CN107109363A - Strengthen the method and pharmaceutical composition to abnormal cell lethality - Google Patents

Strengthen the method and pharmaceutical composition to abnormal cell lethality Download PDF

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CN107109363A
CN107109363A CN201580001069.9A CN201580001069A CN107109363A CN 107109363 A CN107109363 A CN 107109363A CN 201580001069 A CN201580001069 A CN 201580001069A CN 107109363 A CN107109363 A CN 107109363A
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natural killer
killer cells
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high activity
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张明杰
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Abstract

The present invention discloses a kind of method that method for treating patient, pharmaceutical composition and a kind of enhancing are receiving the ADCC effects for the patient that antibody drug is treated.The method for the treatment of patient is included the step of NK cells and antibody drug are given in combination, alleged NK cells include HANK cells, and HANK cells are included：Utilize NK cells in the ghost Activated in Vitro body with cell factor.NK cells comprising HANK cells are combined with antibody drug and grant patient, the lethality to abnormal cell can be mutually reinforcing between HANK cells and antibody drug, hence it is evident that enhancing therapeutic effect.

Description

Enhance the method and pharmaceutical composition to abnormal cell lethality Technical field
The present invention relates to biologic medical fields, specifically, the present invention relates to a kind of enhancings to the method and pharmaceutical composition of abnormal cell lethality.
Background technique
Natural kill (natural killer, NK) cell is the important immunocyte of body, not only related with antitumor, viral infection resisting and immunological regulation, but also participates in the generation of hypersensitivity and autoimmune disease in some cases.
The effect of NK cell is wide spectrum, nonspecific；For specific tumour cell, targeting is not strong；The same as rocket gun, some shells may be beaten less than on target, be wasted.
Monoclonal antibody (monoclonal antibody) targets medicine, the including but not limited to Trastuzumab of the rituximab for the treatment of leukaemia and treatment breast cancer, has clinically used for many years, has achieved preferable curative effect.
The cure mechanism of these monoclonal antibody medicines is mainly to pass through the lethal effect for mediating NK cell, i.e., antibody-mediated cytotoxic effect (Antibody dependent cytotoxicity, ADCC).But since the NK cell activity of cancer patient itself is often all relatively low, after the conventional therapies such as chemotherapy or radiotherapy, the activity of NK cell would generally will limit naturally the curative effect of monoclonal antibody targeting medicine by serious damage in this way.
How disease effectively or more effectively is treated using NK cell and antibody drug, had great importance.
Summary of the invention
The present invention is directed to solve one of above-mentioned technical problem at least to a certain extent or provide a kind of business selection.
One side according to the present invention provides a kind of method for treating patient.The patient suffers from disease, the method includes NK cell and antibody drug are given in combination, the NK cell includes high activity NK (HANK) cell, and the acquisition of the HANK cell includes: to utilize the amplifying activated NK cell ex vivo in vitro of the ghost with cell factor.
So-called HANK cell is that inventor is obtained by the NK cell of cultured and amplified in vitro activation ex vivo.It should be noted that so-called HANK cell, non-generic internal NK cell.Not only quantity is few for common NK cell, and is generally in holddown, kill abnormal cell activity it is low, such as killing cancer cell or virus infected cell activity it is low.In addition, the present invention to the antibody drug being given in combination with no restriction, can be the medicine of existing monoclonal antibody targeting currently on the market, monoclonal antibody targeted drug newly developed after being also possible to.
It should be noted that, the cell factor that the ghost has, it can be the cell factor that cell itself is naturally expressed on its surface, it is also possible to pass through gene engineering method, such as using transiently transfecting or stablizing expression for specific cells factor expression in cell surface, it can also be the cell factor adsorbed or be crosslinked in cell surface.In addition, the cell for being used to prepare ghost can be primary cell, such as peripheral blood mononuclear cells (PBMC), or passage cell, such as K562 cell etc..
Utilize the method for this aspect of the invention, NK cell and antibody drug comprising HANK cell are combined and grant patient, the curative effect of NK cell and antibody drug can be made to be mutually reinforcing, specific manifestation includes: that (1) antibody drug assigns HANK cell-specific, for example being used in combination with Rituximab medicine can allow HANK cell specially to kill leukaemia cell and Trastuzumab monoclonal antibody medicine is used in combination that HANK cell can be allowed specially to kill breast cancer cell；(2) HANK cell can provide enough active factors, such as a large amount of granzyme, perforin etc. that can effectively kill cancer cell for monoclonal antibody medicine.
According to an embodiment of the invention, the method for this aspect of the invention can also at least have one of following additional technical feature:
The disease that so-called patient suffers from is at least one of cancer, virus infection and immunological diseases.According to one embodiment of present invention, so-called patient is cancer patient, and so-called antibody drug is monoclonal antibody targeted drug.Monoclonal antibody targeted drug assigns HANK cell-specific, for example being used in combination with Rituximab medicine can allow HANK cell specially to kill leukaemia cell and Trastuzumab monoclonal antibody medicine is used in combination that HANK cell can be allowed specially to kill breast cancer cell；HANK cell provides enough active factors, such as a large amount of granzyme, perforin etc. that can effectively kill cancer cell for the monoclonal antibody medicine.HANK cell and monoclonal antibody targeted drug is used in combination, curative effect can be increased mutually with preferably treating cancer.
The antibody drug is monoclonal antibody or is polyclonal antibody.Two antigen-binding sites on one antibody molecule can be identical, is also possible to different, i.e. bispecific antibody, claims antigen-binding fragment (antigen-binding fragment, Fab) positioned at two-arm end.According to one embodiment of present invention, so-called antibody drug is complete monoclonal antibody medicine.
