CN110623921A - Bispecific antibody injection preparation for resisting CD3 and CD19 - Google Patents

Abstract

The invention relates to the field of biomedicine, and particularly provides an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation which comprises a drug effect molecule and an injection, wherein the drug effect molecule is an anti-CD 3 and anti-CD 19 bispecific antibody, and the injection comprises: buffer salts, isoosmotic adjusting agents, surfactants. The invention keeps the biological activity of the anti-CD 19 antibody and the anti-CD 3 antibody, better identifies tumor cells and effector cells, targets the effector cells to the tumor cells, achieves the specific killing effect and has higher killing capacity; the light chain and the heavy chain of the single-chain unit are connected through a linker, so that the mismatching between the light chain and the heavy chain in the bispecific antibody is avoided; the surfactant, the solution buffering agent and the osmotic pressure regulator are added into the injection for interaction and synergistic cooperation, so that a good storage environment is provided for the double antibody, the generation rate of antibody aggregates in the preparation is effectively reduced, the physical stability of the antibody is improved, and the potential safety risk is reduced.

Description

Bispecific antibody injection preparation for resisting CD3 and CD19

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a bispecific antibody injection preparation for resisting CD3 and CD 19.

Background

Lymphoma is a malignant tumor of origin in the lymphohematopoietic system, ranked seventh among common tumors, accounting for 3-4% of all tumors. It includes two main categories: hodgkin lymphoma and non-hodgkin lymphoma. The 5-year survival rate of hodgkin lymphoma in the united states is 85%, the non-hodgkin lymphoma is 69%, and in china, non-hodgkin lymphoma has increased at a rate of 5% per year, with an incidence of 6.43/10 ten thousand rising from 2/10 ten thousand in the last century, while nearly 60 ten thousand new cases and about 30 ten thousand patient deaths occur globally in 2012. Depending on the different subtypes of lymphoma, it is not uncommon to develop effective treatment regimens. With the continuous research on the biological characteristics of lymphoma, the 5-year survival rate of patients is effectively improved by applying the molecular targeted drug. Therefore, the search for new tumor markers and the research on the mechanism of the tumor markers in the lymphoma are of crucial significance.

With the continued development of biological drugs, therapeutic antibodies have become the primary drug of choice for patients with cancer, autoimmunity, inflammation, and various other diseases. However, monoclonal antibodies have limitations, and these antibodies are directed against a single target, and many patients do not respond adequately to a single therapy, and may develop resistance or no response. Cancer and other diseases are multifactorial diseases, and a variety of signaling pathways are involved in disease development. Single target immunotherapy does not appear to be sufficient to destroy cancer cells. Bispecific antibodies (biabs) are artificial antibodies containing two specific antigen binding sites, capable of specifically recognizing and binding two different antigens or epitopes, which can bridge between target cells and functional molecules (cells), produce a targeted effector function, redirect specific immune cells to tumor cells to enhance killing of tumors, or block two different mediators/pathways simultaneously to exert unique or overlapping functions.

Currently, there are only three bispecific antibody products approved for marketing worldwide, one is cataxomab developed by trion pharma, which is capable of targeting tumor surface antigen EpCAM and T cell surface receptor CD3, another is Blinatumomab developed by Micromet and Amgen, which binds to both CD19 and CD3, and the last is emilizumab developed by genetag, which binds to both coagulation factor IXa and coagulation factor X. The former two reach the purpose of treating tumor by activating and recruiting killer T cells, but the half-life of the drug effective molecules of Blinatumomab is short, and the preparation is lyophilized powder preparation, the process of the lyophilized preparation is complex and difficult to control, the stability is poor, the antibody is easy to form polymer and lose activity, in addition, the lyophilized preparation has long consumption time, the lyophilized equipment needs to be configured, the floor area of a workshop and the number of production personnel are increased, and the production cost is high. In addition, the freeze-dried powder needs to be resuspended before each administration, the use is troublesome, and the activity of the protein after freeze-drying can not be ensured. Therefore, at present, there is an urgent need for an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation and a preparation method thereof, wherein the injection preparation can simultaneously combine a CD3 antigen on an immune T cell and a CD19 antigen on a tumor cell, has long half-life time and long storage life of a drug molecule, can ensure the quality of a traditional Chinese medicine in the long-term storage process of the drug, and has safe and reliable preparation process, simple formula and convenient injection.

Disclosure of Invention

In order to solve the problems in the prior art, the invention discloses an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation, which has high affinity of drug molecules, long half-life, long-term stability of the preparation, convenient use and low production cost.

The specific technical scheme of the invention is as follows:

the invention provides an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation, which comprises a pharmacodynamic molecule and an injection, wherein the pharmacodynamic molecule is an anti-CD 3 and anti-CD 19 bispecific antibody with the protein content of 0.01-10mg/ml, and the injection comprises the following components in percentage by weight:

5-100mM of buffer salt;

isoosmotic adjusting agent 20-300 mM;

0.005-0.2% (w/v) of a surfactant;

wherein the pH value of the injection is 5.0-6.5.

The anti-CD 3 and anti-CD 19 bispecific antibody injection preparation provided by the invention not only considers the effectiveness of the medicament, but also pays attention to the characteristic analysis, similarity and homogeneity of the product in the aspect of quality control, and under the condition of required medicament concentration, preparation, stability and other aspects, the invention screens out the specific content ranges of different components through the interaction and the synergistic coordination of buffer salt, isoosmotic adjusting agent and surfactant in the injection and a large number of times of experimental investigation, thereby providing a good storage environment for the anti-CD 3 and anti-CD 19 bispecific antibody, effectively reducing the generation rate of the aggregate of the antibody in the preparation, improving the physical stability of the antibody, reducing the potential safety risk and effectively ensuring the long-term stability of the preparation in the long-term storage and transportation process.

