CN111411079A

CN111411079A - Cell culture method, modified antibody, cell culture medium, cell product and application thereof

Info

Publication number: CN111411079A
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: 2020-07-14

Abstract

The invention relates to a cell culture method, a modified antibody, a cell culture medium, a cell product and application thereof, wherein the cell culture method comprises the following steps: adding the modified antibody into a culture system; the modified antibody is an antibody for cell culture, wherein the region outside the complementarity determining region is subjected to protein modification, and the affinity of the modified antibody for Fc receptor is lower than that of the antibody for cell culture, which is not subjected to protein modification. The cell culture method and the cell culture medium can be used for in vitro culture of various cells to obtain target cells with higher purity and higher activity, and the obtained cell products can be used for clinical application research.

Description

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

Technical Field

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

Background

At present, a large number of antibodies for cell culture are used in a common in vitro cell culture method to bind specific receptor molecules on the surface of target cells, so as to activate (activating) or inhibit (blocking) signal pathways, and promote directional differentiation, continuous amplification or apoptosis of the target cells. In small scale cultures, these antibodies are usually coated (coating) on the surface of a petri dish or crosslinked to magnetic beads. In large scale cultures, these antibodies are often added directly to the culture medium in order to avoid lengthy and difficult to control coating processes, or to save high bead costs, to avoid the introduction of foreign substances, or for other process optimization purposes. However, the final product thus obtained generally has the problems of low purity and low activity.

The target cells are of low purity, meaning 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 of the non-target cells are usually difficult to control, so that the complexity of scientific experiments, particularly in vivo experiments of animals and human bodies, is greatly increased, and the repeatability of the research is seriously influenced.

Also, in clinical therapeutic applications, for safety and efficacy, it is necessary to obtain cell preparations with the highest possible purity and high viability.

Disclosure of Invention

In view of the above, there is a need for a cell culture method that can improve cell purity and viability.

The invention discloses a cell culture method, which comprises the following steps: adding the modified antibody into a culture system; the modified antibody is an antibody for cell culture, wherein the region outside the complementarity determining region is subjected to protein modification, and the affinity of the modified antibody for Fc receptor is lower than that of the antibody for cell culture, which is not subjected to protein modification.

The invention also discloses a modified antibody, wherein the modified antibody is a cell culture antibody with a protein modified region outside the complementarity determining region, and the affinity of the modified antibody to an Fc receptor is lower than that of the cell culture antibody without the protein modification.

The invention also discloses a cell culture medium, which comprises a basal medium and the modified 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 effects on tumor cells.

The invention also discloses a medicament for treating cancer, which comprises the cell product.

The invention also discloses a medicament for allogeneic cell therapy, 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, does not change the original mechanism of the antibody for cell culture, and does not need to make great changes to the culture process. Is particularly suitable for the condition that the antibody for cell culture is directly added into the culture medium in the large-scale cell culture process which is widely used 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, simultaneously improve the utilization rate of the antibody, and reduce the use amount of the raw material antibody by optimizing the culture process.

The invention avoids immune cells, particularly macrophages, monocytes, NK cells and the like in initial raw material cells in primary cell culture, has ADCP/ADCC effect on target cells, and improves the survival rate and final yield of the target cells. Especially when the cultured and amplified target cells are immune cells such as macrophages, monocytes and NK cells, the purity and the activity of the target cells in the final product are improved more obviously.

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 in example 1;

FIG. 2 is a graph showing the UV absorption curves of the washing and elution steps for the modified Protein A column chromatography of example 1;

FIG. 3 is a reduced SDS-PAGE electrophoretic image of the target protein of engineering example 1;

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

FIG. 5 is a reduced SDS-PAGE electrophoretic image of the target protein of engineering example 2;

FIG. 6 is a schematic diagram of the modification of human CD 3-resistant mouse IgG2a monoclonal antibody OKT3 in example 3;

FIG. 7 is a reduced SDS-PAGE electrophoretic image of the target protein of engineering example 3;

FIG. 8 is a graph of a fit of viability of the prototype antibody OKT3 in culture example 1 and aCD3-SH prepared in engineering example 3 to T cell expansion;

FIG. 9 is a graph of the expansion of NKT cells by culturing the prototype antibody OKT3 of example 2 and the aCD3-SH prepared in modified example 3;

FIG. 10 is a diagram showing flow cytometry analysis of cells obtained by amplification using the engineered antibody panel and cells obtained by amplification using the prototype antibody panel in culture example 3;

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

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

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

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

FIG. 15 is a graph showing survival rate of groups of mice in application example 3;

FIG. 16 is an image of tumors of each group of mice in application example 3.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. 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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" 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: broadly refers to antibodies used in cell culture processes.

