CN112980851A

CN112980851A - Method for efficiently detecting fusion protein/antibody Fc segment incapable of mediating ADCC (ADCC-mediated cancer cell mediated cytotoxicity) and CDC (CDC) activities

Info

Publication number: CN112980851A
Application number: CN202110233928.4A
Authority: CN
Inventors: 肖志华; 伍小春
Original assignee: Shanghai Aopu Mai Biotechnology Co Ltd
Current assignee: Shanghai Aopu Mai Biotechnology Co Ltd
Priority date: 2021-03-03
Filing date: 2021-03-03
Publication date: 2021-06-18

Abstract

The invention belongs to the field of biology, and particularly relates to a method for efficiently detecting that fusion protein or antibody Fc segment cannot mediate to generate ADCC and CDC activities. First, disclosed is a detection method capable of rapidly finding positive antibodies and positive cells that mediate ADCC and CDC effects and directly verifying that the Fc sequence of a target protein or antibody cannot or can only weakly mediate ADCC and CDC effects, the method comprising: s1, construction of a vector: performing codon optimization according to the Fc sequences of Rituximab and IgG4, synthesizing a target gene and inserting the target gene into a plasmid to construct a transient expression vector; s2, plasmid extraction and sequencing; s3, protein transient expression; s4, purifying protein; s5, ADCC and CDC activity assay. The detection method disclosed by the invention can also directly verify that the target protein or the Fc segment of the antibody cannot or only can weakly mediate to generate ADCC (advanced charge-coupled cellular cytotoxicity) and CDC (CDC-mediated cytotoxicity); the detection method is reliable, wide in applicability and efficient.

Description

Method for efficiently detecting fusion protein/antibody Fc segment incapable of mediating ADCC (ADCC-mediated cancer cell mediated cytotoxicity) and CDC (CDC) activities

Technical Field

The invention belongs to the field of biology, and particularly relates to a method for efficiently detecting that an Fc segment of a fusion protein/antibody cannot mediate ADCC (ADCC-mediated cytotoxicity) and CDC (CDC-mediated immunity) activities.

Technical Field

The Fc fusion protein or antibody generally increases the half-life of the target protein drug through human Fc-fragment FcRn (neonatal Fc receptor) -mediated circulation, improves the pharmacokinetic profile, and the mechanism of action is independent of ADCC/CDC activity. Some non-cancer indication antibody drugs, as well as tumor immunotherapy antibody drugs such as the PD-1/PD-L1 antibody, also have mechanisms of action that are independent of ADCC (antibody-dependent cytotoxicity), CDC (complement-dependent cytotoxicity) activity. In this case, the cytotoxicity ADCC and CDC of the human Fc region may cause unnecessary side effects.

Currently, some new marketed antibody drugs and antibody/fusion protein drugs under investigation are being brought to better safety and stability by using IgG2, IgG4 subtypes or engineering Fc (reducing or removing ADCC/CDC effects).

At present, some pharmaceutical enterprises prove that the Fc segment does not have or has weak mediated ADCC and CDC activities:

firstly, positive control: 1) for an antibody or protein with an Fc mutation, the Fc segment of the antibody or protein can not mediate ADCC/CDC effect or only weakly mediate ADCC/CDC effect, and the positive control is the antibody or fusion protein with the same target point and the Fc of the antibody or protein; 2) while the positive control for antibodies of subtype IgG2 or IgG4 is an antibody with the same target subtype IgG 1. Positive cells: cells highly expressing the target.

Defects and deficiencies: 1) positive antibodies, even if able to bind Fc receptors or C1q at the ELISA level, are not necessarily able to mediate ADCC and CDC of effector cells on cells that highly express the target; 2) even if the positive control of the target can mediate ADCC, since tumor cells frequently highly express some complement inhibitors such as CD46, CD55 and CD59 on the cell surface, the molecules can protect the target cells from the killing effect of complement, and the positive antibody only has very weak or basically no CDC effect.

