CN112048476A

CN112048476A - Preparation method of cell strain for stably expressing hFcRn and application of cell strain in drug screening

Info

Publication number: CN112048476A
Application number: CN202010822793.0A
Authority: CN
Inventors: 韩照中; 潘红芽
Original assignee: Linnuo Shanghai Pharmaceutical Technology Co ltd
Current assignee: Linnuo Shanghai Pharmaceutical Technology Co ltd
Priority date: 2020-08-14
Filing date: 2020-08-14
Publication date: 2020-12-08

Abstract

The invention discloses an FcRn stable expression cell line and application thereof in drug screening, and provides a construction method and an application example of the cell line, which comprises (1) inserting an FcRn gene into a eukaryotic expression vector; (2) introducing the vector obtained in the step 1 into host cells, and performing pressurized screening to obtain a cell line for stably expressing FcRn; 3) and (3) using the cell line obtained in the step 2 for drug screening. The invention is unique in that 1) the co-expression of FcRn and beta 2M protein in the same proportion is realized, and the assembly of FcRn heterodimer with functional activity is convenient; 2) cells with polarity characteristics such as MDCK cells are selected as host cells for FcRn expression, so that the capability test of the endocytosis, the exocytosis and the transmembrane transport of FcRn-mediated antibodies or other drug molecules is conveniently carried out; 3) provides a basic method and a technical approach for detecting and screening FcRn-based medicament endocytosis, exocrine and transmembrane transport by using corresponding cell strains.

Description

Preparation method of cell strain for stably expressing hFcRn and application of cell strain in drug screening

Technical Field

The invention relates to a preparation method of a cell strain for stably expressing hFcRn and application thereof in drug screening

Background

The hFcRn is a heterodimer consisting of two subunits, namely an alpha chain with the molecular weight of 45-53 kD and beta 2 microglobulin (beta 2microglobin or beta 2M) with the molecular weight of 14 kD. β 2 microglobulin is essential for the biological function of hFcRn. The crystal structure shows that the binding region of hFcRn to antibody is CH 2-CH 3, the antibody is unbound to FcRn at physiological PH 7.4, and the hFcRn is bound to the antibody at acidic PH 6.0. FcRn is widely distributed in vivo and mediates endocytosis, exocytosis and transmembrane transport of antibodies or antibody analogs, so binding to FcRn determines the half-life of IgG antibodies in vivo. Results of studies by Shreram Akilesh et al using an FcRn deficient mouse indicate that pathogenic IgG is rapidly cleared in blood of the FcRn deficient mouse, thereby reducing the pathogenicity of the pathogenic IgG in the FcRn deficient mouse, suggesting that it can be used for treating autoimmune diseases caused by autoantibodies by inhibiting FcRn (Shreram Akilesh et al, 2004, J Clin invest.113 (9): 1328-33). Meanwhile, in order to prolong the half-life of the drug in vivo, the serum clearance rate of the related drug can be reduced and the in vivo half-life of the drug can be prolonged by combining with an FcRn means, such as an antibody or an Fc fusion protein, and further the injection frequency of the drug can be reduced.

Experiments in the rat-induced autoimmune myasthenia gravis model have shown that treatment with monoclonal antibodies specific for the FcRn heavy chain can reduce the concentration of pathogenic IgG in serum, thereby reducing the extent of the disease (Liu et al, 2007, j.immunol.178: 5390-5398). In a murine arthritis model, high affinity FcRn antibodies can ameliorate disease (Patel et al, 2011, J.Immunol.187: 1015-1022). In many autoimmune diseases, high doses of IgG also alleviate the disease (Jin and Balthasar, 2005, hum. Immunol.66: 403-. The above results are combined to show that the inhibition of FcRn can shorten the half-life of pathogenic IgG in vivo, thereby achieving the purpose of treating autoimmune diseases.

In the screening and evaluation of FcRn inhibitors or binding proteins (including short peptides, genetically engineered proteins or antibodies), there is a need for an in vitro cell model that can effectively mimic FcRn-mediated endocytosis, exocrine and transmembrane transport. The invention establishes an MDCK-hFcRn cell model, and is characterized in that the co-expression of human FcRn and human beta 2M protein in the same proportion is realized, and the assembly of human FcRn heterodimer with functional activity is facilitated; cells with polar characteristics, such as MDCK (Madin-Darby Canine kidney) cells, are selected as host cells for hFcRn expression, so that the capability test of hFcRn-mediated endocytosis, exocrine and transmembrane transport of antibody or other drug molecules is facilitated.