Further, the anti-CD 3 and anti-CD 19 bispecific antibody comprises a single chain unit capable of specifically binding to the CD3 antigen and a monovalent unit capable of specifically binding to the CD19 antigen, both of which comprise a light chain and a heavy chain, the light chain C-terminus of the single chain unit being linked to the heavy chain N-terminus thereof by a linker; the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region.

The bispecific antibody provided by the invention can specifically bind to a T cell surface antigen cluster of differentiation 3, namely a CD3 molecule, and a tumor surface antigen cluster of differentiation 19, namely a CD19 molecule, the anti-CD 19 antibody adopts a complete antibody light and heavy chain combination mode, the biological activity of the anti-CD 19 antibody is kept, the C terminal of the antibody light chain of the anti-CD 3 is connected with the N terminal of the heavy chain through a linker, the binding capacity of the antibody and a CD3 antigen is kept, and the mismatching between the light chain and the heavy chain of the anti-CD 19 antibody and the anti-CD 3 antibody is avoided; the bispecific antibody can better recognize two different target antigens, thereby activating immune cells, targeting the immune cells to tumor cells, improving the concentration of local immune cells, and enhancing the killing capability of the immune cells to the tumor cells. Finally, the single-chain unit and the monovalent unit both comprise complete light chains and heavy chains, and the Fc fragment is reserved, so that the half-life period of the pharmacodynamic molecule can be effectively prolonged.

Further, the amino acid sequence of the linker is (GGGGX) n, wherein X is Gly or Ser, and n is a natural number of 1-6;

preferably, X is Ser;

preferably, n is 6.

In order to avoid the mismatching of the anti-CD 3 antibody light and heavy chains and the anti-CD 19 antibody light and heavy chains, the anti-CD 3 antibody light and heavy chains are connected through the special structural design of a linker, so that the stability of the molecular structure is ensured. Through a large number of experiments, the stability is best when X is Ser and n is 6.

Further, the amino acid sequence of the heavy chain variable region of the single-chain unit is SEQ ID NO 1; the amino acid sequence of the light chain variable region of the single-chain unit is SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the monovalent unit is SEQ ID NO 3; the amino acid sequence of the light chain variable region of the monovalent unit is SEQ ID NO 4.

Further, the heavy chain constant region of the single-chain unit and the heavy chain constant region of the monovalent unit are each one of IgG1, IgG2, IgG3, or IgG 4;

preferably, said heavy chain constant region of said single-chain unit and said heavy chain constant region of said monovalent unit are both IgG 4.

The invention further defines the chain constant region of the monovalent unit and the single chain unit, reserves the Fc fragment and effectively prolongs the half-life period of the drug effective molecules.

Further, the single-chain unit and the monovalent unit both contain a modified CH3 domain, and the modification of the CH3 domain is an asymmetric knob-and-hole structure.

The heterodimer structure formed by the linkage of the domains containing the modified CH3 is very stable, and the homodimer is difficult to form.

Further, the buffer salt comprises one or more of sodium citrate, L-histidine hydrochloride and sodium acetate;

preferably, the buffer salt is present in the formulation in an amount of 10-20 mM.

The buffer salt is mainly used for adjusting the pH value of the injection and simultaneously can provide a proper storage environment for the antibody.

Further, the isoosmotic adjusting agent comprises one or two combinations of protein protective agents and stabilizing agents, the protein protective agents comprise one or more combinations of sucrose, mannitol and trehalose, and the stabilizing agents comprise one or more combinations of arginine hydrochloride, glycine and proline;

preferably, the isoosmotic adjusting agent also comprises sodium chloride, and the content ratio of the sodium chloride to the protein protective agent to the stabilizing agent is 1: 2;

preferably, the content of the isoosmotic adjusting agent in the preparation is 50-200 mM.

The osmotic regulator can regulate the osmotic pressure of the injection, and in order to meet the requirement of the bispecific antibody in a pharmacodynamic molecule, the invention selects one or more combinations of sodium chloride, sucrose, mannitol, trehalose, arginine hydrochloride, glycine and proline, so as to ensure the stability of the preparation.

Further, the surfactant comprises polysorbate 20 and/or polysorbate 80;

preferably, the surfactant is present in the formulation in an amount of 0.01-0.02% (w/v).

The surfactant selected in the invention can ensure the activity of medicinal effective molecules in the injection, the use of the surfactant can play a role in dispersing the medicinal effective molecules in the injection, the polysorbate 20 and/or polysorbate 80 are/is selected in the invention, the content is 0.01-0.02% (w/v), the dispersibility is better, the surfactant content is too low to play a role in dispersing, and the surfactant content is too high to possibly generate side effects and influence the safety of preparation infusion.

The invention also provides application of the bispecific antibody injection preparation for resisting CD3 and CD19 in preparing a medicament for treating malignant tumor diseases of a B lymphocyte system.