Positive control: antibodies or fusion proteins are marketed whose target (which is different from the target of the target sample) is also highly expressed in selected positive target cells; positive cells: and simultaneously, target cells of target targets and positive antibody corresponding targets are highly expressed.

Defects and deficiencies: 1) target cells with high expression of two targets are difficult to find; 2) when ADCC and CDC detection is carried out, even if the positive antibody shows a positive result, the target antibody shows a negative result, and whether the positive antibody shows the positive result or the negative result is caused by the Fab section or the Fc section cannot be proved; 3) positive antibodies, even though binding to Fc receptors or C1q at the ELISA level, some antibodies are unable to mediate ADCC and CDC at the cell level; 4) even if the positive control mediates ADCC, since tumor cells often express high levels of some complement inhibitors such as CD46, CD55, and CD59 on their cell surface, these molecules can protect target cells from complement-mediated killing, resulting in positive antibodies with very little or no CDC effect.

Disclosure of Invention

In order to solve the above problems in the prior art, the present invention provides a method for grafting Fc of a target protein or antibody to a known antibody (e.g., Rituximab), and selecting Raji cells highly expressing CD20 as positive cells. The positive antibody, namely the antibody Rituximab of CD20 can mediate strong ADCC and CDC effects of Raji cells. The detection method disclosed by the invention has the advantages of positive antibodies, well-found positive cells, ADCC and CDC effects when the positive antibodies are determined, and the direct verification that the Fc of the target protein or antibody can not or only weakly mediate the ADCC and CDC effects can be realized. This protocol is applicable to verify that all Fc segments are unable or only weakly mediated to produce ADCC and CDC effects.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the present invention discloses a detection method, which can rapidly find out positive antibodies and positive cells capable of mediating ADCC and CDC effects, and can directly verify that the Fc region of a target protein or antibody cannot or only weakly mediates ADCC and CDC effects, the method comprising:

s1, construction of a vector: performing codon optimization according to the Fc sequences of Rituximab and IgG4, synthesizing a target gene and inserting the target gene into a plasmid to construct a transient expression vector;

s2, plasmid extraction and sequencing;

s3, protein transient expression;

s4, purifying protein;

s5, ADCC and CDC activity assay.

It should be understood that the present invention is not limited to the above steps, and may also include other additional steps, for example, before step S1, between steps S1 and S2, between steps S2 and S3, between steps S3 and S4, between steps S4 and S5, and after step S5, without departing from the scope of the present invention.

Preferably, the nucleotide sequence of the Rituximab light chain is shown in SEQ ID NO: 1, the nucleotide sequence of the Ritx-Fc (IgG4) heavy chain is shown as SEQ ID NO: 2 is shown in the specification;

preferably, in S2, the strain is inoculated into a culture medium containing ampicillin, shake-cultured for 12-24h, centrifuged, and

the operating manual of the Xtra Midi Plus EF carries out mass extraction of plasmids; and finally, measuring the plasmid concentration by a Quickdrop ultramicro spectrophotometer.

Preferably, in S3, Ritx-Fc (IgG4) antibody in HEK293F cells in transient expression.

Preferably, in S4, the protein purification step sequentially comprises: rinsing, disinfecting, balancing, loading, leaching, eluting, regenerating, rinsing, disinfecting and rinsing;

wherein rinsing and equilibration are carried out with 50mM Tris-HAc, 150mM NaCl, pH 7.4.

Preferably, in S5, ADCC and CDC activities are analyzed with Raji cells as target cells.

Preferably, in S3, the extracted plasmid is transfected into HEK293 cells and cultured using OPM-293-CD05 medium.

More preferably, HEK293 cells are transfected at a density of (4-4.5). times.10⁶cells/ml。

In a second aspect, the invention discloses the use of the above method for the treatment of tumors, transplants and/or autoimmune diseases.

Compared with the prior art, the invention has at least the following beneficial effects:

the detection method disclosed by the invention has the advantages of positive antibodies, good finding of positive cells and the like, and positive antibodies certainly have ADCC and CDC effects, and meanwhile, the detection method disclosed by the invention can also directly verify that the target protein or the Fc segment of the antibody can not or only weakly mediate to generate ADCC and CDC effects; the detection method is reliable, wide in applicability and efficient.

