CN111411079B

CN111411079B - Cell culture methods, modified antibodies, cell culture media, cell products and their applications

Info

Publication number: CN111411079B
Application number: CN202010230370.XA
Authority: CN
Inventors: 程源
Original assignee: Beijing Shihe Biotechnology Co ltd
Current assignee: Beijing Shihe Biotechnology Co ltd
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2025-12-12
Anticipated expiration: 2040-03-27
Also published as: CN111411079A

Abstract

This invention relates to a cell culture method, a modified antibody, a cell culture medium, cell products, and their applications. The cell culture method includes the following steps: adding a modified antibody to a culture system; the modified antibody is a cell culture antibody for which regions other than the complementarity-determining region have been protein-modified, and the affinity of the modified antibody for the Fc receptor is lower than that of an unmodified cell culture antibody for the Fc receptor. The cell culture method and cell culture medium of this invention can be used for the in vitro culture of various cell types to obtain target cells with higher purity and higher viability, and the obtained cell products can be used for clinical application research.

Description

Cell culture method, engineered antibody, cell culture medium, cell product and application thereof

Technical Field

The invention relates to the technical fields of molecular biology and cell biology, in particular to a cell culture method, an altered antibody, a cell culture medium, a cell product and application thereof.

Background

Currently, the commonly used in vitro cell culture method uses a large amount of cell culture antibodies to bind to specific receptor molecules on the surface of target cells, so as to achieve the purpose of activating (activating) or inhibiting (blocking) signal pathways, and promote the directional differentiation, continuous expansion or apoptosis of the target cells. In small scale cultures, these antibodies are typically coated on the surface of a culture dish or cross-linked to magnetic beads. In large scale culture, these antibodies are often added directly to the culture medium in order to avoid lengthy and difficult coating processes, or to save high bead costs, to avoid the introduction of foreign substances, or for other optimization purposes. However, the end products thus obtained generally have the problems of lower purity and lower viability.

The low purity of the target cells means that there are too many other types of cells in the final product. The function of these non-target cells is different from that of the target cells, and the components are generally difficult to control, so that the complexity of scientific tests, particularly in vivo tests of animals and human bodies, is greatly increased, and the repeatability of the research is seriously affected.

Also, for safety and effectiveness reasons in clinical therapeutic applications, it is necessary to obtain cell preparations of as high purity and high viability as possible.

Disclosure of Invention

Based on this, it is necessary to provide a cell culture method that can improve the purity and viability of cells.

The invention discloses a cell culture method, which comprises the following steps of adding an engineered antibody into a culture system, wherein the engineered antibody is a cell culture antibody with the region except for a complementarity determining region subjected to protein engineering, and the affinity of the engineered antibody to an Fc receptor is lower than that of the cell culture antibody without the protein engineering.

The present invention also discloses an engineered antibody for cell culture in which a region other than the complementarity determining region is subjected to protein engineering, wherein the affinity of the engineered antibody for Fc receptor is lower than that of the cell culture antibody not subjected to protein engineering.

The invention also discloses a cell culture medium comprising a basal medium and the engineered antibody.

The invention also discloses a cell product, which is obtained according to the cell culture method.

The invention also discloses application of the cell product in preparing medicines and reagents with killing effect on tumor cells.

The invention also discloses a medicine for treating cancers, which comprises the cell product.

The invention also discloses a medicine for treating allogeneic cells, which comprises the cell product.

Based on the technical scheme, the invention has the following beneficial effects:

The invention is suitable for the established cell culture process, has no change to the original mechanism of the antibody for cell culture, and does not need to greatly change the culture process. Is particularly suitable for the condition that the antibody for cell culture is directly added into a culture medium in the widely used large-scale cell culture process at present, and has simple and convenient operation.

The invention can improve the stability of the antibody for cell culture in a cell culture system, and simultaneously improve the utilization rate of the antibody, and can reduce the use amount of the raw material antibody by optimizing the culture process.

The invention avoids ADCP/ADCC action on target cells by immune cells, especially macrophages, monocytes, NK cells and the like in initial raw material cells in primary cell culture, and improves the activity rate and final yield of the target cells. Particularly, when the cultured and expanded target cells are immune cells such as macrophages, monocytes, NK cells and the like, the purity and the activity of the target cells in the final product are obviously improved.

The invention can provide a target cell product with higher purity and higher activity, expands the application range of the cell product in scientific research, and improves the safety and the effectiveness of the cell product in clinical application.

Drawings

FIG. 1 is a schematic representation of the engineering of the heavy chain of the murine IgG1 monoclonal antibody 3G8 against human CD16 of example 1;

FIG. 2 is a graph showing the ultraviolet absorption of the washing and elution steps of Protein A column chromatography in modification example 1;

FIG. 3 is a diagram showing the detection of reduced SDS-PAGE of the target protein in modified example 1;

FIG. 4 is a schematic representation of the engineering of a humanized IgG1 type Campath-1H monoclonal antibody heavy chain against human CD52 of engineering example 2;

FIG. 5 is a diagram showing the detection of reduced SDS-PAGE of the target protein in modified example 2;

FIG. 6 is a schematic diagram of the engineering of the mouse IgG2a monoclonal antibody OKT3 against human CD3 of engineering example 3;

FIG. 7 is a diagram showing the detection of reduced SDS-PAGE of the target protein in modified example 3;

