CN106890330A - DC CIK cells and the antibody compositions of anti-PD 1 and application thereof - Google Patents
DC CIK cells and the antibody compositions of anti-PD 1 and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
The invention belongs to immunology, tumor microenvironment and immunotherapy of tumors field, there is provided DC CIK cells and the antibody compositions of anti-PD 1 are used to prepare treating cancer or delay the purposes in the immune drug of cancer progression.Present invention simultaneously provides a kind of combined immunization composition for treating cancer, it is made up of the DC CIK cells and the antibody of anti-PD 1 of effective dose.Composition immune drug provided by the present invention can effectively strengthen killing tumor cell immunocompetence in patient's body, be expected to improve the clinical therapeutic efficacy of cancer or delay cancer progression.
Description
Technical field
The invention belongs to immunology, tumor microenvironment and immunotherapy of tumors field, it is related to DC-CIK (BMDCs
The cytokine induced kill cell of activation) cell and anti-PD-1 (programmed death acceptor 1) antibody compositions purposes, tool
Body is related to DC-CIK cells and anti-PD-1 antibody compositions for preparing the purposes of immune drug.
Background technology
With industrialized development, the extraneous factor such as the exacerbation of environmental pollution, and smoking, bad life and eating habit
Effect, the incidence of disease of the tumour in worldwide increase year by year, it has also become the first cause of death of China city dweller.
In recent years, due to the development of the general treatment measures such as operation, chemotherapy, radiotherapy and immunization therapy, tumour gradually into
It is a kind of controllability disease.Especially immunization therapy is considered as a kind of ultimate method that can thoroughly cure tumour.Science is miscellaneous
" immunotherapy of tumors " was chosen as ten big sciences then and broken through by will in 2013, and was continued " immunotherapy of tumors " in 2014
It is classified as one of field for most meriting attention for 2015.And in immunotherapy of tumors field, immunologic test orders blocking agent：Anti- PD-1 resists
The application of body, anti-PD-L1 antibody and anti-CTLA-4 antibody in clinical tumor immunization therapy most merits attention, their notable extensions
The life cycle of the patients such as Advanced Malignant melanoma, lung cancer and colon cancer.Immunologic test order blocking agent be mainly by blocking
T cell inhibition signal path in tumor tissues, so that T cell persistent activation, reaches the purpose for killing tumour.At present
FDA has had been approved by anti-CTLA-4 and anti-PD-1 antibody is used for the tumours such as clinical treatment Advanced Malignant melanoma and lung cancer.So
And, during practical clinical, immunologic test is ordered blocking agent and is also challenged by some：One is due to the blocking of immunologic test point
Agent is lasting attack of the induction body immune system to tumour cell by blocking immune response in body " brake system ",
Stronger autoimmune response is often thus produced after patient's application, so as to cause stronger toxic and side effect；Two is to need enough foots
The application of the course for the treatment of, patient is difficult to bear the medical expense of great number.
And used as a kind of adoptive cellular immunotherapy method of high-efficiency low-toxicity, CIK cell and DC-CIK cells are various
Powerful antitumor action is shown in hematological system tumor and entity tumor.CIK cell is one kind by the single core of peripheral blood
Cell in vitro through the polyclonal T cell of cytokine profiles induced amplification culture, mainly with CD3+CD56+, CD3+CD8+ and
Based on CD3-CD56+ cell subsets, wherein CD3+CD56+ cell subsets is its chief active cell mass, non-limiting by MHC
The celliform effect killing tumor cells of NK.DC-CIK cells are in CIK cell culture to certain phase, with autologous or allosome
The a group heterogeneity T cell obtained after DC cell co-cultivations, because it contains higher levels of CD3+CD8+ and CD3+CD56+ is thin
Born of the same parents' subgroup, and with stronger IFN-γ secretion ability, so the ability of the inside and outside killing tumor cell of DC-CIK cells shows
Work is better than CIK cell.Clinical research in recent years finds CIK cell in liver cancer, lung cancer, colorectal cancer, stomach cancer, cervical carcinoma, kidney
There is therapeutic action with the kinds of tumors such as leukaemia, the life quality of patient can be improved, improve the life span of patient.With CIK
Cell is compared, and DC-CIK cells are complicated compared with the former due to its preparation process, and production cost is higher, thus relevant DC-CIK is thin at present
The clinical research of born of the same parents is relatively fewer.Current research confirms, after tumor patient conventional therapy, continues to receive CIK cell and DC-
CIK adoptive cellular immunotherapies, only some patientss can therefrom benefit, and clinical remission power is very low.Accordingly, it would be desirable to carry out new
Immunotherapy method clinical research further improving the therapeutic effect of tumor patient.
