PBS to remove unabsorbed immunotoxins. They were then fixed with 4% PFA and blocked with BSA solution. The phagocytosis results were photographed under a fluorescence microscope and analyzed using Image J software.
  • CCK8 2000 cells were seeded in 96-well plates. Immunotoxin was diluted in culture medium and added to 96-well plates, with at least 3 replicates per group. After 72 hours, the culture medium containing CCK8 detection solution was replaced and the reading at 450 nm was measured by an ELISA reader. A negative control group B12-PE38mutant was set up.
  • Edu experiment 5*10 4 cells were plated and 1 ⁇ M concentration of immunotoxin was added. After 12 hours, cells were fixed with PFA and washed 3 times with PBS. Cells were stained according to the kit instructions and photographed using a fluorescence microscope. The number of EDU-positive cells was counted.
  • Transwell assay 0.5*10 6 cells were seeded in a 6-well plate and pre-treated with 1 ⁇ M immunotoxin for 12 hours. Subsequently, the cells were trypsinized and collected. 6*10 4 cells were seeded into the Transwell chamber and an empty medium without serum was added. In contrast, the lower chamber was provided with complete medium containing 20% FBS. After 24 hours, the chamber was removed and fixed with methanol. Crystal violet staining was performed and photographed.
  • Human invasive prostate cancer cell line PC3 and human colon cancer cell line HT29 were used to construct a transplant model in 5-6 week old nude mice by subcutaneous injection.
  • immunotoxin B12-PE38 was injected into the tail vein for treatment.
  • the treatment group was divided into 2 treatment doses.
  • the high-dose group was given at a dose of 0.6 mg/kg, and the low-dose group was treated at 0.4 mg/kg.
  • the negative control group received tail vein injection of B12 and PBS, respectively. A total of 6 treatments were given.
  • the tumor volume was measured and the mouse body weight was weighed at intervals of 1 day.
  • the animals were euthanized after treatment.
  • the tumors were removed and measured.
  • Tumor and important organ tissue specimens were collected and fixed with PFA, dehydrated with ethanol gradient, and embedded in paraffin. Tissue HE staining was performed to observe the lesions.
  • tumor tissue specimens were further subjected to Ki67 immunohistochemistry and Tunel staining to analyze proliferation and apoptosis.
  • Chemotherapy regimens based on paclitaxel or cisplatin combined with etoposide have limited effect on the extension of survival period in patients with aggressive variant prostate cancer. Therefore, the therapeutic efficacy of immunotoxin B12-PE38 combined with docetaxel was tested.
  • a PC3 nude mouse xenograft model was constructed according to the method described in Example 3, and a NSG mouse model derived from PDX tissue was constructed.
  • Immunotoxin B12-PE38 was administered by tail vein, for a total of 6 injections.
  • Docetaxel was administered intraperitoneally (calculated at 2.5 mg/kg), for a total of 4 injections. The therapeutic effect was evaluated by survival analysis.
  • Bone metastasis is an important pathological feature and a poor prognostic factor for prostate cancer. Therefore, a tibial tumor in situ model was constructed to evaluate the therapeutic effect. 50 ⁇ l of PC3 cell suspension was injected through tibial plateau puncture. After 3 weeks, when osteolytic changes were observed in X-ray analysis, treatment was started. A total of 6 injections were given.
  • FIG. 5 is a diagram showing the construction and identification results of the immunotoxin based on the nanoantibody B12 targeting CEACAM5.
  • FIG. 5C shows the affinity of targeting the PC3 cell line, in which the CEACAM5-positive human colon cancer cell line HT29 was used as a positive reference, and the CEACAM5-negative human prostate cancer 22RV1 and VCAP cell lines were used as negative references.
  • Figure 5D is a specific result diagram of the targeting effect. As shown in the figure, the use of nanoantibody B12 can effectively block the targeting effect of B12-PE38 on the PC3 cell line, while the non-targeted C9-PE38 immunotoxin group lacks fluorescent signals.
  • FIGs 5E and 5F are the results of the analysis of PC3 phagocytosis and uptake of immunotoxin B12-P38 in vitro.
  • the PC3 cell line positive for CEACAM5 antigen can effectively take up immunotoxin B12-PE38, in which all immunotoxins used are pre-labeled with fluorescent dye molecules Cy5.
  • Figure 5E is the result of fluorescence microscopy, in which the fluorescence signal is measured using Image J software.
  • Figure 5F is the result of flow cytometry analysis, in which the fluorescence signal is characterized by the Cy5 cell positivity rate.
  • the results show that the uptake of immunotoxin B12-PE38 is significantly time- and concentration-dependent and can be blocked by the nanoantibody B12 targeting CEACAM5.
  • the uptake rate of the non-targeted immunotoxin C9-PE38 treatment group is lower than that of the B12-PE38 treatment group.
  • Figures 5G, 5H and 5I are experimental analysis results of the killing of PC3 cells by immunotoxin B12-PE38 in vitro. It was confirmed that B12-PE38 can effectively kill the CEACAM5-positive small cell prostate cancer cell line PC3.
  • Figure 5G is a CCK8 experiment result diagram
  • Figure 5H is an Edu proliferation experiment result diagram
  • Figure 5I is a Transwell experiment result diagram.
  • immunotoxin B12-PE38 treatment can significantly reduce the proportion of proliferating cells in the CEACAM5-positive cell line PC3; referring to Figure 5I, immunotoxin B12-PE38 treatment can significantly inhibit the in vitro migration of the PC3 cell line, which is manifested by a significant decrease in the number of PC3 migrating cells in the Transwell experiment.
  • Figure 6 shows that the immunotoxin B12-PE38 can effectively kill CEACAM5-positive tumor cell lines in vivo.
  • Figures 6A and 6B show that the B12-PE38 immunotoxin mutant can effectively target PC3 nude mouse xenografts.
  • the B12-PE38 immunotoxin mutant used was pre-labeled with the fluorescent dye molecule IR800, and the non-targeted immunotoxin C9-PE38 mutant protein was used as a negative control (Note: the mutant part is the toxin PE38 to avoid harm to nude mice).
  • the fluorescently labeled protein was injected into the nude mouse circulation through the tail vein.
  • Figures 6C, 6D, 6E and 6F are the results of constructing a nude mouse PC3 transplant model and treating it with B12-PE38 via the tail vein. The results showed that B12-PE38 can effectively inhibit the growth of PC3 xenografts in vivo.
  • Figure 6C is a treatment cycle diagram. The transplant model was constructed in the form of subcutaneous tumor inoculation, and the tail vein injection was performed at intervals of 2 days, and a total of 6 immunotoxin B12-PE38 injections were given.
  • Figures 6E and 6F are growth curves of tumors in different treatment groups, respectively. Among them, Figure 6E shows the growth curves of different tumors in each treatment group.
  • FIGS. 6G and 6H are analysis diagrams of the efficacy of immunotoxin B12-PE38 in treating human colon cancer xenografts in a nude mouse model.
  • the results showed that B12-PE38 treatment could significantly inhibit the growth of human colon cancer HT29 and improve the prognosis of nude mice.
  • the above results confirm that B12-PE38 is an effective treatment for CEACAM5-positive tumors.
  • FIG7 is the pathological analysis results of PC3 xenografts in different treatment groups in the in vivo experimental analysis of immunotoxin B12-PE38 treatment of CEACAM5 positive cell line transplants.
  • FIG7A is the result of tissue HE staining
  • FIG7B is the result of cell proliferation and apoptosis marker detection.
  • the results shown in FIG7A and FIG7B are consistent with the previous animal experiments, that is, the immunotoxin B12-PE38 treatment group is better than the untreated group, and the high-dose treatment group (0.6 mg) of immunotoxin B12-PE38 is better than the low-dose treatment group (0.4 mg).
  • Figure 8 is a graph showing the results of organ pathology, blood routine and biochemical index analysis of mice treated with immunotoxin B12-PE38 in the in vivo experimental analysis of CEACAM5-positive cell line transplants.
  • Figure 8A is a graph showing the results of organ pathology analysis, and the results show that no significant lesions were found in the important organs of mice treated with immunotoxins.
  • Figures 8B and 8C are graphs showing the results of blood routine and biochemical index analysis, showing that there were no significant abnormalities in the physiological conditions of mice treated with B12-PE38.
  • Figure 9 is a diagram showing the therapeutic effect of immunotoxin B12-PE38 in combination with docetaxel.
  • Figures 9A and 9B show the therapeutic effect of B12-PE38 in combination with docetaxel in PC3 xenografts.
  • Figures 9C and 9D show the effect of B12-PE38 in combination with docetaxel in the treatment of human prostate cancer PDX models. The results all show that immunotoxins and docetaxel have good synergistic efficacy.
  • Figure 10 is an analysis of the efficacy of immunotoxin B12-PE38 in treating PC3 tibial orthotopic tumors.
  • Figure 10A is a modeling and dosing explanation
  • Figure 10B is an X-ray analysis result of tibial orthotopic tumors during the treatment cycle
  • Figure 10C is a CT examination result of tibial orthotopic tumors
  • Figure 10D is a bone pathology analysis.
  • the coronal and CT reconstruction results shown in Figure 10C show that high-dose B12-PE38 can significantly inhibit the growth of PC3 tibial transplant tumors; the bone pathology analysis shown in Figure 10D shows that immunotoxin B12-PE38 can significantly inhibit the growth of tibial transplant tumors.

