CN117024596B - Tumor primary cell specific markers and in vivo imaging - Google Patents

Tumor primary cell specific markers and in vivo imaging Download PDF

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CN117024596B
CN117024596B CN202311041533.XA CN202311041533A CN117024596B CN 117024596 B CN117024596 B CN 117024596B CN 202311041533 A CN202311041533 A CN 202311041533A CN 117024596 B CN117024596 B CN 117024596B
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antibody
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cells
fluorescent
tumor
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CN117024596A (en
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汪悦
李跃兵
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Mirror Image Qidian Shanghai Cell Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
    • C07K16/3092—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated mucins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
    • A61K49/0043—Fluorescein, used in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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
    • 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
    • C07K2317/90—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

Abstract

The invention discloses a method for tumor primary cell specific labeling and in-vivo imaging. The invention discloses an ovarian cancer primary cell surface labeling antibody molecule with high affinity and binding activity. The antibody is coated on magnetic beads, so that the magnetic beads can be used for separating and purifying primary cells. The antibody is compounded with fluorescent dye, and can be used for real-time tracking and observation of primary cells in tumor-bearing mice.

Description

Tumor primary cell specific markers and in vivo imaging
Technical Field
The invention relates to the field of biotechnology, in particular to a primary cell marking and imaging technology.
Background
The primary cells are cells directly separated from tissues, and have wide application in the fields of research of cell biology, tissue engineering, regenerative medicine and the like. In order to track the growth and differentiation of primary cells in real time, researchers need to label and image these cells. The existing primary cell marking and imaging technology mainly comprises the steps of transfecting imaging probes or fluorescent proteins into cells by using chemical or biological methods, so as to mark and image the cells. In addition, there is a technique of performing primary cell living body imaging by an imaging device such as an optical microscope. These techniques have their limitations such as low labeling efficiency, high toxicity of the labeling agent, low imaging resolution, limited imaging time and times, etc.
CA125, also known as MUC16, is one of the members of the mucin family (MUC) and belongs to the type I transmembrane mucin. CA125 is a tumor specific antigen over-expressed in ovarian cancer, and is currently the most widely used important biomarker for diagnosing ovarian cancer in clinic.
The screening of specific markers that can specifically label tumor primary cells and be applied to in vivo imaging is of great value.
Disclosure of Invention
To overcome the above problems, the first aspect of the present invention provides an anti-human CA125 antibody comprising a sequence as set forth in SEQ ID NO:9, HCDR1, SEQ ID NO:10, HCDR2 shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:12, LCDR1, SEQ ID NO:13 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown in 14.
In certain embodiments, the antibody comprises SEQ ID NO:15 and the heavy chain variable region of the antibody shown in SEQ ID NO:16, and a light chain variable region of an antibody shown in seq id no.
In certain embodiments, the antibody comprises a constant region; preferably, the constant region is a human IgG1 constant region.
In a second aspect the invention provides immunomagnetic beads coated with an antibody according to the first aspect of the invention.
The third aspect of the invention provides the use of the immunomagnetic beads according to the second aspect of the invention for cell separation and purification; preferably, the cells are tumor primary cells.
In certain embodiments, the tumor primary cells are ovarian cancer primary cells.
In a fourth aspect the invention provides a fluorescent antibody comprising an antibody according to the first aspect of the invention and a fluorescent dye.
In certain embodiments, the fluorescent dye is selected from the group consisting of Cy series fluorescent dyes, alexaFluor series fluorescent dyes, conjugated binding probes, cellular function probes, fluorescent proteins; preferably, the fluorescent dye is FITC.
In a fifth aspect, the invention provides the use of a fluorescent antibody according to the fourth aspect of the invention for labelling tumour primary cells for in vivo imaging of tumour-bearing mice.
In certain embodiments, the tumor primary cells are ovarian cancer primary cells.
Compared with the prior art, the invention has the beneficial effects that:
the invention screens and obtains the ovarian cancer primary cell surface marked antibody molecule with high affinity and binding activity. The antibody is coated on magnetic beads, so that the magnetic beads can be used for separating and purifying primary cells. The antibody is compounded with fluorescent dye, and can be used for real-time tracking and observation of primary cells in tumor-bearing mice.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 shows flow analysis of ovarian cancer primary cells (2X 10 7 /mL test group) labeling results.