Drawings

FIG. 1 is a schematic representation of the ADCC effect of Raji cells;

FIG. 2 is a schematic representation of CDC effect of Raji cells.

Detailed Description

The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.

The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.

Example 1

The embodiment discloses a method which can quickly find out positive antibodies and positive cells capable of mediating ADCC and CDC effects and can directly verify that the Fc sequence of a target protein or antibody cannot or only can weakly mediate ADCC and CDC effects. The method specifically comprises the following steps:

first, vector construction

1.1 purpose of the experiment

According to Rituximab, IgG4 Fc antibody sequence, carrying out codon optimization, synthesizing a target gene, inserting the target gene into an empty plasmid, constructing transient expression vectors Rituximab heavy chain, Rituximab light chain and Ritx-Fc (IgG4) heavy chain, and sequencing to confirm that the constructed vectors are consistent with the design, thereby providing a correct non-endotoxin plasmid for cell transient transformation.

1.2 reagents and apparatus

1.2.1 reagents

TABLE 1 Main reagents

1.2.2 apparatus

TABLE 2 Instrument Equipment

1.2.3 Experimental design and results

1.2.3.1 Synthesis of the Gene of interest

Based on the amino acid sequence information Rituximab light chain (Table 3) and Ritx-Fc (IgG4) heavy chain (Table 4), the gene synthesis of Kinzhi Biotech, Suzhou was delegated and inserted into the empty vector of the transient pCDNA3.1, respectively, to synthesize a recombinant plasmid.

TABLE 3

TABLE 4

1.2.3.2 plasmid extraction

Inoculating glycerol bacteria into sterilized 200 ml culture medium, and respectively adding 200 microliters of ampicillin; shaking overnight at 37 ℃, 220 rpm; centrifuging the bacterial liquid at 4500rpm for 10min the next day, and then performing centrifugation according to the following steps

The operating manual of the Xtra Midi Plus EF carries out mass extraction of plasmids; finally, the plasmid concentration was measured by QuickDrop.

Operating procedure for Xtra Midi Plus EF:

centrifuging the bacterial culture solution at 4500rpm for 10min, discarding the supernatant, adding RES buffer and LYS buffer into the precipitate to fully lyse the cells, adding NEU buffer to neutralize to colorless, loading the solution onto a DNA binding column (balanced by EQU buffer in advance), washing the column by EQU buffer, washing the column by WASH buffer, and eluting the DNA by ELU buffer. Adding isopropanol, centrifuging at 4 ℃ for 30min, removing the supernatant, washing the precipitate with 70% ethanol, centrifuging at 4 ℃ for 15min, removing the supernatant, drying at room temperature, and dissolving DNA by TE buffer. (please refer to the description in detail)

1.2.3.3 plasmid sequencing

The plasmid Rituximab light chain is sent for sequencing, the sequencing result is compared with the target sequence, and the comparison result shows that the DNA sequence of the expression plasmid is completely consistent with the target sequence.

The plasmid Ritx-Fc (IgG4) heavy chain is sequenced, the sequencing result is compared with the target sequence, and the result shows that the DNA sequence of the expression plasmid is completely consistent with the target sequence.

1.2.3.4 conclusions of the experiment

The construction of the Ritx-Fc (IgG4) antibody light and heavy chains was successful.

II, protein expression

2.1 purpose of the experiment

Transient expression of the Ritx-Fc (IgG4) antibody in HEK293F cells was performed.

