CN106890330B - DC-CIK cells and anti-PD-1 antibody compositions and application thereof - Google Patents
DC-CIK cells and anti-PD-1 antibody compositions and application thereof Download PDFInfo
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Abstract
The invention belongs to immunology, tumor microenvironment and immunotherapy of tumors field, provides DC CIK cells and 1 antibody compositions of anti-PD 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 compositions for treating cancer, it is made of the DC CIK cells and 1 antibody of anti-PD of effective dose.Composition immune drug provided by the present invention can effectively enhance patient's body killing tumor cell immunocompetence, 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 fields, are related to DC-CIK (Dendritic Cells
The cytokine induced kill cell of activation) cell and anti-PD-1 (programmed death receptor 1) antibody compositions purposes, tool
Body is related to DC-CIK cells and anti-PD-1 antibody compositions are used to prepare the purposes of immune drug.
Background technology
With industrialized development, the extraneous factors such as the exacerbation of environmental pollution, and smoking, bad life and eating habit
Effect, incidence 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 at
For 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 of current year in 2013 and broken through by will, and was continued " immunotherapy of tumors " in 2014
It is classified as one of the field that 2015 most merit attention.And in immunotherapy of tumors field, immunologic test orders blocking agent：Anti- PD-1 is anti-
The application of body, anti-PD-L1 antibody and anti-CTLA-4 antibody in clinical tumor immunization therapy most merits attention, they significantly extend
The life cycle of the patients such as Advanced Malignant melanoma, lung cancer and colon cancer.Immunologic test order blocking agent mainly pass through blocking
T cell inhibition signal path in tumor tissues achievees the purpose that kill tumour to make T cell persistent activation.At present
FDA has had been approved by anti-CTLA-4 and anti-PD-1 antibody for tumours such as clinical treatment Advanced Malignant melanoma and lung cancer.So
And during practical clinical, immunologic test orders blocking agent also by some challenges：First, since immunologic test point blocks
Agent is to induce lasting attack of the body immune system to tumour cell by immune response " brake system " in blocking body,
Stronger autoimmune response is often thus generated after patient's application, so as to cause stronger toxic side effect；Second is that needing enough foots
The application of the course for the treatment of, patient are difficult to bear the medical expense of great number.
And as a kind of adoptive cellular immunotherapy method of high-efficiency low-toxicity, CIK cell and DC-CIK cells are a variety of
Powerful antitumor action is all shown in hematological system tumor and entity tumor.CIK cell is one kind by the single core of peripheral blood
Polyclonal T cell of the cell in vitro through 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 self 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 there is stronger IFN-γ secretion ability, so the ability of the inside and outside killing tumor cell of DC-CIK cells is aobvious
Work is better than CIK cell.Clinical research in recent years finds CIK cell in liver cancer, lung cancer, colorectal cancer, gastric cancer, cervical carcinoma, kidney
There is therapeutic effect in 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 since its preparation process is compared with the former complexity, and production cost is higher, thus related 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.It is new therefore, it is necessary to carry out
Immunotherapy method clinical research to further increase the therapeutic effect of tumor patient.
It is carried with adoptive cellular immunotherapy use in conjunction currently, having part research report and immunologic test being ordered blocking agent
The method of high CIK cell killing ability.For example, Poh et al. in vitro use cell killing it is experimentally confirmed that block KIR, LAG-3 and
After the immunologic tests such as PD-1 point, CIK cell enhances the killing ability of Acute myeloid leukemia cell；But this killing energy
(Poh SL, et al.Cancer is not observed in acting on after another immunologic test point CTLA-4 is blocked for power enhancing
ImmunolImmunother.2016；65(5):525-36).Equally, after Dai et al. is it has also been found that block 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 effect associated with blocking agent is made an inventory of, also reports that DC-CIK cells and anti-PD-1 antibody compositions are answered in human body without correlative study
Specific method, therapeutic effect and toxic side effect.
Invention content
The purpose of the present invention is to provide the purposes of a kind of DC-CIK cells and 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.
The further feature of purposes according to the present invention, the immune drug contain 1-1.5 × 1010A DC-CIK is thin
Born of the same parents and the anti-PD-1 antibody of 10mg.
The further feature of purposes according to the present invention, the immune drug is before inputting human body in 37 DEG C of insulating boxs
It is incubated 30 minutes.
Preferably, the cancer includes：Liver cancer, kidney, colorectal cancer, lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or
Nasopharyngeal carcinoma etc..