Figure 2 shows in vivo imaging of ovarian cancer primary cells in nude mice. Fig. 2A, day 5; fig. 2B, day 12.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without creative efforts, based on the described embodiments of the present invention fall within the protection scope of the present invention.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated, the reagents, instruments, devices and methods used in the present invention are those conventionally commercially available in the art.
EXAMPLE 1 preparation and screening of anti-human CA125 antibodies
1.1 preparation and screening of hybridomas
3 wild type BALB/c mice are immunized by using human CA125 full-length protein as antigen, spleen cells and bone marrow cells of the mice which react with antigen positive are selected from plasma, B cells expressing antibodies are enriched, B cells and myeloma cells are fused to prepare hybridomas, the binding condition of hybridoma culture supernatant to human CA125 is examined, positive cells which can be effectively combined with human CA125 in the culture supernatant are screened out, and then antibody variable region sequences in the hybridoma cells are amplified by a molecular cloning means, and sequencing is carried out to obtain 18 independent mouse-source antibody heavy-light chain pairs in total.
The obtained independent sequences are subjected to gene synthesis and molecular cloning to obtain mammal expression vectors of the corresponding antibodies, and the mammal expression vectors are transfected into HEK293T cells and cultured in 24-well plates for 3 days. The binding activity to human CA125 was measured by ELISA from 0.1mL of culture supernatant.
After 24-well plate culture supernatant detection, 5 strains of murine candidate antibody sequences were obtained in total, with an OD405>0.25 on ELISA, and were initially confirmed as positive candidates.
The candidate antibody strains screened will be transiently cultured in HEK293 suspension cell systems and purified antibody proteins obtained for further investigation.
1.2 binding Activity of candidate antibodies
The positive candidate antibody obtained in the previous step is tested for binding activity to human CA125 protein by ELISA. The specific method comprises the following steps: human CA125 protein is coated on an immune plate at 4 ℃ overnight, a gradient diluted candidate antibody is added after blocking, the reaction is carried out for 1 hour at room temperature, and then a secondary antibody anti-HumanIgG (Fc) HRP is added for 1 hour. Color development was performed using TMB color development kit, and after termination of the reaction, the OD405 was read on a microplate reader. The OD405 of each antibody was plotted against the log of its concentration using Graphpad and EC50 values for the binding of the antibody to human CA125 protein were obtained by four parameter fitting. The results are shown in Table 1:
TABLE 1ELISA detection of binding Activity of CA 125-positive candidate antibodies
Positive candidate antibody Binding to human CA125EC50 (nM)
anti-CA125-1 0.725
anti-CA125-2 0.0165
anti-CA125-3 0.147
anti-CA125-4 0.0457
anti-CA125-5 0.418
It can be seen that 5 candidate molecules of the invention are each capable of binding to human CA125, with anti-CA125-2 exhibiting the strongest binding activity.
1.3 affinity of candidate antibodies
The binding kinetics of candidate positive antibodies anti-CA125-1, anti-CA125-2, anti-CA125-3, anti-CA125-4, anti-CA125-5 to human CA125 protein were examined by the method of biological membrane interferometry (BLI). The specific method comprises the following steps: the candidate antibody was diluted to a final concentration of 5. Mu.g/mL and directly immobilized on AHCbiosensor, and the kinetics was measured by dilution of human CA125 antigen protein with a Kinetics buffer to 200nM,100nM,50nM,100 s of sample injection, dissociation time of 400-800s,10mM Mglycine HCl (pH 1.5) for 15s. The binding rate (kon) and dissociation rate (kdis) were calculated. The equilibrium dissociation constant (kD) is calculated as the ratio kdis/kon. The results are shown in Table 2.
TABLE 2 affinity of BLI to detect CA 125-positive candidate antibodies
Positive candidate antibody BLIKD(M)
anti-CA125-1 5.328E-09
anti-CA125-2 1.0E-12
anti-CA125-3 1.427E-08
anti-CA125-4 2.614E-09
anti-CA125-5 4.429E-09
The affinity of the anti-CA125-2 molecule and the CA125 antigen reaches the nanomolar level, which shows that the anti-CA125-2 has very high binding capacity with human CA 125.
The sequences of anti-CA125-1, anti-CA125-2, anti-CA125-3, anti-CA125-4, anti-CA125-5 are shown in Table 3 below, and the antibody CDRs employ the Kabat coding rules.
TABLE 3CA125 Positive candidate antibody sequences (SEQ ID NOs)
EXAMPLE 2 isolation culture and purification of Primary cells
2.1 tissue digestion and preliminary separation