FIG. 8 is a graph showing the fit of the activity of the pro-antibody OKT3 in culture example 1 to the expansion of T cells by the aCD3-SH prepared in modification example 3;

FIG. 9 is a graph showing the amplification of NKT cells by the pro-antibody OKT3 in culture example 2 and aCD3-SH prepared in modification example 3;

FIG. 10 is a flow cytometry analysis of cells obtained by amplification of the engineered antibody set of culture example 3 versus cells obtained by amplification of the prototype antibody set;

FIG. 11 is a graph showing the results of killing Raji cells by the NK cell products of the test group and the NK cell products of the control group in application example 1;

FIG. 12 is a graph showing the results of killing test of BT-474 cells by using the NK cell products of the test group and the NK cell products of the control group in example 1;

FIG. 13 is a graph showing the results of killing MCF7 cells by the NK cell products of the test group and the NK cell products of the control group in application example 1;

FIG. 14 is a graph of tumor imaging signal intensity in two groups of mice of application example 2;

fig. 15 is a graph showing survival of each group of mice in application example 3;

Fig. 16 is a tumor imaging diagram of each group of mice in application example 3.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention, and preferred embodiments of the present invention are set forth. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The terms involved in the present invention are explained as follows:

Antibody for cell culture refers broadly to an antibody used in a cell culture process.

Prototype antibody refers to a commercially available antibody for cell culture, which has not been modified according to the present invention.

Engineered antibodies, particularly antibodies of the invention that have been protein engineered with a prototype antibody, have a lower affinity for Fc receptors than the prototype antibody.

The culture system generally includes a collection of a medium, a growth factor, an antibody for cell culture, cells (target cells and non-target cells), serum (with or without), antibiotics (with or without), and other additional components, but is not limited thereto, and the composition of the culture system may be different depending on the needs.

Antibody receptor, antibody clearance and antibody dependent killing on cell surface

Some cells, such as lymphocytes, DC cells, macrophages, granulocytes, platelets, mast cells, etc., have their surfaces expressing various Receptor Fc receptors (FcR) capable of binding to the Fc portion of different immunoglobulin isotypes (Isotype), including fcα R, fc γ R, fc epsilon R, etc. Among them, fcγr is classified into fcγri (CD 64), fcγrii (CD 32) and fcγriii (CD 16), and binds mainly to IgG. Fcγr has different affinities for human and murine IgG of different subtypes. In general, the affinity for IgG1, igG3 is higher than for IgG2, igG 4.

During cell culture, the concentration of the antibody for cell culture added to the medium is continuously decreased. This is directly related to the ability of some cell surface expressed Fc receptors in the culture system to bind and scavenge antibodies (FCR DEPENDENT endocytosis), in addition to factors such as their constant inactivation (natural degradation, proteolytic degradation by cells, polymeric precipitation, etc.), endocytosis after binding to the target receptor (receptor-mediated endocytosis).

The antibody used for the culture is usually an IgG1 subtype derived from mouse hybridoma selection, e.g.UCHT 1, 3G8, and CD-28, which are antibodies to human CD3, CD16, and CD-28, respectively. In addition, murine antibodies are often humanized to select the IgG1 subtype, e.g., humanized Campath-1H mab derived from rat anti-human CD52, for increased yield and expanded product applications. The problem of rapid decrease in the effective concentration of these IgG1 antibodies in the culture system is further aggravated.

Even after binding to the target cells, antibodies may also trigger the attack of certain effector cells expressing Fc receptors. This includes antibody-dependent cell-mediated phagocytosis by fcyriii expressing macrophages (Antibody Dependent Cellular Phagocytosis, ADCP) and antibody-dependent cell killing by fcyriii expressing NK cells (Antibody Dependent Cellular Cytotoxicity, ADCC). When immune cells such as macrophages, monocytes, NK cells and the like exist in the culture system, the specific ADCP/ADCC effect can rapidly reduce the activity rate and purity of target cells. In particular, if the cultured target cells are immune cells such as macrophages, monocytes, NK cells, etc., such self ADCP/ADCC action rapidly decreases the viability, purity and vigor of the target cells and allows the target cells to advance into the depletion phase.

Principle and method of antibody engineering

The binding sites for fcγr on IgG are concentrated in the Hinge region (Hinge) and Fc-C _H 2 domain, and engineering of antibodies involves this region. The means of engineering include one or more of sequence substitutions, point mutations, sequence insertions, sequence deletions, glycosylation modifications, and chemical modifications.

For example, the hinge region and the Fc fragment of the antibody for cell culture are replaced with those of an antibody subtype having relatively weak binding to Fc receptor, or amino acid residues at key binding sites on the hinge region and the Fc fragment of the antibody for cell culture are mutated, or the complementarity determining region (Complementarity-DETERMINING REGIONS, CDR) of the antibody for cell culture is inserted into the framework of an antibody of another subtype having relatively weak binding to Fc receptor, or the like. It will be appreciated that the various engineering means described above may be used in combination, for example, by inserting CDRs of a cell culture antibody into the backbones of other subtype antibodies subjected to point mutation.

Since the invention is applied to in vitro culture of cells, the selection criteria for engineered antibodies are different from those used for other purposes (e.g., therapeutic antibodies for human input). The invention thus proposes a better retrofitting strategy.