A preferred embodiment according to the present invention, the ratio of HANK cell and antibody drug in the NK cell being given in combination are 2 × 105To 5 × 105: 1 (a/μ g).When antibody drug is administered, doctor or drug specification can provide usage amount according to patient profiles, and in vitro test and animal experiment show the HANK cell that the ratio is given in combination when administration, can enhance the effect for the treatment of cancer significantly.
The present invention is for being used to the individual source of amplifying activated NK cell with no restriction.According to one embodiment of present invention, the HANK cell is obtained by a kind of at least following NK cell ex vivo of Activated in Vitro: the NK cell of the patient itself, the NK cell of the half-matched of the patient and unrelated allogeneic NK cell.The half-matched NK cell of so-called patient refers to the NK cell from patients' relatives.For example, the peripheral blood NK cell of acquisition patient itself, preferably normal Before rule treatment；Acquire patients' relatives, the i.e. peripheral blood NK cell of half-matched or the peripheral blood NK cell of independent individuals or Cord blood NK cell then this not treated limitation, as long as qualification is surveyed in post-transfusion disease blood screening.
Alleged ghost can come from n cell, can be from engineering cell.Engineering cell, which refers to, carries out modification transformation or recombination to the inhereditary material of host cell using technique for gene engineering or cell-fusion techniques, obtains the cell with the unique shape for stablizing heredity.According to an embodiment of the invention, the cell factor that the ghost has includes at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.Preferably, the ghost has IL-15,4-1BBL and IL-21 cell factor.It so, it is possible to obtain HANK cell for efficiently amplifying activated NK cell ex vivo in vitro.
According to an embodiment of the invention, preparing the ghost includes: that cell is carried out carrying out washing treatment, the cell by washing is obtained；By the cell by washing by other methods common in Hypotonic treatment or industry, to obtain the ghost.Inventors have found that can fast and effeciently prepare the ghost using this method, and this method is easy to operate, is easy to control, it is easy to accomplish large-scale production.
It should be noted that, the cell factor that the ghost has, it can be the cell factor in cell itself surface expression, it is also possible to pass through gene engineering method, such as using transiently transfecting or stablizing expression for specific cells factor expression in cell surface, it can also be the cell factor adsorbed or be crosslinked in cell surface.In addition, the cell for being used to prepare ghost can be primary cell, such as PBMC, or passage cell, such as K562 cell etc..
According to an embodiment of the invention, the carrying out washing treatment includes: that the cell is suspended in isotonic solution, cell suspension is obtained；The cell suspension is subjected to centrifugal treating, to obtain the cell by washing.
The isotonic solution is cooled to 4 degrees Celsius in advance before the cell is suspended in isotonic solution by a preferred embodiment according to the present invention in advance.
According to one embodiment of present invention, the isotonic solution is the isotonic phosphate buffer liquid (PBS) that pH is 7.4.
According to one embodiment of present invention, the Hypotonic treatment includes: that the cell by washing is suspended in hypotonic solution according to predetermined volume ratio, and obtained cell suspension is stood 2 hours, obtains cell Hypotonic treatment object；The cell Hypotonic treatment object is subjected to centrifugal treating, obtains the ghost.
According to one embodiment of present invention, the predetermined volume ratio is 1:40, and the hypotonic solution is hypotonic Tris hydrochloride buffer.Be conducive to improve the efficiency of cell hypotonic lysis as a result,.
According to one embodiment of present invention, before the cell by washing is suspended in hypotonic solution, the hypotonic solution is cooled to 4 degrees Celsius in advance in advance.
According to an embodiment of the invention, obtaining the HANK cell, comprising: isolate monocyte from peripheral blood, the PBMC includes the internal NK cell；Using being added to PBMC described in the culture medium culture of the ghost, with NK cell in the amplifying activated PBMC obtains the HANK cell, prepares the ghost using the method for preparing ghost in any of the above-described embodiment.According to an embodiment of the invention, in vitro then the NK cell by technology massive amplification activation ex vivo, acquisition HANK feed back and carry out HANK cell therapy to patient, it has proved that have relatively good curative effect to kinds cancer.
According to an embodiment of the invention, the NK cell includes at least the 50% HANK cell.Preferably, the NK cell includes at least the 90% HANK cell.
In order to reach the content of above-mentioned HANK, a preferred embodiment according to the present invention, the utilization is added to the culture medium culture PBMC of ghost, with the internal NK cell in amplifying activated PBMC, obtain HANK cell, it include: that the PBMC 12-20 days is cultivated in the X-Vivo15 serum-free medium for being added to 200IU/ml IL-2, the ghost and 5% self blood plasma, the ratio of the number of the addition number and monocyte of the ghost is 1:1.
Preferably, at the 4-8 days of the culture, at least adding the primary X-Vivo15 serum-free medium and the ghost to efficiently obtain the HANK cell of high-content.
According to an embodiment of the invention, described the step of NK cell and antibody drug are given in combination are as follows: successively give the NK cell and the antibody drug respectively or give simultaneously.
Another aspect according to the present invention, the present invention provides a kind of pharmaceutical composition, the pharmaceutical composition includes antibody drug and NK cell, the NK cell includes HANK cell, and the acquisition of the HANK cell includes: to utilize the NK cell of ghost amplification in vitro activation ex vivo with cell factor.