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

Modifying an antibody: in particular, the affinity of the antibody of the present invention, which is obtained by protein engineering the prototype antibody, to the Fc receptor is lower than that of the prototype antibody.

The culture system is generally a collection including a medium, a growth factor, an antibody for cell culture, cells (target cells and non-target cells), serum (presence or absence), an antibiotic (presence or absence), and other additives, but is not limited thereto, and the composition of the culture system may be different depending on the need.

Antibody receptors on cell surfaces, antibody clearance and antibody-dependent killing

Certain cells, such as lymphocytes, DC cells, macrophages, granulocytes, platelets, mast cells, etc., express on their surface various Receptor Fc receptors (Fc receptors, fcrs) that bind the Fc portion of different immunoglobulin isotypes (Isotype), including Fc α R, Fc γ R, FcR, etc., wherein Fc γ rs, in turn, are classified as Fc γ RI (CD64), Fc γ RII (CD32), and Fc γ RIII (CD16), bind primarily IgG.

The concentration of the cell culture antibody added to the medium is continuously decreased during the cell culture. This is directly related to the continuous inactivation of antibodies (natural degradation, degradation by proteases released from cells, aggregation and precipitation), endocytosis after the antibodies bind to the target receptor (receptor-mediated endocytosis), and the like, as well as the ability of certain cell surface expressed Fc receptors in the culture system to bind to and scavenge these antibodies (FcR dependent endocytosis).

The antibodies used for the culture are generally of the IgG1 subtype derived from the screening of mouse hybridomas, for example the mouse anti-human CD3 antibody UCHT1, the mouse anti-human CD16 antibody 3G8, the mouse anti-human CD28 antibody CD-28. Furthermore, for purposes of increasing yields and expanding product applications, humanization of murine antibodies will also generally select the IgG1 subtype, e.g., humanized Campath-1H mAb derived from rat anti-human CD 52. The problem of the rapid decline in the effective concentration of these IgG1 antibodies in the culture system is exacerbated.

Even after binding to the target cells, the antibodies provoke the attack of certain effector cells expressing Fc receptors. This includes Antibody-Dependent cell-mediated Phagocytosis by Fc γ RII-expressing macrophages (ADCP) and Antibody-Dependent cell killing by Fc γ RIII-expressing NK cells (ADCC). When immune cells such as macrophages, monocytes, NK cells and the like are present in the culture system, such specific ADCP/ADCC effect rapidly reduces the viability and purity of the target cells. In particular, if the target cells to be cultured are macrophages, monocytes, NK cells and other immune cells, such self ADCP/ADCC effect rapidly reduces the viability, purity and vitality of the target cells and advances the target cells into the depletion phase.

Antibody engineering principles and methods

The sites on IgG that bind to Fc. gamma.R are concentrated in the Hinge region (Hinge) and Fc-C _H2 domain, the alteration of the antibody is primarily directed to this region. The means of modification 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 a subtype of the antibody having a relatively weak binding force to the Fc receptor, or the amino acid residues at the key binding site of the hinge region and the Fc fragment of the antibody for cell culture are mutated, or the Complementarity Determining Regions (CDRs) of the antibody for cell culture are inserted into the framework of another subtype of the antibody having a weak binding force to the Fc receptor, and the like. It is understood that the above-mentioned various modification means can be used in combination, for example, the CDR of the antibody for cell culture is inserted into the framework of the other subtype of antibody which has been subjected to the point mutation.

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

Sequence replacement

In some embodiments, the above sequence substitutions are substitutions of the hinge region and Fc segment derived from the subclass IgG1, IgG3 antibodies to the corresponding sequences of IgG2 or IgG 4. In a preferred embodiment, these sequences are replaced with the corresponding sequences of IgG 4. For example, the hinge region and Fc region of antibodies such as mouse monoclonal antibody UCHT1 against human CD3 of IgG1 subtype, mouse monoclonal antibody 3G8 and MEM-154 against human CD16, mouse monoclonal antibody CD28 against human CD28, mouse monoclonal antibody 4C1A9 against human CD137, 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, IgG1-C is selected when the replacement of the hinge region and Fc region is performed_HThe C-terminal fragment of the 1-Hinge region was as close as possible to IgG4-C_H1-Hinge the same sequence starts a substitution to keep C as much as possible_H1 and V _H1, maintaining the function of the Fab fragment.

Point mutation

In some embodiments, the point mutation is a mutation of an amino acid at one or more sites of a key binding site of the hinge region and Fc region of the antibody for cell culture.