Sequence substitution

In some embodiments, the above sequence substitutions are those from the hinge and Fc segments of subtype IgG1, igG3 antibodies to IgG2 or IgG4 corresponding sequences. In a preferred embodiment, these sequences are replaced by the corresponding sequences of IgG 4. For example, the hinge region and Fc segment of antibodies of the IgG1 subtype anti-human CD3 mouse monoclonal antibody UCHT1, anti-human CD16 mouse monoclonal antibody 3G8 and MEM-154, anti-human CD28 mouse monoclonal antibody CD-28, anti-human CD137 mouse monoclonal antibody 4C1A9, etc. are replaced with the corresponding sequences of mouse IgG 4. In a more preferred embodiment, these sequences are replaced with the corresponding sequences of human IgG 4. Preferably, upon replacement of the Hinge region and Fc segment, the replacement is initiated by selecting a segment of the IgG1-C _H 1-range region as close to the C-terminus as possible to the same sequence as in the IgG4-C _H 1-range to maintain interaction of C _H 1 and V _H 1 as much as possible, maintaining the function of the Fab segment.

Point mutation

In some embodiments, the point mutations described above are mutations of amino acids at one or more positions of the hinge region and the critical binding site of the Fc fragment of the cell culture antibody.

In some embodiments, the cell culture antibody is of the IgG1 subtype, and the point mutation is a mutation of one or more of amino acids 228, 233, 234, 235, 236, 239, 250, 252, 254, 256, 257, 311, 318, 320, 322, 326, 327, 329, 330, 331, 332, 333, 428, 433 and 434 of the cell culture antibody. In some embodiments, adjacent amino acids to the above-described sites may also be mutated.

Of these sites, both the main and side chain groups of L234 and L235 of hIgG1 are involved in binding to hfcyriii, particularly the hydrophobic interactions of the side chain of L235 with fcyriii. In some preferred embodiments, mutation of L235 to other amino acids, particularly charged amino acids (Glu, asp, arg, lys) or sterically hindered large group side chain amino acids (Phe, etc.), can attenuate binding of the antibody to Fc receptors. In some preferred embodiments, mutation of L234 to other amino acids, particularly amino acids that affect backbone conformation (glycine Gly, proline Pro), can attenuate binding of the antibody to Fc receptors. P329 of hIgG1 is involved in binding to hfcyriii, in particular hydrophobic interactions with fcyriii. In some preferred embodiments, mutation of P329 to other amino acids, particularly charged amino acids or uncharged polar amino acids (serine Ser, threonine Thr, etc.), can attenuate binding of antibodies to Fc receptors. In a more preferred embodiment, the hIgG1 is subjected to L234A, L235R, P D and A330S mutations.

Sequence insertion

In some embodiments, the sequence insertion is insertion of CDRs from subtype IgG1, igG3 antibodies onto the framework of an IgG2 antibody or an IgG4 antibody. In a preferred embodiment, these CDRs are inserted into the framework of an IgG4 antibody. In a preferred embodiment, these CDRs are inserted into the framework of an antibody that has undergone other such engineering, with reduced affinity for Fc receptors. In a more preferred embodiment, these CDRs are inserted into the framework of an IgG1 antibody that has been engineered with the point mutation.

Other methods

In some embodiments, the goal of reducing the affinity of an antibody to an Fc receptor may also be achieved by other sequence deletions, glycosylation modifications, chemical modifications, and the like.

In a preferred embodiment, the antibody expression sequence is engineered to express and purify Fab fragments of the antibody.

In a preferred embodiment, a heavy chain aglycosylation modified antibody (Non-Glycosylated HEAVY CHAIN, NGHC) can be obtained by mutating N297 in the antibody expression sequence.

In a preferred embodiment, antibodies with a glycosyl deletion or glycosyl rearrangement can be obtained by treatment of the antibody with an endoglycosidase (Endoglycosidase).

In a preferred embodiment, antibodies with side chain modifications or conjugated sterically hindered groups can be obtained by treatment of the antibody with chemical coupling (Conjugation).

It is to be understood that the method of engineering an antibody according to the present invention is not limited to the above sequence substitution, point mutation, sequence insertion, sequence deletion, glycosylation modification and chemical modification, and any method may be used as long as the affinity with the Fc receptor is reduced by protein engineering and the binding force with the target molecule is not affected.

Antibody engineering scope

In some embodiments, the cell culture antibody is an anti-CD 3 antibody, an anti-CD 16 antibody, an anti-CD 28 antibody, an anti-CD 52 antibody, or an anti-CD 137 antibody. For example, the murine anti-human CD3 antibody UCHT1, the murine anti-human CD16 antibody 3G8, the murine anti-human CD28 antibody CD-28, the murine anti-human CD52 humanized Campath-1H mab and the anti-human CD137 mouse mab 4C1A9, and the like.

In some embodiments, the heavy chain amino acid sequence of the engineered antibody is shown as SEQ ID NO.1, or the heavy chain amino acid sequence of the engineered antibody is shown as SEQ ID NO.2, or the heavy chain amino acid sequence of the engineered antibody is shown as SEQ ID NO.3, and the light chain amino acid sequence is shown as SEQ ID NO. 4.

It is to be understood that the engineered antibody of the present invention is not limited to the above, and any antibody may be used as long as it is a cell culture antibody having reduced affinity for Fc receptor through protein engineering.