HANK cell and antibody drug in the combination can mutual synergy, cell assay in vitro and In-vivo test in mice show that significant therapeutic effect can be obtained with the medicine composite for curing disease.So-called disease includes cancer, virus infection and immunological diseases.The technical characteristic of the above-mentioned method to the treatment patient in one aspect of the present invention or any embodiment and the description of advantage, the pharmaceutical composition of equally applicable this aspect of the present invention, details are not described herein.
The disease that so-called patient suffers from is at least one of cancer, virus infection and immunological diseases.According to one embodiment of present invention, so-called patient is cancer patient, and so-called antibody drug is monoclonal antibody targeted drug.Monoclonal antibody targeted drug assigns HANK cell-specific, for example HANK cell can be allowed specially to kill leukaemia cell with Rituximab medicine, and Trastuzumab monoclonal antibody medicine can allow HANK cell specially to kill breast cancer cell；HANK cell provides enough active factors, such as a large amount of granzyme, perforin etc. that can effectively kill cancer cell for the monoclonal antibody medicine.NK cell and monoclonal antibody targeted drug comprising HANK cell is used in combination, can mutually increase curative effect between HANK and monoclonal antibody medicine and reach preferably treating cancer.
The antibody drug is monoclonal antibody or is polyclonal antibody.Two antigen-binding sites on one antibody molecule can be identical, is also possible to different, i.e. bispecific antibody, claims antigen-binding fragment (antigen-binding fragment, Fab) positioned at two-arm end.According to one embodiment of present invention, so-called antibody drug be include Fc The complete monoclonal antibody of section.
The ratio of a preferred embodiment according to the present invention, the HANK cell and antibody drug in NK cell in described pharmaceutical composition is 2 × 105To 5 × 105: 1 (a/μ g).When antibody drug is administered, doctor or drug specification can provide usage amount according to patient profiles, and in vitro test and animal experiment show the HANK cell that the special ratios are given in combination when administration, can significantly enhance the effect for the treatment of cancer.
The present invention to be used to amplifying activated NK cell source individual with no restriction, as long as post-transfusion disease screening qualification.According to one embodiment of present invention, the HANK cell is obtained by a kind of at least following NK cell ex vivo of Activated in Vitro: the NK cell of the patient, the NK cell of the half-matched of the patient and unrelated allogeneic NK cell.The half-matched NK cell of so-called patient refers to the NK cell from patients' relatives.For example, the PBMC of acquisition patient itself prepares NK cell, preferably before conventional therapy；Or acquisition patients' relatives, i.e., the PBMC of half-matched prepares NK cell；Either the PBMC preparation NK cell of independent individuals or Cord blood prepare NK cell, then are not so limited.
According to an embodiment of the invention, the cell factor that the ghost has includes at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.Preferably, the ghost has IL-15,4-1BBL and IL-21 cell factor.It so, it is possible efficiently amplification in vitro and activate internal NK cell, obtain HANK cell.
Another further aspect according to the present invention, a kind of method for enhancing and receiving the ADCC effect of patient of antibody drug treatment is provided, the method includes NK cell and antibody drug are given in combination, the NK cell includes HANK cell, and the acquisition of the HANK cell includes: to utilize NK cell in the ghost Activated in Vitro body with cell factor.The technical characteristic of the above-mentioned method to the treatment patient in one aspect of the present invention or any embodiment and the description of advantage, the method for the enhancing ADCC effect of equally applicable this aspect of the present invention, details are not described herein.
The disease that so-called patient suffers from is at least one of cancer, virus infection and immunological diseases.According to one embodiment of present invention, so-called patient is cancer patient, and so-called antibody drug is monoclonal antibody targeted drug.Monoclonal antibody targeted drug assigns HANK cell-specific, for example HANK cell can be allowed specially to kill leukaemia cell with Rituximab medicine, and Trastuzumab monoclonal antibody medicine can allow HANK cell specially to kill breast cancer cell；HANK cell provides enough active factors, such as a large amount of granzyme, perforin etc. that can effectively kill cancer cell for the monoclonal antibody medicine.NK cell and monoclonal antibody targeted drug comprising HANK cell is used in combination, curative effect can be increased between HANK cell and monoclonal antibody medicine mutually with preferably treating cancer.
The antibody drug is monoclonal antibody or is polyclonal antibody.Two antigen-binding sites on one antibody molecule can be identical, is also possible to different, i.e. bispecific antibody, claims antigen-binding fragment (antigen-binding fragment, Fab) positioned at two-arm end.According to one embodiment of present invention, so-called antibody drug is the complete monoclonal antibody for including Fc sections.
A preferred embodiment according to the present invention, HANK cell and antibody drug in the NK cell being given in combination Ratio be 2 × 105To 5 × 105: 1 (a/μ g).When being administered with antibody drug, doctor or drug specification can provide usage amount according to patient profiles, and in vitro test and animal experiment show the HANK cell that the ratio is given in combination when administration, can enhance the effect for the treatment of cancer significantly.
The present invention to be used to amplifying activated internal NK cell the source PBMC individual with no restriction, as long as post-transfusion disease screening qualification.According to one embodiment of present invention, the HANK cell is obtained by least activating a kind of following NK cell in vitro: the NK cell of the patient, the NK cell of the half-matched of the patient and unrelated allogeneic NK cell.The half-matched NK cell of so-called patient refers to the NK cell from patients' relatives.For example, the NK cell in the PBMC of acquisition patient itself, preferably before conventional therapy；Patients' relatives are acquired, i.e. NK cell in the PBMC of half-matched or the NK cell in the PBMC of independent individuals or Cord blood NK cell are not limited then by this.