Preferably, the cancer is to be in the middle and advanced stage stage.
It is another object of the present invention to provide a kind of for treating cancer or delays the combined immunization group of cancer progression
Close object.
It is of the present invention to be used to treating cancer or delay the combined immunization composition of cancer progression be by effective dose
DC-CIK cells and anti-PD-1 antibody composition.
The further feature of composition according to claim 1, the composition contain 1-1.5 × 1010A 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, the cancer includes：Liver cancer, kidney, colorectal cancer, lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or
Nasopharyngeal carcinoma etc..
Preferably, the cancer is to be in the middle and advanced stage stage.
The beneficial effects of the present invention are：DC-CIK cells have activation, proliferation and/or the cell lysis activity of enhancing,
Although DC-CIK cells have the activity of killing tumor cell, since DC-CIK cells can express the inhibitions 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 effect of tumours.And in composition of the present invention, DC-CIK cells through anti-PD-1 antibody processing after again
It feeds back in vivo, being tested through inside and outside confirms：The activity for significantly enhancing DC-CIK cell killing tumours provides it and treats tumour
The effect of.In some embodiments, tumor patient obtains apparent curative effect, tumor disappearance occurs, and partial reaction or disease are steady
It is fixed.
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 criteria,
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
It falls off because lost to follow-up, finally has 31 people that can carry out further clinical evaluation.(B) start to receive first 1 month of combined immunization treatment,
Patient stops using every other tumor-specific therapies, starts treatment blood drawing in first 14 days and carries out DC-CIK cell preparations, feeds back
For same day DC-CIK cell with anti-PD-1 antibody after 37 DEG C of insulating boxs are incubated 30 minutes, venous re-transfusion to patient's body is each to suffer from
Person at least carries out the combined immunization treatment of 8 courses for the treatment of, and preceding 4 courses for the treatment of are that 1 times a week, rear 4 courses for the treatment of are 2 weeks 1 time, some patientss
It can continue to receive the treatment of combined immunization composition after the course for the treatment of, until tumour progression.
Fig. 2 shows patient in group's clinic essential characteristic.In evaluable 31 cancer patients, 24 are male, 7 female
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
It loses, starts to receive combined immunization treatment, receive the number at least 8 times of combined immunization composition treatment, most 26 times, middle position is controlled
It is 12 times to treat number.
Fig. 3 shows the clinical effectiveness of DC-CIK cells and anti-PD-1 antibody compositions treatment 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 variation of line level.(B) the total of patient survives 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 DC-CIK cells and the anti-pretherapy and post-treatment size of tumor of PD-1 antibody compositions.Case 1 is that an example liver cancer is suffered from
Person receives combined immunization treatment, and liver inner disease foci disappears；Case 2 is an example patients with renal cell carcinoma Lung metastases lesion, receives combined immunization and controls
After treatment, intrapulmonary major part foci disappearance, remaining lesion is obviously reduced.
Fig. 5 shows the toxic side effect of DC-CIK cells and anti-PD-1 antibody compositions treatment late tumor patient.Patient connects
It is treated by combined immunization, 3 or 4 grades of toxic reactions occur in only 2 people, illustrate that this method safety is good.
The mechanisms of therapeutic action of Fig. 6 display bodies outer experimental analysis DC-CIK cells and anti-PD-1 antibody compositions.(A) it prepares
DC-CIK cell phenotypes detection.(B) lethal effect of DC-CIK cells and anti-PD-1 antibody compositions to primary tumor cell.
(C) the front and back PD-L1 expressions of primary tumor cell killing.(D) level of front and back DC-CIK cell secretion of gamma-IFN is killed.
Specific implementation mode
It is of the present invention that DC-CIK cells and anti-PD-1 antibody compositions are used to prepare treating cancer or delay cancer
The experimental procedure of the immune drug of progress is as follows.
