Humanized cell type prion protein monoclonal antibody and application thereof

Abstract

The invention discloses a monoclonal antibody aiming at humanized cellular prion protein and application of the monoclonal antibody. The invention relates to the technical field of biology, in particular to a monoclonal antibody aiming at humanized cellular prion protein and application of the monoclonal antibody. The antibody is a monoclonal antibody or an antigen binding fragment thereof, and comprises a heavy chain variable region and a light chain variable region, amino acid sequences of LCDR1, LCDR2 and LCDR3 in a light chain variable region of the antibody are sequentially shown as the 24 to 40 , the 56 to 62 and the 95 to 103 of SEQ ID No.1; amino acid sequences of HCDR1, HCDR2 and HCDR3 in a heavy chain variable region of the antibody are sequentially shown as the 26th site to the 32nd site, the 52nd site to the 57th site and the 99th site to the 103th site of SEQ ID No.2. The antibody disclosed by the invention has relatively high affinity with the cellular prion protein, and can be applied to detection or diagnosis of tumors expressing the cellular prion protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a humanized monoclonal antibody against cellular prion protein and its application. Background Art

[0002] Cellular prion protein (PrPc) is a glycoprotein that is covalently bound to GPI (glycosylphosphatidylinositol) buried in the plasma membrane through its C-terminus and anchored to the extracellular surface of plasma membrane lipid rafts. PrPc is highly expressed in various cancers. It is known that the gene PRNP encoding PrPc is upregulated in cancers, and the expression of PrPc can enhance the proliferation, migration, and drug resistance of cancer cells. The expression of PrPc is positively correlated with the development, drug resistance, and poor prognosis of colorectal tumors.

[0003] In previous studies, the inventors have prepared and screened a series of murine monoclonal antibodies against human PrPc, including clones with biological activity that can inhibit the growth and metastasis of tumor cells, indicating that monoclonal antibodies targeting PrPc have the potential to be applied in the development of tumor drugs. However, murine antibodies have high immunogenicity and may produce strong immune responses. Therefore, humanization and evolution of existing murine antibodies may result in humanized antibodies with higher affinity and specificity, providing new solutions for the development of targeted drugs for various tumors. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a PrPc monoclonal antibody with high specificity and affinity in view of the deficiencies of the prior art, so as to be able to more effectively conduct tumor diagnosis, prognosis evaluation, and the development of tumor treatment drugs.

[0005] To solve the above technical problems, the present invention first provides an antibody.

[0006] The antibody provided by the present invention is a monoclonal antibody or its antigen-binding fragment, and the monoclonal antibody or its antigen-binding fragment includes a heavy-chain variable region and a light-chain variable region; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light-chain variable region of the antibody are shown as positions 24 to 40, 56 to 62, and 95 to 103 of SEQ ID No.1 in sequence; the LCDR1, LCDR2, and LCDR3 are complementary determining regions (CDRs).

[0007] The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy-chain variable region of the antibody are shown as positions 26 to 32, 52 to 57, and 99 to 103 of SEQ ID No.2 in sequence; the HCDR1, HCDR2, and HCDR3 are complementary determining regions (CDRs).

[0008] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody, which generally includes at least a portion of the antigen-binding region or variable region of the parental antibody (e.g., one or more CDRs). The above antigen-binding fragment retains at least some of the binding specificities of the parental antibody. Generally, when the activity is expressed based on molarity, the above antigen-binding fragment retains at least 10% of the parental binding activity. Specifically, the above antigen-binding fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parental antibody to the target.

[0009] The term "Fab fragment" is a heterodimer formed by the binding of the heavy-chain Fd and the intact light chain through a disulfide bond, containing only one antigen-binding site. The above heavy-chain Fd refers to about 1 / 2 of the H-chain portion in Fab (about 225 amino acid residues, including VH, CH1 and a portion of the hinge region).

[0010] The term "Fv fragment" means that vectors containing VH and VL genes can be constructed respectively, co-transfected into cells, expressed respectively, and then assembled into a functional Fv antibody; alternatively, a stop codon can be set between VH and VL in the vector, and two small-molecule protein fragments are expressed respectively, and then combined through non-covalent bonds to form an Fv antibody (Fv fragment).

[0011] The term "Fab′ fragment" contains one light chain and a portion of one heavy chain including the VH domain, the CH1 domain and the region between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab′ fragments to form an F(ab′)2 molecule.

[0012] The term "F(ab′)2 fragment" contains two light chains and two heavy chains each containing a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond is formed between the two heavy chains. Therefore, the F(ab′)2 fragment consists of two Fab′ fragments held together by a disulfide bond between the two heavy chains.

[0013] The term "single-chain antibody (ScFv)" refers to a polypeptide obtained by linking the variable region of the light chain and the variable region of the heavy chain. This polypeptide can spontaneously fold into a natural conformation, maintaining the specificity and affinity of Fv.

[0014] The above CDR is a sequence defined according to the Kabat numbering system.

[0015] Among the above antibodies, the antibody can be as follows:

[0016] 1) The amino acid sequence of the variable region of the light chain of the antibody is SEQ ID No.1; and / or the amino acid sequence of the variable region of the heavy chain of the antibody is SEQ ID No.2;

[0017] 2) The amino acid sequence of the light chain variable region of the antibody is SEQ ID No. 3; and / or the amino acid sequence of the heavy chain variable region of the antibody is any one of the following C1)-C3):

[0018] C1) SEQ ID No. 4;

[0019] C2) SEQ ID No. 5;

[0020] C3) SEQ ID No. 6.

[0021] In the present invention, the light chain variable region (VL) or heavy chain variable region (VH) of the antibody consists of "framework" regions separated by three "complementary determining regions" or "CDRs". The framework regions are used to align the CDRs that specifically bind to the antigen epitope. The CDRs include the amino acid residues in the antibody that are mainly responsible for antigen binding. The VL domain and VH domain each contain the following framework regions (FR) and CDR regions from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, CDR2, and CDR3 of the VL domain are also referred to as LCDR1, LCDR2, and LCDR3 herein, respectively; CDR1, CDR2, and CDR3 of the VH domain are also referred to as HCDR1, HCDR2, and HCDR3 herein, respectively.

[0022] In the above antibody, the heavy chain type of the antibody is IgG1; and / or the light chain type of the antibody is kappa.

[0023] The monoclonal antibody is a monoclonal antibody against PrPc, such as antibody 4AA or 4AA-m, Ab6, HAb6, and DV in the examples.

[0024] In the light chain variable region of the 4AA antibody, the amino acid sequence of FR1 is positions 1-23 of SEQ ID No. 3, the amino acid sequence of FR2 is positions 41-55 of SEQ ID No. 3, the amino acid sequence of FR3 is positions 63-94 of SEQ ID No. 3, and the amino acid sequence of FR4 is positions 104-114 of SEQ ID No. 3.

[0025] In the heavy chain variable region of the 4AA antibody, the amino acid sequence of FR1 is positions 1-25 of SEQ ID No. 6, the amino acid sequence of FR2 is positions 33-55 of SEQ ID No. 6, the amino acid sequence of FR3 is positions 62-102 of SEQ ID No. 6, and the amino acid sequence of FR4 is positions 108-117 of SEQ ID No. 6.

[0026] The present invention also provides an antibody obtained by immunizing an animal with a conjugate of the polypeptide shown in SEQ ID No.9 or SEQ ID No.10 and a carrier protein.

[0027] The original murine antibody Ab6 was obtained by immunizing female Balb / c mice with full-length PrPc protein, and the specific method is the same as that in Patent CN103013926B.

[0028] The present invention also provides any of the following biological materials:

[0029] (A1) A humanized antibody obtained by humanizing any of the antibodies described above.

[0030] (A2) An active fragment of the antibody, which is any of the following derived from the antibody described above or the humanized antibody described in (A1): single-chain antibody, Fab region of the antibody, antigen-binding fragment.

[0031] (A3) A nucleic acid molecule encoding the antibody described above, the humanized antibody described in (A1), or the active fragment of the antibody described in (A2).