Cell culture

The cell culture method according to one embodiment of the present invention comprises the step of adding an engineered antibody having a lower affinity for an Fc receptor than an unmodified cell culture antibody to a cell culture antibody having a modified protein in a region other than the complementarity determining region to the cell culture system.

In some embodiments, the cell type is one or more of lymphocytes, granulocytes, mast cells, DC cells, macrophages, monocytes, and platelets. The cells are derived from human blood, tissues, human solid tumors subjected to surgical excision and ascites. Either freshly collected venous blood, umbilical cord blood or cryopreserved PBMC cells. It will be appreciated that the cell type is not limited thereto, as long as there is a portion of the cells in the culture system that express Fc receptors on their surface.

In some embodiments, the cytobiological assay is designed to quantitatively detect functional viability of the engineered antibody against a target cell in a particular starting material cell population, including cell expansion assays, cytotoxicity assays, cell killing assays, cell migration assays, and the like. In a specific example, engineered antibodies are used to stimulate proliferation of T cells (cd3+) in human PBMCs and the viability of the antibodies is quantitatively determined, i.e., half the effective dose (ED ₅₀). This viability data can be used in other cell culture methods that require stimulation of T cells (cd3+) in human PBMCs. In some embodiments, higher-activity engineered antibodies may also be obtained by screening.

Culture medium

In some embodiments, the cell culture medium comprises a basal medium and the engineered antibody described above. In some preferred embodiments, after quantitative determination of the viability of the engineered antibody, a standardized medium or kit may be formulated to meet the needs of a particular cell culture.

Cell products

In some embodiments, the cell product is a cell product obtained according to the cell culture methods described above. This includes one or more of lymphocytes, granulocytes, mast cells, DC cells, macrophages, monocytes and platelets.

In some embodiments, immune cell products such as T cells, NKT cells, NK cells, CTL cells, TIL cells, and the like can be obtained. In some embodiments, cell transduction and culture may be continued to obtain cell products such as CAR-T cells, CAR-NK cells, and the like.

Cell product application

In some embodiments, the cell products described above may be used in vitro cell killing assays, in vivo animal killing assays, in vivo human assays.

In some embodiments, the cell products described above, including immune cell products such as DC cells, T cells, NKT cells, NK cells, CTL cells, TIL cells, CAR-T cells, CAR-NK cells, etc., can identify and kill viruses that invade humans, host cells transformed by virus infection, tumor cells, etc., and can be used for antiviral therapy and targeted tumor therapy. Since the foreign immune cells accumulate first in the lung and then in the liver after intravenous administration to a patient, in some preferred embodiments, the immune cell products described above are used for the treatment of lung cancer and liver cancer.

NK cell surface rich in FcgammaRIII (CD 16) is the main effector cell for ADCC. In some embodiments, NK cells may be used in combination with therapeutic antibodies for ADCC killing studies. In some preferred embodiments, NK cell products may be used in combination with the therapeutic antibody Rituximab for the treatment of lymphomas. In some preferred embodiments, NK cell products can be used in combination with the therapeutic antibody Trastuzumab for the treatment of breast and gastric cancer. In some preferred embodiments, NK cell products can be used in combination with the therapeutic antibody Cetuximab for the treatment of head and neck cancer and colorectal cancer.

NK cells can also recognize and kill targets by non-self or self-failure response (missing-self). This cell killing is Non-MHC restricted (Non-MHC-RESTRICTED CYTOTOXICITY) and does not cause Graft Versus Host Disease (GVHD), and therefore NK cells can be used in allograft therapy. In some more preferred embodiments, the high purity NK cell products and CAR-NK cell products described above are used for human allogeneic cell therapy, but it will be appreciated that the cell products used for allogeneic cell therapy are not so limited.

The present invention will be described in further detail by way of specific examples, but embodiments of the present invention are not limited thereto.

1. Antibody engineering

Modification example 1

Engineering of anti-human CD16 cell culture antibodies

In this example, the V _H-C_H 1 fragment sequence of the mouse IgG1 monoclonal antibody 3G8 expressing anti-human CD16 was spliced with the C _H2-C_H fragment sequence of the human IgG4, and then expressed and purified together with the light chain sequence of the antibody 3G8 to give the engineered antibody aCD16-SH.

The sequence of the same fragment (T ₂₁₄KVDK₂₁₈) of mouse IgG1-C _H 1 as that of human IgG4-C _H 1 was selected for antibody sequence splicing, i.e., the sequence before T ₂₁₄ was the sequence of mouse IgG1-3G8 and the sequence after K ₂₁₈ was the sequence of human IgG 4. The signal peptide sequence of human CD33 was also used.

First, as shown in FIG. 1, the murine 3G8 antibody heavy chain expression DNA sequence was amplified with Primer pair Primer1-1F/Primer1-1R to obtain its V _H-C_H 1 fragment sequence (FIG. 1A). Then, the human IgG4 heavy chain expression DNA sequence was amplified using Primer pair Primer1-2F/Primer1-2R to obtain its finger-C _H2-C_H fragment sequence (FIG. 1B). The 2 sequences were then spliced by overlap PCR (Overlap-PCR) (FIG. 1C) by mixing the purified 2 PCR products equimolar, performing 5 PCR cycles at 60℃annealing temperature, adding Primer1-3F and Primer1-2R, and performing 30 PCR cycles at 72℃annealing temperature. Primer sequences are shown in the following table.