Enter group conventional therapy unsuccessfully patient with advanced cancer through definitive pathological diagnosis, and is starting to receive combined immunization treatment preceding 1
Every other specific tumour treatment is deactivated within a month, peripheral blood in patients is extracted since treating first 14 days, uses density gradient centrifugation
Method isolates mononuclearcell (PBMC), the PBMC for the separation that suspended using Quanta-007 serum free mediums, with 1 × 108/ml
Quantity be laid on 75cm2Culture bottle in, be placed in CO2Stationary culture 1 hour in incubator.Culture bottle is gently shaken, suction is not pasted
The lymphocyte of wall is placed in new culture bottle, and 1000U/ml IFN-γ culture 24 hours is added, and 100ng/ml is added 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 culture solutions are cultivated.Full dose is mended every other day
Fresh medium is primary, and OK-432 (0.1KE/ml) and IFN-γ (1000U/ is added in the 5th day harvest immature DC cell (iDC)
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 centrifuging, 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 the combined immunization composition treatment of 8 courses for the treatment of, preceding 4 courses for the treatment of are that 1 times a week, rear 4 courses for the treatment of are 2 weeks 1 time, 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 fallen 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 is stablized, and total effective percentage reaches 22.5%, disease control rate 64.5%, and the middle total life span in position is 288 days, middle position without into
It is 162 days to open up life span.There are 3 or 4 grades of adverse reactions in only 2 people, are fever and Neuroleptic Leukocytopenia respectively.These results are said
Bright combined immunization composition therapy of the present invention can significantly improve disease control rate, extend patient with advanced cancer life
At the time, (operation instruction of the anti-PD-1 antibody of U.S.'s listing is pressed with anti-PD-1 antibody is used alone：Tumor patient presses 2mg/kg
Weight uses, and usual ampoule is more than 100mg) it compares, have dosage few (each 10mg), Small side effects are 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, in relation to case selection conventional therapy (hand
Art, chemicotherapy, targeted therapy) the advanced refractory malignant tumor patient that is in progress afterwards, observation DC-CIK cells and anti-PD-1 are anti-
Body composition treats safety, feasibility and the validity of these patient clinicals application.
(1) research object is included in：
1) inclusion criteria:
1. advanced refractory malignant tumor patient, including primary hepatoma, clear cell carcinoma of kidney, carcinoma of urinary bladder, colon
Cancer, non-small cell lung cancer etc.；2. age 18-75 Sui；3. estimated life cycle>3 months；4. ECOG scores 0 or 1 point；5. cardiopulmonary liver
Normal renal function；6. sufficient bone marrow reserve ability and factor is normal；7. the women of child-bearing age urinate gestation examination before administration starts
Feminine gender is tested, and is intended to together between experimental period until effective contraceptives person is taken in last time follow-up；8. according to RECIST standards
There is evaluable lesion；9. this experiment of voluntary participation simultaneously signs informed consent form.
2) exclusion criteria：
1. the patient treated using CTLA-4 or PD-1/PD-L1 blocking agents；2. the age<18 or>75 years old；3. the gestational period
Or women breast-feeding their children, effective contraceptives person is not taken in breeding time；4. there are serious Cardial or cerebral vascular diseases, cannot control
Hypertension and diabetes, renal insufficiency or failure person；5. the HIV positives or other immune deficiency disorders；6. there is autoimmunity
Property Disease；7. there is the patient of organ transplant history to use excess dosage glucocorticoid or other immunosuppressor within 4 weeks
Patient；8. there is the patient for clearly infecting or generating heat；9. t cell lymphoma, patients with malignant myeloma.
3) standard is exited：
1. doctor thinks stopped treatment to benefits subjects according to patient profiles；2. patient's initiative exits or not by research
Implementing plan；3. needing to carry out other chemotherapy other than this programme, operative treatment or experimental drug treatment during experiment；4. sending out
Raw serious adverse events, complication or special physiological variation, should not receive continual cure.
(2) therapeutic scheme：
It is treated the previous moon with DC-CIK cells and anti-PD-1 antibody compositions, it is related that enrolled patient stops all tumours
Treatment, the DC-CIK cells of preparation are fed back after being incubated 30 minutes in 37 DEG C of insulating boxs with anti-PD-1 antibody, once a week, at least
4 times are fed back to treat for previous stage；It is once assessed after the completion, if patient is effectively or stable, it is contemplated that carry out the 2nd rank
The maintaining treatment of section, 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 having treatment
Effect can continue with maintaining treatment once every 2 weeks, and every 3 months carry out clinical assessment.
(3) interpretation of result：
1) patient is included in and basic analysis of clinical：
Shown in Figure 1A, patient is included in analysis result and shows, incorporates 37 late period difficulties in the design of clinical test altogether
The property controlled 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 patients carry out subsequent analysis research.As shown in Fig. 2, 31 patients include male 24, women 7.Wherein primary
Hepatocellular carcinoma 9, clear cell carcinoma of kidney 8, colorectal cancer 6, non-small cell lung cancer 3, carcinoma of urinary bladder 2 and breast cancer, ovum
Each 1 of nest cancer, nasopharyngeal carcinoma.