[0032] (A4) An expression cassette, recombinant vector, recombinant cell, or recombinant bacterium containing the nucleic acid molecule described in (A3).

[0033] (A5) A kit containing the antibody described above, the humanized antibody described in (A1), the active fragment of the antibody described in (A2), the nucleic acid molecule described in (A3), or the expression cassette, recombinant vector, recombinant cell, or recombinant bacterium described in (A4).

[0034] Among the above biological materials, in the nucleic acid molecule described in (A3), the nucleotide sequences encoding LCDR1, LCDR2, and LHCDR3 in the light chain variable region of the antibody are successively as shown in positions 70 to 120, 166 to 186, and 283 to 309 from the 5'-end of SEQ ID No.7.

[0035] Among the above biological materials, in the nucleic acid molecule described in (A3), the nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are successively as shown in positions 76 to 96, 166 to 183, and 307 to 321 from the 5'-end of SEQ ID No.8.

[0036] In the present invention, the kit includes immunohistochemistry kit, immunofluorescence kit, flow cytometry detection kit, and immunoblotting kit.

[0037] The present invention also provides a pharmaceutical composition, which comprises the antibody described above, or the humanized antibody (A1) or the active fragment of the antibody (A2) in the biological material.

[0038] The present invention also provides the use of the antibody described above in any one of the following:

[0039] (B1) Identifying or assisting in identifying cellular prion protein;

[0040] (B2) Binding or assisting in binding to cellular prion protein;

[0041] (B3) Detecting or assisting in detecting whether a test sample contains cellular prion protein;

[0042] (B4) Preparing a product for detecting or assisting in detecting whether a test sample contains cellular prion protein; (B5) Detecting or assisting in detecting the content of cellular prion protein in a test sample;

[0043] (B6) Preparing a product for detecting or assisting in detecting the content of cellular prion protein in a test sample;

[0044] (B7) Detecting or assisting in detecting a tumor or tumor cell expressing cellular prion protein;

[0045] (B8) Preparing a product for detecting or assisting in detecting a tumor or tumor cell expressing cellular prion protein; (B9) Identifying or assisting in identifying a tumor or tumor cell;

[0046] (B10) Binding or assisting in binding to a tumor or tumor cell;

[0047] (B11) Preparing a product for identifying or assisting in identifying a tumor or tumor cell;

[0048] (B12) Preparing a product for binding or assisting in binding to a tumor or tumor cell;

[0049] (B13) Preparing a product targeting cellular prion protein;

[0050] (B14) Preparing a product for diagnosing or treating a disease related to cellular prion protein;

[0051] (B15) Preparing an anti-tumor drug;

[0052] (B16) Preparing a product for inhibiting tumor cell metastasis.

[0053] The present invention also provides the use of the antibody in the biological material in any one of the following:

[0054] (B1) Identifying or assisting in identifying cellular prion protein;

[0055] (B2) Binding or assisting in binding to cellular prion protein;

[0056] (B3) Detect or assist in detecting whether a sample to be tested contains cellular prion protein;

[0057] (B4) Prepare a product for detecting or assisting in detecting whether a sample to be tested contains cellular prion protein; (B5) Detect or assist in detecting the content of cellular prion protein in a sample to be tested;

[0058] (B6) Prepare a product for detecting or assisting in detecting the content of cellular prion protein in a sample to be tested;

[0059] (B7) Detect or assist in detecting a tumor or tumor cells expressing cellular prion protein;

[0060] (B8) Prepare a product for detecting or assisting in detecting a tumor or tumor cells expressing cellular prion protein; (B9) Identify or assist in identifying a tumor or tumor cells;

[0061] (B10) Bind or assist in binding to a tumor or tumor cells;

[0062] (B11) Prepare a product for identifying or assisting in identifying a tumor or tumor cells;

[0063] (B12) Prepare a product for binding or assisting in binding to a tumor or tumor cells;

[0064] (B13) Prepare a product targeting cellular prion protein;

[0065] (B14) Prepare a product for diagnosing or treating diseases related to cellular prion protein;

[0066] (B15) Prepare an anti-tumor drug;

[0067] (B16) Prepare a product for inhibiting the metastasis of tumor cells.

[0068] The present invention also provides the use of the antibody in the drug composition described above in any one of the following: (B1) Identify or assist in identifying cellular prion protein;

[0069] (B2) Bind or assist in binding to cellular prion protein;

[0070] (B3) Detect or assist in detecting whether a sample to be tested contains cellular prion protein;

[0071] (B4) Prepare a product for detecting or assisting in detecting whether a sample to be tested contains cellular prion protein;

[0072] (B5) Detect or assist in detecting the content of cellular prion protein in a sample to be tested;

[0073] (B6) Prepare a product for detecting or assisting in detecting the content of cellular prion protein in a sample to be tested;

[0074] (B7) Detecting or assisting in detecting tumors or tumor cells expressing cellular prion protein;

[0075] (B8) Preparing products for detecting or assisting in detecting tumors or tumor cells expressing cellular prion protein;

[0076] (B9) Identifying or assisting in identifying tumors or tumor cells;

[0077] (B10) Binding or assisting in binding to tumors or tumor cells;

[0078] (B11) Preparing products for identifying or assisting in identifying tumors or tumor cells;

[0079] (B12) Preparing products for binding or assisting in binding to tumors or tumor cells;

[0080] (B13) Preparing products targeting cellular prion protein;

[0081] (B14) Preparing products for diagnosing or treating diseases related to cellular prion protein;

[0082] (B15) Preparing anti-tumor drugs;

[0083] (B16) Preparing products with the function of inhibiting tumor cell metastasis.

[0084] In the specific embodiments of the present invention, the tumor cells may be colorectal cancer cells, esophageal squamous cell carcinoma cells or hepatocellular carcinoma cells.

[0085] In the specific embodiments of the present invention, the colorectal tumor cells may specifically be colorectal cancer cells P6C.

[0086] In the specific embodiments of the present invention, the esophageal squamous cell carcinoma cells may specifically be KYSE150, KYSE450 and KYSE510.

[0087] In the specific embodiments of the present invention, the hepatocellular carcinoma cells may specifically be RBE, HCCLM3, SMMC7721, HepG2, BEL7407 and HepB.

[0088] The PrPc monoclonal antibody 4AA of the present invention is evolved and mutated from the murine PrPc monoclonal antibody Ab6. 4AA has the ability to bind to PrPc antigen and antigen fragments; 4AA has the characteristic of significantly higher affinity than the parental murine monoclonal antibody Ab6 and commercial murine monoclonal antibodies, and can recognize PrPc antigen or antigen fragments in colorectal cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma cells; and 4AA has the function of significantly inhibiting tumor cell metastasis. Therefore, 4AA can be used to prepare reagents for detecting PrPc expression in colorectal cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma, and can also be used to prepare drugs for inhibiting tumor metastasis, providing a new solution for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 For the humanization and affinity maturation of a murine PrPc monoclonal antibody. Among them, A. Flow cytometry data of 293T and 293T-PrPc cells labeled with murine PrPc monoclonal antibody clone 6 (Ab6). Ab6 strongly binds to 293T-PrPc and weakly binds to wild-type 293T cells. IgG was used as a negative control; B. Flow cytometry data comparing the humanized anti-PrPc antibody HAb6 of Ab6. 293T-PrPc was labeled with Ab6 and HAb6 antibodies. The binding of HAb6 to 293T-PrPc is better than that of other antibodies; C. Generation and screening of the mutant library. CHO-PuroR-14 is a wild-type cell without the insertion of the HAb6 single-chain antibody gene. No-AID is the parental cell inserted with the HAb6 scFv gene. AID is the cell inserted with the HAb6 scFv mutant gene. Cells binding to FITC-PrPc-35aa in AID were enriched by flow sorting; D. The affinity-matured cells were enriched from AID cells. The mature cells consisted of 3 subpopulations, among which the sub1 and sub2 antibody mutants showed higher affinity; E. Flow cytometry detection of the binding of HAb6 mutants (A16D, A97V, A16D-A97V) to PrPc-35aa-FITC. A16D and A97V were obtained by sequencing the mutant antibody clones of sub1 and sub2 cells in D. The results showed that the cells displaying the A16D, A97V, and A16D-A97V mutant antibodies bound to FITC-PrPc-35aa higher than the cells displaying HAb6.