Disclosure of Invention

The invention provides a preparation method and application of a cell strain for stably expressing hFcRn.

The preparation method of the cell strain for stably expressing hFcRn provided by the invention comprises the following specific steps:

cloning human FcRn and human beta 2M genes into a eukaryotic expression vector.

The eukaryotic expression vector has the following characteristics:

the human FcRn and the human beta 2M coding gene are under the control of the same transcription regulatory element and can be co-expressed in mammalian cells; the eukaryotic expression vector has all regulation elements required for transcription and translation for driving exogenous gene expression in eukaryotic cells, including a promoter, an enhancer, a transcription initiation region, a polyA processing and transcription termination signal, a ribosome binding region, a translation initiation signal, a translation termination signal and the like; making it under the control of the same transcription and translation regulatory elements, i.e. in the same transcript or mRNA;

the expression vector contains self-splicing peptide P2A and T2A sequences between the encoding genes of the human FcRn and the human beta 2M, so that the CMV promoter and the gene transcription regulatory element thereof drive the co-expression of the FCGRT, the B2M and the eGFP in the same Open Reading Frame (ORF). The translated polypeptide sequences were cleaved from the cleavage sites at P2A and T2A into the α chain FCGRTN, β chain B2M, and eGFP of hFcRn, respectively. The α chain FCGRTN and β chain B2M constitute the mature heterodimeric hFcRn receptor by non-covalently binding forms. To facilitate observation of transfection efficiency and gene expression in cells, eukaryotic expression vectors may be supplemented with genes for enzymes or light-emitting compounds that produce a color change, such as GFP. A schematic diagram of the open reading frame of the vector is shown in the attached figure 7:

and (3) introducing the expression vector in the step (1) into host cells, and screening to obtain the hFcRn cell strain which is stably expressed and has functional activity.

The mode of introducing the expression vector into the cells comprises introducing the expression vector into mammalian cells by a chemical mode (cell transfection mediated by a microsphere structure taking lipid components such as liposome, lipofectamine and the like as a main body), a physical mode or a biological mode;

physical means of introducing the expression vector into a cell include, but are not limited to, introducing the DNA into a mammalian cell by electroporation;

the biological method for introducing the expression vector into the cell includes, but is not limited to, introducing the foreign gene segment by encapsulating the gene segment described in step 1 into a viral particle and infecting a mammalian cell with the viral particle;

the host cell is an epithelial or endothelial cell (epithelial or endothelial cell) with polar characteristics; for example, Madin-Darby Canine kidney (MDCK) cells, human epithelial vein endothelial cells (HUVEC), human colon adenocarcinoma cells Caco-2, colon epithelial cells T84, and the like.

Screening of Positive cell lines

The screening of the above cells can be achieved by:

based on single cell separation technology such as flow cytometry sorting, high-expression cell strains can be selected by fluorescence intensity through expression of fluorescein genes (such as green, yellow and red fluorescent proteins GFP, YFP, RFP and the like) related to the gene segments in the step 1;

the cells can be selected by expressing antibiotic resistance genes associated with gene segments in the expression vector and removing cells without gene integration or with relatively low expression by using antibiotics; high-expression cell strains can be selected by gradually increasing the concentration of antibiotics; obtaining the monoclonal cells by a limiting dilution method.

Identification of hFcRn-stably expressing cell lines

The expression level of hFcRn in the hFcRn high-expression cell strain can be determined by immunostaining the cells by using an hFcRn specific antibody, or detecting the content of hFcRn in cell lysates by using a corresponding antibody through western printing and dyeing (western blot) or enzyme-linked immunosorbent assay (ELISA);

functional characterization of hFcRn cell lines

The functional activity of hFcRn in hFcRn high expressing cell lines can be determined by:

IgG endocytosis, IgG exocrine, and transmembrane transport of IgG refer to: a schematic of the pulse-wise chamber is shown in FIG. 8.

MDCK-hFcRn cells were seeded in 6-well plates, 1X 10⁵Cells/well, cultured for 24 hours. Add 200. mu.g/mL biotinylated hIgG and incubate at 37 ℃ for 1 hour. The cells were washed thoroughly 5 times with pre-chilled PBS on ice. Mu.l of DMEM without fetal bovine serum was added to each well and incubated at 37 ℃ for 0.5 hour, 1 hour and 2 hours, respectively. Cell culture supernatants were collected, centrifuged at 12000rpm for 10min, and the biotinylated hIgG concentration in the supernatants was quantified by ELISA.