2) patient treatment protocol's design result：
Shown in Figure 1B, studies the patient being included in and carry out DC-CIK cells and anti-P D-1 according to the therapeutic scheme of design
Antibody compositions are treated.The analysis result of Fig. 2 shows that the treatment of 31 patients at least all reaches a course for the treatment of and (at least feeds back 8
It is secondary), preceding 4 courses for the treatment of are that 1 times a week, rear 4 courses for the treatment of are 2 weeks 1 time, and it is 8-26 times to feed back numbers range, and middle position feeds back number and is
12 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 detach PBMC, carry out the culture of DC-CIK cells,
Patient's body is fed back to after being incubated altogether with anti-PD-1 antibody to be 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, peripheral blood in patients 50ml is extracted, after 800g × 8min is centrifuged,
Autologous plasma is collected, 56 DEG C of water-baths inactivate 30min, and after placing 4 DEG C of 15min, 800g × 8min takes supernatant blood plasma (about 20ml) to wait for
With.It is under superclean bench normal operating conditions, the peripheral blood cells after being collected by centrifugation are dilute with 0.9% injection physiological saline
It releases to 60ml, it is labelled with 2 disposable 50ml sterile centrifugation tubes, indicate patient's name, gender, age and admission number etc.
Relevant information is separately added into 20ml lymphocyte separation mediums, is detached with density-gradient centrifugation method and obtains peripheral blood mononuclear cells
(PBMC)。
Using the PBMC of Quanta-007 serum free mediums suspension separation, with 1 × 108The quantity of/ml is laid on 75cm2's
In culture bottle, it is placed in CO2Stationary culture 1 hour in incubator.Culture bottle is gently shaken, not adherent lymphocyte is sucked out, sets
In new culture bottle, 1000U/ml IFN-γ culture 24 hours is added, be added 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 culture solutions are cultivated.Full dose is mended every other day
Fresh medium is primary, and OK-432 (0.1KE/ml) and IFN-γ (1000U/ is added in the 5th day harvest immature DC cell (iDC)
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, DC-CIK cells were collected in disposable sterilized Centrifuge Cup (250ml), 2300rpm ×
8min is collected by centrifugation, and brine is used in combination 2 times, by 0.9% injection of human serum albumin of the cell containing 5ml 20%
It is resuspended in physiological saline.The cell suspension prepared keeps sample 2ml in glass ampule, seals ampoule mouth, labelled, indicates patient
Name, gender, the age, admission number and treatment project, 4 DEG C storage three months it is for future reference.By the DC-CIK cells prepared with contain
The 200ml physiological saline of the anti-PD-1 antibody of 10mg send lesion to be fed back after being incubated 30min in 37 DEG C, 5%CO2 incubators.
2) flow cytometry analysis DC-CIK cell phenotypes
Collect the above-mentioned DC-CIK cells prepared of sub-fraction, flow cytomery CD3+, CD3+CD4+, CD3+
The ratio of CD8+, CD3+CD56+ and CD3-CD16+CD56+ cell.Wherein CD3+CD8+ cells should be not less than 60%, CD3+
CD56+ cells should be not less than 10%.
(2) interpretation of result：
1) quality measurements of DC-CIK cells and anti-PD-1 antibody compositions：
Physical inspection：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 × 1010A cell, range are about
1-1.5×1010It is a；
Cell survival rate：It is detected with Trypan Blue, cell survival rate is all not less than 95%；
It is sterile, detected without mycoplasma and endotoxin-free：By current edition《Chinese Pharmacopoeia》Biological products testing regulations is to feeding back
Cell carries out bacterium, mycoplasma and endotoxin detection, and result is feminine gender.
2) flow cytometry analysis result：
Using flow cytomery analyze DC-CIK cells after, CD3+, CD3+CD4+, CD3+CD8+, CD3+CD56+ and
The ratio of CD3-CD16+CD56+ cells.As a result referring to shown in Fig. 6 (A), in the DC-CIK cells of 30 patients, CD3+T lymphs
Cell average proportions reach 94.1%, and main component is CD3+CD8+T lymphocytes, and the ratio averagely accounted for is 66.1%；CD3
+ CD56+T lymphocytes, the ratio averagely accounted for are 20.2%.