According to an embodiment of the invention, the cell factor that the ghost has includes at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.Preferably, the ghost has IL-15,4-1BBL and IL-21 cell factor.It so, it is possible efficiently amplifying activated internal NK cell in vitro, obtain HANK cell.
HANK cell and antibody drug especially monoclonal antibody targeting medicine is used in combination in the various aspects of aforementioned present invention, it is not only able to improve the lethality of monoclonal antibody medicine, and the non-specific HANK cell of wide spectrum is made to obtain the function of target killing tumor cell and virus infected cell, the two can be mutually improved.
Detailed description of the invention
The description of embodiment will be apparent from and is more easily to understand in conjunction with following accompanying drawings of the present invention and/or additional aspect and its advantage, in which:
Fig. 1 shows that HANK cell joint Rituximab is to the ADCC effect of SUDHL-4 lymphoma cell in one embodiment of the present of invention.
Fig. 2 shows the joint Rituximab of the HANK cell in one embodiment of the present of invention to the curative effect of mouse lymph lymphoma.
Fig. 3 shows the joint Trastuzumab monoclonal antibody of the HANK cell in one embodiment of the present of invention to the ADCC effect of MDA-MB-435 breast cancer cell.
Fig. 4 shows the joint Trastuzumab monoclonal antibody of the HANK cell in one embodiment of the present of invention to the curative effect of mouse breast cancer.
Fig. 5 shows the joint GPC3 monoclonal antibody of the HANK cell in one embodiment of the present of invention to the ADCC effect of HepG2 liver cancer cells.
Fig. 6 shows the joint GPC-3 monoclonal antibody of the HANK cell in one embodiment of the present of invention to the curative effect of rat liver cancer.
Fig. 7 shows the joint mC3 monoclonal antibody of the HANK cell in one embodiment of the present of invention to the ADCC effect of the bhk cell of JEV infection.
Fig. 8 shows protective effect of the joint mC3 monoclonal antibody of the HANK cell in one embodiment of the present of invention to JEV infection mouse.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and for explaining only the invention, and is not considered as limiting the invention.Particular technique or condition are not specified in embodiment, described technology or conditions or is carried out according to the literature in the art according to product description.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
Embodiment one
Prepare 8866 ghost of RPMI
According to the following steps, ghost is prepared using continuous cell line RPMI 8866, wherein RPMI 8866 is people's B lymph matricyte system.It is specific as follows:
RPMI8866 cell is suspended in 3 times of head for precooling into the isotonic PBS of 4 degrees Celsius of pH 7.4, is centrifuged 10 minutes under 4 degrees Celsius 1500 revs/min, supernatant is removed, washes repeatedly 1-3 times, obtains 8866 cell of RPMI by washing；Then will by washing 8866 cell of RPMI be by volume 1:40 ratio be added in advance be cooled to 4 degrees Celsius, concentration be in the hypotonic Tris hydrochloride buffer of 10mmol/L, side edged is slowly stirred, then, obtained mixed liquor is stood 2 hours in 4 degrees Celsius of refrigerator, cracks cell completely；Then, it is centrifuged 10 minutes under 4 degrees Celsius 9000 revs/min, precipitates ghost, further washed repeatedly, be centrifuged 3-5 times to get RPMI8866 ghost, i.e. RPMI 8866- ghost.Then, by 2 × 107The concentration of a ghost/ml dispenses, in subzero 80 degrees Celsius of refrigerator cold-storages；Or after freezing dried, saved in 4 degrees Celsius of refrigerator freezings.
Embodiment two
1, prepared by HANK cell, and conventional method is as follows:
1) Healthy People periphery anticoagulation is acquired, separates PBMC with lymphocyte separation medium；
2) PBMC is cultivated in the X-Vivo15 serum-free medium for being added to IL-2 and RPMI 8866- ghost and 5% self blood plasma, NK cell massive amplification therein can be made to activate；
3) when cultivating 14 days or so, NK cell number can expand up to a hundred to thousands of times, and NK cell purity can act on from 10% in PBMC and increase to 80-90% or more；
This amplifying activated NK cell in vitro is exactly HANK cell, fresh can use, can also -80 degree refrigerators or liquid nitrogen in freeze it is spare, in subsequent killing experiment in vitro be used as effector cell, in animal experiment individually or with monoclonal antibody use in conjunction, play the antiviral effect of anticancer.
2, it is expanded using NK cell in RPMI 8866- ghost stimulation PBMC, the peripheral blood mononuclear cells of 3 normal persons is tested altogether, specific as follows:
1) prepared by autologous plasma: extracting anticoagulation cirumferential blood 50ml, 700G, the centrifugation of 20min room temperature；Blood plasma is drawn, is placed in water-bath 56 DEG C, 30min；Then 4 DEG C of standing 15min；Last 4 DEG C, 900G, it is centrifuged 30min, takes 4 DEG C of autologous plasma to save backup.