Primer1-1F	ATCCTTTTTCTAGTAGCAACTGCAACCGGTGTACATTCTcaggttactctgaaagagtctg
		Primer1-1R	CTCAACTCTcttgtccaccttggtgctgctggc
Primer1-2F	gccagcagcaccaaggtggacaagAGAGTTGAG
		Primer1-2R	GGGCTTGCCGGCCGTCGCACtcatttacccagagacaggga
Primer1-3F	CTATCGATTGAATTCCACCATGGGATGGTCATGTATCATCCTTTTTCTAGTAGCAACT

The PCR product obtained by purification was subjected to treatment with restriction enzymes EcoRI (GAATTC) and NaeI (GCCGGC), and then inserted into an expression plasmid (pMD 18-T-based, comprising expression elements such as CMV promoter and polyA Tail) to obtain a heavy chain-expressing plasmid pM-3G8-HC-M1 (FIG. 1D). Sequencing confirmed that the pM-3G8-HC-M1 sequence was correct.

Plasmid pM-3G8-LC for expression of the 3G8 light chain was equimolar mixed with plasmid pM-3G8-HC-M1 for expression of the engineered 3G8 heavy chain described above, and 293F cells in the logarithmic phase were co-transfected with linearized polyethylenimine (Polyethylenimine Linear, PEI). After 5 days of culture at 37℃with 5% CO ₂, the medium was collected by centrifugation. The ultraviolet absorption curve of the washing and elution steps for purifying the target antibody Protein by Protein A affinity chromatography is shown in FIG. 2. The samples were subjected to an electrophoretic examination, and the results are shown in FIG. 3, wherein M is a molecular weight standard (MW Ladder), lane 1 and 2 are samples of the column wash section, and Lane 3 and 4 are samples of the elution peak. Then the high-purity antibody product is obtained through ion exchange chromatography and desalting column chromatography, which is named as aCD16- (3G 8-Fab) - (hIgG 4-Fc), abbreviated as aCD16-SH, and the heavy chain sequence is shown as SEQ ID NO.1.

Modification example 2

Engineering of anti-human CD52 cell culture antibodies

In this example, the sequence of humanized IgG1 type Campath-1H monoclonal antibody expressing anti-human CD52 was mutated, then expressed and purified to obtain the engineered antibody aCD52-SH.

As shown in FIG. 4, 3 fragments were obtained by PCR using the sequences expressing the Campath-1H monoclonal antibody heavy chain (FIG. 4A) as templates and Primer pairs Primer2-3F/Primer2-ARR, primer2-ARF/Primer2-DSR, and Primer2-DSF/Primer2-3R, respectively, and then spliced by overlap PCR. Primer pair sequences are shown in the following table.

As shown in FIG. 4B, the PCR product obtained by purification was subjected to treatment with restriction enzymes EcoRI (GAATTC) and NaeI (GCCGGC), and then inserted into an expression plasmid to obtain a plasmid (pMA-C1H-HC) for expressing a heavy chain. The L234A, L235R, P329D and A330S mutations were completed in the sequence. Sequencing confirmed that the pMA-C1H-HC sequence was correct.

Plasmid pM-C1H-LC for expressing the Campath-1H light chain was equimolar mixed with plasmid pMA-C1H-HC for expressing the engineered Campath-1H heavy chain described above, and 293F cells in the logarithmic growth phase were co-transfected with PEI. After 5 days of culture at 37℃with 5% CO ₂, the medium was collected by centrifugation. The Protein A affinity chromatography, ion exchange chromatography and desalting column chromatography are adopted to obtain a high-purity antibody product, which is named as aCD52- (C1H-Fab) - (FcA), abbreviated as aCD52-SH, and the heavy chain sequence is shown as SEQ ID NO.2. The products were checked by electrophoresis and the results are shown in FIG. 5, where M is the molecular weight standard (MW Ladder) and Lane 1 and 2 are the target antibodies.

Modification example 3

Engineering of anti-human CD3 cell culture antibodies

In this example, the CDR sequence of the mouse IgG2a mab OKT3 against human CD3 was inserted into the IgG1 backbone of engineered example 2, and then expressed and purified to give engineered antibody aCD3-SH.

The protein sequences of OKT3 light and heavy chains were aligned with the hIgG1-kappa (kappa) light and hIgG1 heavy chains, respectively (FIGS. 6A, B), confirming the 6 CDR sequences. The CDRs expressing DNA sequences after codon optimization were chemically synthesized and spliced by a genetic engineering method such as overlap PCR to obtain plasmid pM-OKT3h-LC for expressing the light chain (FIG. 6C) and plasmid pMA-OKT3h-HC for expressing the heavy chain comprising 4 point mutations in modification example 2 (FIG. 6D).

Expressed in 293F cells, and subjected to Protein A affinity chromatography, ion exchange chromatography and desalting column chromatography to obtain high-purity antibody products, which are named as aCD3- (OKT 3 h-Fab) - (FcA), abbreviated as aCD3-SH, the heavy chain sequence and the light chain sequence of which are respectively shown as SEQ ID NO.3 and SEQ ID NO.4. The products were checked by electrophoresis and the results are shown in FIG. 7, where M is the molecular weight standard (MW Ladder) and Lane 1 and 2 are the target antibodies.