Embodiment 3：The clinical efficacy and safety of DC-CIK cells and anti-PD-1 antibody compositions treating cancer are evaluated
(1) curative effect evaluation：
The patient for being included in this clinical experimental study has measurable targeted site.The evaluation of lesion is by CT images
Follow-up period (every 6 months) in baseline after (treating the previous moon), treatment stage (every 3 months) and treatment end into
Row.
1) curative effect evaluation standard
Using (the Response Evaluation Criteria in Solid of evaluation criterion the effect of solid tumor
Tumors, RECIST) carry out therapeutic evaluation.First determine length summation of all measurable targeted sites in baseline, as
The reference baseline of record is effectively relieved, then according to the follow-up period targeted site versus baseline after treatment stage or treatment end
Situation is effectively relieved in the horizontal Assessment of Changes state of an illness.
2) standard being effectively relieved
The standard of alleviation includes CR：All targeted sites disappear；PR：The diminution of baseline lesion major diameter summation is more than or equal to
30%；SD：Baseline lesion major diameter summation has diminution but does not reach PR or have increase but do not reach PD；PD：Baseline lesion major diameter summation increases
Add more than 20% or occur new lesion.
3) follow-up
After since treatment, follow-up record is carried out to each patient, and check in every 3 months is once to determine the progress of patient
Situation, check content includes blood routine, blood biochemistry, tumor marker, hepatic and renal function, blood electrolyte, thyroid function and image
Learn assessment etc..Enter existence follow-up after to Tumor response stabilization or progress, every half a year is primary.According to the treatment time of patient and just
Secondary 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
Adverse reaction observes and records the treatment correlation toxic side effect of patient's appearance that is, in follow-up during treatment and after treatment,
And according to national cancer institute adverse events generic term standard (NCI-CTCAE) the 4.0th edition be classified.DC-CIK
Cell and the anti-treatment-related adverse reaction of PD-1 antibody compositions include mainly：Fever, fatigue, anaemia, hepatic and renal function be abnormal,
Allergic reaction, Neuroleptic Leukocytopenia and hypothyroidism etc..
(3) interpretation of result：
1) DC-CIK cells and the clinical objective of anti-PD-1 antibody compositions treatment react assessment result
Patients target's lesion is evaluated by CT images, determine patient's treatment is effectively relieved situation.Referring to figure
Shown in 3A, in 31 advanced refractory malignant tumor patients, 20 are effectively relieved (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, the therapeutic effect of liver cancer and patients with renal cell carcinoma is preferable.Wherein 64 years old male Hepatocellular liver
Cancer patient, after multiple TACE and RFA treatments, patient's lesion still has progressed earlier above, by 8 course for the treatment of DC-CIK cells and resists
After the treatment of PD-1 antibody compositions, patient's liver inner disease foci disappears.33 years old multiple pulmonary of another example, 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 the treatment of anti-PD-1 antibody compositions, patient many places metastatic lesion
It is obviously reduced or disappears, especially the multiple transfer stove of lung largely disappears, some lesions 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.Ginseng
See shown in Fig. 3 B, follow up time up to now, the total life span in middle position of 31 patients is 288 days, middle position Progression free survival
Time is 162 days, wherein 8 deaths, 11 conditions of patients progress.
3) safety evaluation result：
Shown in Figure 5,17 there are treatment related reactions (54.8%) over the course for the treatment of, predominantly generate heat,
Fatigue and Neuroleptic Leukocytopenia etc..Wherein, 15 patients show 1 or 2 grade of adverse reaction (88.2%), and only 2 show 3 or 4 grades
Adverse reaction (11.8%) shows that DC-CIK cells and anti-PD-1 antibody compositions treatment advanced refractory malignant tumor patient are
Safety.
Embodiment 4：The lethal effect of DC-CIK cells and anti-PD-1 antibody compositions to tumour cell
Treat the validity in clinical application to verify DC-CIK cells and anti-PD-1 antibody compositions, the present invention into
One step detects the lethal effect of DC-CIK cells and anti-PD-1 antibody compositions to autologous patient tumour cell in vitro.