A schematic diagram of a Transwell chamber-based assay is shown in FIG. 9

MDCK-hFcRn cells were seeded into 96-well trans-well cell culture plates at 1X 10⁵Cells/well, incubated at 37 ℃ for 48 hours. 200. mu.g/mL hIgG-biotin was added to the inner chamber of the trans-well and incubated at 37 ℃ for 24 h. 90 minutes before the sample was collected, 2ul/ml Lucifer Yellow fluorochrome was added to the trans-well chamber to evaluate the cell layer for the presence of leakage. And after the incubation is finished, collecting the culture solution in the extracellular chamber for ELSIA quantitative determination of the amount of hIgG-biotin transported through the cell membrane.

A cell strain MDCK-hFcRn for stably expressing hFcRn is preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 4 days of 2020, and the code of the preservation unit: CCTCC China center for type culture Collection; and (4) storage address: wuhan university in China; preservation state: survival; the preservation date is as follows: 8, month 4 in 2020; the preservation number is: c2020116; and (3) classification and naming: human FcRn overexpresses the canine kidney epithelial cell line MDCK-hFcRn.

Description of the drawings:

figure 1hFcRn heterodimer co-expression plasmid: the pcDNA3.1 multiple cloning site has inserted into it the gene coding sequence (CDS) of HUMAN FCGRT (UniProtKB-P55899, FCGRTN _ HUMAN), B2M (UniProtKB-P61769, B2MG _ HUMAN) and eGFP (P42212.1). The three are respectively connected through self-cutting peptide P2A and T2A sequences, so that the CMV promoter and a gene transcription regulatory element thereof drive the co-expression of FCGRT, B2M and eGFP in the same Open Reading Frame (ORF).

FIG. 2 Western-blot identification of MDCK-hFcRn stable expression clones: MDCK-hFcRn C #1, C #2, C #3 and C #5hFcRn expression level is obviously higher than that of control MDCK cells.

FIG. 3 shows the result of the experiment on the biological function of the MDCK-hFcRn stable expression cell line, Pulse-chase assay: the circulating secreted biotin IgG amount in the cell supernatant of two MDCK-hFcRn positive clone strains is more than that of MDCK of a control cell. The amount of biotin IgG in the exocrine circulation gradually increased with the incubation time.

FIG. 4 results of the biological function chamber-based assay of MDCK-hFcRn stably expressing cell lines: the transmembrane transport efficiency of MDCK and MDCK-hFcRn C #1 cells is 0.13 percent and 0.73 percent respectively. The transmembrane transport efficiency of MDCK-hFcRn C #1 cells is 5 times that of the original MDCK cells.

Figure 5. effect of hFcRn inhibitors compound 1, compound 2 in inhibiting cell transmembrane transport of IgG mediated by hFcRn: the compound 2 has the functions of inhibiting an FcRn receptor and reducing the transport of IgG cells. The amount of IgG transported by the cells under the action of Compound 2 is 46% of that of Compound 1.

FIG. 6. cell transmembrane transport capacity mediated by hFcRn by Fc fusion proteins compound 3, compound 4: compound4 has the function of inhibiting FcRn receptor. The circulating amount of secreted IgG in the cell supernatant under the action of compound4 was 64.8% of that of compound 3.

FIG. 7 schematic drawing of open reading frame of vector

FIG. 8 is a schematic diagram of pulse-chase

FIG. 9 is a schematic diagram of a Transwell chamber-based assay

Detailed Description

The present invention is further illustrated by the following specific examples.

The methods used in the following examples are conventional methods and some key reagents used in experiments unless otherwise specified.