2. Cell culture and antibody viability assay

Cultivation example 1

T cell culture and detection of amplification activity of anti-human CD3 antibody

The test uses the engineered antibody aCD3-SH obtained in engineered example 3 and its prototype antibody OKT3, respectively, to stimulate proliferation of T cells (CD3+) in human PBMC, and uses MTS (CAS# 138169-43-4) to stain the cells to quantitatively determine the viability of the antibody, i.e., half the effective dose (ED ₅₀).

Material

Human venous blood, human peripheral blood lymphocyte isolates (Tianjin Zhiyang, LTS 1077), IL-2 (Beijing Shuanglu, recombinant human interleukin-2 for injection), basal medium X-VIVO5 (Lonza, 04-418Q), complete medium (X-VIVO 5, IL-2 1000 IU/mL), CELLTITER AQUEOUS ONE SOLUTION (Promega, G3580, i.e., MTS solution), OKT3 (Takara Bio, T210).

Preparation of antibody concentration gradient

96-Well flat bottom cell culture plates were taken and 100. Mu.L of complete medium was added to the wells. The antibody was diluted to 6.40. Mu.g/mL with complete medium, 100. Mu.L of each was added to column 1 wells, and mixed well. Gradient dilution (100. Mu.L) was performed in the wells from column 1 to column 11, and after mixing, the mixture was continued to the next column. Finally, each well contained 100. Mu.L of complete medium and formed a 1:2 dilution gradient of antibody from 1.60. Mu.g/mL in column 1 to 1.56ng/mL in column 11.

Cell culture

Human PBMC cells were isolated using a white blood cell isolate and the density was adjusted to 3E5/mL with basal medium. 100. Mu.L of the cell suspension was added to each of the wells 1 to 11 of the 96-well plate, and the wells were mixed well. Finally, each well contained 200. Mu.L of complete medium and 3E4 cells and formed a 1:2 dilution gradient of antibody from 800ng/mL in column 1 to 0.781ng/mL in column 11. Incubated at 37℃for 5 days with 5% CO ₂.

Data acquisition and processing

Mu.L of MTS solution was added to each well. The plates were placed in a cell incubator for a further 6 hours. The absorbance at 490nm was read using a microplate reader. The readings were averaged and the Blank (i.e., the average of the 12 th column of readings) was subtracted. ED ₅₀ was calculated using a 4-parameter fitting method (Four Parameter Logistic Regression) with a semi-log curve of antibody concentration/OD.

Discussion of results

ED ₅₀ for aCD3-SH was 18.9ng/mL (FIG. 8, open circles), ED ₅₀ for prototype antibody OKT3 was 130ng/mL (FIG. 8, filled circles), indicating that target cell T cells are more sensitive to aCD 3-SH. At the same time, the maximum amplified signal of aCD3-SH was also significantly stronger than that of OKT3 at the saturated antibody concentration. The results show that the engineered antibody aCD3-SH has significantly higher activity of stimulating T cell expansion.

Cultivation example 2

NKT cell culture and detection of amplification activity of anti-human CD3 antibody

The test uses the engineered antibody aCD3-SH obtained in engineered example 3 and its prototype antibody OKT3, respectively, to stimulate proliferation of NKT cells (CD3+CD56+) in human PBMC, and the viability is compared by counting cells.

Material

Human venous blood, basal medium GT-T551 (TaKaRa, WK 551T), amplification medium (GT-T551, IL-2 1000 IU/mL), cell culture flask T75 (CORNING, 430720)/T175 (CORNING, 431080), IFN-gamma (Shanghai Kailong, recombinant human interferon gamma for injection).

Cell culture and detection method

Human PBMC cells were isolated using a white blood cell separation solution, adjusted to a cell density of 2E6/mL with basal medium, and placed in a culture flask. The monocytes were attached by incubation at 37 ℃ with 5% co ₂ for 2 hours. Suspension cells were collected and the cell concentration was adjusted to 1E6/mL with basal medium. Recombinant human IFN-gamma (1000 IU/mL) was added. Incubated at 37℃with 5% CO ₂ for 24 hours. Anti-human CD3 antibody (30 ng/mL) and IL-2 (1000 IU/mL) were added and incubation was continued for 48 hours. Amplification medium was added at a 1:1 volume ratio and culture was continued for 48 hours. Then sampling and counting every 48 hours, regulating the cell density to 0.7E6/mL by using an amplification culture medium, and continuing culturing. And culturing until the 12 th to 20 th days, harvesting the cells, and counting.

Discussion of results

In one 12 day trial, PBMC from volunteer 1 (Donor 1) were amplified 146.9-fold with aCD3-SH (FIG. 9A, open circles) and 50.4-fold with OKT3 (FIG. 9A, filled circles). In the 19-day amplification test on PBMCs of volunteer 2 (Donor 2), 491-fold and 251-fold, respectively (fig. 9B). The results show that the engineered antibody aCD3-SH has higher activity of stimulating the expansion of NKT cells.

Cultivation example 3

NK cell specific amplification and purity detection

The test takes blood samples from volunteers, after separation of PBMC, the samples were divided into 2 parts, and the modified antibodies aCD3-SH, aCD16-SH and aCD52-SH (test group) obtained by modification and prototype antibodies OKT3, aCD16-3G8 and Campath-1H (control group) were used for culturing, respectively, and proliferation of NK cells (CD 3-CD16+CD56+) in human PBMC was stimulated, and the functions of the antibody groups were compared by analysis of cell components.