[0090] Figure 2For the directed evolution of PrPc-FC-FITC and the identification of 4AA. Among them, A. Flow cytometry was used to detect the binding ability of the antibody to 293T-PrPc. PrPc was expressed as full-length in 293T; B. Directed evolution of the DV antibody labeled with PrPc-FC-FITC. DV-S1 is a cell expressing the 16D-A97V mutation in the scFv region of the PrPc antibody. AID-plus was transfected into DV-S1 to induce DV mutations, and mutant cells DV-M1 with higher binding ability were obtained. DV-M1 cells were transfected with AID to obtain DV-M2 with higher binding ability to PrPc-FC; C. The 4AA antibody was identified from DV-M2 cells. BLI measurement results between PrPc-FC and the anti-PrPc antibody 4AA. 4AA was expressed as a full-length antibody in Expi293F. The affinity between 4AA and PrPc-FC was 2.03E-10 M; D. KinExA measurement results between 293T-PrPc and DV, 4AA. The affinities of 293T-PrPc for DV and 4AA were 1.01 nM and 92.68 pM, respectively.

[0091] Figure 3 For the specificity and sensitivity of the 4AA antibody. Among them, A. Western blotting was performed on the cell lysates of PrPc-KO-209 and P6C cells using 4AA-m. Loading amount: 10 μg of cell lysate; B. Western blotting was performed on the cell lysates of 293T-PrPc and 293T using 4AA-m. Loading amount: 10 μg of cell lysate; C. Western blotting experiments were performed on the cell lysates of 293T-PrPc with the indicated loading amounts using 4AA-m, 8H4, or 3F4; D. Immunohistochemistry IHC was performed on P6C or PrPc-KO-209 cells using the indicated concentrations of 4AA-m, 8H4, or 3F4 (left), and the OD ratio of P6C / PrPc-KO-209 was semi-quantified (right); E. IHC was performed on human cerebellum using the indicated concentrations of 4AA-m, 8H4, and 3F4 antibodies.

[0092] Figure 4Expression of PrPc in esophageal squamous cell carcinoma. Among them, A. Flow cytometry was used to detect the expression of PrPc in the four esophageal squamous cell carcinoma cell lines shown in the figure (solid line) is the negative control labeled with isotype IgG, and (dashed line) is the result stained with 4AA-m; B. Expression of PrPc in tissues of patients with esophageal squamous cell carcinoma (ESCC). Data were from 17 different patients. The leftmost column shows the expression of PrPc in the basal cells of the para-carcinoma tissue. The second left column shows negative expression of PrPc in 4 ESCC tissues. The third left column shows low expression of PrPc in 4 ESCC cases. The right column shows high-level expression of PrPc in 4 ESCC cases; C. Kaplan-Meier overall survival analysis was performed according to the expression of PrPc in ESCC. (D) Relationship between PrPc expression and patient prognosis. The upper figure shows the correlation analysis between LNR and the tumor DAB index, and the lower figure shows the correlation analysis between LODS and the tumor DAB index. Two metastatic lymph node parameters, LNR (lymph node ratio) and LODS (logarithmic ratio of positive lymph nodes), are important indicators for the prognosis of ESCC patients. LNR = (number of lymph nodes containing cancer cells) ÷ (total number of lymph nodes examined); LODDS = Ln((number of metastatic lymph nodes + 0.5) ÷ (number of non-metastatic lymph nodes + 0.5)).

[0093] Figure 5 Expression of PrPc in liver cancer. Among them, A. Flow cytometry was used to detect the expression of PrPc in several liver cancer cell lines. The solid line is the negative control labeled with isotype human IgG, and the dashed line is the result stained with 4AA-m; B. Expression of PrPc in hepatocellular carcinoma tissues. The left column shows the expression of PrPc, the middle column shows hepatocellular carcinoma tissues without the expression of PrPc, and the right column shows the expression of PrPc in hepatic stellate cells; C. Kaplan-Meier progression-free survival analysis was performed according to the expression of PrPc in liver cancer; D. Kaplan-Meier overall survival analysis was performed according to the expression of PrPc in liver cancer.

[0094] Figure 6 Inhibitory effect of 4AA antibody on tumor migration. The inhibitory effect of 4AA on the migration of P6C is stronger than that of the wild-type clone Ab6. A: Representative images of the migration results of P6C cells after treatment with different antibodies; B: Statistical chart of the migration results of P6C cells after treatment with different antibodies. Detailed implementation manners

[0095] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following examples provided can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0096] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications.

[0097] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0098] The cell lines and main reagents used in the following examples:

[0099] The cell lines used in the present invention include: 293T cells (purchased from ATCC and stored in our laboratory), P6C cells (this cell line was isolated from the tumor tissue of a colon cancer patient by our laboratory, patent number ZL201210033124.0), and were cultured in DMEM medium (HyClone, catalog number SH30243.01) containing 10% FBS (HyClone).

[0100] CHO / dhFr cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in IMDM medium (HyClone, catalog number SH30228.01) containing 10% FBS (HyClone), 0.1 mM hypoxanthine and 0.016 mM thymidine (HT, Gibco). Expi293F (Invitrogen, Carlsbad, CA, USA) was cultured in suspension in SMM293-TII medium (Sino Biological Inc, Beijing, China). All cells were cultured at 37 °C and 5% CO2.

[0101] The tissue chips used in the following examples:

[0102] The cancer tissue chip was purchased from Shanghai OUTDO Biotechnology Co., Ltd. The catalog number of the primary liver cancer tissue chip is HLivH180Su17, and the patient underwent surgical resection from June 2007 to October 2008 and was followed up until June 2016.

[0103] The esophageal cancer tissue chip - 1 has a catalog number of HEsoS180Su06, and the patient underwent surgical resection from January 2011 to April 2013 and was followed up until June 2015. The esophageal cancer tissue chip - 2 has a catalog number of HEsoS180Su11, and the patient underwent surgical resection from July 2006 to September 2008 and was followed up until July 2015.

[0104] 180 cases of primary esophageal squamous cell carcinoma were staged according to the 7th edition of the AJCC Cancer Staging Manual.

[0105] The commercial PrPc monoclonal antibodies 3F4 (Catalog No. MAB1562) and 8H4 (Catalog No. P0110) were purchased from Sigma.

[0106] Restriction endonucleases were purchased from Biolabs.

[0107] dNTP and Pyrobest DNA Polymerases were purchased from Takara.

[0108] Lipofectemin2000, OPTI-MEM, and Mitotracker Red CMXROS were purchased from Invitrogen.

[0109] Unless otherwise specified, other reagents were of analytical grade made in China.

[0110] All flow cytometry analyses were performed on a FACSAriaIII (BD).

[0111] The pcDNA3.1 plasmid in the following examples was purchased from Invitrogen.

[0112] The PFRT vector, pCI-Flp-2A-Cre plasmid, and pFRL-antibody-scFv-TM plasmid in the following examples were described in: Coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody affinity maturation in CHO cells; Accession Number: 26235363 DOI: 10.1002 / bit.25541. The biomaterial can be obtained from the applicant. This biomaterial is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0113] The pCEP4-Ig-Ek-Neo-mAID plasmid in the following examples was described in: Simultaneous Maturation of Single Chain Antibody Stability and Affinity by CHO Cell Display; Accession Number: 36004885 PMCID: PMC9404881 DOI: 10.3390 / bioengineering9080360. The biomaterial can be obtained from the applicant. This biomaterial is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0114] The CHO cell line in the following examples: Coupling recombinase-mediated cassette exchange with somatic hypermutation for antibody affinity maturation in CHO cells; Accession Number: 26235363 DOI: 10.1002 / bit.25541. The biological material can be obtained from the applicant by the public. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0115] In the quantitative experiments in the following examples, unless otherwise specified, three repeated experiments were set up.