Under aseptic conditions, ovarian cancer specimens are collected within half an hour after surgical excision, a 50mL plastic aseptic centrifuge tube is taken, 20mL of RPMI1640 cell culture solution precooled at 4 ℃ is filled in, and the culture solution contains 10% fetal bovine serum, 100U/mL penicillin and 100 mug/mL streptomycin. And cutting a tumor sample at a non-necrotic part, placing the tumor sample into the sterile centrifuge tube, and transporting the centrifuge tube on ice to quickly bring the tumor sample back to a laboratory. The tumor sample is transferred into a cell culture dish, washed three times by PBS, and blood cells are washed away to remove non-tumor tissues such as fascia on the surface. Transferring the treated tumor tissue into a new cell culture dish, and cutting the tissue into 1mm with sterile surgical blade, surgical scissors and forceps 3 Left and right blocks. The minced tumor tissue was transferred to a centrifuge tube, centrifuged at 300g for 120s, the supernatant removed, resuspended in the digestion solution shown in Table 4 below, and subjected to shaking digestion on a constant temperature shaker at 37℃for 1 hour. The digested tumor tissue suspension was centrifuged at 300g for 5 minutes, the supernatant was discarded, and the digested tumor tissue was resuspended in 10mL of 1 XPBS, ground through a 100 mesh sieve, and collected in a 100mm dish. The collected cell suspension was sieved through a 40 μm pore size cell sieve and collected in a 50mL centrifuge tube and counted with a hemocytometer. It was then centrifuged at 1200 rpm for 5min, the supernatant was discarded and the pellet was resuspended in serum-free RPMI1640 at a density of 1X 10 5 -1×10 6 /mL cell/mL.
Table 4 digestive juice
2.2 magnetic bead separation and purification
The cells are separated by immunomagnetic beads, and the specific steps are as follows: adding CA125 antibody coupled nanometer magnetic beads (prepared by adopting the antibody anti-CA125-2 of example 1 according to a conventional method), uniformly mixing at 4-8 ℃ and standing for 15-60 minutes. Cells were washed with PBS buffer and centrifuged (300 g,10min, 4-8deg.C); re-suspending the cells, and adding a proper amount of PBS buffer solution; preparing a separation column, washing the separation column by using a buffer solution, pouring a cell suspension into the column, and washing the column by using 500ul of PBS buffer solution for three times; the column was removed from the magnetic field into a suitable vessel and rinsed with 1ml pbs buffer slightly with force to obtain ca125+ cells, i.e. ovarian cancer primary cells.
2.3 cell culture and expansion
Primary cells were prepared to a final concentration of 1X 10 in primary cell culture medium (RPMI 1640,2% penicillin/streptomycin diabody, 6mg/ml EGF,18mg/ml FGF10, 10% foetal calf serum) 5 ml of cell suspension. Adding the cell suspension into a culture flask, and placing at 37deg.C and 5% CO 2 Culturing in an incubator. After 48h, the cell growth reached log phase, and the cell growth was observed under an inverted phase contrast microscope and photographed. And (3) after the cells grow to more than 80% and fuse, observing again by a microscope, and repeating subculturing for 3 times to obtain the ovarian cancer primary cells.
EXAMPLE 3 labelling of Primary cells and in vivo imaging
3.1 specific markers for Primary cells
Counting ovarian cancer primary cells, and taking 2×10 7 Cells were centrifuged in 50mL centrifuge tube for 5min at 1000g, the supernatant was discarded, after the cells were resuspended in PBS, centrifuged again at 1000g for 5min, the supernatant was discarded, cells were resuspended in 100. Mu.LPBS, 30. Mu.L of FITC fluorescent-labeled anti-human CA125 antibody (antibody prepared using anti-CA125-2, FITC fluorescent-labeled antibody according to methods conventional in the art) was added to the cells, mixed well and incubated in ice bath for 30min, 600. Mu.LPBS was added to resuspend cells, 300g centrifuged for 5min, the supernatant was discarded, washing was repeated, and cells were resuspended in an appropriate amount of PBS to 5X 10 6 /mL、2×10 7 /mL、5×10 7 /mL, as 3 experimental groups. The control group used PBS buffer instead of human CA125 antibody. Flow cytometry analysis of human CA125 antibody-labeled ovarian cancer primary cells, 2×10 7 the/mL group showed the best effect of specific labeling. FIG. 1 shows 2X 10 7 The flow analysis result of the/mL experimental group shows that the anti-CA125-2 antibody can realize the specific labeling of ovarian cancer primary cells.
3.2 in vivo imaging of Primary cells
The method comprises the following specific steps of injecting labeled primary cells into the abdomen of a nude mouse in the skin: the cell resuspension of example 3.1 was injected intradermally 2X 10 with a sterile syringe 7 Individual cells, fluorescence intensity was measured using a small animal imager. A regular shape fluorescence signal was seen at the site of injection (fig. 2), and no apparent fluorescence signal was detected after the mice were turned over.
Besides belly injection, the primary cells marked by the invention can be injected into the back of a nude mouse in a percutaneous way or injected into the nude mouse in a tail vein way, and the distribution and migration and quantity information of ovarian cancer cells in the animal body can be known by detecting the form change of fluorescent signals by adopting a small animal imager.
While the fundamental and principal features of the invention and advantages of the invention have been shown and described, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and the description is provided for clarity only, and those skilled in the art will recognize that the embodiments of the disclosure may be combined appropriately to form other embodiments that will be understood by those skilled in the art.