In some embodiments, the antibody for cell culture is an IgG1 subtype, and the point mutation is a mutation at 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 antibody for cell culture. In some embodiments, adjacent amino acids to the above-mentioned positions may also be mutated.

In some preferred embodiments, L235 is mutated to another amino acid, particularly an amino acid that affects backbone conformation (glycine Gly, proline Pro), which can reduce binding of the antibody to the Fc receptor.p 329 of hIgG1 is involved in binding to hfcyriii, particularly the hydrophobic interaction of the sidechain of L with Fc γ RIII. in some preferred embodiments, P329 is mutated to another amino acid, particularly a charged amino acid or an uncharged polar amino acid (serine, threonine, etc.), in some preferred embodiments, Thr is mutated to Fc γ RIII. in some preferred embodiments, P329 is mutated to another amino acid, particularly to a charged amino acid or to an uncharged polar amino acid (Ser, threonine, etc.), in some preferred embodiments, P63234 is mutated to hfcyriii, in some preferred embodiments, a 46234A is mutated to another amino acid, particularly to a charged amino acid (glutamic acid Glu, aspartic acid Asp, arginine Arg, lysine L ys) or to a sterically hindered bulky sidechain amino acid (phenylalanine Phe, etc.).

Sequence insertion

In some embodiments, the sequence insertion is insertion of CDRs derived from the subclass IgG1, IgG3 antibody onto the backbone 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 otherwise engineered antibody having reduced affinity for Fc receptors. In a more preferred embodiment, these CDRs are inserted into the backbone of the IgG1 antibody that has been engineered by the point mutation.

Other methods

In some embodiments, the affinity of the antibody for the Fc receptor can be reduced by other means such as sequence deletions, glycosylation modifications, and chemical modifications.

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

In a preferred embodiment, N297 in the expression sequence of the antibody is mutated to obtain a Heavy Chain Non-Glycosylated Heavy Chain antibody (NGHC).

In a preferred embodiment, the antibody is treated with Endoglycosidase (Endoglycosidase) to obtain an antibody with a deletion or rearrangement of glycosyl groups.

In a preferred embodiment, the antibody is treated with chemical Conjugation (Conjugation) to obtain an antibody with side chain modifications or conjugated steric groups.

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

Scope of antibody modification

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, a mouse anti-human CD3 antibody UCHT1, a mouse anti-human CD16 antibody 3G8, a mouse anti-human CD28 antibody CD-28, a mouse anti-human CD52 humanized Campath-1H monoclonal antibody, and an anti-human CD137 mouse monoclonal antibody 4C1A 9.

In some embodiments, the heavy chain amino acid sequence of the engineered antibody is set forth in SEQ ID No. 1; or the heavy chain amino acid sequence of the modified antibody is shown as SEQ ID NO. 2; or the heavy chain amino acid sequence of the modified 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 modified antibody of the present invention is not limited to the above antibodies, and any antibody for cell culture may be used as long as the affinity for Fc receptor is reduced by protein modification.

Cell culture

The cell culture method according to an embodiment of the present invention comprises the steps of: adding the modified antibody into a culture system; the modified antibody is an antibody for cell culture, wherein the region outside the complementarity determining region is subjected to protein modification, and the affinity of the modified antibody for Fc receptor is lower than that of the antibody for cell culture, which is not subjected to protein modification.

In some embodiments, the cell type is one or more of lymphocytes, granulocytes, mast cells, DC cells, macrophages, monocytes, and platelets. The cell is derived from human blood, tissue, human solid tumor removed by operation and ascites. May be freshly collected venous blood, umbilical cord blood, or may be cryopreserved PBMC cells. It is understood that the cell type is not limited thereto, as long as the Fc receptor is expressed on the surface of a part of the cells in the culture system.

In some embodiments, the cell biology assay is designed to quantitatively detect functional viability of the engineered antibody against target cells 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, an engineered antibody is used to stimulate T cell proliferation (CD3+) in human PBMCs and the viability of the antibody, i.e., half the Effective Dose (ED), is quantitatively determined₅₀). This viability data is useful for other cell culture methods that require stimulation of T cells (CD3+) in human PBMCs. In some embodiments, more active engineered antibodies may also be obtained through 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 quantitatively determining the viability of the engineered antibody, standardized media or kits can be formulated to meet the needs of a particular cell culture.

Cell product

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

In some embodiments, T cells, NKT cells, NK cells, CT L cells, TI L cells, and the like immune cell products can be obtained.

Cell product applications

In some embodiments, the cell product can be used in vitro cell killing assays, in vivo animal killing assays, and in human assays.