[0116] The experimental data in the present invention are expressed as mean ± SD and analyzed by T-test. GraphPad Prism 9 software was used for multiple comparisons to determine the differences. P < 0.05 was considered to be significantly different. Pathological-related statistical analysis was performed using SPSS 26.0 software (SPSS, Chicago, IL). Chi-square test and rank correlation analysis were used to determine the correlation between PrPc expression and pathological factors. Log-rank test and cox regression analysis were used to determine the correlation between PrPc expression and other pathological factors and the survival rate of patients. The Cox proportional hazards regression model was used to include variables with P values less than 0.2 in the multivariable survival analysis. P < 0.05 was considered to be statistically significant, and all P values were two-sided.

[0117] Example 1: Obtaining and humanizing a mouse anti-PrPc monoclonal antibody

[0118] 1. Obtaining a mouse anti-PrPc monoclonal antibody

[0119] In the previous preparation and screening of PrPc monoclonal antibodies, a laboratory obtained a mouse anti-PrPc monoclonal antibody named Ab6 ( Figure 1 in A). The preparation method of Ab6 monoclonal antibody is referred to Patent CN103013926A. The specific information of Ab6 (see Table 2 for details) is as follows: The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of Ab6 antibody are the 24th to 40th, 56th to 62nd, and 95th to 103rd positions of SEQ ID No. 1 in sequence. LCDR1, LCDR2, and LCDR3 are complementary determining regions (CDRs); The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of Ab6 antibody are shown as the 26th to 32nd, 52nd to 57th, and 99th to 103rd positions of SEQ ID No. 2 in sequence; HCDR1, HCDR2, and HCDR3 are complementary determining regions (CDRs).

[0120] 2. Synthesis of Genes and Peptides, Construction of Vectors, and Expression of Proteins

[0121] The codon-optimized synthesis of Ab6 and HAb6 antibody genes was completed by GenScript Biotech Corporation.

[0122] The polypeptide fragments PrPc-35aa of cellular prion protein (PrPc) and the sequence of FITC-PrPc-35-aa (i.e., PrPc-35aa conjugated with FITC at the N-terminus) were synthesized by GenScript Biotech Corporation. The sequence of PrPc-35aa is SEQ ID No.9, and the sequence of FITC-PrPc-35-aa is SEQ ID No.10.

[0123] The sequences PrPc-Fc and all antibodies were expressed in Expi293F and purified by Protein A column (BBI Life Sciences). The primer sequences for constructing the vectors are shown in Table 1. The specific method for constructing the vectors is as follows:

[0124] Table 1. Primer Sequences for Constructing Vectors

[0125]

[0126]

[0127] PCR was performed using primers PrPc-NHE1-F and PrPc-XHO1-R to amplify the PrPc-encoding gene PRNP. The vector was the pcDNA3.1 plasmid, and the restriction enzyme sites were NHE1 and XHO1.

[0128] The structure of the pcDNA3.1-hypromycin-PrPc vector is described as follows: A recombinant vector obtained by replacing the fragment between the NHE1 and XHO1 recognition sites of the pcDNA3.1 vector with a DNA molecule having the nucleotide sequence of SEQ ID No.11, while keeping other nucleotide sequences on the pcDNA3.1 vector unchanged.

[0129] PCR was performed using primers PrPc-Fc-F1 and PrPc-Fc-R1 to amplify the PrPc-encoding gene PRNP; PCR was performed using primers PrPc-Fc-F2 and PrPc-Fc-R2 to amplify the human Fc sequence. The two overlapped to obtain the nucleotide sequence of the 12PrPc-Fc fragment, which was cloned between the Hind3 and Xho1 restriction enzyme sites of the pcDNA3.1 vector to obtain a recombinant vector capable of expressing the PrPc-Fc fragment (nucleotide sequence: SEQ ID No.12).

[0130] The structure of the pcDNA3.1-PrPc-Fc vector is described as follows: a recombinant vector obtained by replacing the fragment between the Hind3 and Xho1 recognition sites of the pcDNA3.1 vector with a DNA molecule having a nucleotide sequence of SEQ ID No.12, and keeping other nucleotide sequences on the pcDNA3.1 vector unchanged.

[0131] pcDNA3.1-Antibody-HC: The antibody mutant was generated by stepwise site-directed mutagenesis using pcDNA3.1-HAb6-HC as a template. The primers used for the A16D site-directed mutagenesis of HAb6 were PrPc-hu-heavy-A16D-F and PrPc-hu-heavy-A16D-R.

[0132] The structure of the pcDNA3.1-Antibody-HC vector is described as follows: a recombinant vector obtained by replacing the fragment between the Hind3 and Xho1 recognition sites of the pcDNA3.1 vector with a DNA molecule having a nucleotide sequence of SEQ ID No.13, and keeping other nucleotide sequences on the pcDNA3.1 vector unchanged.

[0133] The structure of the pFRL-Antibody-scFv-TM vector is described as follows: a recombinant vector obtained by replacing the fragment between the EcoR1 and Xho1 recognition sites of the pFRL vector with a DNA molecule having a nucleotide sequence of SEQ ID No.14, and keeping other nucleotide sequences on the pFRL vector unchanged. SEQ ID No.14 contains the following sequences: EcoR1 cleavage site, antibody signal peptide, heavy chain variable region of antibody, (G4S)3 hinge, light chain variable region of antibody, HA tag, (G4S)3 hinge, Xho1 cleavage site.

[0134] 3. Cell Transfection

[0135] Cells were passaged and seeded into 6-well plates at a density of 70% 24 hours in advance. At the time of transfection, 4 μg of plasmid and 6 μg of transfection reagent PEI MAX 40K (Polysciences, catalog number 24765-1) were added to each well. Fresh culture medium was replaced 6 hours after transfection, and antibiotics were added 48 hours after transfection to screen for positive clones.

[0136] Preparation of 293T-PrPc cells overexpressing PrPc: The pcDNA3.1-hypomycin-PrPc plasmid obtained in step 2 was transfected into 293T cells, cultured with 100 μg / mL hygromycin for 15 days, and single cells were sorted by flow cytometry and seeded into 96-well plates to culture and expand monoclonal cells. By flow cytometry analysis, the monoclonal with the highest PrPc expression was selected as 293T-PrPc cells.

[0137] Preparation of PrPc knockout cells P6C-PrPc cells: Obtained by knocking out the PrPc gene using Crisp Cas9. The sequence of sgRNA is shown in Table 1.

[0138] Specific experimental procedures, knockout methods, and knockout vector information for knocking out PrPc using Crisp Cas9 can be obtained from the literature Shalem O*, Sanjana NE*, Hartenian E, Shi X, Scott DA, Mikkelsen T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014). Science, 343, 83 - 7. DOI: 10.1126 / science.1247005. Successful knockout of PrPc was verified by first-generation sequencing and immunoblotting.

[0139] Preparation of PrPc overexpressing cells P6C-PrPc: Transfect the pcDNA3.1-hypomycin-PrPc obtained in step 2 into P6C cells to obtain PrPc overexpressing cells, and screen for positive clones with 100 μg / mL hygromycin drug.

[0140] 4. Antibody humanization

[0141] First, clone the CDR regions of the Ab6 antibody and sequence them. Then, use Ab6 to retrieve similar human germline sequences. The most similar light chain sequence is >Z00023HomsapIGKV4-1*01F (80% similarity). The most similar heavy chain sequence is >KF698734HomsapIGHV1-69-2*01F (78% similarity). Design humanization based on these sequences to obtain the sequence of HAb6. The amino acid sequence of antibody HAb6 is shown in Table 2.

[0142] Express the full-length clone 6 antibody (Ab6 for short) using Expi293F cells. The sequence was synthesized by Genscript and replaced into the pcDNA3.1 vector by enzymatic digestion.