At present, immunologic test is ordered blocking agent and is carried with adoptive cellular immunotherapy use in conjunction by existing part research report
The method that CIK cell high kills ability.For example, Poh et al. in vitro with cell killing it is experimentally confirmed that blocking KIR, LAG-3 and
After the immunologic tests such as PD-1 point, CIK cell strengthens the killing ability of Acute myeloid leukemia cell；But this killing energy
Enhanced the acting on after another immunologic test point CTLA-4 is blocked of power is not observed (Poh SL, et al.Cancer
ImmunolImmunother.2016；65(5):525-36).Equally, Dai et al. is it has also been found that after blocking PD-1, CIK cell is to stomach
The killing ability of cancer cell significantly increases (Dai C, et al.Oncotarget.2016；7(9):10332-44).Due to CIK
Cell there is no research report DC-CIK cells and immune inspection at present in many differences such as subsets distribution and cytokine secretion
Therapeutic action associated with blocking agent is made an inventory of, also reports that DC-CIK cells and anti-PD-1 antibody compositions should in human body without correlative study
Specific method, therapeutic effect and toxic and side effect.
The content of the invention
It is an object of the invention to provide a kind of DC-CIK cells and the purposes of anti-PD-1 antibody compositions.
Purposes of the present invention is：DC-CIK cells and anti-PD-1 antibody compositions are used to prepare treating cancer or delay
Purposes in the immune drug of cancer progression.
According to the further feature of purposes of the present invention, the immune drug contains 1-1.5 × 1010Individual DC-CIK is thin
Born of the same parents and the anti-PD-1 antibody of 10mg.
According to the further feature of purposes of the present invention, the immune drug is before human body is input into 37 DEG C of insulating boxs
It is incubated 30 minutes.
Preferably, described cancer includes：Liver cancer, kidney, colorectal cancer, lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or
Nasopharyngeal carcinoma etc..
Preferably, described cancer is in the middle and advanced stage stage.
For treating cancer or delay the combined immunization group of cancer progression it is another object of the present invention to provide a kind of
Compound.
It is of the present invention for treating cancer or to delay the combined immunization composition of cancer progression be by effective dose
DC-CIK cells and anti-PD-1 antibody are constituted.
The further feature of composition according to claim 1, the composition contains 1-1.5 × 1010Individual DC-
CIK cell and the anti-PD-1 antibody of 10mg.
The further feature of composition according to claim 1, the DC-CIK cells are with anti-PD-1 antibody in group
It is incubated 30 minutes in 37 DEG C of insulating boxs after conjunction.
Preferably, described cancer includes：Liver cancer, kidney, colorectal cancer, lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or
Nasopharyngeal carcinoma etc..
Preferably, described cancer is in the middle and advanced stage stage.
The beneficial effects of the present invention are：DC-CIK cells have enhanced activation, propagation and/or cell lysis activity,
Although DC-CIK cells have the activity of killing tumor cell, because DC-CIK cells can express the inhibition molecule such as PD-1,
After feeding back in body, the PD-L1 molecular actions with tumor cells expression reduce the killing activity of DC-CIK cells, limitation
They treat the curative effect of tumours.And in composition of the present invention, DC-CIK cells through anti-PD-1 antibody process after again
Feed back in vivo, being tested through inside and outside confirms：Significantly enhance the activity of DC-CIK cell killing tumours, there is provided its treatment tumour
Curative effect.In some embodiments, tumor patient obtains obvious curative effect, tumor disappearance occurs, and partial reaction or disease are steady
It is fixed.
Brief description of the drawings
Fig. 1 is DC-CIK cells and anti-PD-1 antibody compositions therapeutic scheme.(A) according to inclusion criteria and exclusion standard,
37 patient with advanced cancer meet into a group condition, enter group and receive DC-CIK cells and the treatment of anti-PD-1 antibody compositions, wherein 6 people
Come off because lost to follow-up, finally there are 31 people to carry out further clinical evaluation.(B) start to receive combined immunization treatment first 1 month,
Patient stops using every other tumor-specific therapies, and starting treatment blood drawing in first 14 days carries out DC-CIK cell preparations, feeds back
With anti-PD-1 antibody after 37 DEG C of insulating boxs are incubated 30 minutes, in venous re-transfusion to patient's body, each suffers from same day DC-CIK cell
Person at least carries out 8 combined immunization treatments of the course for the treatment of, and preceding 4 courses for the treatment of are for 1 times a week, 4 courses for the treatment of are 1 time, some patientss in 2 weeks afterwards
Can continue to receive combined immunization composition treatment after the course for the treatment of terminates, until tumour progression.