In some embodiments, the above cell products, including immune cell products such as DC cells, T cells, NKT cells, NK cells, CT L cells, TI L cells, CAR-T cells, CAR-NK cells, and the like, can recognize and kill viruses invading the human body, host cells transformed by viral infection, tumor cells, and can be used for antiviral therapy and targeted tumor therapy.

NK cells express Fc gamma RIII (CD16) on their surface and are the main effector cells for ADCC. In some embodiments, NK cells can be used in ADCC killing studies in combination with therapeutic antibodies. In some preferred embodiments, the NK cell product may be used in combination with the therapeutic antibody Rituximab for the treatment of lymphoma. In some preferred embodiments, the NK cell product may be used in combination with the therapeutic antibody Trastuzumab for the treatment of breast and gastric cancer. In some preferred embodiments, the NK cell product may 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 either non-self or self-depleting effects (missing-self). This cell killing is Non-MHC Restricted (Non-MHC-Restricted cytoxicity) and does not cause Graft-versus-Host Disease (GVHD), and therefore NK cells can be applied to allograft therapy. In some more preferred embodiments, the high purity NK cell product and CAR-NK cell product described above are used for human allogeneic cell therapy, but it is understood that the cell product used for allogeneic cell therapy is not limited thereto.

The present invention is described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto.

First, antibody modification

Modification example 1

Modification of anti-human CD16 cell culture antibody

This example will express V of the mouse IgG1 monoclonal antibody 3G8 against human CD16_H-C _H1 sequence and C expressing human IgG4_H2-C _H3, then expressed and purified together with the light chain sequence of the antibody 3G8 to obtain the modified antibody aCD 16-SH.

Selection of mouse IgG1-C _H1 last paragraph (T)₂₁₄KVDK₂₁₈) With human IgG4-C _H1, i.e. at T₂₁₄The preceding sequence was that of mouse IgG1-3G8, at K₂₁₈The subsequent sequence was that 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 _H1 fragment sequence (fig. 1A). Then, Primer pair Primer1-2F/Primer1-2R is used for amplifying human IgG4 heavy chain expression DNA sequence to obtain Hinge-C thereof_H2-C _H3 fragment(s)Sequence (FIG. 1B). Splicing of 2 sequences was then accomplished by Overlap PCR (Overlap-PCR) (FIG. 1C), which was performed as follows: after mixing the purified 2 PCR products in equal molar numbers, 5 PCR cycles were carried out at an annealing temperature of 60 ℃, Primer1-3F and Primer1-2R were added, and 30 PCR cycles were carried out at an annealing temperature of 72 ℃. The 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 treated with restriction enzymes EcoRI (GAATTC) and NaeI (GCCGGC), and inserted into an expression plasmid (pMD18-T as a base, containing expression elements such as CMV promoter and polyA Tail) to obtain a plasmid pM-3G8-HC-M1 for expressing the heavy chain (FIG. 1D). Sequencing confirmed that the pM-3G8-HC-M1 sequence was correct.

The plasmid pM-3G 8-L C for expressing the light chain of 3G8 was used in combination with the plasmid described abovePlasmid pM-3G8-HC-M1 expressing the modified 3G8 heavy chain was mixed in equimolar amounts, 293F cells in logarithmic growth phase were cotransfected with linearized Polyethyleneimine (PEI) L initial, 5% CO at 37 deg.C₂After 5 days of culture, the culture medium is collected by centrifugation, the target antibody Protein is purified by Protein A affinity chromatography, and the ultraviolet absorption curve of the washing and elution steps is shown in figure 2. the sample is subjected to electrophoresis inspection, and the result is shown in figure 3, wherein M is a molecular weight standard (MW L adder),

L ane

1 and 2 are samples of a column washing part, and

L ane

3 and 4 are samples of an elution peak, and then the high-purity antibody product is obtained by ion exchange chromatography and desalting column chromatography, and is named aCD16- (3G8-Fab) - (hIgG4-Fc), which is called aCD16-SH for short, and the heavy chain sequence of the antibody product is shown in SEQ ID NO. 1.

Modification example 2

Modification of anti-human CD52 cell culture antibody

In this example, the sequence of humanized IgG1 Campath-1H monoclonal antibody expressing anti-human CD52 was mutated, and then expressed and purified to obtain the engineered antibody aCD 52-SH.

As shown in FIG. 4, the sequences expressing the heavy chain of Campath-1H monoclonal antibody (FIG. 4A) were used as templates, and Primer pairs Primer2-3F/Primer2-ARR, Primer2-ARF/Primer2-DSR, and Primer2-DSF/Primer2-3R were subjected to PCR to obtain 3 fragments, which were then subjected to overlap PCR for splicing. The primer pair sequences are shown in the table below.