The invention has the following beneficial effects: the drug effect molecule of the invention is a bispecific antibody which can be specifically combined with a T cell surface antigen cluster 3, namely CD3 molecule, and a tumor surface antigen cluster 19, namely CD19 molecule, and completely retains the biological activities of an anti-CD 19 antibody and an anti-CD 3 antibody, so that the bispecific antibody can better identify tumor antigen and effector cells, target immune cells to tumor cells, achieve specific killing effect and stronger killing capability, and in addition, the invention also completely retains an Fc fragment, can effectively prolong the half-life period of the drug molecule, improve the solubility and stability of the antibody, simultaneously, a light chain and a heavy chain of a single-chain unit of the anti-CD 3 antibody are connected through a linker, thereby effectively avoiding direct mismatch between the light chain and the heavy chain of the anti-CD 3 antibody and the anti-CD 19 antibody macromolecular structure, and solving the difficulty in the purification process, provides a solid foundation for the later-stage scale-up production of the bispecific antibody;

the injection disclosed by the invention has the advantages that the surfactant, the solution buffering agent and the osmotic pressure regulator are added into the injection, and are cooperated with each other, so that a good storage environment is provided for the anti-CD 3 and anti-CD 19 bispecific antibody, the generation rate of an aggregate of the antibody in the preparation can be effectively reduced in the processes of storage and transportation, the physical stability of the antibody is improved, and the potential safety risk is reduced; in addition, compared with a freeze-dried preparation, the bispecific antibody injection preparation provided by the invention has the advantages of simple preparation process, low cost, no need of special equipment and harsh conditions, easiness in realization of large-scale production and the like, has strong practical value, has significant progress compared with the prior art, and is lack of stable preparations of bispecific antibodies against CD3 and CD19 in the current market.

Drawings

FIG. 1 is a schematic diagram of the molecular structure of an anti-CD 3 and anti-CD 19 bispecific antibody provided by the invention;

FIG. 2 is a plasmid vector map of PTSE-anti CD19H in example 30 of the present invention;

FIG. 3 is a plasmid vector map of PTSE-anti CD19L in example 30 of the present invention;

FIG. 4 is a plasmid vector map of PTSE-anti CD3 in example 30 of the present invention;

FIG. 5 is an SDS-PAGE electrophoresis of the purified bispecific antibody in example 31 of the present invention;

FIG. 6 is a graph showing the binding capacity of bispecific antibody to Raji cells in the first experiment of the present invention;

FIG. 7 is a graph showing the binding capacity of bispecific antibodies to Jukart cells in experiment one of the present invention;

FIG. 8 is a graph showing the potency of bispecific antibody mediated killing of Raji cells by PBMC in experiment two of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples.

Examples 1 to 9

The embodiments 1-9 of the present invention respectively provide an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation, which comprises a pharmacodynamic molecule and an injection, wherein the pharmacodynamic molecule is an anti-CD 3 and anti-CD 19 bispecific antibody with a protein content of 2 mg/ml;

the anti-CD 3 and anti-CD 19 bispecific antibodies comprise a single chain unit capable of specifically binding to the CD3 antigen and a monovalent unit capable of specifically binding to the CD19 antigen, both of which comprise a light chain and a heavy chain, the C-terminus of the light chain of the single chain unit being linked to the N-terminus of its heavy chain by a linker; the heavy chain includes a heavy chain variable region and a heavy chain constant region, and the light chain includes a light chain variable region and a light chain constant region.

The amino acid sequence of the linker is (GGGGX) n, wherein X is Gly or Ser, and n is a natural number of 1-6;

preferably, X is Ser;

preferably, n is 6.

The amino acid sequence of the heavy chain variable region of the single-chain unit is SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the single-chain unit is SEQ ID NO. 2;

SEQ ID NO 1 (amino acid sequence of heavy chain variable region of Single-chain Unit)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGY INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHY CLDYWGQGTTLTVSS；

SEQ ID NO 2 (amino acid sequence of light chain variable region of Single-chain Unit)

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTS KVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK 。

The amino acid sequence of the heavy chain variable region of the monovalent unit is SEQ ID NO 3; the amino acid sequence of the variable region of the light chain of the monovalent unit is SEQ ID NO 4.

SEQ ID NO 3 (amino acid sequence of heavy chain variable region of monovalent unit)

QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQ IWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTV GRYYYAMDYWGQGTTVTVSS；

SEQ ID NO 4 (amino acid sequence of light chain variable region of monovalent unit)

DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIY DASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKL EIK。

The heavy chain constant region of the single chain unit and the heavy chain constant region of the monovalent unit are each one of IgG1, IgG2, IgG3, or IgG 4; preferably, the heavy chain constant region of the single chain unit and the heavy chain constant region of the monovalent unit are both IgG 4.

Both the single-chain unit and the monovalent unit contain a modified CH3 domain, and the modification of the CH3 domain is an asymmetric knob-and-hole structure.

The components and contents of the injections in the preparations provided in examples 1 to 9 are shown in table 1.

TABLE 1 EXAMPLES 1-9 the contents of the ingredients in the injection

Examples 10 to 20

Examples 10 to 20 of the present invention provide an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation, respectively, the preparation comprising a drug effect molecule and an injection, the drug effect molecule being an anti-CD 3 and anti-CD 19 bispecific antibody with a protein content of 1.2 mg/ml;

the anti-CD 3 and anti-CD 19 bispecific antibodies comprise a single chain unit capable of specifically binding to the CD3 antigen and a monovalent unit capable of specifically binding to the CD19 antigen, both of which comprise a light chain and a heavy chain, the C-terminus of the light chain of the single chain unit being linked to the N-terminus of its heavy chain by a linker; the heavy chain includes a heavy chain variable region and a heavy chain constant region, and the light chain includes a light chain variable region and a light chain constant region.

The amino acid sequence of linker is (GGGGS)₆。

The amino acid sequence of the heavy chain variable region of the single-chain unit is SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the single-chain unit is SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the monovalent unit is SEQ ID NO 3; the amino acid sequence of the variable region of the light chain of the monovalent unit is SEQ ID NO 4.

