CN116286633A

CN116286633A - B cell expansion method

Info

Publication number: CN116286633A
Application number: CN202310440789.1A
Authority: CN
Inventors: 张凤禹; 高斌; 谷为岳; 晏涛; 臧航
Original assignee: Chineo Medical Technology Co ltd
Current assignee: Chineo Medical Technology Co ltd
Priority date: 2023-04-23
Filing date: 2023-04-23
Publication date: 2023-06-23

Abstract

The invention discloses a B cell expansion method. The invention discloses a method for amplifying B cells, which comprises the following steps: co-culturing the B cells and the feeder cells to realize the expansion of the B cells; feeder cells are recombinant animal cells expressing CD40L protein and COMP-SCD40L protein, or fusion proteins obtained by ligating CD40L protein and COMP-SCD40L protein. The invention simultaneously expresses CD40L multimeric protein on the basis of 293T-CD40L feeder cells, and the feeder cells and primary B cells are used for co-culture, so that the stable B cell expansion of more than 200 times can be obtained in two to three weeks under the existence of IL-4 and IL-10. The feeder cells and B cells amplification method has good application prospect.

Description

B cell expansion method

Technical Field

The invention relates to a method for expanding B cells in the biomedical field.

Background

B cells are important members of the immune system, mainly responsible for humoral immunity. After being stimulated by antigen, B cells can be converted into plasma cells, and anti-stimulated antigen antibodies are secreted, so that an important defense line for resisting invasion of external microorganisms is constructed. In addition, B cells, like dendritic cells and macrophages, serve as a member of professional antigen presenting cells, and can present specific antigens to primary T cells in the form of MHC complexes, which activate and proliferate them, playing an important role in modulating the immune system, killing invading pathogens and in vivo mutant cells. Thus, expansion of B cells in vitro is of great importance for antibody discovery, neoantigen vaccine development, and development of cell factories capable of secreting antibodies and therapeutic proteins.

The current method for expanding B cells in vitro mainly adopts the method for stimulating the CD40 of the B cell receptor to activate and differentiate the B cells, thereby obtaining rapid proliferation. Common methods are anti-CD 40 antibodies and soluble CD40L stimulation protocols, but these methods typically differentiate B cells into plasma cells rapidly, depleting the original B cells, and limiting the number of B cell proliferation.

To compensate for the limitations of the soluble ligand, B cells were expanded and CD40L was used to switch to NIH3T3 cells in the 90 s. T cells were expanded in vitro with this feeder cell. However, in view of the fact that the mouse-derived cells can cause heterologous tissue immune reaction in vivo, clinical research is difficult, attempts are made to replace mouse-derived cells with human 293 cells, express CD40L to stimulate B cells, proliferate and differentiate the B cells, and solve the problem that mouse-derived 3T3 cells are difficult to be used clinically, but the feeder cells reach a platform after a period of time for stimulating the B cells, and cause apoptosis of the B cells, so that proliferation of the B cells is reduced.

Tension in Zhongshan university and the like allows feeder cells to simultaneously express soluble BAFF protein for in vitro expansion of B cells based on membrane CD40L, but researches show that BAFF only prevents further apoptosis of B cells differentiated into plasma cells, but does not actually promote proliferation of B cells.

Disclosure of Invention

The technical problem to be solved by the invention is how to expand B cells.

To solve the above technical problems, the present invention provides a method for expanding B cells, the method comprising: co-culturing the B cells and the feeder cells to realize the expansion of the B cells;

the feeder cells are recombinant animal cells expressing CD40L protein and COMP-SCD40L protein or recombinant animal cells expressing fusion protein obtained by connecting CD40L protein and COMP-SCD40L protein.

In the above method, the CD40L protein may be A1), A2), or A3) as follows:

a1 1-261 of SEQ ID No. 2;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequences shown in the 1 st to 261 st positions of SEQ ID No.2 in the sequence table and has the same function;

a3 Fusion proteins obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2);

the COMP-SCD40L protein may be B1), B2) or B3) as follows:

b1 283-523 of SEQ ID No. 2;

b2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequences shown in 283-523 of SEQ ID No.2 in the sequence table and has the same function;

b3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of B1) or B2).

The protein of A2) has 75% or more identity with the amino acid sequence of the protein shown in positions 1-261 of SEQ ID No.2 and has the same function. The protein B2) is a protein having 75% or more identity and the same function as the amino acid sequence of the protein shown in positions 283 to 523 of SEQ ID No. 2. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

In the above method, the recombinant animal cell can be obtained by introducing a recombinant vector expressing CD40L protein and COMP-SCD40L protein into a recipient animal cell.