2) cellular layer after taking blood plasma is added D-PBS and mixes, and separates PBMC with lymphocyte separation medium 800G, 20min；
3) 1, T75 culture bottle is selected, 40ml lymphocyte complete culture solution (containing about 200IU/ml IL-2, autologous plasma 1-10%) is added to the PBMC isolated, cell suspension (about 5 × 10 is made6A lymphocyte), it is added to T75 culture bottle；2 × 10 are added simultaneously7RPMI 8866- ghost is placed in saturated humidity, 37 DEG C, 5.0%CO2It is cultivated in incubator；
4) at the about the 4th day of the culture, about 40ml lymphocyte complete culture solution is added；
5) at the 7th day or so of the culture, the cell in the T75 culture bottle is transferred in culture bag, adds the lymphocyte complete culture solution to about 400ml, and adds about 8 × 107A obtained ghost in front；
6) at the tenth day or so of the culture, culture is passaged in two culture bags, wherein contain lymphocyte complete culture solution described in about 640ml in each culture bag；And
7) at the 12nd day or so of the culture, cultured products are collected, are tested differently to the amplifying activated effect of NK cell in PBMC.Wherein, according to the quantity of required cell, the culture of different time can be carried out to NK cell in PBMC, such as can continuously cultivate 18 days or 20 days etc..
Embodiment three
Killing experiment in vitro, conventional method:
1) measurement one pipe HANK effector cell of the previous day recovery, the culture in NK cell culture fluid (NKEM, the X-Vivo15 containing IL-2 and 5% self blood plasma)；
2) every time measurement when use a kind of cell line as target cell, and using K562 cell as positive control cell.Need 6X10^5 effector cell's cell and 3X10^5 target cell；
3) with NKEM culture solution dilution Calcein-AM (calcein-AM), CAM liquid is prepared；
4) 10^6 target cell suspension in 1mL CAM liquid.37 DEG C are cultivated 1 hour, are shaken in due course.Then it is washed 2 times with NKEM culture solution, each 1200rpm is centrifuged 5min.It counts, adjustment concentration is 1X10^5/mL；
5) effector cell is diluted to 1X10^6/mL, 3 holes is set in 96 porocyte culture plates of U-shaped bottom, every hole adds target cell 200uL, corresponding effect target ratio (E:T) 10:1；
6) test sets that maximum relief hole, negative control hole, natural kill be anti-and the hole ADCC；
7) hole 100uL 2%Triton X-100, ADCC is added to add appropriate diluted monoclonal antibody to be measured, remaining hole to maximum relief hole Add 100uL complete culture solution；
8) effector cell is done 5 doubling dilutions, the effect target ratio (E:T) of the last one dilution is 0.3125:1；
9) every hole adds 100 μ L target cells, and 100g is centrifuged 1min, guidance cell contact.37 DEG C of 5%CO2Buoyancy tank culture 4 hours；
10) it is gently inhaled with 100 μ L sample injectors and beats cell, with the calcein of suspension and release；100g is centrifuged 5min, with sedimentation cell.100 μ L of gentle aspiration supernatant, is transferred to a new culture plate, prevents foam.If there is formation of foam, just punctured with needle；
11) with fluorescence plate reader (exciting light 485nm emits light 530nm) read plate；
12) specific cytotoxicity percentage is calculated.[(test group-Spontaneous release group)/(maximum release group-Spontaneous release group)] X100.
Example IV
Mouse tumor and encephalitis model are established, conventional method:
A) foundation of people's diffusivity large B cell lymphoid tumor mouse tumor model: everybody diffusivity large B cell lymphoid tumor (DLBCL) Transplanted tumor model can be successfully set up by inoculating 10^7 cell using SCID mice, the histological appearance of tumor formation rate 70%, tumour is similar to people DLBCL.
1) cell culture: SUDHL-4 is people GCB sample DLBCL cell strain.By initial density be 2.5 × 10^5/ml cell be placed in containing 10%FBS, 100U/ml penicillin, 100 μ g/ml streptomysins, 30 μ g/ml glutamine RPMI1640 culture solution T25 Tissue Culture Flask in, cultivated in 37 DEG C, 5%CO2, saturated humidity incubator；After 2-3d the 1st time passage, it is transferred to T75 Tissue Culture Flask, moves into T150 Tissue Culture Flask in due course according to cell growth status later；
2) SPF grades of SCID mices, female, 5 week old, weight 16-20g is grouped at random, raise and test constant temperature (20-26 DEG C), constant humidity (50%-56%) SPF grade mouse room in progress.Mouse is placed in laminar-flow rack mouse box with cover, and air is through medium air filtration, feeding standard particle feed, all goods contacted with mouse sterilized processing in advance；
3) cell inoculation: the SUDHL-4 cell of logarithmic growth phase is suspended in serum-free PBS after being rinsed 2 times with serum-free PBS.
Experimental mice (every group 10) inoculates 0.1ml in one side flank and contains 107The cell suspension of cell；0.1ml PBS is subcutaneously injected in right side flank in Normal group (every group 10).
4) mouse ordinary circumstance, tumor formation and tumour growth situation mice with tumor Indexs measure: are observed during experiment daily.Daily measurement weight and the tumour line of apsides and height, and calculate gross tumor volume (calculation method: π/6 × length × width × height)；When knurl reaches 1200mm3When be considered as human terminal.After mouse is anaesthetized and neck is drawn to put to death, each position tumor formation situation of body surface is first observed；Then animal is dissected, each internal organs and lymphatic metastasis situation are observed.
B) human breast carcinoma MDA-MB-435 cell strain Nude Mouse Model: with gentamicin containing 50U/ml, 10% inactivation calf serum MEM culture solution, culture Breast cancer lines MDA-MB-435 is spare under the conditions of 37 DEG C and 5%C02 abundant humidifyings.Adopt BALB/C female nude mice, SPF grades, 20 ± 2g of weight；35~40 ages in days, take the breast cancer cell MDA-MB-435 of the in vitro culture of logarithmic growth, and cell concentration is adjusted to 1 × 107/ml.Under aseptic condition, MDA-MB-435 human breast cancer cell is inoculated in the fat pad of the second nipple on the left of nude mouse, inoculum concentration is that only (cell number is 1 × 10 to 0.1ml/6/ only).It is grouped at random when subcutaneously obviously touching tumor growth after 2 weeks.Administration time is 10 weeks, animal is taken off neck and is put to death within 3 days after drug withdrawal.