Both the heavy chain constant region of the single chain unit and the heavy chain constant region of the monovalent unit are IgG 4.

Both the single-chain unit and the monovalent unit contain a modified CH3 domain, and the modification of the CH3 domain is an asymmetric knob-and-hole structure.

The components and contents of the injections in the preparations provided in examples 10 to 20 are shown in table 2.

TABLE 2 contents of ingredients in examples 10 to 20

Examples 21 to 28

Examples 21 to 28 of the present invention provide an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation, respectively, which comprises a drug effect molecule and an injection, wherein the drug effect molecule is an anti-CD 3 and anti-CD 19 bispecific antibody with a protein content of 5 mg/ml;

the anti-CD 3 and anti-CD 19 bispecific antibodies comprise a single chain unit capable of specifically binding to the CD3 antigen and a monovalent unit capable of specifically binding to the CD19 antigen, both of which comprise a light chain and a heavy chain, the C-terminus of the light chain of the single chain unit being linked to the N-terminus of its heavy chain by a linker; the heavy chain includes a heavy chain variable region and a heavy chain constant region, and the light chain includes a light chain variable region and a light chain constant region.

Amino acids of linkerThe sequence is (GGGGS)₆。

The amino acid sequence of the heavy chain variable region of the single-chain unit is SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the single-chain unit is SEQ ID NO. 2;

the amino acid sequence of the heavy chain variable region of the monovalent unit is SEQ ID NO 3; the amino acid sequence of the variable region of the light chain of the monovalent unit is SEQ ID NO 4.

Both the heavy chain constant region of the single chain unit and the heavy chain constant region of the monovalent unit are IgG 4.

Both the single-chain unit and the monovalent unit contain a modified CH3 domain, and the modification of the CH3 domain is an asymmetric knob-and-hole structure.

The components and contents of the injections in the preparations provided in examples 21 to 28 are shown in table 3.

TABLE 3 contents of ingredients in the injections of examples 21 to 28

Example 29

The embodiment 29 of the invention provides application of an anti-CD 3 and anti-CD 19 bispecific antibody injection preparation in preparing a medicament for treating malignant tumor diseases of a B lymphocyte system.

Example 30 construction of expression vectors for bispecific antibody molecules

Corresponding gene sequences were designed according to any of examples 1 to 28 with reference to the structural form of the bispecific antibody shown in FIG. 1, pTSE was selected as an expression vector, the antibody genes were synthesized by Zhongmeitai and Biotechnology (Beijing) Ltd, Sall and BamHI cleavage sites were introduced on both sides of the light chain of the anti-CD 19 antibody, the heavy chain of the anti-CD 19 antibody and the single chain gene of the anti-CD 3, then carrying out Sall and BamHI double enzyme digestion on both the pTSE expression vector and the synthesized gene, and the enzyme digestion products of the vector and the gene are subjected to agarose gel electrophoresis and target fragment recovery, and finally the recovered target fragments are respectively connected to the pTSE expression vector, transforming into TOP competence (Huitian Orient, cat number HT702-03), obtaining expression vector containing light chain, heavy chain and single chain gene after correct sequencing (as shown in figure 2-4), and respectively naming the plasmids as: pTSE-anti CD19L, pTSE-anti CD19H and pTSE-anti CD 3. The plasmid is greatly extracted by using an endotoxin-free large extraction kit (kang century, CW2104), the specific operation steps are operated according to the instructions provided by the kit, and the plasmid is stored at the temperature of minus 20 ℃ after the concentration of the plasmid is finally determined.

Example 31 expression and purification of bispecific antibody molecules

1) Expression of bispecific antibodies

Human embryonic kidney cells (HEK293ES suspension cells) were cultured in FreeStyle 293Expression Medium (Gibco) and passaged every other one to two days with the initial cell density maintained at 0.2-0.6X 10⁶The cell/ml, the cell culture volume is 15-35% of the volume of the shake flask, the cell culture flask is placed in a shaking table (shaking table rotation speed: 135rpm, temperature: 37 deg.C, CO)₂5%) in the culture medium. HEK293ES cells in logarithmic growth phase and good growth status were passaged to a cell density of 0.5X 10 the day before transfection⁶Cells/ml, placed in a shaker (135rpm, 37 ℃, 5% CO)₂) Incubate overnight, wait for the next day for transfection.

1X 10 will be prepared before transfection⁶Cells/ml cell suspension in a shaker (135rpm, 39 ℃, 5% CO)₂) After culturing for 2h, pTSE-anti CD19L (final concentration 0.3. mu.g/ml), pTSE-anti CD19H (final concentration 0.3. mu.g/ml), pTSE-anti CD3 (final concentration 0.3. mu.g/ml) and polyethyleneimine PEI (final concentration 2. mu.g/ml) were added in this order and mixed well, and then CO-transfected into HEK293ES suspension cells, followed by placing in a shaker (135rpm, 39 ℃, 5% CO)₂) Culturing for 40 min. The transfected cells were continued at 135rpm, 37 ℃ and 5% CO₂The bispecific antibodies against CD3 and CD19 were expressed by culturing in a shaker. Supernatants were harvested by centrifugation 96 hours after transfection.