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

Plasmid pM-C1H-L C for expression of Campath-1H light chain is as described above for the tablesPlasmid pMA-C1H-HC to achieve the engineered Campath-1H heavy chain was mixed in equimolar amounts and 293F cells in log phase were co-transfected with PEI. At 37 deg.C, 5% CO₂After 5 days of culture, the culture medium is collected by centrifugation, and a high-purity antibody product is obtained by Protein A affinity chromatography, ion exchange chromatography and desalting column chromatography, and is named as aCD52- (C1H-Fab) - (FcA), which is called aCD52-SH for short, and the heavy chain sequence of the product is shown in SEQ ID NO. 2. the product is subjected to electrophoresis inspection, and the result is shown in figure 5, wherein M is a molecular weight standard (MW L adder), and

L ane

1 and 2 are target antibodies.

Modification example 3

Modification of anti-human CD3 cell culture antibody

In this example, the CDR sequences of OKT3 of mouse IgG2a monoclonal antibody against human CD3 were inserted into the IgG1 framework of modified example 2, and then expressed and purified to obtain modified antibody aCD 3-SH.

The protein sequences of OKT3 light chain and heavy chain were aligned with the sequences of hIgG1-kappa (kappa) light chain and hIgG1 heavy chain, respectively (FIG. 6A, B), and 6 CDR sequences were confirmed.A codon-optimized CDR-expressing DNA sequence was chemically synthesized and spliced by a genetic engineering method such as overlap PCR to obtain plasmid pM-OKT3 h-L C (FIG. 6C) for expressing light chain and plasmid pMA-OKT3h-HC (FIG. 6D) for expressing heavy chain containing 4 point mutations in modification example 2.

Expressing in 293F cell, obtaining high purity antibody product named aCD3- (OKT3h-Fab) - (FcA) for short aCD3-SH by Protein A affinity chromatography, ion exchange chromatography and desalting column chromatography, wherein the heavy chain sequence and the light chain sequence of the antibody product are respectively shown in SEQ ID NO.3 and SEQ ID NO.4, and performing electrophoresis inspection on the product, wherein the result is shown in figure 7, wherein M is molecular weight standard (MW L adder), and

L ane

1 and 2 are target antibodies.

Secondly, cell culture and antibody activity detection

Culture example 1

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

The assay stimulated proliferation of T cells (CD3+) in human PBMC with the engineered antibody aCD3-SH obtained in engineering example 3 and its prototype antibody OKT3, respectively, and MTS (CAS # 13816)9-43-4) staining of cells to quantitatively determine the viability of the antibody, i.e., the half Effective Dose (ED)₅₀)。

Material

Human venous blood, human peripheral blood lymphocyte separation fluid (Tianjin top ocean, L TS1077), I L-2 (Beijing Erlu, recombinant human interleukin-2 for injection), basic medium X-VIVO5 (L naza, 04-418Q), complete medium (X-VIVO5, I L-21000 IU/m L), CellTiter AQueous One Solution (Promega, G3580, MTS Solution), OKT3(Takara Bio, T210).

Preparation of antibody concentration gradient

A96-well flat-bottom cell culture plate was taken and 100. mu. L complete medium was added to the wells, the antibody was diluted to 6.40. mu.g/m L with complete medium, 100. mu. L was added to each well in column 1, and the wells were mixed together, and a gradient dilution (serial dilution) was performed from column 1 to column 11, i.e., 100. mu. L was transferred, and after mixing, the transfer was continued to the next column, finally, 100. mu. L complete medium was contained in each well, and a 1:2 dilution gradient of antibody was formed, from 1.60. mu.g/m L in column 1 to 1.56ng/m L in column 11.

Cell culture

Human PBMC cells were isolated using leukocyte isolation, adjusted to density 3E5/m L using basal medium, 100. mu. L cell suspension was added to each of the 1 st to 11 th wells of a 96-well plate, mixed well, and finally 200. mu. L complete medium and 3E4 cells were contained in each well, and a 1:2 dilution gradient of antibody was established from 800ng/m L in column 1 to 0.781ng/m L in column 11, 5% CO at 37 ℃, 5%₂The culture was carried out for 5 days.