[0143] The clone 6 antibody Ab6 was humanized to obtain the humanized antibody HAb6 (Table 2). The specific information of the humanized antibody HAb6 (see Table 2 for details) is as follows: The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the HAb6 antibody are the 24th to 40th, 56th to 62nd, and 95th to 103rd positions of SEQ ID No. 3 respectively. LCDR1, LCDR2, and LCDR3 are complementarity-determining regions (CDRs); The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the HAb6 antibody are shown as the 26th to 32nd, 52nd to 57th, and 99th to 103rd positions of SEQ ID No. 4 respectively; HCDR1, HCDR2, and HCDR3 are complementarity-determining regions (CDRs). The sequence was synthesized by Genscript and replaced into the pcDNA3.1 vector by restriction enzyme digestion.

[0144] Antibody Ab6 strongly binds to 293T-PrPc cells and weakly binds to 293T control cells ( Figure 1 in A). HAb6 can bind to 293T PrPc cells, and its binding ability to cells is higher than that of the original mouse antibody Ab6 ( Figure 1 in B).

[0145] Table 2. Sequence information of the antibodies and binding proteins of the present invention

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152] Example 2. Affinity Maturation of Humanized Anti-PrPc Monoclonal Antibody HAb6

[0153] 1. Affinity Maturation Scheme and Flow Cytometry

[0154] The pFRL-antibody-scFv-TM plasmid and the pCI-Flp-2A-Cre plasmid were co-transfected into CHO-PuroR-14 cells. The recombinases Flpo and iCre expressed by the pCI-Flp-2A-Cre plasmid were able to cleave the FRT, LOXP sequences in the genome and the pFRL-antibody-scFv-TM plasmid. Therefore, the antibody sequence in pFRL-antibody-scFv-TM could be replaced into the genome of CHO-PuroR-14.

[0155] The cells with the antibody gene replacement were sorted by anti-HA-APC (Columbia Biosciences).

[0156] The cells No-AID expressing the antibody were transfected with pCEP4-Ig-Ek-Neo-mAID, and were screened with G418 sulfate drug (molecular formula C 20 H 40 N4O 10. 2H2SO4, catalog number E859-5G, Amresco). After 10 days of screening, flow sorting was performed. The cells with AID mutation were incubated with anti-HA-APC antibody (Columbia Biosciences) and PrPc-35aa-FITC (or PrPc-Fc-FITC) on ice for 30 minutes. Under the condition of binding the same anti-HA-APC antibody, the cells binding more ligands (FITC-PrPc-35aa or PrPc-Fc-FITC) were sorted. The genomic DNA of the sorted cells was purified using the Genomic DNA Purification System (Promega, Madison, WI, USA). The antibody gene was amplified using Hind3-kozak-SP-F and genomic R as primers and sequenced.

[0157] 2. Antibody Affinity Measurement

[0158] The BLI experiment was conducted by Octet Red96 (ForteBio Octet, Menlo Park, CA, USA). The antibody was captured by an anti-human IgG antibody capture (AHC) biosensor (ForteBio Octet, Menlo Park, CA, USA) in an affinity assay using PrPc-35aa as the antigen, and PrPc-35aa was bound for 60 seconds and dissociated for 300 seconds. An affinity assay was performed using a streptavidin (SA) biosensor (ForteBio Octet, Menlo Park, CA, USA) with PrPc-Fc as the antigen to capture biotinylated PrPc-Fc, the antibody was bound for 300 s and dissociated for 900 s. KinExA analysis was performed using KinExA 4000 (Sapidyne Instruments Co., Ltd.). Serial dilutions of 293T-PrPc and a constant concentration of antibody were incubated for 18 hours, and the affinity was calculated from the free antibody concentration.

[0159] 3. Affinity Maturation of HAb6 Antibody to PrPc-35aa Peptide

[0160] There are 11 and 6 amino acid residues different in the heavy chain variable region and light chain variable region of HAb6 and Ab6, respectively (specific sequence information is shown in Table 2). The maturation of HAb6 was carried out as follows:

[0161] The HAb6 scFv gene (nucleotide sequence is SEQ ID No. 14) was constructed and inserted into the genome by homologous recombination at the chromosomal locus of the CHO cell line previously developed in our laboratory. HAb6 scFv can be displayed on the cell surface ( Figure 1 in C in no-AID).

[0162] The mouse AID (AID-plus) gene was constructed and stored in our laboratory. In this study, AID-plus was transfected into cells displaying HAb6 scFv. By culturing CHO cells, random mutations of the gene HAb6scFv were induced by AID-plus in CHO cells.

[0163] PrPc-35aa is a polypeptide of amino acids 127 - 161 in the PrPc sequence (nucleotide sequence is SEQ ID No. 11), containing 35 amino acids, which is the epitope of Ab6. The following cells were labeled with FITC-PrPc--35aa: 1) cells without HAb6 (abbreviated as CHO-PuroR-14), 2) cells displaying parental HAb 6 (without AID) (abbreviated as No-AID), and 3) cells displaying the HAb 6 mutant (AID) library (abbreviated as AID) ( Figure 1 in C).

[0164] The results showed that there was a small population of cells in AID cells with higher FITC-PrPc-35aa binding ability than other AID cells and all no-AID cells. This part of the cells ( Figure 1 the cells within the rectangular box in C) were sorted and enriched by flow cytometry, and the cells were cultured and amplified to obtain mature cells.

[0165] The mature cells contained three subsets, and their binding ability to FITC-PrPc-35aa was higher than that of No-AID cells ( Figure 1 in D). Two subsets (sub1 and sub2) were further enriched by flow sorting, and antibody genes were cloned from the enriched cells.

[0166] Sequencing of 20 clones of sub1 and 27 clones of sub2 found that: all 20 sub1 clones contained a single mutation A16D, 25 sub2 clones contained a single mutation A97V. In addition, two sub2 clones contained double mutations A97V-G107D and single mutation S76I respectively. The above four mutations were all located in the heavy chain variable region.

[0167] To further identify mutants A16D and A97V, full-length A16D, A97V and A16D-A97V antibody (abbreviated as DV antibody) mutants were constructed. The specific method was as follows: the A16D mutant was obtained by site-directed mutagenesis with PrPc hu heavy A16D F and PrPc hu heavy A16D R primers. The A97V mutant was obtained by site-directed mutagenesis with A97V F and A97V R primers. And they were transiently displayed on PrPc-35aa-FITC-labeled CHO cells. The HAb6 antibody was used as a positive control, and CHO-PuroR-14 was used as a negative control (abbreviated as Negative). Flow cytometry analysis showed that the binding of A16D and A97V to PrPc-35aa-FITC was slightly higher than that of HAb6, and the binding of A16D-A97V antibody to PrPc-35aa-FITC was significantly higher than that of A16D or A97V ( Figure 1 in E).

[0168] 4. Affinity maturation of HAb6 antibody to full-length PrPc

[0169] Full-length HAb6 and A16D, A97V, and A16D-A97V mutant antibodies were synthesized, and their affinities for full-length PrPc were detected. The affinities of anti-PrPc antibodies for PrPc-35aa and PrPc-Fc were determined by the BLI method. The affinities of anti-PrPc antibodies for 293T PrPc were determined by KinExA. The binding of the original murine antibody Ab6, humanized antibody HAb6, and A16D-A97V antibody to PrPc-35aa polypeptide, soluble full-length PrPc-Fc, and full-length PrPc expressed on 293T cells (abbreviated as 293T-PrPc) was detected.

[0170] The results showed that all mutants had higher affinities than the wild-type antibody (HAb: 1.24E-08, A16D: 3.36E-09 M, A97V: 5.15E-09), and the affinity of the A16D-A97V antibody was 5.4 times that of the wild-type antibody HAb6 (Table 3).

[0171] The results indicated that the binding ability of all antibodies to PrPc-35aa polypeptide was higher than the affinities of soluble full-length PrPc and full-length PrPc expressed on 293T cells for the polypeptide (Table 3).

[0172] Table 3. Affinity results between antibodies and different forms of PrPc

[0173]

[0174] Note: N.D. indicates below the detection limit and no valid data could be obtained.