2) Purification of bispecific antibodies

The supernatant was filtered through a 0.22uM filter, and an antibody having an Fc domain was obtained from the expression supernatant by using an affinity column. The equilibration buffer and elution buffer were 50mM Tris-HCl, 0.15M NaCl pH7.0 and 0.1M citric acid-sodium citrate pH3.0, respectively. Target bispecific antibody (JY039) is obtained through cation exchange column HiTrap-SPFF, and finally, solution exchange concentration is carried out by PBS buffer solution. The purified bispecific reduced SDS-PAGE is shown in FIG. 5, the right side is bispecific antibody, which is anti-CD 3, anti-CD 19 antibody heavy chain, anti-CD 19 light chain from top to bottom, and the left side is protein molecular weight Marker; the results show that the purified bispecific antibody contains a single chain unit of anti-CD 3, a heavy chain of anti-CD 19 antibody, and a light chain of anti-CD 19, and the molecular weight size of each band is consistent with theory.

Comparative example 1

Comparative example 1 of the present invention is based on example 1, and further defines that the buffer salt is a mixture of 10mM sodium phosphate and 10mM sodium citrate, and the other components are all the same.

Comparative example 2

Comparative example 2 of the present invention is further defined in example 17 that the isotonic reagent is a mixture of 50mM sodium chloride, 50mM arginine hydrochloride and 50mM fructose, and the other components are all the same.

Comparative example 3

Comparative example 3 of the present invention further defines, on the basis of example 23, a mixture of polysorbate 80 and polysorbate 40 at a concentration of 0.01% (w/v) as surfactants, and the other ingredients are all the same.

Experimental Primary and bispecific antibody molecules binding to cells overexpressing CD19 or CD3

The experiment provided by the invention adopts an over-expressed lymphoma cell line Raji (purchased from ATCC, CCL-86) as a CD19 positive cell; the over-expressed T cell line Jurkat (Jurkat, TIB-152) was used as CD3 positive cells, and human IgG (hIgG) was used as an isotype control.

1. The binding activity of the bispecific antibody to Raji cells was tested by flow assay.

The binding condition of the bispecific antibody and cell surface CD19 is detected by using a lymphoma cell line (Raji) over expressing CD19, a monoclonal antibody is constructed by using an anti-CD 19 sequence of JY039, and the monoclonal antibody of the anti-CD 19, a v5851 bispecific antibody in a patent CN105531374A and a v1661 bispecific antibody in CN 106062206A are compared with the bispecific antibody JY039 purified in the example 31 of the invention by adopting the following steps:

enough Raji cells were cultured and centrifuged to collect the cells. The antibodies were diluted simultaneously, starting at 1. mu.M, in 5-fold gradient dilutions, to obtain 12 concentration gradients for use. The collected cells were washed three times with PBS + 1% FBS, and the cells were resuspended to 4X 10 with PBS + 1% FBS⁶Cells/ml, then plated in 96-well plates at 50ul (2X 10) per well⁵Individual cells), 50ul of diluted bispecific antibody was added, and incubation was performed at 4 ℃ for 1 hour; the supernatant was centrifuged off, the cells were washed twice with PBS, resuspended with diluted Alexa 488-labeled anti-human IgG-Fc antibody (Biolegend, 409304), incubated for 30 minutes at room temperature in the dark, washed three times with PBS, resuspended with 100ul PBS, the fluorescence intensity was detected with flow cytometer, and the corresponding EC50 value was calculated by calculating the binding affinity of each antibody to Raji cells with software graphpadprism5.0, using the average fluorescence intensity, as follows:

item	JY039	v5851	v1661	AntiCD19McAb	hIgG
						EC50(nM)	1.023	2.486	7.923	0.1910	—

From the above data and as shown in fig. 6, with a non-related antibody isotype human igg (higg) as a negative control, the anti-CD 3 and anti-CD 19 bispecific antibody JY039 obtained by purification in example 31 of the present invention had slightly weaker binding activity to CD19 positive Raji cells than the anti-CD 19 monoclonal antibody had to CD19 positive Raji cells, and the JY039 bispecific antibody had binding activity to Raji cells comparable to the v5851 bispecific antibody in patent CN105531374a, but the JY039 of the present invention had stronger activity than the v1661 bispecific antibody in CN 106062206 a. The bispecific antibody JY039 provided by the invention is proved to better maintain the activity of the parent antibody specifically binding with cell surface CD 19.

2. Detection of binding Activity of bispecific antibody with Jurkat cells by flow assay

The combination of bispecific antibody and cell surface CD3 is detected by using a T cell line (Jurkat) over expressing CD3, a monoclonal antibody is constructed by using an anti-CD 3 sequence of JY039, and the monoclonal antibody of the anti-CD 3, a v5851 bispecific antibody in a patent CN105531374A and a v1661 bispecific antibody in a patent CN 106062206A are compared with the bispecific antibody JY039 purified in the example 31 of the invention by adopting the following steps:

sufficient Jurkat suspension cells were cultured and harvested by centrifugation. Simultaneously diluting various antibodies, wherein the concentration is from 5 mu M, and diluting by 5 times of gradient to obtain 12 concentration gradients for later use. The following experiment was performed as described in the previous example, 100. mu.l of PBS-resuspended cells were tested on the machine, the binding affinity of the diabodies to Jurkat cells was calculated by analysis with the software GraphPadprism5.0, and the corresponding EC50 value was calculated as follows:

item	JY039	v5851	v1661	AntiCD3McAb	hIgG
						EC50(nM)	21.57	59.84	117.9	8.560	—

By the above data and as shown in fig. 7, with the unrelated antibody human igg (hIgG) as a negative control, the binding activity of the JY039 bispecific antibody obtained in example 31 of the present invention to CD3 positive Jurkat cells is slightly weaker than that of the monoclonal antibody against CD3 to CD3 positive Jurkat cells, but the binding activity of the JY039 bispecific antibody to CD3 positive Jurkat cells is stronger than that of the v5851 bispecific antibody in patent CN105531374A and the v1661 bispecific antibody in CN 106062206a to CD3 positive Jurkat cells, while the bispecific antibody JY039 provided by the present invention does not bind to the control (hIgG), so the bispecific antibody JY039 provided by the present invention preferably maintains the cell surface CD3 binding activity of the parent antibody.