Material

Human venous blood, basal medium X-VIVO15 (Lonza, 04-418Q), amplification medium (X-VIVO 15, IL-2 1000 IU/mL), human serum (Genimi, 100-512).

Cell culture and detection method

PBMC cells were isolated and cell density was adjusted to 1E6/mL with basal medium. Test or control antibodies, IL-2 (1000 IU/mL), human serum (5%) were added. Incubated at 37℃with 5% CO ₂ for 72 hours. An equal volume of amplification medium was added and culture continued for 48 hours. The cells were then sampled and counted every 48 hours, diluted to 0.7E6/mL with expansion medium, and cultured continuously. Cells were harvested from culture until day 14 and flow cytometric detection was performed with fluorescent antibodies PerCP Mouse Anti-Human-CD3(BD Biosciences,552851)、APC-Cy7 Mouse Anti-Human-CD16(BD Biosciences,557758)、PE Mouse Anti-Human-CD56(BD Biosciences,555516).

Discussion of results

The NK cell ratio of CD3-CD56+ in the cells obtained by the amplification of the test group was 97.6% (FIG. 10A) higher than that obtained by the control group (FIG. 10B). The NK cell fraction of CD3-CD16+CD56+ in this population was 96.2% (FIG. 10C) and 71.7% (FIG. 10D), respectively. Then, NK cells of CD3-CD16+CD56+ obtained by amplification of the test group can reach 93.9% of the total cells, which is better than 51.5% obtained by the control group. The results show that the purity of the NK cell product obtained with the test group antibodies was higher.

3. Cell product application

Application example 1

In vitro killing activity detection of NK cell products on tumor cells

The present test uses NK cell products (test group) obtained by amplification of the test group in culture example 3 and NK cell products (control group) obtained by amplification of the control group to conduct direct killing and ADCC killing tests on lymphoma cell line Raji, breast cancer cell line BT-474, breast cancer cell line MCF7, respectively, and the viability of the modified antibody and the prototype antibody is compared by the viability analysis of the final product.

Material Raji cells (ATCC, CCL-86), BT-474 cells (ATCC, CRL-3247), MCF7 cells (ATCC, HTB-22), detection kit CytotoxNon-Radioactive Cytotoxicity Assay (Promega, G1780), medium RPMI 1640 (ThermoFisher, 11879020), rituximab, trastuzumab.

Detection method

Tumor cells were subcultured, the cells were collected and then the density was adjusted to 1E5/mL with medium, and 100. Mu.L was added to each well of a 96-well plate. NK cells were collected, and the density was adjusted to 1E5/mL or 5E5/mL with medium, and 100. Mu.L was added to the corresponding wells. Rituximab was adjusted to a medium concentration of 2mg/mL and 5. Mu.L was added to the corresponding wells. Trastuzumab was adjusted to 2mg/mL with medium concentration and 5 μl was added to the corresponding wells. The kit instructions were prepared for spontaneous cell release wells (containing only one cell of Raji, BT-474, MCF7 or NK), maximum target cell release wells (containing only one cell of Raji, BT-474 or MCF7 cells), and volume correction wells (without any cells).

Target cell killing test wells (raji+nk, BT-474+nk, or mcf7+nk), ADCC killing test wells (raji+nk+rituximab, BT-474+nk+trastuzumab, mcf7+nk+trastuzumab) were prepared. Wherein, 100 μl of NK cells of 1E5/mL, i.e. E/T=1:1, was added to the killing test of Raji cells. While killing experiments with BT-474 and MCF7 added 100 μl of NK cells at 5E5/mL, i.e. E/t=5:1. Incubated at 37℃with 5% CO ₂ for 3 hours. To the target cell maximum release well, 20. Mu.L of lysate (Lysis Solution) was added to the volume-corrected well. Incubation was continued for 45 minutes.

Transfer 50 μl of supernatant from each well to another new 96-well plate, prepare substrate Solution according to the detection kit method, add 50 μl of substrate Solution per well (Substrate Solution), incubate at room temperature for 30min in the dark, and add 50 μl of reaction Stop Solution per well (Stop Solution). The 490nm absorbance was read on a plate reader (Molecular Devices, spectraMax M5 Microplate Readers).

The killing rate calculation formula is as follows:

killing ratio = (killing test signal-effector cell spontaneous release signal-target cell spontaneous release signal)/(target cell maximum release signal-target cell spontaneous release signal) ×100%

Discussion of results

The results of the killing test on Raji cells are shown in fig. 11. It can be seen that NK cells directly kill Raji cells on an existing basis at E/t=1:1, but the killing rates of the two groups are not very different. After Rituximab addition, NK cells were activated and the killing rate of NK in the test group increased from 14.7% to 85.0%. Control NK was also activated but only increased from 12.5% to 56.2% with a clear gap from the test group. ADCC killing was specific, with Trastuzumab added, NK cells were not activated, and the direct killing rates of the two NK cells under this condition were not very different.

The results of the killing test on BT-474 and MCF7 cells are shown in FIGS. 12 and 13. It can be seen that the killing rate of the NK cells of the test group to the BT-474 cells is obviously superior to that of the control group, namely direct killing and ADCC killing. Also, the killing rate of the NK cells of the test group on the MCF7 cells is obviously superior to that of the control group, namely direct killing and ADCC killing.