[0175] To improve the affinity of the A16D-A97V antibody (abbreviated as DV antibody) for full-length PrPc, cells were labeled with PrPc-Fc conjugated with FITC. The A16D-A97V scFv gene was inserted into the genome of CHO cells, and two rounds of evolutionary maturation were carried out. The procedure for the first round was the same as that for the maturation of the HAb6 antibody in step 4 above. Then the enriched cells were transfected with a plasmid expressing murine AID to generate mutations, and cells that bound to full-length PrPc were enriched by flow sorting; for the second round of evolution, murine AID with a different mutation type distribution instead of highly active murine AID-plus was used to induce new mutations in subsequent rounds of evolution. After two rounds of evolution, the main cell subset had a higher binding ability than the cells displaying A16D-A97V scFv ( Figure 2 in A). Figure 2In B, DV-S1 is a cell expressing the 16D-A97V mutant of the scFv region of the anti-PrPc antibody. Transfecting AID-plus into DV-S1 induces DV mutations, resulting in mutant cells DV-M1 with higher binding ability. Transfecting AID into DV-M1 cells yields DV-M2 with even higher binding ability to PrPc-Fc.

[0176] Fifty-seven antibody gene clones of the cells after two rounds of evolution were sequenced (Table 4), and full-length antibodies of these mutant genes were constructed. The antibodies were expressed in Expi293F in the form of human IgG1, and their affinities were tested (Table 5).

[0177] Table 4. Sequencing of antibody genes cloned from DV-M2

[0178] Mutation type Number of antibodies A40T 27 Q39K + S76I 10 Insertion of W between W104 and G105 4 Q39K 2 S76I 2 Insertion of A40T + W between W104 and G105 2 Insertion of APGK between Q39 and A40 2 Wild type 2 A40T + S76I 1 C96S 1 S76N 1 L4V 1 A40T F59L 1 S76N + S58N 1

[0179] Biolayer Interferometry (BLI) was used to measure the KD values of these mutant antibodies except for mutants C96S and L4V (not expressed) and mutant A40T plus F59L (unable to bind PrPc-Fc). It was found that the only clone that bound better to full-length PrPc was the mutant with the insertion of four amino acids APGK between Q39 and A40 ( Figure 2 in C), and this clone was named 4AA.

[0180] The specific information of antibody DV (see Table 2 for details) is as follows: The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the DV antibody are positions 24 to 40, positions 56 to 62, and positions 95 to 103 of SEQ ID No. 3, respectively. LCDR1, LCDR2, and LCDR3 are complementarity-determining regions (CDRs); The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the DV antibody are shown as positions 26 to 32, positions 52 to 57, and positions 99 to 103 of SEQ ID No. 5, respectively; HCDR1, HCDR2, and HCDR3 are complementarity-determining regions (CDRs).

[0181] The specific information of antibody 4AA (see Table 2 for details) is as follows: The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the 4AA antibody are the 24th to 40th, 56th to 62nd, and 95th to 103rd positions of SEQ ID No. 3, respectively. LCDR1, LCDR2, and LCDR3 are complementarity-determining regions (CDRs); the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the 4AA antibody are shown as the 26th to 32nd, 56th to 61st, and 103rd to 107th positions of SEQ ID No. 6, respectively; HCDR1, HCDR2, and HCDR3 are complementarity-determining regions (CDRs).

[0182] Table 5. Affinities between various antibody mutants and PrPc-Fc

[0183]

[0184]

[0185] Binding of DV and 4AA to full-length PrPc expressed on 293T-PrPc cells was analyzed by KinExA ( Figure 2 in D). The affinity of antibody DV was 1.01 nM, and the affinity of antibody 4AA was 92.68 pM, which was approximately 10-fold smaller than the KD value of its parental clone A16D-A97V (DV antibody) (Table 5). Two rounds of evolution against full-length PrPc increased the affinity of DV for full-length PrPc (Prpc Fc) on the cell surface by approximately 10-fold.

[0186] The increase in the affinity of the antibody for full-length PrPc (Prpc Fc) is very important for its applications in detection and treatment. Two-step maturation increased the affinity of the PrPc monoclonal antibody for full-length PrPc by approximately 100-fold, with a KD value of 2.03E-10 M. This indicates that antibody 4AA can bind well to PrPc expressed in cells and fixed tissue specimens (Table 3).

[0187] Example 3. Specificity and Sensitivity of 4AA Antibody

[0188] The variable region of 4AA was linked to the mouse constant region by the overlap method to construct 4AA-m (4AA with the human IgG constant region replaced by the mouse constant region).

[0189] 1. Immunoblot Detection of 4AA Antibody

[0190] Protein blotting experiments were performed on human colon cancer P6C cells and P6C cells with PRNP (the coding gene of PrPc protein is PRNP) knocked out. 4AA-m (4AA with the human IgG constant region replaced by the mouse constant region) was used to label the blot.

[0191] The results showed that PrPc protein was detected in P6C cells, but not in PRNP knockout P6C cells ( Figure 3 in A), indicating that the 4AA-m antibody specifically binds to PrPc.

[0192] The 4AA-m antibody was also able to detect PrPc in wild-type 293T cells and 293T cells overexpressing PrPc (293T-PrPc). PrPc protein was detected in both types of cells ( Figure 3 in B). 293T cells express low levels of PrPc protein, while high levels of PrPc can be detected in 293T-PrPc cells. Quantitative results showed that the PrPc in 293T-PrPc was 500 times that of wild-type 293T cells. The above data demonstrated that the 4AA-m antibody is specific for human PrPc.

[0193] 2. Comparison of the sensitivity of 4AA antibody and commercial antibodies in immunoblotting

[0194] Immunoblotting was performed on cell lysates of 293T-PrPc to analyze and compare the antibodies 4AA-m and commercial antibodies 8H4 and 3F4. 4AA detected strong PrPc signals on both 5 and 0.5 μg of cell lysates, 8H4 could only detect weak signals on 5 μg of lysate, and 3F4 could also show strong and weak signals on 5 and 0.5% lysates, respectively. Subsequently, the signals from 5 μg of lysate were quantified, and the signals detected by 4AA were 9 times and 3 times those detected by 8H4 and 3F4, respectively. This result indicated that 4AA is more sensitive than 8H4 and 3F4 in Western blot analysis ( Figure 3 in C).

[0195] 3. Comparison of the specificity and sensitivity of 4AA antibody and commercial antibodies in immunohistochemical staining

[0196] Immunohistochemical staining was performed on fixed human colon cancer cells P6C and PRNP knockout P6C cells (abbreviated as P6C-KO-209) to detect the specificity of 4AA-m. The results showed that P6C cells were positive for 4AA-m staining, while PRNP knockout P6C cells were negative for immunohistochemistry. It is known that α-cleavage occurs during the maturation process of PrPc protein, and its cleavage site is at amino acid positions 111 / 112. The binding regions of the two commercial anti-PrPc antibodies 3F4 and 8H4 are located at amino acid residues 106-112 and 145-180 of human PrPc, respectively. Therefore, 3F4 cannot bind to most PrPc in cells, while 8H4 should be able to bind to most PrPc. 8H4 and 3F4 showed smaller signal differences between wild-type P6C and PRNP knockout P6C cells (P6C-KO-209) Figure 3D). The staining density of the samples was quantified, and it was found that the signal ratio of P6C / P6C-PrPc KO was significantly higher than that of 8H4 and 3F4, especially when the antibody concentration was low. This result indicates that antibody 4AA-m has better specificity than 3F4 and 8H4 in immunohistochemistry.

[0197] PrPc is highly expressed in the human and other mammalian cerebella. Immunohistochemical staining was performed on serial sections of paraffin-embedded human cerebellum (Bioaitech). 4AA-m showed stronger positive signals than 3F4 and 8H4 in the granular and molecular layers of the cerebellum ( Figure 3 E), and its expression pattern was consistent with the results of previous studies, and the working concentrations of 4AA-m were 29-fold and 166-fold lower than those of 8H4 and 3F4, respectively. The difference in sensitivity between the 4AA-m antibody and the commercial 3F4 and 8H4 antibodies indicates that 4AA-m has higher specificity and sensitivity for studying the expression of PrPc in human cells and tissues.