Fig. 2 display patient in group's clinic essential characteristics.In evaluable 31 cancer patients, it is male, 7 female to have 24
Property, the median age is 52 years old (31-71 Sui).The cancer for the treatment of include 9 liver cancer, 8 kidneys, 6 colorectal cancers, 3 lung cancer,
2 carcinomas of urinary bladder, each 1 of breast cancer, oophoroma, nasopharyngeal carcinoma.These patients lose after the complex treatments such as operation, chemotherapy, radiotherapy
Lose, start to receive combined immunization treatment, receive the number of times at least 8 times of combined immunization composition treatment, most 26 times, middle position is controlled
It is 12 times to treat number of times.
Fig. 3 shows that DC-CIK cells and anti-PD-1 antibody compositions treat the clinical effectiveness of late tumor patient.(A) 31
After patient receives DC-CIK cells and the treatment of anti-PD-1 antibody compositions, according to RECIST evaluation criterions, compare target focus compared with base
The change of line level.(B) total existence of patient and without progression of disease life span.(C) patient receives DC-CIK cells and anti-PD-
After the treatment of 1 antibody compositions, 1/31 acquisition complete incidence graph, 7/31 patient obtains part and alleviates, and disease effective percentage reaches
22.5%；A total of 20/31 patient disease gets nowhere, and disease control rate reaches 64.5%.
Fig. 4 shows size of tumor before and after DC-CIK cells and the treatment of anti-PD-1 antibody compositions.Case 1 is that a liver cancer is suffered from
Person receives combined immunization treatment, and liver inner disease foci disappears；Case 2 is a patients with renal cell carcinoma Lung metastases focus, receives combined immunization and controls
After treatment, intrapulmonary major part foci disappearance, remaining focus is obviously reduced.
Fig. 5 shows that DC-CIK cells and anti-PD-1 antibody compositions treat the toxic and side effect of late tumor patient.Patient connects
Treated by combined immunization, only 2 people occur in that 3 or 4 grades of toxic reactions, illustrate that the method security is good.
Fig. 6 shows the mechanisms of therapeutic action of external experimental analysis DC-CIK cells and anti-PD-1 antibody compositions.(A) prepare
DC-CIK cell phenotypes detection.(B) lethal effect of DC-CIK cells and anti-PD-1 antibody compositions to primary tumor cell.
(C) PD-L1 expressions before and after primary tumor cell is killed.(D) level of DC-CIK cell secretion of gamma-IFN before and after killing.
Specific embodiment
It is of the present invention to be used to prepare treating cancer by DC-CIK cells and anti-PD-1 antibody compositions or delay cancer
The experimental procedure of the immune drug of progress is as follows.
Enter group conventional therapy unsuccessfully through the patient with advanced cancer of definitive pathological diagnosis, and starting to receive combined immunization treatment preceding 1
Every other specific tumour treatment is disabled within individual month, peripheral blood in patients is extracted since treatment is first 14 days, use density gradient centrifugation
Method isolates mononuclearcell (PBMC), and suspended separate PBMC using Quanta-007 serum free mediums, with 1 × 108/ml
Quantity be laid on 75cm2Blake bottle in, be placed in CO2Quiescent culture 1 hour in incubator.Blake bottle is gently rocked, sucking-off is not pasted
The lymphocyte of wall, is placed in new blake bottle, adds 1000U/ml IFN-γs culture 24 hours, adds 100ng/ml within the 2nd day
OKT, 1ng/ml IL-1 α and 1000U/ml IL-2 continue to cultivate, and the fresh culture containing IL-2 is added every other day.Attached cell adds
Enter GM-CSF containing 1000U/ml, 400U/ml IL-4 and Cellgro serum-free DC nutrient solutions are cultivated.Full dose is mended every other day
Fresh medium once, harvests immature DC cell (iDC) on the 5th day, adds OK-432 (0.1KE/ml) and IFN-γ (1000U/
Ml after) cultivating 24 hours, you can obtain ripe DC cells.7th day by ripe DC cells with above-mentioned CIK cell according to 1:20
Ratio be mixed 7 days, you can obtain DC-CIK cells.The DC-CIK cells after centrifugation, washing, are resisted with containing the anti-PD-1 of 10mg
The physiological saline of the 200ml of body is resuspended, after being incubated 30 minutes, by DC-CIK cells through venous re-transfusion to patient.Patient at least connects
By 8 combined immunization composition treatments of the course for the treatment of, preceding 4 courses for the treatment of are for 1 times a week, 4 courses for the treatment of are 2 weeks 1 time afterwards, if patient tumors
Stabilization can continue to receive program treatment, until tumour progression.