Experiment II, killing effect of PBMC mediated by bispecific antibody molecule on Raji cell

a) PBMC isolation

Taking four SepMate separation tubes, adding about 15mL of human mononuclear cell separation solution by using a pipette to avoid generating bubbles, wherein the liquid level just submerges a bracket in the tube; carefully transferring the whole blood into two 50mL centrifuge tubes, wherein each centrifuge tube is 25mL, and adding 25mL PBS to mix evenly; slowly adding the whole blood diluted by 1:1 into a SepMate separation tube along the tube wall without damaging liquid layering; centrifuging at 1200g for 10 minutes; pipette the PBMC layer above the tray, transfer to a new 50mL centrifuge tube, fill volume to 50mL with PBS, centrifuge for 10 minutes at 1200 g; discarding the supernatant, adding 25ml PBS for resuspension, and centrifuging for 10 minutes at 1200 g; discarding the supernatant, adding 2mL of erythrocyte lysate, incubating at room temperature for 5 minutes, adding about 20mL of PBS, and centrifuging at 1200g for 10 minutes; resuspend and remove large clumps using a 40 μm cell sieve. Cells were counted in trypan blue and PBMC total recorded.

b) LDH detection

In 1640 medium (Gibico, A10491-01) + 10% FBS (VisTech, SE200-ES), 37 ℃, 5% CO₂Raji cultured in an incubator as a target cell, the inoculation density of which is 1 × 10⁴Per well, corresponding cells were diluted to 1X 10⁵Per mL, 100. mu.L of cell suspension was added per well; when the PBMC obtained by separation is used as effector cells, the ratio of effective targets is 25:1, and the PBMC is diluted to 5X 10⁶mL, 50 μ L of cell suspension per well; adding antibody drugs with gradient concentration into the experimental holes, wherein the initial concentration is 0.1 mu M, the gradient dilution is 5 times, 12 gradients are adopted, and 50 mu L of antibody diluted samples are added into each hole; in addition, a background group (target cells + effector cells) and a mixed maximum group (target cells + effector cells) were set at 37 ℃ and 5% CO₂After culturing in an incubator for 18h, adding 20 mu L of Lysis lysate into the maximum mixed hole, and incubating for 90 minutes at 37 ℃; after centrifugation at 3000rpm for 5 minutes, 50. mu.L of supernatant was taken and 50. mu.L of LDH substrate was added; incubate 20 minutes at room temperature in the dark, detect absorbance at 490nm, and calculate the corresponding EC50 value, the data are as follows:

cell killing rate (%) (experimental-background wells)/(mixed largest wells-background wells) × 100;

item

JY039

v1661

v5851

AntiCD19McAb

AntiCD3McAb

hIgG

EC50(uM)

1.26E-6

9.24E-5

3.00E-5

2.22E-4

—

—

As shown in fig. 8, the data and the results show that the ability of the JY039 bispecific antibody purified in example 31 of the present invention to kill target cells is significantly better than that of the bispecific antibodies (v5851, v1661) of patents CN105531374a and CN 106062206a, and is more significantly better than that of the CD19 monoclonal antibody and CD3 monoclonal antibody, which proves that the JY039 bispecific antibody provided by the present invention has significant killing effect on the basis of maintaining the binding ability of the JY039 bispecific antibody with a lymphoma cell line (Raji) over-expressing CD19 and with a T cell line (Jurkat) expressing CD 3.

The above examples demonstrate that the bispecific antibody of the present invention maintains the in vivo biological activities of the original anti-CD 19 antibody and anti-CD 3 antibody, and simultaneously targets the immune effector cells to tumor cells, thereby increasing the effect of the immune effector cells in killing tumor cells. The bispecific antibodies of the invention are in the form of single chain unit-monovalent units, which can help VH and VL form stable structures compared to ScFv, CH1 and CL, and thus are more structurally stable than ScFv forms. The monovalent unit and the single-chain unit are connected by introducing asymmetric heterodimer mutation design through a CH3/CH3 interface of IgG4, the structure is different from a common KiH structure, and the formed heterodimer is more stable and is more difficult to form homodimers. The bispecific antibody of the invention can be used for preparing medicines for treating abnormally expressed B lymphocyte related diseases and tumor related diseases, wherein the tumor related diseases are B cell malignant tumors positive to CD19 antigen.

Experiment III, stability experiment test

The preparation method of the antibody preparation comprises the following steps: the purified JY039 anti-CD 3 and anti-CD 19 bispecific antibody from example 31 was exchanged into the target injection through an ultrafiltration tube, the injection was prepared according to the different components set in examples 1-28 and comparative examples 1-3, and the concentrated sample was diluted to 2mg/ml to prepare 31 bispecific antibody preparations which were filled in 2ml penicillin bottles and 1 ml/bottle respectively.

Samples of the 31 bispecific antibodies prepared above, placed at 4 ℃ and 40 ℃ simultaneously, were removed at weeks 2 and 4 for analytical testing, including SEC-HPLC, SDS-PAGE and CEX-HPLC.

The analysis and detection method comprises the following steps: and (3) detecting purity and concentration: molecular size exclusion high performance liquid chromatography analysis and SDS-PAGE electrophoresis analysis; charge isomers: the charge isomer main peak content is determined by cation exchange chromatography.