[0198] Example 4. Application of antibody 4AA in detecting PrPc expression in esophageal squamous cell carcinoma

[0199] 1. Flow cytometry detection of esophageal squamous cell carcinoma cells

[0200] Four esophageal squamous cell carcinoma cell lines (KYSE150, KYSE450, KYSE510, and TE-1 cell lines) were labeled with 4AA-m for flow cytometry. It was found that three cell lines, KYSE150, KYSE450, and KYSE510 (gifted by the research groups of YAN Xiyun and YANG Pengyuan, Institute of Biophysics, Chinese Academy of Sciences), expressed PrPc, while PrPc expression was not detected in the TE-1 cell line ( Figure 4 A). Therefore, 4AA can be used for flow cytometry to detect differential expression of PrPc in esophageal squamous cell carcinoma.

[0201] 2. Immunohistochemical staining of esophageal squamous cell carcinoma

[0202] An automated immunohistochemistry and in situ hybridization stain, Bond RX, was used to perform immunohistochemical staining on an esophageal squamous cell carcinoma tissue microarray. The working concentration of the 4AA-m antibody was 0.6 μg / mL, and it was incubated at room temperature for 1 hour. The chip array was scanned by a pathology slide scanner, CS2. Developed and manufactured by Indica Labs, Inc. Staining data analysis was performed on a high-throughput whole-layer pathological image analysis platform. The Multiplex IHC v3.1 module was used to analyze the PrPc staining intensity in tumor tissues, and the tumor-DAB-H-Score was calculated. Tumor-DAB-H-SCORE = (percentage of weakly stained cells × 1) + (percentage of moderately stained cells × 2) + (percentage of strongly stained cells × 3). It was found that the expression levels of PrPc differed among different esophageal squamous cell carcinomas ( Figure 4 in B).

[0203] Examination of the para-cancerous tissues of patients with esophageal squamous cell carcinoma revealed that the 4AA-m antibody could detect positive PrPc expression in the basal cells of para-cancerous tissues ( Figure 4 in B).

[0204] 3. 4AA was used to detect the correlation between PrPc expression and cancer progression

[0205] Immunohistochemical studies were performed on tissue microarrays of 179 patients with primary ESCC using 4AA-m. The tumor-DAB-H-score data were obtained from the measured arrays and semi-quantitatively represented the expression level of PrPc in the cancer tissues of patients. According to the Tumor-DAB-H core data, the patients were divided into two groups: high and low values of the Tumor-DAB-H core represented strong and weak expression of PrPc in the cancer tissues of patients, respectively.

[0206] Multivariate COX regression analysis was performed on the pathological information of the patients. The results showed that high PrPc expression was an independent prognostic factor affecting overall survival (HR: 0.545, 95% CI: 0.316 - 0.941, P = 0.029). Compared with the low PrPc group, the prognosis of the high PrPc expression group was better (Table 6, Figure 4 in C). The tumor-DAB-H-score, which reflects the semi-quantitative expression of PrPc, was also an independent prognostic factor for the overall survival of primary ESCC (HR: 0.995, 95% CI: 0.991 - 1000, P = 0.035). Patients with high PrPc expression had a better prognosis (Table 7, Figure 4 in D). The above analysis indicated that the expression of PrPc in esophageal squamous cell carcinoma was correlated with the prognosis of patients. The 4AA antibody could detect the expression of PrPc in esophageal squamous cell carcinoma, suggesting that 4AA could be used to prepare products for the prognosis assessment of esophageal squamous cell carcinoma.

[0207] Table 6. Multivariate COX analysis of the overall survival of patients with primary ESCC

[0208]

[0209]

[0210] Note: 95% CI, 95% confidence interval

[0211] Table 7, Multivariate COX analysis of overall survival in patients with primary ESCC (semi - quantitative analysis of PrPc)

[0212] Variable Hazard ratio 95% CI p - value Tumor - DAB - H - Score 0.995 0.991-1.000 0.035 Gender (male / female) 1.372 0.804-2.341 0.245 <![CDATA[Tumor size (cm 3 )]]> 1.009 1.004-1.014 0.001 T stage 1.613 1.046-2.489 0.031 N stage 1.517 1.251-1.841 0.001 M stage 2.950 0.697-12.486 0.142

[0213] Example 5, Immunohistochemical staining of 4AA is negatively correlated with the metastasis of hepatocellular carcinoma

[0214] 1. 4AA is used for flow cytometry detection of hepatoma cells

[0215] The expression of PrPc in liver tumor cell lines was detected by flow cytometry. PrPc was highly expressed in RBE, HCCLM3, and SMMC7721 cells (gifted by the research groups of YAN Xiyun and YANG Pengyuan from the Institute of Biophysics, Chinese Academy of Sciences), and was also positively expressed at a lower level in HepG2, BEL7407, and HepB3 cells (gifted by the research groups of YAN Xiyun and YANG Pengyuan from the Institute of Biophysics, Chinese Academy of Sciences). PrPc was not detected in PLC - PRF5, HuH - 7, and SK - HEP1 (gifted by the research groups of YAN Xiyun and YANG Pengyuan from the Institute of Biophysics, Chinese Academy of Sciences) ( Figure 5 in A). It shows that 4AA can be used for flow cytometry to detect the expression of PrPc in hepatoma cell lines.

[0216] 2. 4AA is used for immunohistochemical staining of hepatocellular carcinoma

[0217] Immunohistochemistry was performed on the tumor tissue microarray of 92 patients with primary hepatocellular carcinoma using the 4AA - m antibody. 14% of the patients' cancer tissues (13 cases) showed positive staining, while all adjacent hepatocytes showed negative staining. A typical example is shown as Figure 5 shown in B.

[0218] 3. Immunohistochemical staining of 4AA is negatively correlated with the metastasis of hepatocellular carcinoma

[0219] Hepatoma patients were divided into a PrPc - positive group and a PrPc - negative group. Through chi - square analysis, the expression of PrPc was related to gender, age, degree of differentiation, number of cirrhotic nodules, cirrhosis, clinical stage, tumor size, vascular invasion, tumor capsule, HBsAg (hepatitis B surface antigen), HBcAb (hepatitis B core antibody), anti - HCV (hepatitis C virus), TB (total bilirubin), ALT (alanine aminotransferase), ALB (albumin), AFP (alpha - fetoprotein), GGT (gamma - glutamyl transferase) as shown in Table 8.

[0220] Table 8, Multivariate Cox regression analysis of clinicopathological parameters and RFS in patients with primary hepatocellular carcinoma

[0221] Variable Hazard ratio 95% CI p - value Age (>=50 vs. <50*) 1.273 0.703-2.306 0.425 Grade 1.357 0.949-1.939 0.094 Number of cirrhotic nodules 1.623 0.996-2.644 0.052 TNM staging 0.863 0.436-1.708 0.672 Tumor size (>5 cm vs. <=5 cm*) 2.521 1.281-4.958 0.007 Number of tumors (>=2 vs. 1*) 2.626 0.988-6.981 0.053 Glutamyl transferase (U / L) 1.004 1.000-1.009 0.047 PrPc expression (positive vs. negative*) 2.646 1.271-5.509 0.009

[0222] Note: CI: Confidence Interval; *: Control group.

[0223] Using the Kaplan–Meier survival curve and log-rank test, the recurrence-free survival (RFS) and overall survival (OS) of patients with positive PrPc expression were shorter than those of patients with negative PrPc (P = 0.03) ( Figure 5 in C and D). The median RFS time in the PrPc-negative liver cancer group was 48 months, and the median RFS time in the group of patients with positive PrPc expression was 19 months. The three-year RFS in the PrPc-negative liver cancer group was 52.6%, and the three-year RFS in the group of PrPc-positive patients was 38.5%. Through further integration of other pathological information, a multivariate Cox analysis of recurrence-free survival time was performed, indicating that PrPc expression was an independent prognostic factor for RFS (P = 0.009; HR = 2.646; 95% CI = 1.271 - 5.509). The above experiments showed that PrPc was an adverse prognostic factor for liver cancer recurrence. 4AA could detect the expression of PrPc in liver cancer, indicating that 4AA could be used to prepare products for the prognosis assessment of liver cancer.