The present invention enters to have organized 37 patient with advanced cancer, wherein 6 people are come off due to lost to follow-up, is receiving combined immunization composition
In 31 people for the treatment of, according to RECIST evaluation criterions, there is 1 people to obtain complete incidence graph, 6 people obtain part and alleviate, and 13 people obtain disease
Disease stabilization, total effective percentage reaches 22.5%, and disease control rate is 64.5%, and the total life span of middle position is 288 days, and middle position is without entering
Exhibition life span is 162 days.There are 3 or 4 grades of adverse reactions in only 2 people, are respectively heating and Neuroleptic Leukocytopenia.These results are said
Bright combined immunization composition treatment method of the present invention, can significantly improve disease control rate, extension patient with advanced cancer life
Into the time, (operation instruction of the anti-PD-1 antibody that the U.S. lists is pressed with anti-PD-1 antibody is used alone：Tumor patient presses 2mg/kg
Body weight is used, and usual ampoule is more than 100mg) compare, Small side effects few (each 10mg) with consumption, it is preferably safe
Property and validity.
Embodiment 1：The design of clinical test
What the present embodiment was carried out is the prospective clinical trial research of I phase single armeds, relevant case selection conventional therapy (operation,
Chemicotherapy, targeted therapy) the advanced refractory malignant tumor patient that is in progress afterwards, observation DC-CIK cells and anti-PD-1 antibodyomes
Compound treats security, feasibility and the validity of these patient clinical applications.
(1) research object is included：
1) inclusion criteria:
1. it is advanced refractory malignant tumor patient, including primary hepatoma, clear cell carcinoma of kidney, carcinoma of urinary bladder, colon cancer, non-
ED-SCLC etc.；2. age 18-75 Sui；3. it is expected life cycle>3 months；4. ECOG scores 0 or 1 point；5. cardiopulmonary hepatic and renal function
Normally；6. sufficient bone marrow reserve ability and factor are normal；7. women of child-bearing age's urine pregnancy test before administration starts is negative,
And until effective contraceptives person is taken in last time follow-up between being intended to experimental period together；8. have evaluable according to RECIST standards
Focus；9. voluntary participation is originally tested and signs Informed Consent Form.
2) exclusion standard：
1. the patient for being treated using CTLA-4 or PD-1/PD-L1 blocking agents；2. age<18 or>75 years old；3. the gestational period or the food in one's mouth
Newborn phase women, effective contraceptives person is not taken in breeding time；4. there are serious Cardial or cerebral vascular diseases, it is impossible to the height of control
Blood pressure and diabetes, renal insufficiency or exhaustion person；5. HIV is positive or other immune deficiency disorders；6. there is LADA disease
Patient；7. the patient for having organ transplant history uses excess dosage glucocorticoid or the trouble of other immunodepressant within 4 weeks
Person；8. there is the patient for clearly infecting or generating heat；9. t cell lymphoma, patients with malignant myeloma.
3) standard is exited：
1. physician in view patient profiles think stopped treatment to benefits subjects；2. patient's initiative is exited or not by project
Implement；3. need to carry out other chemotherapy beyond this programme, operative treatment or experimental drug during testing to treat；4. occur tight
Weight adverse events, complication or special physiological change, and should not receive continual cure.
(2) therapeutic scheme：
Treated the previous moon with DC-CIK cells and anti-PD-1 antibody compositions, enrolled patient stops all tumour correlations and controls
Treat, the DC-CIK cells of preparation feed back after being incubated 30 minutes in 37 DEG C of insulating boxs with anti-PD-1 antibody, once in a week, at least return
Treated for previous stage for defeated 4 times；After the completion of once assessed, if patient effectively or stabilization, it is contemplated that carry out the 2nd stage
Maintaining treatment, i.e., scheme once every 2 weeks, totally 4 times；It is evaluated after completing the 8th course for the treatment of, if stable disease or effective in cure,
Maintaining treatment once every 2 weeks can be continued with, and every 3 months carry out clinical assessment.