2.2 reagents, materials and devices

2.2.1 reagents

TABLE 5

Name of reagent	Brand	Goods number
			Glucose	Sigma	G7021-10KG
L-Glutamine	Sigma	59202C
			PEI	Polyscience	23966
Trypan blue solution	Sigma	T8154-100ml
			OPM-293 CD05 Medium	OPM	81075-001
OPM-CHO PFF06 Medium	OPM	1265F05-001
			Opti-MEM	Gibico	51985-034

2.2.2 materials

TABLE 6

2.2.3 instruments

TABLE 7

Instrumentation and equipment	Brand	Model number	Numbering
				Biological safety cabinet	Thermo	1300	/
Shaking table	Inforce	Multitron Pro	L01180400214
				Cell counter	CountStar	IC1000	L02170402704
Cell counter	Beckman	Vi-Cell XR	L01180505301
				Centrifugal machine	Beckman	X12R	L01170400607
Miniature table centrifuge	Thermo	Pico17	L01180400610
				Refrigerator with a door	Haier	BCD-290W	L01140801101
-80 ℃ refrigerator	Thermo/Forma	902	L01140801001
				Liquid nitrogen tank	Thermo	8143	L01140800901
Liquid nitrogen storage system	Thermo	7403	L04170802503
				Biochemical analyzer	Hieman (Hieman)	M 100	/
-80 ℃ refrigerator	Thermo/Forma	902	L01140801001

2.3 content of the experiment

2.3.1 plasmid preparation

And (3) extracting a large amount of escherichia coli from the successfully constructed plasmid in the step one to obtain a sufficient amount of non-endotoxin plasmid, and preparing for transient expression. Sample information is shown in table 8 below.

TABLE 8

Sample numbering	Sample name	Expression volume (L)
			1	Ritx-Fc(IgG4)	0.1

2.3.1.1 Medium preparation

The OPM-293-CD05 medium was stored in a refrigerator at 4 ℃ and preheated in a 37 ℃ water bath before use.

2.3.1.2 cell preparation

2.3.1.2.1 cell domestication

The HEK293 cells were thawed to OPM-293-CD05 and cultured in 125mL shake flasks at 30mL, 130rpm, 37 ℃ and 8% CO₂And (5) culturing. After more than three passages, the seed cells were maintained.

2.3.1.2.2 cell expansion

HEK cell density was diluted to 2-2.2X 10⁶cell/ml, activity greater than 95%, 130rpm, 37 ℃, 8% CO₂And (5) culturing.

2.3.2 cell transfection expression

(1) Cell dilution

The cell transfection density is 4-4.4 multiplied by 10⁶cell/ml, if the cell density is too high, diluted to the appropriate density with the corresponding Medium (OPM-293-CD05 Medium).

(2) Cell transfection

The DNA and PEI are diluted and mixed according to a proper proportion, and after the mixture is gently mixed, the mixture is kept stand for 20 minutes at room temperature. A defined amount of DNA-PEI mixture was poured into the corresponding volume of HEK293F cells and mixed well. Placing in a shaking table at 130rpm, 37 deg.C and 8% CO₂And (5) culturing.

2.3.3 protein expression

(1)Day1-Day6

18-24 h after transfection, PFF06 was added in an initial volume of 10%. Sampling every day, recording cell density and cell activity, measuring glucose concentration in the culture medium, and if the sugar content is less than 2g/L, supplementing 8g/L glucose.

(2) On day 6 post-transfection, cells were harvested and retained. The sample was transferred to the purification department for purification.

2.3.4 results of the experiment are shown in Table 9 below

TABLE 9

Ritx-Fc (IgG4) antibodies were harvested on day 6 post-transfection.

2.3.5 summary of the experiments

The expression process is smooth, the cell state is not abnormal, and the cell is delivered to the downstream for purification and sample preparation after centrifugal filtration.

Thirdly, protein purification

3.1 purpose of the experiment

And (3) capturing the sample obtained in the second step by using Protein A.

3.2 apparatus and instruments are shown in Table 10:

watch 10

Serial number	Name (R)	Instrument numbering	Model number
				1	Protein purification instrument	6(03)-01-004	AKTA PureM150
2	Micro ultraviolet spectrophotometer	6(03)-09-005	Nano-300
				3	Balance with a movable handle	6(03)-09-001	BSA3202S-CW
4	PH conductivity meter	6(03)-09-003	S470-K

3.3 Experimental methods and procedures are shown in Table 11:

TABLE 11

Fourth, data analysis after purification

The analysis of the purified data is shown in Table 12.