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Abstract

L'invention concerne un nanoanticorps B12 ciblant CEACAM5 et une utilisation du nanoanticorps B12. Le nanoanticorps B12 comprend trois régions déterminant la complémentarité (CDR), qui sont respectivement CDR1, CDR2 et CDR3 ; la séquence d'acides aminés de CDR1 est telle que représentée dans SEQ ID NO. 6, la séquence d'acides aminés de CDR2 est telle que représentée dans SEQ ID NO. 7, et la séquence d'acides aminés de CDR3 est telle que représentée dans SEQ ID NO. 8. Le nanoanticorps B12 et CEACAM5 ont une affinité et une force de liaison relativement élevées, et le nanoanticorps B12 cible un marqueur membranaire spécifique du cancer de la prostate neuroendocrinien CEACAM5, de telle sorte que le développement de médicaments associés à des nanoanticorps du marqueur membranaire spécifique du cancer de la prostate neuroendocrinien CEACAM5 est facilité, et un traitement ciblé sur un cancer de la prostate neuroendocrinien résistant à la castration est obtenu ; et en outre, le développement de médicaments pour des maladies associées au CEACAM5 est également facilité, et le diagnostic et le traitement de maladies associées au CEACAM5 sont obtenus. De plus, le nanoanticorps peut être largement utilisé en raison d'un faible poids moléculaire.
PCT/CN2024/071306 2023-01-09 2024-01-09 Nanoanticorps b12 ciblant ceacam5 et utilisation du nanoanticorps b12 Ceased WO2024149232A1 (fr)

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