(3) interpretation of result：
1) patient includes and basic analysis of clinical：
Referring to shown in Figure 1A, patient includes analysis result and shows, incorporates 37 advanced refractories in the design of clinical test altogether
Malignant tumor patient, has 6 patients lost to follow-up after carrying out DC-CIK cells and the treatment of anti-PD-1 antibody compositions, final remaining 31
Patient carries out subsequent analysis research.As shown in Fig. 2 31 patients include male 24, women 7.Wherein Primary Hepatic is thin
Born of the same parents' cancer 9, clear cell carcinoma of kidney 8, colorectal cancer 6, non-small cell lung cancer 3, carcinoma of urinary bladder 2, and breast cancer, ovary
Each 1 of cancer, nasopharyngeal carcinoma.
2) patient treatment protocol's design result：
Referring to shown in Figure 1B, the patient that research is included carries out DC-CIK cells and anti-P D-1 antibody according to the therapeutic scheme of design
Composition treatment.The analysis result of Fig. 2 shows that 31 treatments of patient at least all reach a course for the treatment of (at least feeding back 8 times), preceding
4 courses for the treatment of are for 1 times a week, 4 courses for the treatment of are 2 weeks 1 time afterwards, and it is 8-26 times to feed back numbers range, and it is 12 times that middle position feeds back number of times.
Embodiment 2：The specific method of DC-CIK cells and anti-PD-1 antibody compositions treating cancer
After determining research object, the present embodiment will extract peripheral blood in patients and separate PBMC, carry out the culture of DC-CIK cells, and anti-
PD-1 antibody feeds back in patient's body after being incubated altogether and is treated.
(1) material and method：
1) preparation of DC-CIK cells and anti-PD-1 antibody compositions
After conscientiously article and patient-relevant informa are drawn blood in verification, after extracting peripheral blood in patients 50ml, 800g × 8min centrifugation, collect
Autologous plasma, 56 DEG C of water-baths inactivate 30min, and after placing 4 DEG C of 15min, 800g × 8min takes supernatant blood plasma (about 20ml) stand-by.
Under superclean bench normal operating conditions, PBC after being collected by centrifugation is with 0.9% injection normal saline dilution
It is labelled with 2 disposable 50ml sterile centrifugation tubes to 60ml, indicate the phases such as patient's name, sex, age and admission number
Pass information, is separately added into 20ml lymphocyte separation mediums, is separated with density-gradient centrifugation method and obtains PMNC
(PBMC)。
Suspended separate PBMC using Quanta-007 serum free mediums, with 1 × 108The quantity of/ml is laid on 75cm2's
In blake bottle, CO is placed in2Quiescent culture 1 hour in incubator.Blake bottle is gently rocked, not adherent lymphocyte is suctioned out, put
In new blake bottle, add 1000U/ml IFN-γs culture 24 hours, add within the 2nd day final concentration of 100ng/ml OKT,
1ng/ml IL-1 α and 1000U/ml IL-2 continue to cultivate, and the fresh culture containing IL-2 is added every other day.Above-mentioned attached cell adds
Enter GM-CSF containing 1000U/ml, 400U/ml IL-4 and Cellgro serum-free DC nutrient solutions are cultivated.Full dose is mended every other day
Fresh medium once, harvests immature DC cell (iDC) on the 5th day, adds OK-432 (0.1KE/ml) and IFN-γ (1000U/
Ml after) cultivating 24 hours, you can obtain ripe DC cells.7th day by ripe DC cells with above-mentioned CIK cell according to 1:20
Ratio be mixed 7 days, you can obtain DC-CIK cells.
14th day or the 15th day, collect DC-CIK cells in disposable sterilized Centrifuge Cup (250ml), 2300rpm ×
8min is collected by centrifugation, and with brine 2 times, by cell 0.9% injection of the human serum albumin containing 5ml 20%
It is resuspended in physiological saline.The cell suspension for preparing keeps sample 2ml in glass ampule, and envelope ampoule mouthful is labelled, indicates patient
Name, sex, the age, admission number and treatment project, 4 DEG C storage three months it is for future reference.The DC-CIK cells that will be prepared with contain
The 200ml physiological saline of the anti-PD-1 antibody of 10mg send lesion to be fed back after 30min is incubated in 37 DEG C, 5%CO2 incubators.
2) flow cytometry analysis DC-CIK cell phenotypes
Collect the above-mentioned DC-CIK cells for preparing of sub-fraction, flow cytomery CD3+, CD3+CD4+, CD3+CD8+,
The ratio of CD3+CD56+ and CD3-CD16+CD56+ cells.Wherein should to be not less than 60%, CD3+CD56+ thin for CD3+CD8+ cells
Born of the same parents should be not less than 10%.