(1) Effect of different buffer salts on formulation stability

The JY039 bispecific antibody preparations prepared in the above examples 1-9 and control example 1 were examined by SEC-HPLC method for protein purity and concentration after 2 weeks and 4 weeks at 40 deg.C, and the JY039 preparations prepared in the above examples 1-9 and control example 1 were examined by CEX-HPLC method for main peak content after 2 weeks and 4 weeks at 40 deg.C.

The rate of decline (%/week) was calculated as the purity and main peak content of the initial (0w), resting 2w and resting 4w of the test; the stability data are summarized in table 4 below.

Table 4 summary of stability data for bispecific antibodies provided in examples 1-9 and control 1 at different pH values

From the above data, it can be seen that the stability of the preparation provided by the present invention is much higher than that of comparative example 1, and compared with comparative example 1, in examples 2, 5 and 8 of the present invention, in the same pH environment, after 4 weeks, the buffer solution added in the antibody preparation provided by the embodiment 2 of the invention is sodium citrate, and the purity and the main peak change are small and the stability is best when the pH value is 6.0, therefore, it can be shown that any addition of one buffer component in the preparation provided by the present invention may result in a decrease in the amount of the active ingredient and a decrease in the stability, and that examples 1 to 3, examples 4 to 6, and examples 7 to 9 show that the present invention has a higher stability at a pH of 5.5 to 6.0, therefore, the pH value of the bispecific antibody preparation provided by the invention is preferably 5.5-6.0.

(2) Effect of different isoosmotic Agents on formulation stability

The JY039 bispecific antibody preparations prepared in the above examples 10-20 and the control example 2 were examined by SEC-HPLC method for protein purity and concentration after 2 weeks and 4 weeks at 40 ℃, and the JY039 preparations prepared in the above examples 10-20 and the control example 2 were examined by CEX-HPLC method for main peak content after 2 weeks and 4 weeks at 40 ℃.

The rate of decline (%/week) was calculated as the purity and main peak content of the initial (0w), resting 2w and resting 4w of the test; the stability data are summarized in table 5 below.

TABLE 5 stability data summary of bispecific antibodies under different isotonicity modulators provided in examples 10-20 and control example 2

As can be seen from Table 5, combining the results of SEC-HPLC and CEX-HPLC, the purity and the main peak content of comparative example 2 decreased faster and less stable after being placed at 40 ℃ for 2 weeks and 4 weeks by comparative example 2 compared with other examples by adding different isoosmotic adjusting agents at pH6.0, the surfactant added in other examples was more stable than comparative example 2, especially example 17, the purity and the main peak content of which were less changed, so that any replacement of the isoosmotic adjusting agents provided by the present invention would possibly result in decreased stability, whereas the bispecific antibody preparation provided by example 17 contains 50mM of sodium chloride and 100mM of arginine hydrochloride, which decreased less purity and main peak content after being placed for 4 weeks by comparing with examples 10-20, to illustrate the high stability, the preferred surfactants for this purpose are 50mM sodium chloride and 100mM arginine hydrochloride.

(3) Effect of different surfactants on formulation stability

JY039 bispecific antibody formulations prepared in examples 21-28 and control example 3 above were examined by SEC-HPLC method for protein purity and concentration after 2 weeks and 4 weeks at 40 deg.C, and for main peak content by CEX-HPLC method for JY039 formulations prepared in examples 21-28 and control example 3 above after 2 weeks and 4 weeks at 40 deg.C.

The rate of decline (%/week) was calculated as the purity and main peak content of the initial (0w), resting 2w and resting 4w of the test; the stability data are summarized in table 6 below.

Table 6 stability data for bispecific antibodies at different surfactants provided in examples 21-28 and control 3 are summarized

Table 6 shows that the surfactants provided by the present invention comprise polysorbate 80 and polysorbate 20, which are capable of ensuring the stability of the formulation provided by the present invention, and it is possible to show that any addition of one of the surfactants will result in a decrease in purity and main peak content of the formulation after 4 weeks, as compared with comparative example 3, and that examples 21 to 28 show that the preferred surfactant of the present invention is polysorbate 80, which has a smaller decrease in purity and main peak content after 4 weeks, that is, is relatively stable with respect to the selection of polysorbate 20, and examples 21 to 24 show that the preferred surfactant of the present invention is polysorbate 80, which has a smaller change in main peak content and purity when the surfactant selected in example 23 is polysorbate 80 and the content is 0.02% (w/v), and example 23 shows that the preferred surfactant of the present invention is polysorbate 80, and at a content of 0.02% (w/v), the formulation could remain stable after 4 weeks of storage, and the addition of one would likely result in a decrease in stability, and in addition, too low a content of the surfactant could not result in dispersion, and too high a content could cause side effects, affecting the safety of infusion of the formulation.

Therefore, the most stable injection liquid in the antibody preparation provided by the embodiment 23 of the present invention is selected from the stability tests of the above different embodiments, and comprises the following components: 20mM sodium citrate, pH6.0, 50mM NaCl, 100mM arginine hydrochloride, 0.02% polysorbate 80.

(4) Effect of different antibody protein concentrations on the formulations:

through the experimental screening verification, the JY039 bispecific antibody provided by the embodiment 23 of the invention is an optimal preparation formula, and the influence of different antibody concentrations on the stability is further researched. The main comparative protein concentrations were 0.005mg/ml, 0.01mg/ml, 1mg/ml, 2mg/ml, 5mg/ml, 8mg/ml, 10mg/ml, and the purity and main peak content were determined after 2 weeks and 4 weeks at 40 ℃, and the stability data are summarized in table 7 below.