Example 1 construction of recombinant eukaryotic expression vector pcDNA3.1-FCGRT-P2A-B2M-T2A-eGFP

HUMAN FCGRT (UniProtKB-P55899, FCGRTN _ HUMAN) is inserted into the multi-cloning site of the eukaryotic expression vector pcDNA3.1, and the sequence is Seq 1; HUMAN B2M (UniProtKB-P61769, B2MG _ HUMAN), the sequence Seq 2; the eGFP sequence is derived from the gene coding sequence (CDS) of P42212.1, which is Seq 3. The three are respectively connected through self-cutting peptide P2A and T2A sequences, so that the CMV promoter and a gene transcription regulatory element thereof drive the co-expression of FCGRT, B2M and eGFP in the same Open Reading Frame (ORF). The sequence of P2A is Seq 4, and the sequence of T2A is Seq 5. The translated polypeptide sequences were cleaved from the cleavage sites at P2A and T2A into FcRn alpha chain FCGRTN, beta chain B2M, and eGFP, respectively. The α chain FCGRTN and β chain B2M constitute the mature heterodimeric FcRn receptor by non-covalently binding forms. As shown in figure 1.

Host cell MDCK, cell species dog. The expression sequence was codon optimized for the cell. The synthesized fragment and the pcDNA3.1 vector are subjected to double enzyme digestion by BamH I and Xba I, T4 ligase connection, DH 5 alpha competent cell transformation and amplification, and plasmids are extracted for standby. FIG. 1hFcRn heterodimer co-expression plasmid

Example 2 establishment of MDCK-hFcRn Stable cell line

MDCK cells were cultured in high-glucose DMEM containing 4.0mM L-Glutamine, Sodium Pyruvate (HyClone, SH30243.01), 10% FBS (GIBCO,10099-141 c). Digested with conventional 0.25% Trypsin-EDTA (GIBCO,25200056), subcultured, and stored in liquid nitrogen (frozen stock solution: 5% DMSO, 40% FBS, 55% medium).

pcDNA3.1-FCGRT-P2A-B2M-T2A-eGFP transfected MDCK cells: digesting and centrifuging to collect MDCK cells, and collecting 2 × 10⁵Cells were seeded into 6-well plates. The cell density of D1 was as high as 80%. The cells were washed three times with PBS and Opti-MEM (Invitrogen, Cat #31985062) was added at 700 ul/well. According to Lipofectamine^TM2000Transfection Reagent (Invitrogen, Cat #11668027), DNA Lipofectamine^TMMix at room temperature for 5min at 2000 ═ 1:5, add to 6 well plates.

Establishment of a stably expressing MDCK-hFcRn cell line: d2 transiently transfected MDCK-FcRn cells were transfected at 1: passage 6 (P1). D3, adding 500ug/ml G418 to screen under pressure. (MDCK cells G418 selection concentration titration was done before selection: concentration was increased from 200ug/ml to 600ug/ml, cells were all dead on day 7 at 500 ug/ml; cells were all dead on day 10 at 400 ug/ml). D7, cell change fluid, G418500 ug/ml. D10, control cells all dead, MDCK-hFcRn cells were instead cultured in medium containing G418250 ug/ml. And (4) carrying out cell passage, inoculating the cells into a 96-well plate by adopting a limiting dilution method, and screening MDCK-hFcRn monoclonal cells. D11 cells were observed and wells containing single cells in 96-well plates were labeled. D15, single clone cells were passaged into 24-well plates (P2) for a total of 8 clones, labeled MDCK-hFcRn (P3) C #1, C #2, C #3, … C #8, respectively. D18, and the monoclonal cells were individually passaged into 6-well plates (P3). D21, respectively passaging the monoclonal cells to a culture dish (P4) with the diameter of phi 9cm, and culturing the cells in a culture medium containing G418250 ug/ml. And identifying the clone with the FcRn positive expression by Western-blot according to conventional passage, amplification, culture and liquid nitrogen preservation for later use.

EXAMPLE 3 identification of stably expressing MDCK-hhFcRn cell lines

Extracting total cell protein: MDCK, MDCK-hFcRn C #1, 2-8 (P5) cells were seeded into 6-well plates. Culturing for 24h, washing cells for 3 times with PBS, extracting total cell protein according to RIPA cell lysate specification, centrifuging at 12000rpm for 10min, collecting supernatant, and freezing at-80 deg.C for use. western blot assay for expression of hFcRn: 20ug of each protein sample was denatured at 100 ℃ for 5min and placed on ice. SDS-PAGE, 100V, 1.5 h. 80mA, wet-transfer overnight, and transfer the protein to PVDF membrane. The PVDF membrane with 1% BSA PBS buffer room temperature blocking for 1 hours. Adding primary Anti-FCGRT/FCRN, RabMAB (Abcam, ab228975) 1: 1000, Actin rabbit monoclonal antibody (Biolynx, BX00063) and incubation at room temperature for 1 hour. PBST membrane washing 3 times. HRP anti-rabbit IgG (H + L) (SD0039), 1: 5000, incubation at room temperature for 1 hour. PBST membrane washing 3 times. DAB color development kit color development (SD 0145). The results (fig. 2) show that: MDCK-hFcRn C #1, C #2, C #3 and C #5hFcRn expression level is obviously higher than that of control MDCK cells.