C) rat liver cancer model: 10,000,000 HepG2 cells of mouse bare subcutaneous injection are given, 14 days start to treat.With cubic centimetre (mm3) it is that the calculation formula of tumor size of unit is: (a) X (b) X 0.5；Wherein a is length of tumor, and b is tumor width.
D) mouse encephalitis model:
1) Strain: JEV SA14 continuously passed in suckling rat brain 3 generations increase poison after for；
2) mouse infection: the Balb/c mouse of 8 grams or so 3 week old is selected, is inoculated with, is measured as 10^5LD50 through abdominal cavity with the mouse brain suspension of JEV infection；
3) curative effect is calculated with infecting mouse survival rate.
Embodiment five
1, monoclonal antibody targets medicine
Rituximab is a kind of anti-CD20 monoclonal antibody, can kill CD20 positive cell in conjunction with the Fc receptor CD16 on NK cell, in multiple countries for clinical treatment leukaemia many years.
2, HANK cell joint rituximab treats leukaemia
(1) killing experiment in vitro: being effector cell with HANK cell, K562 cell and SUDHL-4 cell are target cell, the ADCC effect that the natural kill effect of 2 kinds of target cells and Rituximab are mediated according to the progress that embodiment three describes, measurement HANK cell.
As a result HANK cell is 92% or so to the killing rate of K562 cell as shown in Figure 1:, and the killing rate to SUDHL-4 cell is 52% or so；Rituximab does not influence HANK cell to the lethal effect of K562 cell, but substantially increases HANK cell to the lethal effect of SUDHL-4 cell, rises to 87% or so from 52%.
(2) In vivoprotective test: the 15th day after inoculation SUDHL-4 cell, tumor-bearing mice gross tumor volume about 524mm^3.Tumor-bearing mice is divided into: 4 groups, every group 10.One group is applied alone HANK cell therapy, and every mouse vein is transfused 1X10^7HANK cell, once a week；Another group of rituximab alone treatment, every mouse vein are transfused 40 micrograms, once a week；3rd group is the joint rituximab treatment of HANK cell, and venoclysis 1X10^7HANK cell adds 40 micro- Gram Rituximab, once a week；4th group is saline control group.
As a result see Fig. 2: 14 days after treatment, saline control group gross tumor volume 1129mm^3, HANK cell therapy group gross tumor volume 224mm^3, Rituximab targeted therapy group 315mm^3, HANK cell+rituximab combination therapy group 83mm^3.Lotus knurl volume is smaller after treatment shows that curative effect is better.
Embodiment six
1, monoclonal antibody targets medicine
Trastuzumab is a kind of anti-EGFR Her2 monoclonal antibody, can kill EGFR positive cell in conjunction with Her2 positive cell, in multiple countries for clinical treatment breast cancer many years.
2, HANK cell joint Trastuzumab treats breast cancer
(1) killing experiment in vitro: being effector cell with HANK cell, and K562 cell and MDA-MB-435 cell are target cell, and measurement HANK cell is to the natural kill effect of 2 kinds of target cells and the ADCC effect of Trastuzumab monoclonal antibody mediation.
As a result as shown in Figure 3: HANK cell is 92% or so to the killing rate of K562 cell, and the killing rate to MDA-MB-435 cell is 63% or so；Trastuzumab monoclonal antibody does not influence HANK cell to the lethal effect of K562 cell, but substantially increases HANK cell to the lethal effect of MDA-MB-435 cell, rises to 91% or so from 63%.
(2) In vivoprotective test: the 15th day after inoculation MD-MB-435 cell, tumor-bearing mice gross tumor volume about 311mm^3.Tumor-bearing mice is divided into: 4 groups, every group 10.One group is applied alone HANK cell therapy, and every mouse vein is transfused 1X10^7HANK cell, once a week；Another group is applied alone Trastuzumab monoclonal antibody to treat, and every mouse vein is transfused 40 micrograms, once a week；3rd group is the joint Trastuzumab treatment of HANK cell, and venoclysis 1X10^7HANK cell adds 40 microgram Trastuzumab monoclonal antibodies, once a week；4th group is saline control group.
As a result see Fig. 4: 14 days after treatment, saline control group gross tumor volume 724mm^3, HANK cell therapy group gross tumor volume 121mm^3, Trastuzumab monoclonal antibody targeted therapy group 312mm^3, HANK cell+Trastuzumab combination therapy group 51mm^3.Lotus knurl volume is smaller after treatment shows that curative effect is better.
Embodiment seven
1, monoclonal antibody targeted drug
1G12 is a kind of resisting GPC 3 monoclonal antibody, can inhibit the liver cancer cells in-vitro multiplication of the GPC3 positive, also has good therapeutic effect to the intracorporal transplantability hepatocellular carcinoma of nude mice (HCC).
2, HANK cell joint GPC3 monoclonal antibody treats liver cancer
(1) killing experiment in vitro: being effector cell with HANK cell, and K562 cell and HepG2 cell are target cell, the ADCC effect that measurement HANK cell mediates the natural kill effect of 2 kinds of target cells and resisting GPC 3 monoclonal antibody 1G12.