TABLE 12

4.1 sample Conditioning Process

Concentrated to a concentration >1mg/mL for assay as shown in Table 13 below.

Watch 13

Concentrated to a concentration >1mg/mL for analysis.

Fifth, ADCC and CDC Activity assay

5.1ADCC

5.1.1 sample information

TABLE 14 sample information

Sample numbering	Sample name	Concentration of
			1	Rituximab	10mg/ml
2	Ritx-Fc(IgG4)	0.8mg/ml
			3	hIgG1	1.88mg/ml

5.1.2 reagents, consumables, apparatus

5.1.2.1 reagents and materials

TABLE 15 list of reagents and materials

5.1.2.2 consumables

TABLE 16 consumable List

Name of consumable	Manufacturer of the product	Goods number/model
			96-hole U-shaped plate	Corning	3799

5.1.2.3 apparatus

TABLE 17 device List

Device name	Manufacturer of the product	Goods number/model
			Biological safety cabinet	Thermo	1379
Carbon dioxide incubator	Thermo	311
			Cell counter	Countstar	S2
Microscope	AolinBass	CKX41SF
			Centrifugal machine	Eppendorf	5810R
Enzyme-linked immunosorbent assay (ELISA) instrument	Molecular Devices	M5

5.1.2.4 Experimental methods

(1) The experimental layout is shown in table 18 below.

Table 18 type table for experiment

Tm target cell maximum release

Ts target cell splantaneous release (spontaneous release of target cell)

(T + E) s target cell and effector cell splantaneous release (spontaneous release of target and effector cells)

Es, effector cell splantaneous release (spontaneous release of effector cells)

(2) Procedure of experiment

A. The experimental culture medium is as follows: MEM alpha containing 1% FBS

B. Raji cell preparation and enumeration

Raji cells in logarithmic growth phase were centrifuged at 1000rpm for 5 minutes. After washing once with the test medium and centrifugation at 1000rpm for 5 minutes. Resuspending the cells in 5mL of experimental medium, and taking a proper amount of cell suspension to a 1.5mL EP tube; after mixing, 20 μ L of cell suspension is taken from the EP tube and mixed with the same amount of trypan blue (namely diluted 2 times), and counting is carried out; 1.1mL of the resuspended Raji cells were mixed with 4.9mL of the experimental medium to adjust the density of the Raji cells to 5.0X 10⁵Per mL; the diluted Raji cells above were added at 25. mu.L/well to 96-well U-well plate sample wells and (T + E) s (0. mu.g/ml), Tm, and Ts control wells, and 25. mu.L of experimental medium was added to the remaining experimental wells. The cell information is shown in table 19 below.

Watch 19

Generation of cell	Number of viable cells (. times.10)⁶one/mL)	Cell viability (%)
			P5	2.77E6/mL	98.61％

C. Preparation of antibody solutions

Samples were pre-diluted with experimental medium to 4-fold final concentration 0.8. mu.g/mL (final concentration 0.2. mu.g/mL) A1, and then diluted down 9 points, including zero, with a 5-fold gradient for a total of 10 points, with a maximum dilution of no more than 50-fold, depending on the amount of antibody labeled.

Watch 20

D. Sample application

Mu.l of diluted antibody was added to each well in the corresponding assay wells, each sample was double-plated, Raji cells were incubated with antibody in a cell incubator at 37 ℃ for 20-30 minutes, and 25. mu.l of assay medium was added to Ts, Tm, (T + E) s and Blank assay wells.

E. NK92MI-CD16a cell preparation and enumeration

NK92MI-CD16a cells in logarithmic growth phase were taken and centrifuged at 1000rpm for 5 minutes. After washing once with the test medium and centrifugation at 1000rpm for 5 minutes. Resuspending the cells in 5mL of experimental medium, and taking a proper amount of cell suspension to a 1.5mL EP tube; after mixing, 20 μ L of cell suspension is taken from the EP tube and mixed with the same amount of trypan blue (namely diluted 2 times), and counting is carried out; 3.1mL of the resuspended NK92MI-CD16a cells were mixed with 4.9mL of the experimental medium to obtain a dense mixture of NK92MI-CD16a cellsThe degree is adjusted to 1.5 multiplied by 10⁶Per mL; the diluted NK92MI-CD16a cells were added at 50. mu.L/well to 96-well U-well plate sample wells such that the ratio of effector cells to target cells was 6: 1.