In the method, the recombinant vector contains the DNA fragment shown in SEQ ID No.1 and can express and express CD40L protein and COMP-SCD40L protein or express a fusion protein obtained by connecting CD40L protein and COMP-SCD40L protein.

The recombinant vector contains an expression cassette encoding the fusion protein. The expression cassette refers to a DNA capable of expressing the fusion protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the fusion protein, but also a terminator for terminating transcription of the gene encoding the fusion protein. Further, the expression cassette may also include an enhancer sequence.

Recombinant vectors containing the expression cassette can be constructed using existing expression vectors.

In the above method, the vector may be a plasmid, cosmid, phage, or viral vector (e.g., lentivirus).

The recombinant vector can be specifically obtained by packaging plasmid required by packaging the Planti-CD 40L-COMP-SCD40L and other lentiviruses.

The plasmid-CD 40L-COMP-SCD40L is a recombinant plasmid obtained by replacing a DNA fragment between XbaI and SalI recognition sequences of a plasmid for packaging the plasmid of the plasmid into a DNA shown in SEQ ID No. 1.

In the above method, the recipient animal cell may be a 293T cell.

In the above method, the culture system for co-culture may further comprise IL-10 and IL-4.

Further, the concentration of IL-10 in the culture system may be 5-40ng/mL (e.g., 10 ng/mL), and the concentration of IL-4 in the culture system may be 5-40ng/mL (e.g., 20 ng/mL).

In the above method, the B cells may be CD19 positive B cells.

In the above method, the ratio of the feeder cells to the B cells may be (1-4): 1-5, such as 1 (1-5), 2 (1-5), 4 (1-5), etc.

The recombinant animal cell, or the DNA fragment shown in SEQ ID No.1, or the expression cassette, the recombinant vector, the recombinant microorganism or the recombinant animal cell line containing the DNA fragment shown in SEQ ID No.1 also belong to the protection scope of the invention.

Wherein the recombinant animal cell line does not include propagation material.

The invention also provides any one of the following uses of the recombinant animal cell, the DNA fragment, the expression cassette, the recombinant vector, the recombinant microorganism or the recombinant animal cell line:

x1) expanding B cells;

x2) preparing a B cell expansion product.

The invention simultaneously expresses CD40L multimeric protein on the basis of 293T-CD40L feeder cells. The feeder cells are used for co-culturing with primary B cells, and stable B cell expansion of more than 200 times can be obtained in two to three weeks in the presence of IL-4 and IL-10. The feeder cells and B cells amplification method has good application prospect.

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

Drawings

FIG. 1 is an identification of the expression of CD40L membrane in 293T-CD40L-COMP-SCD40L cell line, A:293T cell negative control; b:293T-CD40L-COMP-SCD40L cell line experimental group.

FIG. 2 is an identification of soluble protein COMP-SCD40L expression in 293T-CD40L-COMP-SCD40L cell line, A: raji cell negative control; b:293T-CD40L-COMP-SCD40L cell line culture supernatant experimental group.

FIG. 3 shows the proportion of CD19+ B cells isolated from B cells by magnetic beads, A: negative control (B cells not stained with antibody); and B, APC anti-human CD19 anti-body Antibody staining group.

FIG. 4 shows the effect of different feeder cells and B cell culture densities on fold expansion of B cells.

FIG. 5 shows the effect of different feeder cell and B cell culture densities on the proportion of CD19+ cells during B cell expansion.

FIG. 6 shows the results of B cell proliferation stimulation by 293T-CD40L-COMP-SCD40L feeder cells. D: and (3) days.

FIG. 7 shows the proportion of CD19+ cells during B cell proliferation stimulated by 293T-CD40L-COMP-SCD40L cells. D: and (3) days.

Detailed Description

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The plasmid vector packaging (adedge), helper plasmid pMD2G (adedge, 12259), REV (adedge, 12253), gag-pol (adedge, 12251) plasmids of the following examples are publicly available from the applicant, and are used only for repeated experiments related to the present invention, but are not used for other purposes.

Wherein the PMD2G, REV, gag-pol plasmid can be replaced with a helper plasmid in a three-plasmid or four-plasmid lentiviral packaging system.