As a result as shown in Figure 5: HANK cell is 92% or so to the killing rate of K562 cell, to SUDHL-4 cell Killing rate is 52% or so；1G12 monoclonal antibody does not influence HANK cell to the lethal effect of K562 cell, but substantially increases HANK cell to the lethal effect of HepG2 cell, rises to 85% or so from 48%.
(2) In vivoprotective test: the 15th day after inoculation HepG2 cell, tumor-bearing mice gross tumor volume about 435mm^3.Tumor-bearing mice is divided into 4 groups, every group 10.One group is applied alone HANK cell therapy, and every mouse vein is transfused 1X10^7HANK cell, once a week；Another group is applied alone 1G12 monoclonal antibody to treat, and every mouse vein is transfused 40 micrograms, once a week；3rd group is the joint 1G12 treatment of HANK cell, and venoclysis 1X10^7HANK cell adds 40 microgram 1G12 monoclonal antibodies, once a week；4th group is saline control group.
As a result see Fig. 6: 14 days after treatment, saline control group gross tumor volume 936mm^3, HANK cell therapy group gross tumor volume 185mm^3,1G12 monoclonal antibody targeted therapy group 423mm^3, HANK cell+1G12 combination therapy group 77mm^3.Lotus knurl volume is smaller after treatment shows that curative effect is better.
Embodiment eight
1, monoclonal antibody targeted drug
MC3 is a kind of anti-JEV (japanese encephalitis virus) monoclonal antibody, and being used alone has the activity for neutralizing JEV, has protective effect to the mouse of infection JEV.
2, HANK cell joint japanese encephalitis virus monoclonal antibody treats encephalitis
(1) killing experiment in vitro: being effector cell with HANK cell, and the bhk cell of K562 cell and JEV infection is target cell, the ADCC effect that measurement HANK cell mediates the natural kill effect of 2 kinds of target cells and anti-JEV monoclonal antibody mC3.
As a result as shown in Figure 7: HANK cell is 92% or so to the killing rate of K562 cell, and the killing rate to JEV-BHK cell is 62% or so；MC3 monoclonal antibody does not influence HANK cell to the lethal effect of K562 cell, but substantially increases HANK cell to the lethal effect of JEV-BHK cell, rises to 88% or so from 62%.
(2) In vivoprotective test: the 5th day after JEV infection, some animals are already close to death.Infecting mouse is divided into: 4 groups, every group 10.One group is applied alone HANK cell therapy, and every mouse vein is transfused 1X10^7HANK cell, once a week；Another group is applied alone mC3 monoclonal antibody to treat, and every mouse vein is transfused 40 micrograms, once a week；3rd group is the joint mC3 monoclonal antibody treatment of HANK cell, and venoclysis 1X10^7HANK cell adds 40 microgram mC3 monoclonal antibodies, once a week；4th group is saline control group.
As a result see Fig. 8: 14 days after treatment, saline control group mouse all biological, HANK cell therapy group mouse survival rate 72%, mC3 monoclonal antibody targeted therapy group mouse survival rate 62%, HANK cell+mC3 combination therapy group mouse survival rate 90%.Protective rate is higher, and curative effect is better.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " tool The description of body example " or " some examples " etc. means that particular features, structures, materials, or characteristics described in conjunction with this embodiment or example are included at least one embodiment or example of the invention.In the present specification, the schematic representation of the above terms does not necessarily have to refer to the same embodiment or example.Moreover, particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more of the embodiments or examples.In addition, without conflicting with each other, the feature of different embodiments or examples described in this specification and different embodiments or examples can be combined by those skilled in the art.
Although the embodiments of the present invention has been shown and described above, it can be understood that, above-described embodiment is exemplary, and is not considered as limiting the invention, and those skilled in the art can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.

Claims (39)

  1. A method of patient being treated, the patient suffers from disease, which is characterized in that the method includes natural killer cells and antibody drug are given in combination,
    The natural killer cells includes high activity natural killer cells,
    The acquisition of the high activity natural killer cells includes: to utilize natural killer cells in the ghost Activated in Vitro body with cell factor.
  2. Method of claim 1, which is characterized in that the disease is selected from least one of cancer, virus infection and immunological diseases.
  3. Method of claim 1, which is characterized in that the antibody drug is monoclonal antibody or polyclonal antibody.
  4. Method of claim 1, which is characterized in that the natural killer cells includes at least the 50% high activity natural killer cells.
  5. Method of claim 1, which is characterized in that the natural killer cells includes at least the 90% high activity natural killer cells.
  6. Method of claim 1, which is characterized in that the ratio of high activity natural killer cells and antibody drug in the natural killer cells being given in combination is 2 × 105To 5 × 105: 1 (a/μ g).
  7. Method of claim 1, it is characterized in that, the high activity natural killer cells is obtained by internal natural killer cells at least one below Activated in Vitro: natural killer cells, the natural killer cells of the half-matched of the patient and the unrelated allogeneic natural killer cells of the patient.
  8. Method of claim 1, which is characterized in that the ghost comes from n cell and/or engineering cell.
  9. Method in claim 8, which is characterized in that the cell factor that the ghost has includes at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.
  10. Method for claim 8, which is characterized in that the ghost has IL-15,4-1BBL and IL-21 cell factor.
  11. Method for claim 8, which is characterized in that preparing the ghost includes:
    Cell is subjected to carrying out washing treatment, obtains the cell by washing；
    The cell by washing is subjected to Hypotonic treatment, to obtain the ghost.