TABLE 21

Generation of cell	Number of viable cells (. times.10)⁶one/mL)	Cell viability (%)
			P5	3.89E6 pieces/mL	95.15％

F. And (3) incubation: the cell culture plate was placed at 37 ℃ in 5% CO₂The incubation was carried out in an incubator for 2 h.

G. Preparation of Working solution: working solution was equilibrated to room temperature from 4 ℃ refrigerator half an hour in advance.

Lysis buffer (lysate): mu.l of lysis buffer was added to the Tm control well of the plate, mixed well, and then placed in the incubator again for 30 min.

Adding Working Solution and Stop Solution: add 100. mu.L of working Solution to each well, then stand 30min at room temperature in the dark for 50. mu.l of Stop Solution to each well and read immediately.

And (3) plate reading operation: OD490 values were read and reference OD630 was set for data analysis.

H. The killing rate was calculated using the formula cytoxicity% (Sample- (T + E) s)/(Tm-Ts) × 100%.

Tm:target cell maximum release

Ts:target cell spantaneous release

(T+E)s:target cell and effector cell spantaneous release

Es:effector cell spantaneous release

(3) Results and discussion

The results are shown in FIG. 1 and Table 22.

TABLE 22

Sample name	R²	EC50(ng/mL)
			huIgG1(NC)	-	-
Ritx-Fc(IgG4)	-	-
			Rituximab	0.9781	0.607

Discussion of the related Art

The Rituximab antibody shows stronger ADCC killing effect on Raji cells, while the Ritx-Fc (IgG4) sample has no or weak ADCC effect, which indicates that the Fc region of the human IgG4 enables the Ritx-Fc (IgG4) not to or only weakly induces the NK-92MI CD16a cells to kill the Raji cells.

5.2CDC

5.2.1 sample information is shown in Table 23.

TABLE 23

5.2.2 reagents, consumables, apparatus

Reagents and materials are shown in table 24.

Watch 24

Name of reagent Material	Manufacturer of the product	Goods number/model
			Raji	Cell bank of Chinese academy of sciences	TCHu 44
FBS	Gibco	10099-141C
			RPMI1640	Gibco	A10491-01
Cytotoxicity LDH Assay Kit-WST	DOJINDO	CK12
			Human Serum Complement	Quidel	A113

Consumables are shown in table 25.

TABLE 25 consumable List

The apparatus is shown in table 26.

Table 26 device list

5.2.3 Experimental methods

The experimental layout is shown in table 27.

Table 27 type table for experiment

5.2.4 Experimental procedures

A. The experimental culture medium is RPMI1640 basic culture medium

B. Raji cell preparation and enumeration

Raji cells in logarithmic growth phase were centrifuged at 1000rpm for 5 minutes. After washing 2 times with the test medium and centrifugation at 1000rpm for 5 minutes. Resuspending the cells in 5mL of experimental medium, and taking a proper amount of cell suspension to a 1.5mL EP tube; after mixing, 20 μ L of cell suspension is taken from the EP tube and mixed with the same amount of trypan blue (namely diluted 2 times), and counting is carried out; taking 2.5mL of resuspended Raji cells, and uniformly mixing the Raji cells with 5.5mL of experimental culture medium, thereby adjusting the density of the Raji cells to 5.0 multiplied by 10^5 cells/mL; the diluted Raji cells above were added at 50 μ L/well to 96 well U-well plate sample wells and Tm and Ts wells, and the remaining experimental wells (Blk) were added with 25 μ L of experimental medium.