Example 1, 293T-CD40L-COMP-SCD40L cell lines can be used for in vitro expansion of B cells

1. Vector construction

The CD40L-COMP-SCD40L gene shown in SEQ ID No.1 was synthesized, the DNA fragment between the XbaI and SalI recognition sequences of the plasmid for packaging the Planti virus was replaced with the obtained CD40L-COMP-SCD40L gene, and the obtained recombinant plasmid was designated as Planti-CD 40L-COMP-SCD40L. Planti-CD 40L-COMP-SCD40L expresses the CD40L-COMP-SCD40L protein shown in SEQ ID No. 2.

The DNA sequence of SEQ ID No.1 is CD40L at positions 1-783, T2A at positions 793-846 and COMP-SCD40L at positions 847-1569.

The amino acid sequence of CD40L at positions 1-261, T2A at positions 265-282 and COMP-SCD40L at positions 283-523 of SEQ ID No. 2.

2. Construction of 293T-CD40L-COMP-SCD40L cell line

1. 293T cells in T175 flasks were cultured overnight to 70% -80% density for transfection; mixing pMD2G, REV, gag-pol and the Plenti-CD40L-COMP-SCD40L prepared in the first step according to the mole ratio of 1:1:1:1 of each plasmid to obtain 70 mug of cotransfection plasmid;

2. respectively marking a tube 1 and a tube 2 by two 15mL centrifuge tubes, adding the cotransfected plasmid of the tube 1, supplementing a serum-free DMEM culture medium to 1mL, adding 140 mu l of a 1mg/mL PEI aqueous solution (PEI is a sigma product, the commodity number is GF 70215825) into the tube 2, supplementing the serum-free DMEM culture medium to 1mL, fully and uniformly mixing the materials respectively, dropwise adding the liquid in the tube 2 into the tube 1 to obtain a plasmid-PEI mixed solution, and standing the plasmid-PEI mixed solution at room temperature for 30min; the transfected cells were placed in 293T for transfection at 37℃with 5% CO ₂ Incubate overnight in incubator. The cell culture broth was then discarded and 35ml of DMEM medium containing 10% FBS was added again at 37℃with 5% CO ₂ Incubator culture overnight for 48 hours.

3. Collecting the cell culture supernatant obtained in the step 2, centrifuging for 10min at 3000g/min, filtering the supernatant by a 0.45 mu m filter to obtain a virus stock solution, centrifuging for 120 min at 100 g/min, and adding 1mL of DMEM medium containing 10% FBS into the supernatant to resuspend virus precipitate to obtain a virus suspension.

4. Taking 1×10 ⁵ About 24 hours, then 200. Mu.L of the concentrated virus suspension obtained in step 3, 37℃and 5% CO ₂ Incubator overnight culture then change 10% FBS DMEM medium after about 1d culture, get 293T-CD40L-COMP-SCD40L cells.

5. The 293T-CD40L-COMP-SCD40L cells obtained in step 4 were digested, and monoclonal selected, and 293T-CD40L-COMP-SCD40L cell lines stably expressing membrane proteins CD40L and COMP-SCD40L were selected.

6. Identification of 293T-CD40L-COMP-SCD40L cell line Membrane protein CD40L and soluble protein COMP-SCD40L expression

1) Identification of 293T-CD40L-COMP-SCD40L cell line CD40L Membrane expression:

negative control: taking 5×10 ⁵ 2. Mu.L of FITC anti-human CD154Antibody (biolegend) antibody was added to 293T cells of (B), incubated at room temperature for 30min, washed twice with 1mL PBS, and subjected to flow analysis;

experimental group: taking 5×10 ⁵ 2. Mu.L of FITC anti-human CD154Antibody (biolegend) antibody was added to 293T-CD40L-COMP-SCD40L cells, incubated at room temperature for 30min, and washed twice with 1mL PBS for flow analysis.

As a result, as shown in FIG. 1, it can be seen that the CD40L membrane protein was stably expressed on the surface of 293T-CD40L-COMP-SCD 40L;

2) Identification of soluble protein COMP-SCD40L expression in 293T-CD40L-COMP-SCD40L cell line.

The expression of the soluble protein COMP-SCD40L was detected using a CD40 positive cell raji cell line (B cell line, surface expressed CD40 receptor protein binding to CD40L protein).