Claims (11)

1. An anti-human CA125 antibody comprising a sequence as set forth in SEQ ID NO: HCDR1, seq id NO:10, HCDR2 shown in SEQ ID NO:11, HCDR3 as shown in SEQ ID NO:12, LCDR1, SEQ id no:13 and LCDR2 as set forth in SEQ ID NO: LCDR3 as shown in 14.
2. The antibody of claim 1, wherein the antibody comprises the amino acid sequence of SEQ ID NO:15 and the heavy chain variable region of the antibody shown in SEQ ID NO:16, and a light chain variable region of an antibody shown in seq id no.
3. The antibody of claim 2, wherein the antibody comprises a constant region.
4. The antibody of claim 3, wherein the constant region is a human IgG1 constant region.
5. Immunomagnetic beads characterized in that they are coated with an antibody according to any one of claims 1-4.
6. Use of the immunomagnetic beads according to claim 5 for the isolation and purification of cells.
7. The use according to claim 6, wherein the cells are primary tumor cells.
8. The use according to claim 7, wherein the primary tumor cells are primary ovarian cancer cells.
9. A fluorescent antibody, comprising an antibody according to any one of claims 1 to 4 and a fluorescent dye.
10. The fluorescent antibody of claim 9, wherein the fluorescent dye is selected from the group consisting of Cy series fluorescent dyes, alexa Fluor series fluorescent dyes, conjugated probes, cellular function probes, fluorescent proteins.
11. The fluorescent antibody of claim 10, wherein the fluorescent dye is FITC.
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