Data acquisition and processing

Add 20 u L MTS solution into each well, place the plate into the cell culture box for further culture for 6 hours, read 490nm absorbance with microplate reader, average the reading, subtract Blank (Blank, the average of 12 th row reading), make semi-log curve with antibody concentration/OD, calculate ED with 4 Parameter fitting method (Four Parameter L g) method₅₀。

Discussion of results

ED of aCD3-SH₅₀18.9ng/m L (FIG. 8, open circle), ED of prototype antibody OKT3₅₀Is 130ng/m L (FIG. 8, true)Heart circle), indicating that the target cell T cells are more sensitive to aCD 3-SH. At the same time, the maximum amplification signal for aCD3-SH was also significantly stronger than that of OKT3 at saturating antibody concentrations. The results show that the engineered antibody aCD3-SH has significantly higher activity in priming T cell expansion.

Culture example 2

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

The assay stimulated proliferation of NKT cells (CD3+ CD56+) in human PBMCs with the engineered antibody aacd 3-SH obtained in engineered example 3 and its prototype antibody OKT3, respectively, and viability was compared by counting cells.

Material

Human venous blood, basal medium GT-T551(TaKaRa, WK551T), amplification medium (GT-T551, I L-21000 IU/m L), cell culture flask T75(CORNING, 430720)/T175(CORNING, 431080), IFN-gamma (Shanghai Kamao, recombinant human interferon gamma for injection).

Cell culture and detection method

Separating human PBMC cells with leukocyte separation liquid, adjusting cell density to 2E6/m L with basal medium, placing in culture flask, and culturing at 37 deg.C and 5% CO₂Culturing for 2 hr to allow monocytes to adhere, collecting the suspension cells, adjusting the cell concentration to 1E6/m L with a basal medium, adding recombinant human IFN-gamma (1000IU/m L), 5% CO at 37 deg.C₂Culturing for 24 hours, adding an anti-human CD3 antibody (30ng/m L) and I L-2 (1000IU/m L), continuing to culture for 48 hours, adding an amplification culture medium according to the volume ratio of 1:1, continuing to culture for 48 hours, sampling and counting every 48 hours, adjusting the cell density to 0.7E6/m L by using the amplification culture medium, continuing to culture, harvesting cells after culturing to 12-20 days, and counting.

Discussion of results

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

Culture example 3

NK cell specific amplification and purity detection

Blood samples provided by volunteers are taken in the test, the blood samples are evenly divided into 2 parts after PBMC is separated, modified antibodies aCD3-SH, aCD16-SH and aCD52-SH (test group) and prototype antibodies OKT3, aCD16-3G8 and Campath-1H (control group) obtained by modification are respectively used for culturing, the proliferation of NK cells (CD3-CD16+ CD56+) in human PBMC is stimulated, and the functions of the antibody groups are compared by analyzing cell components.

Material

Human venous blood, basal medium X-VIVO15 (L onza, 04-418Q), amplification medium (X-VIVO15, I L-21000 IU/m L), human serum (Genimi, 100-.

Cell culture and detection method

PBMC cells were isolated and cell density was adjusted to 1E6/m L with basal medium test or control antibody, I L-2 (1000IU/m L), human serum (5%). 5% CO was added at 37 deg.C₂After 72 hours of culture, an equal volume of amplification medium was added and culture was continued for 48 hours, after which time samples were taken and counted every 48 hours, the cells were diluted with amplification medium to 0.7E6/m L and cultured further, cells were harvested by culture to day 14 and flow cytometric assays were performed with the 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 proportion of CD3-CD56+ in the cells obtained by the expansion of the test group was 97.6% (FIG. 10A), which is higher than 71.8% (FIG. 10B) obtained by the control group. Among this population of cells, the NK cell occupancy ratio of CD3-CD16+ CD56+ was 96.2% (FIG. 10C) and 71.7% (FIG. 10D), respectively. Then, among the total cell population, the NK cells of CD3-CD16+ CD56+ obtained by the expansion of the test group could 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 experimental group antibodies is higher.

Application of cell product

Application example 1

In-vitro killing activity detection of NK cell product on tumor cells

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

Raji cell (ATCC, CC L-86), BT-474 cell (ATCC, CR L-3247), MCF7 cell (ATCC, HTB-22), and detection kit CytoTox

Non-Radioactive cytoxicity Assay (Promega, G1780), medium RPMI 1640(ThermoFisher, 11879020), Rituximab, Trastuzumab Trastuzumab.

Detection method

Subculturing tumor cells, collecting cells, adjusting the density to 1E5/m L with a culture medium, adding 100 μ L to each well of a 96-well plate, collecting NK cells, adjusting the density to 1E5/m L or 5E5/m L with a culture medium, adding 100 μ L to the corresponding well, adjusting the concentration of Rituximab Rituximab to 2mg/m L with a culture medium, adding 5 μ L to the corresponding well, adjusting the concentration of Trastuzumab Trastuzumab to 2mg/m L with a culture medium, adding 5 μ L to the corresponding well, preparing a cell spontaneous release well (containing only Raji, BT-474, MCF7 or NK one cell), a target cell maximum release well (containing only one of Raji, BT-474 or MCF7 cells), and a volume correction well (containing no cell) according to kit instructions.