[0224] Example 6. Inhibition of tumor cell migration by 4AA

[0225] The single-cell suspension of colon cancer cells P6C obtained by trypsin digestion was counted and inoculated on the microporous membrane of the upper chamber of a transwell (Corning, 3422) in serum-free medium, and the medium in the lower culture dish contained 10% FBS (Hyclone). The medium in both the upper chamber and the lower culture dish contained 5 μg / ml of specific antibody. After culturing for 24 hours, some cells migrated through the 8-μm pores at the bottom of the transwell chamber into the culture medium containing 10% FBS. The migrated cells were fixed and stained with Coomassie blue ( Figure 6 in A). The migrated cells were counted under a microscope.

[0226] The results showed that: the inhibitory effect of the mature humanized antibody 4AA on the migration of colon cancer cells P6C was significantly better than that of Ab6 and DV antibodies, and the inhibitory rate of DV on migration was also higher than that of Ab6 ( Figure 6 in B), indicating that each step of affinity maturation increased the anti-migration function of the antibody.

[0227] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. An antibody, characterized in that: The antibody is a monoclonal antibody or an antigen-binding fragment thereof, and the monoclonal antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light-chain variable region of the antibody are successively as shown in positions 24 to 40, 56 to 62, and 95 to 103 of SEQ ID No.1, and LCDR1, LCDR2, and LCDR3 are complementarity-determining regions; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy-chain variable region of the antibody are successively as shown in positions 26 to 32, 52 to 57, and 99 to 103 of SEQ ID No.2; HCDR1, HCDR2, and HCDR3 are complementarity-determining regions.

2. The antibody according to claim 1, wherein: The antibody is as follows (1) or (2): (1) The amino acid sequence of the light-chain variable region of the antibody is SEQ ID No.1; and / or the amino acid sequence of the heavy-chain variable region of the antibody is SEQ ID No.2; (2) The amino acid sequence of the light-chain variable region of the antibody is SEQ ID No.3; and / or the amino acid sequence of the heavy-chain variable region of the antibody is any one of the following (C1)-(C3): (C1) SEQ ID No.6; (C2) SEQ ID No.4; (C3) SEQ ID No.

5.

3. The antibody according to claim 1 or 2, characterized in that: The heavy-chain type of the antibody is IgG1; and / or the light-chain type of the antibody is kappa.

4. An antibody obtained by immunizing an animal with a conjugate of a polypeptide shown in SEQ ID No.9 or SEQ ID No.10 and a carrier protein.

5. Any of the following biological materials: (A1) A humanized antibody obtained by humanizing any of the antibodies described in claims 1-3 or the antibody described in claim 4; (A2) An active fragment of an antibody, which is any of the following derived from any of the antibodies described in claims 1-3 or the antibody described in claim 4 or the humanized antibody described in (A1): single-chain antibody, Fab region of an antibody, and antigen-binding fragment; (A3) A nucleic acid molecule encoding any of the antibodies described in claims 1-3 or the antibody described in claim 4 or the humanized antibody described in (A1) or the active fragment of the antibody described in (A2); (A4) An expression cassette, recombinant vector, recombinant cell, or recombinant bacterium containing the nucleic acid molecule described in (A3); (A5) A kit containing any of the antibodies described in claims 1-3 or the antibody described in claim 5 or the humanized antibody described in (A1) or the active fragment of the antibody described in (A2) or the nucleic acid molecule described in (A3) or the expression cassette, recombinant vector, recombinant cell, or recombinant bacterium described in (A4).

6. The biomaterial according to claim 7, characterized in that: In the nucleic acid molecule described in (A3), the nucleotide sequences encoding LCDR1, LCDR2, and LHCDR3 in the light-chain variable region of the antibody are successively as shown in positions 70 to 120, 166 to 186, and 283 to 309 from the 5'-end of SEQ ID No.7; and / or In the nucleic acid molecule described in (A3), the nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are successively as shown at positions 76 to 96, 166 to 183, and 307 to 321 from the 5'-end of SEQ ID No.

8.

7. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody described in any one of claims 1 - 3, or the antibody described in claim 4, or the humanized antibody described in (A1) in claim 5 or 6, or the active fragment of the antibody described in (A2).

8. Use of the antibody described in any one of claims 1 - 4 in any of the following: (B1) Identifying or assisting in identifying cellular prion protein; (B2) Binding or assisting in binding to cellular prion protein; (B3) Detecting or assisting in detecting whether a test sample contains cellular prion protein; (B4) Preparing a product for detecting or assisting in detecting whether a test sample contains cellular prion protein; (B5) Detecting or assisting in detecting the content of cellular prion protein in a test sample; (B6) Preparing a product for detecting or assisting in detecting the content of cellular prion protein in a test sample; (B7) Detecting or assisting in detecting a tumor or tumor cell expressing cellular prion protein; (B8) Preparing a product for detecting or assisting in detecting a tumor or tumor cell expressing cellular prion protein; (B9) Identifying or assisting in identifying a tumor or tumor cell; (B10) Binding or assisting in binding to a tumor or tumor cell; (B11) Preparing a product for identifying or assisting in identifying a tumor or tumor cell; (B12) Preparing a product for binding or assisting in binding to a tumor or tumor cell; (B13) Preparing a product targeting cellular prion protein; (B14) Preparing a product for diagnosing or treating a disease related to cellular prion protein; (B15) Preparing an anti-tumor drug; (B16) Preparing a product for inhibiting tumor cell metastasis.

9. Use of the antibody in the biological material described in any one of claims 5 or 6 in any of the following: (B1) Identifying or assisting in identifying cellular prion protein; (B2) Binding or assisting in binding to cellular prion protein; (B3) Detecting or assisting in detecting whether a test sample contains cellular prion protein; (B4) Preparing a product for detecting or assisting in detecting whether a test sample contains cellular prion protein; (B5) Detecting or assisting in detecting the content of cellular prion protein in a test sample; (B6) Preparing a product for detecting or assisting in detecting the content of cellular prion protein in a test sample; (B7) Detecting or assisting in detecting a tumor or tumor cell expressing cellular prion protein; (B8) Preparing a product for detecting or assisting in detecting a tumor or tumor cell expressing cellular prion protein; (B9) Identifying or assisting in identifying a tumor or tumor cell; (B10) Binding or assisting in binding to a tumor or tumor cell; (B11) Preparing a product for identifying or assisting in identifying a tumor or tumor cell; (B12) Preparing a product for binding or assisting in binding to a tumor or tumor cell; (B13) Preparing a product targeting cellular prion protein; (B14) Preparing a product for diagnosing or treating a disease related to cellular prion protein; (B15) Preparing an anti-tumor drug; (B16) Preparing a product for inhibiting tumor cell metastasis.

10. Use of the antibody in any of the pharmaceutical compositions according to claim 7 in any of the following: (B1) identifying or assisting in the identification of cellular prion protein; (B2) binding or assisting in the binding of cellular prion protein; (B3) detecting or assisting in the detection of whether a test sample contains cellular prion protein; (B4) preparing a product for detecting or assisting in the detection of whether a test sample contains cellular prion protein; (B5) detecting or assisting in the detection of the content of cellular prion protein in a test sample; (B6) preparing a product for detecting or assisting in the detection of the content of cellular prion protein in a test sample; (B7) detecting or assisting in the detection of tumors or tumor cells expressing cellular prion protein; (B8) preparing a product for detecting or assisting in the detection of tumors or tumor cells expressing cellular prion protein; (B9) identifying or assisting in the identification of tumors or tumor cells; (B10) binding or assisting in the binding of tumors or tumor cells; (B11) preparing a product for identifying or assisting in the identification of tumors or tumor cells; (B12) preparing a product for binding or assisting in the binding of tumors or tumor cells; (B13) preparing a product targeting cellular prion protein; (B14) preparing a product for diagnosing or treating diseases related to cellular prion protein; (B15) preparing an anti-tumor drug; (B16) preparing a product having the effect of inhibiting the metastasis of tumor cells.

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