TABLE 28 Raji cell information Table

Generation of cell	Number of viable cells (. times.10)⁶one/mL)	Cell viability (%)
			P6	1.58E6/mL	98.5％

C. Preparation of antibody solutions

Samples were pre-diluted with experimental medium to 4-fold final concentration of 20 μ g/mL (final concentration of 5 μ g/mL) to a1, and then diluted down to 8 points with a 4-fold gradient, including zero, for a total of 9 points, with a maximum dilution of no more than 50-fold, depending on the labeled amount of antibody.

Watch 29

D. Co-incubation of Raji cells with antibody: 25ul of antibody was added to the corresponding assay wells, Raji cells were incubated with antibody in a 37 ℃ cell incubator for 20-30 minutes, and 25. mu.l of assay medium was added to Ts, Tm and Blank assay wells.

Preparation of complement: complement was diluted to 10% (final concentration 2.5%) with assay medium and 25 μ l per well was added to the assay wells.

And (3) incubation: the cell culture plate was placed at 37 ℃ in 5% CO₂The incubation was carried out in an incubator for 2 h.

Preparation of the Working solution: the Working solution was equilibrated to room temperature from 4 ℃ refrigerator half an hour in advance.

Lysine buffer 10. mu.l of lysine buffer was added to the Tm control well of the plate, mixed well, and then placed in the incubator again for 30 min.

Adding the Working Solution and Stop Solution: add 100. mu.L of working Solution to each well, and then let stand for 30min at room temperature in the dark. Each well was immediately read with 50ul of stop solution.

E. And (3) plate reading operation: OD490 values were read and reference OD630 was set for data analysis.

F. The killing rate was calculated using the formula cytoxicity% (Sample-Ts)/(Tm-Ts) × 100.

Tm:target cell maximum release

Ts:target cell spantaneous release

5.2.5 results and discussion

The results are shown in FIG. 2 and Table 30.

Watch 30

Sample name	R²	EC50(ng/mL)
			huIgG1(NC)	-	-
Ritx-Fc(IgG4)	-	-
			Rituximab	0.978	210.2

Discussion of the related Art

The Rituximab antibody shows a strong CDC effect on Raji cells, while the Ritx-Fc (IgG4) sample has no or weak CDC effect, which indicates that the Fc region of Ritx-Fc (IgG4) cannot be combined with C1q molecules in complement and cannot start the complement cascade.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Shanghai Apu Mey Biotech Co., Ltd

<120> method for efficiently detecting fusion protein/antibody Fc segment incapable of mediating ADCC and CDC activities

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 213

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 2

<211> 448

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly

210 215 220

Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

260 265 270

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

Claims

1. A method for detecting positive antibodies and positive cells capable of mediating ADCC and CDC effects rapidly and directly verifying that the target protein or Fc fragment of the antibody cannot or only weakly mediate ADCC and CDC effects, the method comprising:

s2, plasmid extraction and sequencing;

s3, protein transient expression;

s4, purifying protein;

s5, ADCC and CDC activity assay.

2. The method according to claim 1, wherein the nucleotide sequence of the Rituximab light chain is as set forth in SEQ ID NO: 1, the nucleotide sequence of the Ritx-Fc (IgG4) heavy chain is shown as SEQ ID NO: 2 is shown in the specification;

3. the method according to claim 1, wherein the strain is inoculated into a medium containing ampicillin at S2, shake-cultured for 12-24 hours, centrifuged, and then cultured as follows

4. The method of claim 1, characterized by transient expression of Ritx-Fc (IgG4) antibody in HEK293F cells in S3.

5. The method of claim 1, wherein in S4, the protein purification steps sequentially comprise: rinsing, disinfecting, balancing, loading, leaching, eluting, regenerating, rinsing, disinfecting and rinsing;

6. The method according to claim 1, wherein ADCC and CDC activities are analyzed in S5 using Raji cells as target cells.

7. The method of claim 1, wherein the extracted plasmid is transfected into HEK293 cells and cultured in OPM-293-CD05 medium in S3.

8. The method of claim 7, wherein the HEK293 cell has a transfection density of (4-4.5) x 10⁶cells/ml。

9. Use of the method according to any one of claims 1-8 for the treatment of tumors, transplants and/or autoimmune diseases.