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, where NK cells 100 μ L of 1E5/m L, i.e., E/T ═ 1:1, were added for killing tests of Raji cells, and NK cells 100 μ L of 5E5/m L, i.e., E/T ═ 5:1, were added for killing tests of BT-474 and MCF7 at 37 ℃ with 5% CO₂Culturing for 3 hr, adding 20 μ L lysis solution to the maximum release hole and volume correction hole of target cell(L sis Solution.) the incubation was continued for 45 minutes.

Transfer 50 μ L supernatant from each well to a new 96-well plate, prepare Substrate Solution according to assay kit method, add 50 μ L Substrate Solution (Substrate Solution) to each well, incubate in the dark for 30min at room temperature, add 50u L Stop Solution (Stop Solution) to each well, read 490nm absorbance on a plate reader (Molecular Devices, spectra Max 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 assay on Raji cells are shown in fig. 11. It can be seen that the NK cells had a basic direct killing on Raji cells at E/T ═ 1:1, but the killing rates of the two groups were not very different. After addition of Rituximab, NK cells were activated and the killing rate of NK in the test group was increased from 14.7% to 85.0%. The control group NK was also activated, but only increased from 12.5% to 56.2%, with a clear difference from the test group. ADCC killing is specific, NK cells cannot be activated when Trastuzumab is added, and the direct killing rate of two groups of NK cells under the condition is not very different.

Results of the killing assay on BT-474 and MCF7 cells are shown in FIGS. 12 and 13. As can be seen, 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 in both direct killing and ADCC killing. Similarly, the killing rate of the test group NK cells on MCF7 cells is obviously superior to that of the control group in both direct killing and ADCC killing.

Application example 2

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

The test determines the in vivo viability of NK cells by performing an in vivo killing test on a mouse Raji-L uc hemangioma model with NK cell products obtained by expansion of the panel of culture example 3.

Material

NSG mice (6-8 weeks old), Raji-L uc cells (Raji cells containing stably transfected luciferase).

Detection method

Raji-L uc cells were subcultured, collected, adjusted to density of 5E6/m L with PBS, injected 100 μ L via tail vein into each NSG mouse, mice were randomized into 2 groups 48 hours later based on body weight, and tumor growth was measured on an in vivo imager (PerkinElmer, IVIS L ulina L T InVivo Imaging System) by tail vein injection of physiological saline 100 μ L (control group) and NK cells 1E7 (test group), respectively.

Discussion of results

In a 19-day experiment, the mean imaging signal intensity of the control mice increased to 9.16E6 p/sec/cm²The/sr, and the test group reached 1.63E6 p/sec/cm²The/sr is 17.8% of the control group (FIG. 14). The results show that NK cells have obvious killing activity in vivo.

Application example 3

Detection of ADCC killing activity of NK cell product on tumor cells in animal body

The NK cell viability was determined in vivo by the test using the NK cell product obtained by amplification of the test group in culture example 3 and the therapeutic antibody by performing an in vivo ADCC killing test on a mouse Raji-L uc hemangioma model.

Material

NSG mice (6-8 weeks old), Raji-L uc cells (Raji cells containing stably transfected luciferase), Rituximab Rituximab.

Detection method

After establishing a mouse Raji-L uc blood tumor model according to the method of application example 2, dividing the model into 4 groups, injecting physiological saline (G1, blank group), rituximab (G2), NK cells (G3) and combination drug (G4, namely simultaneously inputting rituximab and NK cells) respectively, continuously feeding, measuring the growth condition of the tumor at regular intervals, and observing the growth and survival state of the animals.

Discussion of results

From the survival results (fig. 15) and the tumor image results (fig. 16) of the mice in each group, it was seen that the fluorescence signal derived from the tumor cells gradually increased and the mice gradually died. The G1 group reached median survival on day 26 and all died at day 30. In group G2, median survival was achieved at 51 days, with 2 surviving at the end of the 75 day trial. In the group G3, 3 survived at 75 days. The G4 group survived 6 at the last imaging test day 61 and 5 at day 75.

On the comparison of the overall survival rate and tumor imaging signals, the treatment effect data of the combined drug group is obviously superior to that of the blank group and the single drug group. The in vivo ADCC killing effect of the rituximab and the NK cells obtained by the invention is obvious when the rituximab and the NK cells are used in a combined way.