Application example 2

Detection of in-vivo direct killing activity of NK cell products on tumor cells

The NK cell products obtained by amplification of the test group in culture example 3 were subjected to an in vivo killing test on a mouse Raji-Luc hematological tumor model to determine the in vivo viability of NK cells.

Material

NSG mice (6-8 weeks old), raji-Luc cells (Raji cells containing stable transfected luciferase).

Detection method

Raji-Luc cells were subcultured, the cells were collected and then the density was adjusted to 5E6/mL with PBS, and 100. Mu.L was injected into each NSG mouse via tail vein. After 48 hours, the mice were randomly divided into 2 groups according to body weight, and 100 μl of physiological saline was injected via tail vein (control group) and NK cell 1E7 (test group), respectively. Tumor growth was measured on an in vivo imager (PERKINELMER, IVIS Lumina LT In Vivo IMAGING SYSTEM) and imaging signal patterns and signal intensities were collected.

Discussion of results

In one 19 day trial, the average imaging signal intensity of the control mice increased to 9.16E6p/sec/cm ²/sr, whereas the test mice reached 1.63E6p/sec/cm ²/sr, which was 17.8% of the control group (FIG. 14). The results show that NK cells have obvious in vivo killing activity.

Application example 3

Detection of ADCC killing activity of NK cell products on tumor cells in animals

NK cell products and therapeutic antibodies obtained by amplification of the test group in culture example 3 were subjected to an in vivo ADCC killing test on a mouse Raji-Luc hematological tumor model to determine the in vivo viability of NK cells.

Material

NSG mice (6-8 weeks old), raji-Luc cells (Raji cells containing stable transfected luciferase), rituximab.

Detection method

After establishing a mouse Raji-Luc hematological tumor model according to the method of application example 2, the mice were divided into 4 groups, and physiological saline (G1, blank group), rituximab (G2), NK cells (G3) and combination drug (G4, i.e., rituximab and NK cells were simultaneously infused) were injected respectively. And (5) continuing feeding, periodically measuring the growth condition of the tumor, and observing the growth and survival state of the animal.

Discussion of results

As can be seen from the survival results (fig. 15) and tumor imaging results (fig. 16) of each group of mice, the fluorescence signal derived from tumor cells gradually increased, and the mice gradually died. The G1 group reached median survival at 26 days and all died at 30 days. In the G2 group, a median survival period was reached at day 51 and 2 survived at the end of the 75 day trial. In the G3 group, 3 survived at 75 days. Group G4 survived 6 at the last imaging test at day 61 and 5 at day 75.

The treatment effect data of the combination group are obviously superior to that of the blank group and the independent group in comparison of the overall survival rate and the tumor imaging signals. Indicating that rituximab has remarkable in vivo ADCC killing effect when used in combination with NK cells obtained by the present invention.

Application example 4

Clinical study of NK cell products in the treatment of hepatocellular carcinoma

Patients with advanced hepatocellular carcinoma (Hepatocellular Carcinoma, HCC) were selected for this study, and NK cell products prepared according to the expansion method in culture example 3 were infused, and the short-term and long-term responses of the patients were observed to initially explore the safety and efficacy of treatment protocols using autologous high-purity human NK cells.

Material

NK cells are prepared into cell products after being collected and cleaned, the cell activity rate is more than 90%, and the purity of the NK cells is more than 90%.

Research method

The intended patient, after screening by inclusion and exclusion, begins 1-2 NK cell therapy sessions, each session being spaced 1-3 months apart. Each course of treatment comprises 2 NK cell treatments, 5-10 days apart. 1-2E 9 NK cells are input in each treatment, and the change of the patient symptoms and the main description are recorded before and after the input. Follow-up was initiated after the end of the first course of treatment until 2 years after treatment or the patient had exited the study for various reasons.

Discussion of results

In a clinical trial that passed the clinical trial ethical review board review, 33 people underwent a total of 38 courses of treatment. After NK cell infusion, most patients had no uncomfortable symptoms, and few patients (4 times, 5.3% of total) had fever response, and the next day after the antipyretic treatment was resumed. The results show that the high-purity NK cell product obtained by the invention can ensure the safety of the treatment of the homologous allogeneic cells, and has potential to solve the dilemma of low immunity of patients.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

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Claims

1. A cell culture method, characterized by comprising the following steps: adding a modified antibody to the culture system;

The heavy chain amino acid sequence of the modified antibody is shown in SEQ ID NO.1, and the light chain of the modified antibody is the light chain of the mouse IgG1 monoclonal antibody 3G8 against human CD16.

Or the heavy chain amino acid sequence of the modified antibody is as shown in SEQ ID NO.2, and the light chain of the modified antibody is the light chain of the humanized IgG1 type Camppath-1H monoclonal antibody against human CD52;

Alternatively, the heavy chain amino acid sequence of the modified antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence of the modified antibody is shown in SEQ ID NO.4;

Some cells in the culture system have Fc receptors on their surface.

2. The cell culture method according to claim 1, wherein the cells having Fc receptors on their surface are selected from one or more of lymphocytes, granulocytes, mast cells, dendritic cells, macrophages, and monocytes.