Example 4 Stable expression of MDCK-hFcRn cell biological function assay (Pulse-chase assay)

MDCK and MDCK-hFcRn cells were seeded into 6-well plates at 1X 10⁵Cells/well, cultured for 24 hours.

Add 200. mu.g/mL biotinylated hIgG and incubate at 37 ℃ for 1 hour. The cells were washed thoroughly 5 times with pre-chilled PBS on ice. Mu.l of DMEM without fetal bovine serum was added to each well and incubated at 37 ℃ for 0.5 hour, 1 hour and 2 hours, respectively. Cell culture supernatants were collected, centrifuged at 12000rpm for 10min, and the biotinylated hIgG concentration in the supernatants was quantified by ELISA.

Coating 1ug/ml streptavidin overnight at 4 ℃ according to the conventional ELISA method; blocking the sample the next day, adding the sample, diluting biotinylated human IgG (1ug/ml,2 times of gradient dilution) in a gradient way to make a standard curve, and incubating for 1h at room temperature; the plate was washed 3 times, goat Fab anti-human IgG Fab horseradish peroxidase-labeled secondary antibody (ab98535) was added, TMB was developed, absorbance was read at 450nm after termination, and analysis was performed using GraphPad 8.0 software. The experimental results are shown in fig. 3: the circulating secreted biotin IgG amount in the cell supernatant of two MDCK-hFcRn positive clone strains is more than that of MDCK of a control cell. The amount of biotin IgG in the exocrine circulation gradually increased with the incubation time. The results are shown in FIG. 3: the circulating secreted biotin IgG amount in the cell supernatant of two MDCK-hFcRn positive clone strains is more than that of MDCK of a control cell. The amount of biotin IgG in the exocrine circulation gradually increased with the incubation time.

Example 5 Stable expression of MDCK-hFcRn assay for cellular biological Activity (Chamber-based assays)

D1: MDCK and MDCK-hFcRn cells were seeded into 96-well trans-well cell culture plates at 1X 10⁵Cells/well, incubated at 37 ℃ for 48 hours. D4, trans-well chamber was added with 200. mu.g/mL hIgG-biotin and incubated at 37 ℃ for 24 h. D5, 2ul/ml Lucifer Yellow fluorochrome was added to the trans-well chamber 90 minutes before the sample was collected to evaluate the presence of leakage in the cell layer. And after the incubation is finished, collecting the culture solution in the extracellular chamber for ELSIA quantitative determination of the amount of hIgG-biotin transported through the cell membrane. And detecting the content of the Lucifer Yellow in the outer chamber and the inner chamber of the trans-well simultaneously, wherein if the content of the Lucifer Yellow in the outer chamber is less than or equal to 0.1 percent of that in the inner chamber, the trans-well is indicated to have no leakage.

ELISA quantitative detection of biotin hIgG: ELISA96 well plates were coated with conventional streptavidin at 1ug/ml and incubated overnight at 4 ℃. And (4) sealing for 2 hours after washing the plate the next day, adding the sample and the standard curve sample after washing the plate, and incubating for 1 hour at room temperature. Adding 1: a5000 dilution of the goat Fab anti-human IgG-Fab conjugated to HRP (Abcam, ab98535), incubation at room temperature for 1 hour, TMB color development, after termination the microplate reader reading. Data were analyzed using GraphPad 8.0 software. Experimental results figure 4 shows: the transmembrane transport efficiency of MDCK and MDCK-hFcRn C #1 cells is 0.13 percent and 0.73 percent respectively. The transmembrane transport efficiency of MDCK-hFcRn C #1 cells is 5 times that of MDCK cells. The results show that the MDCK-hFcRn C #1 cell strain is a good in-vitro model of cell transport mediated by hFcRn, and can be used for evaluating or predicting the influence of different drugs on the in-vivo half-life of IgG mediated by FcRn.