Application example 4

Clinical study of NK cell products on treatment of hepatocellular carcinoma

In the study, patients with advanced Hepatocellular Carcinoma (HCC) were selected, NK cell products prepared by the amplification method in culture example 3 were input, and short-term and long-term reactions of the patients were observed to preliminarily explore the safety and effectiveness of the treatment scheme using allogeneic high-purity human NK cells.

Material

NK cells are collected and cleaned to be prepared into cell products, the cell survival rate is greater than 90%, and the purity of the NK cells is greater than 90%.

Research method

After screening by enrollment and exclusion, the intended patient begins 1-2 courses of NK cell therapy, each course separated by 1-3 months. Each treatment course comprises 2 times of NK cell treatment, and the interval is 5-10 days. 1-2E 9 NK cells were infused into each treatment, and changes in patient signs and chief notes were recorded before and after infusion. Follow-up was started after the end of the first treatment period until 2 years after treatment or the patient was withdrawn from the study for various reasons.

Discussion of results

In a clinical trial approved by the ethical review committee of clinical trials, a total of 38 treatments were administered to 33 people. After NK cell infusion, most patients had no symptoms of discomfort, and a few patients (4 patients, 5.3% of total) had febrile reactions, and recovered the next day after the antipyretic treatment. The result shows that the high-purity NK cell product obtained by the invention can ensure the safety of homologous allogeneic cell therapy and has potential to solve the problem of low immunity of patients.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Claims

1. A method of cell culture comprising the steps of: adding the modified antibody into a culture system; the modified antibody is an antibody for cell culture, wherein the region outside the complementarity determining region is subjected to protein modification, and the affinity of the modified antibody for Fc receptor is lower than that of the antibody for cell culture, which is not subjected to protein modification.

2. The cell culture method of claim 1, wherein the protein is engineered by one or more of sequence substitutions, point mutations, sequence insertions, sequence deletions, glycosylation modifications, and chemical modifications.

3. The cell culture method according to claim 2, wherein the sequence replacement replaces the hinge region and the Fc fragment of the antibody for cell culture with the hinge region and the Fc fragment of an IgG2 antibody or an IgG4 antibody; the point mutation is to perform mutation on amino acids at one or more sites in a hinge region and an Fc segment of the antibody for cell culture; the sequence insertion is to insert the complementarity determining region of the cell culture antibody into the backbone of an IgG2 antibody, an IgG4 antibody, or other engineered antibody.

4. The cell culture method according to claim 2, wherein the antibody for cell culture is an IgG1 antibody, and the point mutation is a mutation of one or more amino acids selected from the group consisting of 228 th, 233 th, 234 th, 235 th, 236 th, 239 th, 250 th, 252 th, 254 th, 256 th, 257 th, 311 th, 318 th, 320 th, 322 th, 326 th, 327 th, 329 th, 330 th, 331 th, 332 th, 333 th, 428 th, 433 th and 434 th amino acids of the antibody for cell culture.

5. The cell culture method according to any one of claims 1 to 4, wherein the antibody for cell culture 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.

6. The cell culture method according to any one of claims 1 to 4, wherein the amino acid sequence of the heavy chain of the modified antibody is shown in SEQ ID No. 1; or the heavy chain amino acid sequence of the modified antibody is shown as SEQ ID NO. 2; or the heavy chain amino acid sequence of the modified antibody is shown as SEQ ID NO.3, and the light chain amino acid sequence of the modified antibody is shown as SEQ ID NO. 4.

7. The cell culture method according to any one of claims 1 to 4, wherein Fc receptors are present on the surface of a part of cells selected from one or more of lymphocytes, granulocytes, mast cells, DC cells, macrophages, monocytes and platelets in the culture system.

8. An engineered antibody for cell culture, wherein a region other than the complementarity determining region is protein engineered, wherein the affinity of the engineered antibody for Fc receptor is lower than that of the cell culture antibody without protein engineering.

9. A cell culture medium comprising a basal medium and the engineered antibody of claim 8.

10. A cell product obtained by the cell culture method according to any one of claims 1 to 7.

11. Use of the cell product of claim 10 for the preparation of medicaments and agents with a killing effect on tumor cells.

12. A medicament for antiviral and cancer therapy, comprising the cell product of claim 10.

13. The medicament of claim 12, wherein the cancer comprises hepatocellular carcinoma, lung cancer, lymphoma, breast cancer, gastric cancer, head and neck cancer, and colorectal cancer.

14. A medicament for allogeneic cell therapy, comprising the cell product of claim 10.