(2) interpretation of result：
1) quality measurements of DC-CIK cells and anti-PD-1 antibody compositions：
PE：Outward appearance is milky cell suspension, and loading amount is about 200ml；
Cell quantity：Using white blood cell count(WBC) method, each patient's cell number is all no less than 1 × 1010Individual cell, scope is about 1-
1.5×1010It is individual；
Cell survival rate：Detected with Trypan Blue, cell survival rate is all not less than 95%；
It is aseptic, detected without mycoplasma and endotoxin-free：By current edition《Chinese Pharmacopoeia》Biological products testing regulations is to feeding back cell
The detection of bacterium, mycoplasma and endotoxin is carried out, feminine gender is as a result.
2) flow cytometry analysis result：
After flow cytomery analysis DC-CIK cells, CD3+, CD3+CD4+, CD3+CD8+, CD3+CD56+ and CD3-
The ratio of CD16+CD56+ cells.Result referring to Fig. 6 (A) Suo Shi, in 30 DC-CIK cells of patient, CD3+T lymphocytes
Average proportions reach 94.1%, and main component is CD3+CD8+T lymphocytes, and the ratio for averagely accounting for is 66.1%；CD3+
CD56+T lymphocytes, the ratio for averagely accounting for is 20.2%.
Embodiment 3：The clinical efficacy and safety evaluation of DC-CIK cells and anti-PD-1 antibody compositions treating cancer
(1) curative effect evaluation：
Including the patient of this clinical experimental study has measurable targeted site.The evaluation of focus is in base by CT iconographies
Follow-up period (every 6 months) during line after (the treatment previous moon), treatment stage (every 3 months) and treatment end is carried out.
1) curative effect evaluation standard
Using solid tumor the standard of curative effect evaluation (Response Evaluation Criteria in Solid Tumors,
RECIST therapeutic evaluation) is carried out.Length summation of all measurable targeted sites in baseline is first determined, as effectively alleviation
The reference baseline of record, then according to the change of the follow-up period targeted site versus baseline level after treatment stage or treatment end
Change the evaluation state of an illness and effectively alleviate situation.
2) standard effectively alleviated
The standard of alleviation includes CR：All targeted sites disappear；PR：Baseline focus major diameter summation reduces and is more than or equal to 30%；SD：
Baseline focus major diameter summation has diminution but does not reach PR or have increase but do not reach PD；PD：Baseline focus major diameter summation is increased above
20% there is new focus.
3) follow-up
After since the treatment, follow-up record is carried out to each patient, and check in every 3 months is once determining the progress feelings of patient
Condition, check content includes blood routine, blood biochemistry, tumor marker, hepatic and renal function, blood electrolyte, thyroid function and iconography
Assessment etc..Enter existence follow-up after to Tumor response stabilization or progress, every half a year is once.Treatment time according to patient with it is first
Evolution time and death time calculate the progression free survival phase (DFS) and Overall survival (OS) of patient.
(2) safety evaluation：
The safety evaluation of this experimental study mainly monitors DC-CIK cells and anti-PD-1 antibody compositions are treatment-related bad
Reaction, i.e., in the follow-up during the treatment and after treatment, the therapy-related toxic and side effect that observed and recorded patient occurs, and root
According to national cancer institute adverse events generic term standard (NCI-CTCAE), the 4.0th edition is classified.DC-CIK cells
Mainly include with the anti-treatment-related adverse reaction of PD-1 antibody compositions：Heating, fatigue, anaemia, hepatic and renal function exception, allergy
Reaction, Neuroleptic Leukocytopenia and hypothyroidism etc..
(3) interpretation of result：
1) DC-CIK cells and the clinical objective reaction assessment result of anti-PD-1 antibody compositions treatment
Patients target's focus is evaluated by CT iconographies, determines effective alleviation situation of patient's treatment.Referring to Fig. 3 A institutes
Show, in 31 advanced refractory malignant tumor patients, 20 obtain effectively alleviation (64.5%), wherein 1 acquisition CR
(3.2%), 6 obtain PR (19.4%), and 13 obtain SD (41.9%).Remaining 11 there is progress (35.5%).Referring to 3C
Shown, in all kinds of tumor patients, liver cancer is preferable with the therapeutic effect of patients with renal cell carcinoma.Wherein one 64 years old male Hepatocellular liver
Cancer patient, after being treated through multiple TACE and RFA, patient's focus still gets along with earlier above, by 8 course for the treatment of DC-CIK cells and anti-
After the treatment of PD-1 antibody compositions, patient liver inner disease foci disappears.Another example 33 years old multiple pulmonary, left clavicle superior gluteal lymph node and centrum
The patients with renal cell carcinoma of transfer, after 8 course for the treatment of DC-CIK cells and anti-PD-1 antibody compositions are treated, patient many places metastatic lesion
It is obviously reduced or disappears, particularly the multiple MET of lung is most of disappears, and some focuses are obviously reduced (Fig. 4).These knots
Fruit shows that DC-CIK cells and anti-PD-1 antibody compositions treatment advanced refractory malignant tumor patient are safe and effective.