TABLE 7 summary of stability data for bispecific antibodies at different antibody concentrations

As can be seen from Table 7, the antibody protein at the above concentrations had a high stability in purity and in the content of the main peak of the charge isomer, except for the concentration of 0.005 mg/ml. However, by contrast, the optimal antibody protein concentration provided by the invention is 1-2mg/ml, which is the optimal preparation concentration.

Experiment four, prescription verification experiment

The prescription verification test comprises a freeze-thaw stability test and a shaking stability test.

1) And (3) freeze-thaw stability test:

JY039 bispecific antibody exchange solutions provided by examples 23, 17 and 2 were concentrated to desired concentrations, and freeze-thaw stability tests were performed for 1, 2 and 3 times of freeze-thawing, respectively. The protein purity and the charge isomer main peak content were examined with emphasis and the results are summarized in Table 8.

TABLE 8 summary of antibody formulation freeze thaw stability results

2) And (3) shaking stability test:

JY039 bispecific antibody exchange solutions provided in examples 23, 17, 2 were concentrated to desired concentrations and subjected to shaking stability tests, horizontal shaking (80-120rpm) and circular shaking (30rpm) for 3 days at room temperature, respectively. We focused on protein purity and charge isomer main peak content and summarized the results in Table 9.

TABLE 9 summary of the results of the shaking stability test of the antibody formulations

From tables 8 and 9, it can be seen that from the investigation of protein purity and charge isomer main peak content, the purity of the JY039 antibody preparation provided in the embodiment of the invention can reach more than 97% after a freeze-thaw test and a shaking test, and the concentration of the pharmacodynamic molecule bispecific antibody is not changed greatly basically, which indicates that the preparation provided by the invention has good stability.

The present invention is not limited to the above-mentioned preferred embodiments, and any other products in various forms can be obtained by anyone in the light of the present invention, but any changes in the shape or structure thereof, which have the same or similar technical solutions as those of the present application, fall within the protection scope of the present invention.

Sequence listing

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Claims (10)

1. An anti-CD 3 and anti-CD 19 bispecific antibody injection preparation, which is characterized in that the preparation comprises a pharmacodynamic molecule and an injection, wherein the pharmacodynamic molecule is an anti-CD 3 and anti-CD 19 bispecific antibody with a protein content of 0.01-10mg/ml, and the injection comprises the following components in percentage by weight:

5-100mM of buffer salt;

isoosmotic adjusting agent 20-300 mM;

0.005-0.2% (w/v) of a surfactant;

wherein the pH value of the injection is 5.0-6.5.

2. The anti-CD 3 and anti-CD 19 bispecific antibody injection formulation of claim 1, wherein the anti-CD 3 and anti-CD 19 bispecific antibody comprises a single chain unit capable of specifically binding to the CD3 antigen and a monovalent unit capable of specifically binding to the CD19 antigen, both of which comprise a light chain and a heavy chain, the light chain C-terminus of the single chain unit being linked to the heavy chain N-terminus thereof by a linker; the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region.

3. The anti-CD 3 and anti-CD 19 bispecific antibody injection formulation of claim 2, wherein the linker amino acid sequence is (GGGGX) n, wherein X is Gly or Ser, and n is a natural number from 1 to 6;

preferably, X is Ser;

preferably, n is 6.

4. The anti-CD 3 and anti-CD 19 bispecific antibody injection formulation of claim 2, wherein the amino acid sequence of the heavy chain variable region of the single chain unit is SEQ ID NO: 1; the amino acid sequence of the light chain variable region of the single-chain unit is SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of the monovalent unit is SEQ ID NO 3; the amino acid sequence of the light chain variable region of the monovalent unit is SEQ ID NO 4.

5. The anti-CD 3 and anti-CD 19 bispecific antibody injection formulation of claim 2, wherein said heavy chain constant region of said single chain unit and said heavy chain constant region of said monovalent unit are each one of IgG1, IgG2, IgG3, or IgG 4;

preferably, said heavy chain constant region of said single-chain unit and said heavy chain constant region of said monovalent unit are both IgG 4.

6. The injectable anti-CD 3 and anti-CD 19 antibody formulation of claim 5, wherein the single chain unit and the monovalent unit both comprise a modified CH3 domain and the modification of the CH3 domain is an asymmetric knob and hole structure.

7. The anti-CD 3 and anti-CD 19 bispecific antibody injectable formulation of claim 1, wherein the buffer salt comprises a combination of one or more of sodium citrate, L-histidine hydrochloride, and sodium acetate;

preferably, the buffer salt is present in the formulation in an amount of 10-20 mM.

8. The anti-CD 3 and anti-CD 19 bispecific antibody injection formulation of claim 1, wherein the isoosmotic adjusting agent comprises one or a combination of two of a protein protectant comprising one or a combination of sucrose, mannitol, trehalose, and a stabilizer comprising one or a combination of arginine hydrochloride, glycine, proline;

preferably, the isoosmotic adjusting agent also comprises sodium chloride, and the content ratio of the sodium chloride to the protein protective agent to the stabilizing agent is 1: 2;

preferably, the content of the isoosmotic adjusting agent in the preparation is 50-200 mM.

9. The anti-CD 3 and anti-CD 19 bispecific antibody injectable formulation of any one of claim 1, wherein the surfactant comprises polysorbate 20 and/or polysorbate 80;

preferably, the surfactant is present in the formulation in an amount of 0.01-0.02% (w/v).

10. The application of the bispecific antibody injection preparation of anti-CD 3 and anti-CD 19 in preparing medicines for treating malignant tumor diseases of B lymphocyte system.