Negative control: about 5X 10 ⁵ 1. Mu.L of Strep-like cells was added to the raji cells of (E)

DY-649Conjugatepdf (IBA Life Sciences, 2-1568-050) antibody, incubated at room temperature for 30min, washed twice with 1mL PBS, and subjected to flow analysis;

experimental group: about 5X 10 ⁵ 200. Mu.L of 293T-CD40L-COMP-SCD40L culture supernatant was added, incubated at room temperature for 30min, and 1mL of PBS was washed once; then 1. Mu.L of Strep-

DY-649Conjugatepdf (IBA Life Sciences, 2-1568-050) antibody was incubated at room temperature for 30min, washed twice with 1mL PBS, and subjected to flow analysis.

As a result, as shown in FIG. 2, it can be seen that the 293T-CD40L-COMP-SCD40L cell line can exogenously express the soluble protein COMP-SCD40L.

3. Isolation of B cells Using magnetic negative beads

1. 500g of monoculture human blood cells (informed consent from the collection subject) were centrifuged for 10min, the supernatant was discarded, 10 volumes of ACK lysate (Themo, A1049201; cryopreservation resuscitation does not require lysis of erythrocytes) was added, 5min incubation at 37℃was performed, 500g was centrifuged for 10min, the supernatant was discarded, 30mL of sorting buffer (PBS containing 2% FBS) was added to wash the cells, 500g was centrifuged for 5min, and the cells were collected.

2. Counting, and adjusting cell density to 1×10 with sorting buffer ⁸ Each cell/mL to give a cell suspension, and adding the cell suspension to a 15mL centrifuge tube using EasySep ^TM Human B CellIsolation Kit (STEMCELL, # 17954) B cells were isolated; the operation steps are as follows:

a) Adding Cocktail Enhancer to the cell suspension, adding 50 mu l Cocktail Enhancer per milliliter of the cell suspension, uniformly mixing, then adding Isolation Cocktail, adding 50 mu l Isolation Cocktail per milliliter of the cell suspension, and incubating for 5 mm at room temperature to obtain a cell mixed solution;

b) Vortex mixing RapidSpheres ^TM Magnetic beads, adding RapidSpheres to the cell suspension ^TM Magnetic beads, 50. Mu.l of RapidSpheres per ml of cell suspension ^TM Magnetic beads incubated at room temperature for 3 mm;

c) After the step b) is finished, less than 4mL of sample is supplemented and sorted into buffer to 5mL, and more than 4mL of sample is supplemented and sorted into buffer to 10mL, and the buffer is placed into a centrifuge tube;

d) Placing the centrifuge tube on a stemcell magnetic rack, and adsorbing for 5min at room temperature;

e) Transferring the cell suspension to a new 15mL centrifuge tube again, and adsorbing the cell suspension on a magnetic rack for 5min at room temperature;

f) After the step e) is completed, the cell suspension is transferred to a new 15mL centrifuge tube again, namely the B cell suspension;

3. selecting 5×10B cells ⁵ The antibody of APC anti-human CD19 Antibody (biolegend) was labeled and flow-through detection was performed using cells not stained with the antibody as a negative control. The results are shown in FIG. 3.

The results show that successful sorting yields B cells (cd19+).

4. B cell expansion

Effect of feeder cells and B cell culture Density on B cell expansion

1. Preparing feeder cells:293T-CD40L-COMP-SCD40L cells were 1X 10, respectively ⁵ Per well, 2×10 ⁵ Individual/well, 4×10 ⁵ The cells were stopped by plating the cells/wells into 12-well plates, culturing them in DMEM medium containing 10% fetal bovine serum for 5-7 hours, and then adding 4mM mitomycin for 16-20 hours. The cells were observed for 12-24h and stopped.

2. B cells were then plated into 12-well plates and co-cultured with feeder cells: b cells obtained in the third step were cultured according to a ratio of 0.5X10 ⁵ Per ml, 1X 10 ⁵ Per ml, 2.5X10) ⁵ Each of the B cell suspensions was prepared by suspending 2mL of B cells in RPMI 1640 medium containing 10% fetal bovine serum, transferring the B cell suspensions into 12-well plates plated with new feeder cells according to the conditions shown in the following table, and adding IL-10 and IL-4 thereto to give the B cell suspensions having IL-10 and IL-4 concentrations of 10ng/mL and 20ng/mL, respectively. The culture medium in the feeder cells of step 1 was aspirated, and then the B cell suspension was added to the 12-well plate of step 1 at a volume of 2ml per well to co-culture with the feeder cells at 37℃under 5% CO ₂ 。

TABLE 1,

3. Every 3-5 days, B cells were uniformly blown and counted, B cells were transferred into 12-well plates plated with new feeder cells according to the conditions shown in the above table, and the proliferation results of the cells were examined on days 6, 10, and 13 of culture (FIG. 4), and the density of 293T-CD40L-COMP-SCD40L cells in the 12-well plates was localized to 1-4X 10 ⁵ The density of B cells per well is 1-5×10 ⁵ B cells can obtain better amplification effect under the condition of each cell/hole.