2) Overall survival of patient and progression free survival phase analysis result
According to the follow-up record carried out to each patient, the Overall survival and progression free survival phase of all patients are counted.Referring to figure
Shown in 3B, follow up time up to now, the total life span of middle position of 31 patients is 288 days, middle position Progression free survival time
It it is 162 days, wherein 8 deaths, 11 conditions of patients progress.
3) safety evaluation result：
Shown in Figure 5,17 there is therapy-related adverse reaction (54.8%) over the course for the treatment of, predominantly heating, fatigue
And Neuroleptic Leukocytopenia etc..Wherein, 15 patients show 1 or 2 grade of adverse reaction (88.2%), and only 23 or 4 grades of performances are bad
Reaction (11.8%), shows that DC-CIK cells and anti-PD-1 antibody compositions treatment advanced refractory malignant tumor patient are safety
's.
Embodiment 4：The lethal effect of DC-CIK cells and anti-PD-1 antibody compositions to tumour cell
In order to verify the validity of DC-CIK cells and the treatment of anti-PD-1 antibody compositions in clinical practice, the present invention is further
The lethal effect of DC-CIK cells and anti-PD-1 antibody compositions to autologous patient tumour cell is detected in vitro.
(1) material and method：
1) separation and culture of autologous tumor cell
Collect patients with renal cell carcinoma fresh tumor tissue to be placed in clean bench, with without calcium, magnesium PBS 2-3 times in plate.Tissue block
It is put into conical flask after shredding, adds 30-50 times more than tissue mass of 10% type Ⅳ collagenase digestive juice, is placed in 37 DEG C of water
Bath slightly shakes 1-2 hours.After tissue dispersion into after cell mass or individual cells, terminate digestion.1500r/min is centrifuged
5min, it adds the cleaning of the culture mediums of RPMI 1640, and the cell strainer elimination not yet fully digestion for passing through 100um after abandoning supernatant
Not yet tissue block.Supernatant is abandoned after 1500r/min centrifugations, a certain amount of resuspended list being centrifuged of the culture mediums of RPMI 1640 is added
Individual tumour cell, and it is placed in 37 DEG C, 5.0%CO with the culture mediums of RPMI 1640 containing 20% hyclone2Incubator is trained
Support.
2) autologous DC-CIK cells in vitro killing experiments
Above-mentioned autologous patient tumour cell is digested with 0.25% tryptic digestive juice, re-suspended cell after 1500r/min centrifugations, and
Cell is blown and beaten with pipette uniform.After counting concentration of cell suspension with tally, the cell concentration 10x that experiment needs is configured to
104Cell/ml.Cytotoxicity in vitro detection is carried out using RTCA systems.50 μ l culture mediums are added in the hole of E-Plate 16 first
After be put on RTCA Station and detect baseline values.The detection plates of E-Plate 16 are then taken out, adds 100 μ l to mix in hole
Uniform cell suspension, cell number is 10x 10 in making every hole3Cell/well.E-Plate 16 is placed in room in super-clean bench
Temperature is put on the RTCA Station in incubator after placing 30min, carries out the Real-time and Dynamic Detection of cell propagation.Second day,
By 50ul DC-CIK cells and anti-PD-1 antibody compositions or simple DC-CIK cell suspensions according to 3:1、10:1、30:1 effect
Target separately sets a hole and is not added with the Target cell wells of effector cell as a control group than adding in the detection plates of E-Plate 16.By E-
Plate16 detection plates are placed in real-time monitoring on RTCA monitor stations, compare two groups of lethal effects of cells against tumor cells.Finally kill
The computing formula for hindering effect is：Killing rate=(control group CI values-experimental group CI values)/control group CI values.
3) detection of DC-CIK cell killing activities and the change of tumor cells expression PD-L1 levels
Using the anti-PD-1 antibody of Cytomics FC500 flow cytometry analysis to the shadow of DC-CIK cell secretion of gamma-IFN levels
Ring, to judge the killing activity of cell.The level of tumor cells expression PD-L1 is detected simultaneously, and analysis tumour cell is to DC-CIK
The influence of cell killing activity.For the analysis of DC-CIK cell killing activities, cell is through 50ng/mL PMA, 500ng/mL
After ionomycin and 10ng/mL brefeldin A stimulate 4h, collect cell and be incubated 30min in AntiCD3 McAb/IFN-γ antibody
Afterwards, upper machine testing.For tumor cells expression PD-L1 horizontal analysis, after cell is incubated into 30min in anti-PD-L1 antibody, on
Machine testing.