TABLE 1 concentration of Bio-IgG transported across the membrane of the cell (ug/ml)

Example 6 use of stably expressing MDCK-hFcRn cells in drug screening

Example 1: the effect of the hFcRn inhibitors compound 1, compound 2 in inhibiting IgG endocytosis, exocrine recycling and transmembrane transport mediated by hFcRn was evaluated:

d1: MDCK-hFcRn cells were seeded into 96-well trans-well cell culture plates at 1X 10⁵Cells/well, incubated at 37 ℃ for 48 hours.

D4: 200. mu.g/mL hIgG-biotin/

compound

1, 200. mu.g/mL hIgG-biotin/

compound

2, 3 wells were added to the trans-well chamber, respectively. Incubate at 37 ℃ for 24 h. D5, 2ul/ml Lucifer Yellow 20uM fluorescent dye was added to the trans-well chamber 90 minutes before the sample was collected to evaluate the presence of leakage in the cell layer. And (4) collecting the culture solution in the extracellular chamber after the incubation is finished, and quantitatively detecting the amount of hIgG-biotin transported through the cell membrane by ELSIA. And detecting the content of the Lucifer Yellow in the outer chamber and the inner chamber of the trans-well simultaneously, wherein if the content of the Lucifer Yellow in the outer chamber is less than or equal to 0.1 percent of that in the inner chamber, the trans-well is indicated to have no leakage.

ELISA quantitative detection of biotin hIgG: 96-well E LISA plates were coated with conventional 1ug/ml streptavidin and incubated overnight at 4 ℃. And (3) sealing for 2 hours after washing the plate the next day, adding the sample and the standard curve sample after washing the plate, incubating for 1 hour at room temperature, and adding 1: the goat Fab anti-human IgG-Fab conjugated to HRP (Abcam, ab98535) was diluted 5000-fold, incubated at room temperature for 1 hour, TMB developed, and after termination, plate read was carried out with a microplate reader at 450 nm. GraphPad 8.0 software analyzed the data. Experimental results figure 5 shows: the compound 2 has the functions of inhibiting an FcRn receptor and reducing the transport of IgG cells. The amount of IgG transported by the cells under the action of Compound 2 is 46% of that of Compound 1.

Example 2: testing the capacity of antibodies or fusion proteins comprising different Fc mutants to transport through cells mediated by hFcRn for endocytosis, exocrine recycling and transmembrane transport:

MDCK-hFcRn cells were seeded into 6-well plates, 1X 10⁵Cells/well, cultured for 24 hours.

200. mu.g/mL hIgG-biotin/

compound

3, 200. mu.g/mL hIgG-biotin/

compound

4, 3 wells were added and incubated at 37 ℃ for 1 hour. The cells were washed thoroughly 5 times with pre-chilled PBS on ice. Mu.l of DMEM without fetal bovine serum was added to each well and incubated at 37 ℃ for 2 hours. Cell culture supernatants were collected, centrifuged at 12000rpm for 10min, and the biotinylated hIgG concentration in the supernatants was quantified by ELISA.

Coating 1ug/ml streptavidin overnight at 4 ℃ according to the conventional ELISA method; blocking the sample the next day, adding the sample, diluting biotinylated human IgG (1ug/ml,2 times of gradient dilution) in a gradient way to make a standard curve, and incubating for 1h at room temperature; the plate was washed 3 times, goat Fab anti-human IgG Fab horseradish peroxidase-labeled secondary antibody (ab98535) was added, TMB was developed, absorbance was read at 450nm after termination, and analysis was performed using GraphPad 8.0 software. Experimental results figure 6 shows: compound4 has the function of inhibiting FcRn receptor. The circulating amount of secreted IgG in the cell supernatant under the action of compound4 was 64.8% of that of compound 3.

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Claims

1. A cell line stably expressing functionally active FcRn.

2. The cell line of claim 1, which is obtained by inserting a foreign gene fragment into the genome of the cell by genetic engineering means.

3. The cell line of claim 1 which expresses both human FcRn and human β 2M protein.

4. The cell line of claim 1 which is MDCK or other mammalian derived epithelial cells of polar character.

5. The cell line of claim 1 for use in FcRn-mediated endocytosis, exocrine and transmembrane transport assays of antibody or other drug molecules.

6. The assay of claim 5 comprising pulse-wise, chamber-based transcytosis and immunofluorescence-based microscopic intracellular tracking technologies.

7. The drug molecule of claim 5 comprising an antibody, an Fc fusion protein, and an FcRn binding protein.