4. About 5X 10 was taken at each passage ⁵ The individual cells were incubated with 2. Mu.L of APC anti-human CD19 Antibody (biolegend) for 30min at room temperature, washed twice with 1mL of PBS, and subjected to flow analysis.

As a result, as shown in FIG. 5, it can be seen that the cells proliferatedDensity localization of 293T-CD40L-COMP-SCD40L cells in Process 1-4X 10 ⁵ The density of B cells per well is 1-5×10 ⁵ The CD19 positive B cells were greater than 90% on day 10 of culture at each/well.

(II) B cell expansion experiment

1. Preparing feeder cells: 293T-CD40L-COMP-SCD40L cell line was plated into 12-well plates. When the cell density is about 50%, 4mg/L mitomycin C is treated for 16-20 hours to stop the growth of the cells. The cells were observed for 12-24h and stopped.

2. Sorting B cells by the method of the third step, paving the B cells into a 12-hole plate and culturing the feeder cells together: mapping the density of 293T-CD40L-COMP-SCD40L cells to 4X 10 ⁵ And/or holes. B cells were prepared according to 2X 10 ⁵ Each of the cells was suspended in a medium containing 10% fetal bovine serum RPMI 1640, and IL-10 and IL-4 were added thereto to obtain a B cell suspension having IL-10 and IL-4 concentrations of 10ng/mL and 20ng/mL, respectively. The culture medium in the feeder cells was aspirated, and the B cell suspension was then added to the 12-well plate of step 1 at a rate of 2ml per well to co-culture with the feeder cells at 37℃under 5% CO ₂ 。

3. B cells were evenly counted every 4-5 days, according to 4 x 10 ⁵ The density of cells/well was transferred to a 12-well plate with new feeder cells, and the proliferation results of the cells are shown in FIG. 6, and the proliferation of B cells was 50-200-fold in 15 days and 200-2000-fold in about 22 days.

The results are shown in FIG. 7, and it can be seen that the percentage of CD19 positive B cells was greater than 90% during cell proliferation.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

The sequences involved in the above examples are as follows:

SEQ ID NO.1 (amino acid sequence of CD40L-T2A-COMP-SCD 40L):

SEQ ID NO.2 (amino acid sequence of CD40L-T2A-COMP-SCD 40L):

Claims

1. a method of expanding B cells comprising: co-culturing the B cells and the feeder cells to realize the expansion of the B cells;

the feeder cells are recombinant animal cells expressing both CD40L protein and soluble COMP-SCD40L protein.

2. The method according to claim 1, characterized in that: the CD40L protein is A1), A2) or A3) as follows:

a1 1-261 of SEQ ID No. 2;

the COMP-SCD40L protein is B1), B2) or B3) as follows:

b1 283-523 of SEQ ID No. 2;

3. The method according to claim 1 or 2, characterized in that: the recombinant animal cells are obtained by introducing a recombinant vector expressing CD40L protein and COMP-SCD40L protein into recipient animal cells.

4. A method according to claim 3, characterized in that: the recombinant vector contains a DNA fragment shown in SEQ ID No. 1.

5. The method according to any one of claims 1-4, wherein: the recipient animal cells are 293T cells.

6. The method according to any one of claims 1-5, wherein: the co-cultured culture system also contains IL-10 and IL-4;

further, the concentration of IL-10 in the culture system is 5-40ng/mL, and the concentration of IL-4 in the culture system is 5-40ng/mL.

7. The method according to any one of claims 1-6, wherein: the B cells are CD19 positive B cells.

8. The recombinant animal cell of any one of claims 1 to 5, or the DNA fragment shown in SEQ ID No.1, or an expression cassette, recombinant vector, recombinant microorganism or recombinant animal cell line comprising the DNA fragment shown in SEQ ID No. 1.

9. The recombinant animal cell, the DNA fragment, the expression cassette, the recombinant vector, the recombinant microorganism, or the recombinant animal cell line of claim 8, for use in any of the following:

x1) expanding B cells;

x2) preparing a B cell expansion product.