(2) interpretation of result：
1) autologous DC-CIK cells in vitro kills autologous tumor cell experimental result
Referring to shown in Fig. 6 B, TC19 represents the patients with renal cell carcinoma that PR is reached after treating, and it is the patients with renal cell carcinoma of SD that TC17 is represented after treating,
There is the patients with renal cell carcinoma of PD after representing treatment in TC13.The Mortaility results for choosing 24 hours point RTCA system detectios are counted
Analysis, as a result shows, the autologous DC-CIK cells that effective reduction of patient is obtained after treatment have significantly to autologous tumor cell
The effect of DC-CIK cell killing tumour cells can be significantly increased after lethal effect, and the anti-PD-1 antibody of addition.For not obtaining
Remission patient, no matter its autologous DC-CIK cell have plus with anti-PD-1 antibody, all without substantially killing function of tumor.
2) DC-CIK cell killing activities and tumor cells expression PD-L1 analysis results
The autologous DC-CIK cells for further inquiring into the effective reduction of patient of acquisition show stronger anti-swollen after the treatment of anti-PD-1 antibody
The mechanism of tumor activity finds that autologous tumor cell can raise the expression of PD-L1, enter while by DC-CIK cell killings
And negative-feedback suppresses the immunocompetence of DC-CIK cells by PD-1/PD-L1 paths.It is effectively slow referring to shown in Fig. 6 C, obtaining
Two patients TC19 and TC17 of solution, its tumour cell have substantially raised PD-L1's after by autologous DC-CIK cell killings
Expression, respectively from 21.9% and 3.9%, increases to 97% (98.1%) and 34.2% (41%).And do not obtain disease and delay
The patient TC13 of solution, is not affected by the obvious killing of autologous DC-CIK cells without substantially raising the expression of PD-L1, only from
2.7%, increase to 4.4% (5.6%).Additionally, referring to shown in Fig. 6 D, to the testing result table of DC-CIK cell killing activities
Bright, TC19 is subject to the PD-L1 negative immunes that tumour cell is raised to adjust with TC17 autologous patient DC-CIK cells during killing
Save and reduce the secretion of IFN-γ, respectively from 18.5% and 11.8%, be reduced to 10.7% and 6.8%.And it is anti-through anti-PD-1
After body treatment, DC-CIK cells are not influenceed by PD-L1, continue to secrete more IFN-γs, respectively from 20.8% and 12.5%,
Increase to 39.6.7% and 21.9%, so as to play the activity of stronger killing tumor cell.The DC-CIK of TC13 autologous patient
The secretion of cell then unobvious secretion of gamma-IFN.
Claims (10)
- During 1.DC-CIK cells and anti-PD-1 antibody compositions are used to prepare treating cancer or delay the immune drug of cancer progression Purposes.
- 2. purposes according to claim 1, it is characterised in that：The immune drug contains 1-1.5 × 1010Individual DC-CIK is thin Born of the same parents and the anti-PD-1 antibody of 10mg.
- 3. purposes according to claim 1, it is characterised in that：The immune drug is before human body is input into 37 DEG C of insulating boxs It is incubated 30 minutes.
- 4. purposes according to claim 1, it is characterised in that described cancer is to be selected from：Liver cancer, kidney, colorectal cancer, Lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or nasopharyngeal carcinoma etc..
- 5. purposes according to claim 1, it is characterised in that：Described cancer is in early stage or middle and advanced stage rank Section.
- 6. it is a kind of for treating cancer or to delay the combined immunization composition of cancer progression, it is characterised in that the composition by The DC-CIK cells of effective dose and anti-PD-1 antibody are constituted.
- 7. composition according to claim 6, it is characterised in that：The composition contains 1-1.5 × 1010Individual DC-CIK is thin Born of the same parents and the anti-PD-1 antibody of 10mg.
- 8. composition according to claim 6, it is characterised in that：The DC-CIK cells are with anti-PD-1 antibody after combining It is incubated 30 minutes in 37 DEG C of insulating boxs.
- 9. composition according to claim 6, it is characterised in that：Described cancer is to be selected from：Liver cancer, kidney, Colon and rectum Cancer, lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or nasopharyngeal carcinoma etc..
- 10. composition according to claim 6, it is characterised in that：Described cancer is in early stage or middle and advanced stage Stage.
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