  12. The method of claim 11, which is characterized in that the carrying out washing treatment includes:
    The cell is suspended in isotonic solution, cell suspension is obtained；
    The cell suspension is subjected to centrifugal treating, to obtain the cell by washing.
  13. The method of claim 12, which is characterized in that before the cell is suspended in isotonic solution, the isotonic solution is cooled to 4 degrees Celsius in advance.
  14. The method of claim 12, which is characterized in that the isotonic solution is the isotonic phosphate buffer liquid that pH is 7.4.
  15. The method of claim 11, which is characterized in that the Hypotonic treatment includes:
    The cell by washing is suspended in hypotonic solution according to predetermined volume ratio, obtained cell suspension is stood 2 hours, obtains cell lysate；
    The cell lysate is subjected to centrifugal treating, obtains the ghost.
  16. The method of claim 15, which is characterized in that the predetermined volume ratio is 1:40.
  17. The method of claim 15, which is characterized in that before the cell by washing is suspended in hypotonic solution, the hypotonic solution is cooled to 4 degrees Celsius in advance in advance.
  18. The method of claim 15, which is characterized in that the hypotonic solution is hypotonic Tris hydrochloride buffer.
  19. Method of claim 1, which is characterized in that obtain the high activity natural killer cells, comprising:
    Monocyte is isolated from peripheral blood, the monocyte includes the internal natural killer cells；
    Using monocyte described in the culture medium culture of the ghost is added to, with the internal natural killer cells in the amplifying activated monocyte, the high activity natural killer cells is obtained,
    The ghost is prepared using claim 11-18 either method.
  20. The method of claim 19, which is characterized in that the utilization is added to the culture medium culture monocyte of ghost, with the internal natural killer cells in amplifying activated monocyte, obtains high activity natural killer cells, comprising:
    The monocyte is cultivated in the X-Vivo15 serum-free medium for being added to 200IU/ml IL-2, the ghost and 5% self blood plasma 12-20 days, the addition number of the ghost and the ratio of the monocyte are 1:1.
  21. The method of claim 20, which is characterized in that at the 4th to the 8th day of the culture, at least add the primary X-Vivo15 serum-free medium and the ghost.
  22. Claim 1-21 either method, which is characterized in that described the step of natural killer cells and antibody drug are given in combination are as follows:
    The natural killer cells and the antibody drug are successively given respectively or given simultaneously.
  23. A kind of pharmaceutical composition, which is characterized in that including antibody drug and natural killer cells, the natural killer cells includes high activity natural killer cells,
    The acquisition of the high activity natural killer cells includes: to utilize natural killer cells in the ghost Activated in Vitro body with cell factor.
  24. The pharmaceutical composition of claim 23, which is characterized in that the antibody drug is monoclonal antibody or polyclonal antibody.
  25. The pharmaceutical composition of claim 23, which is characterized in that the natural killer cells includes at least the 50% high activity natural killer cells.
  26. The pharmaceutical composition of claim 23, which is characterized in that the natural killer cells includes at least the 90% high activity natural killer cells.
  27. The pharmaceutical composition of claim 23, which is characterized in that the ratio of high activity natural killer cells and antibody drug in the natural killer cells in described pharmaceutical composition is 2 × 105To 5 × 105: 1 (a/μ g).
  28. The pharmaceutical composition of claim 23, it is characterized in that, the high activity natural killer cells is obtained by natural killer cells in vivo at least one below Activated in Vitro: natural killer cells, the natural killer cells of the half-matched of the patient and the unrelated allogeneic natural killer cells of the patient.
  29. The pharmaceutical composition of claim 23, which is characterized in that the cell factor that the ghost has includes at least one of IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL.
  30. The pharmaceutical composition of claim 29, which is characterized in that the ghost has IL-15,4-1BBL and IL-21 cell factor.
  31. A method of enhancing is receiving the ADCC effect of the patient of antibody drug treatment, which is characterized in that the method includes natural killer cells and antibody drug are given in combination,
    The natural killer cells includes high activity natural killer cells,
    The acquisition of the high activity natural killer cells includes: to utilize natural killer cells in the ghost Activated in Vitro body with cell factor.
  32. The method of claim 31, which is characterized in that the antibody drug is monoclonal antibody or polyclonal antibody.
  33. The method of claim 31, which is characterized in that the natural killer cells includes at least the 50% high activity natural killer cells.
  34. The method of claim 31, which is characterized in that the natural killer cells includes at least the 90% high activity natural killer cells.
  35. The method of claim 31, which is characterized in that the ratio of high activity natural killer cells and antibody drug in the natural killer cells being given in combination is 2 × 105To 5 × 105: 1 (a/μ g).
  36. The method of claim 31, it is characterized in that, the high activity natural killer cells is obtained by internal natural killer cells at least one below Activated in Vitro: natural killer cells, the natural killer cells of the half-matched of the patient and the unrelated allogeneic natural killer cells of the patient.
  37. The method of claim 31, which is characterized in that the ghost comes from n cell and/or engineering cell.
  38. The method of claim 37, which is characterized in that the cell factor that the ghost has include at least IL-4, IL-7, IL-15, IL-21, CD19, CD64, CD86 and 4-1BBL in one of.
  39. The method of claim 37, which is characterized in that the ghost has IL-15,4-1BBL and IL-21 cell factor.
CN201580001069.9A 2015-11-09 2015-11-09 Strengthen the method and pharmaceutical composition to abnormal cell lethality Pending CN107109363A (en)

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