(1) material and method：
1) separation and culture of autologous tumor cell
It collects patients with renal cell carcinoma fresh tumor tissue to be placed in clean bench, be cleaned 2-3 times with no calcium, magnesium PBS in plate.Group
It knits after block shreds and is put into conical flask, 30-50 times more than tissue mass of 10% type Ⅳ collagenase digestive juice is added, is placed in 37
DEG C water-bath slightly shakes 1-2 hours.After tissue dispersion is at cell mass or individual cells, digestion is terminated.1500r/min is centrifuged
5min is added the cleaning of 1640 culture mediums of RPMI after abandoning supernatant, and filters off not yet fully digestion by the cell strainer of 100um
Not yet tissue block.Supernatant is abandoned after 1500r/min centrifugations, 1640 culture mediums of a certain amount of RPMI is added, the list centrifuged is resuspended
A tumour cell is used in combination 1640 culture mediums of RPMI containing 20% fetal calf serum to be placed in 37 DEG C, 5.0%CO2Incubator is trained
It supports.
2) self DC-CIK cells in vitro killing experiments
Above-mentioned autologous patient tumour cell is digested with 0.25% tryptic digestive juice, is resuspended after 1500r/min centrifugations thin
Born of the same parents are used in combination pipette to blow and beat cell uniform.After counting concentration of cell suspension with tally, it is dense to be configured to the cell that experiment needs
Spend 10x 104Cell/ml.Cytotoxicity in vitro detection is carried out using RTCA systems.50 μ l are added in the hole of E-Plate 16 first
It is put into after culture medium on RTCA Station and detects baseline level.16 detection plates of E-Plate are then taken out, 100 are added in hole
Cell suspension uniformly mixed μ l, it is 10x 10 to make cell number in every hole3Cell/well.E-Plate 16 is placed in ultra-clean
It is placed at room temperature in platform on the RTCA Station being put into incubator after 30min, carries out the Real-time and Dynamic Detection of cell Proliferation.The
Two days, 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 than be added 16 detection plates of E-Plate in, separately set a hole and be not added with the Target cell wells of effector cell as a control group.By E-
Plate16 detection plates are placed on RTCA monitor stations and monitor in real time, compare the lethal effect of two groups of cells against tumor cells.Finally kill
The calculation 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 variation of tumor cells expression PD-L1 levels
It is horizontal to DC-CIK cells secretion of gamma-IFN using the anti-PD-1 antibody of Cytomics FC500 flow cytometry analysis
Influence, 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-
The influence of CIK cell killing activity.DC-CIK cell killing activities are analyzed, cell is through 50ng/mL PMA, 500ng/mL
After ionomycin and 10ng/mL brefeldin A stimulations 4h, collects 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 30min in anti-PD-L1 antibody, on
Machine testing.
(2) interpretation of result：
1) self DC-CIK cells in vitro kills autologous tumor cell experimental result
Shown in Fig. 6 B, the patients with renal cell carcinoma of PR, the kidney for being SD after TC17 representative treatments are reached after TC19 representative treatments
There is the patients with renal cell carcinoma of PD after representing treatment in patient, TC13.Choose 24 hour time point RTCA system detectio Mortaility results into
Row statistical analysis, the results showed that, the self DC-CIK cells that patient is effectively relieved are obtained after treatment has autologous tumor cell
Apparent lethal effect, and the effect of DC-CIK cell killing tumour cells can be significantly increased after the anti-PD-1 antibody of addition.It is right
In the patient for not obtaining remission, self DC-CIK cells no matter have plus use anti-PD-1 antibody, all without obviously kill it is swollen
Tumor acts on.
2) DC-CIK cell killing activities and tumor cells expression PD-L1 analysis results
Further inquire into obtain be effectively relieved the self DC-CIK cells of patient showed after the processing of anti-PD-1 antibody it is stronger
The mechanism of antitumor activity finds that autologous tumor cell can raise the table of PD-L1 while by DC-CIK cell killings
It reaches, and then negative-feedback inhibits the immunocompetence of DC-CIK cells by PD-1/PD-L1 accesses.Shown in Fig. 6 C, had
Two the patients TC19 and TC17 alleviated are imitated, tumour cell has obviously raised PD- after by self DC-CIK cell killings
The expression of L1 increases to 97% (98.1%) and 34.2% (41%) respectively from 21.9% and 3.9%.And disease is not obtained
The patient TC13 that disease is alleviated is not affected by the apparent expression killed without obviously raising PD-L1 of self DC-CIK cells, only from
2.7%, increase to 4.4% (5.6%).In addition, referring to shown in Fig. 6 D, to the testing result table of DC-CIK cell killing activities
PD-L1 negative immune tune bright, that TC19 is raised during killing by tumour cell with TC17 autologous patient DC-CIK cells
The secretion of IFN-γ is saved and reduced, respectively from 18.5% and 11.8%, is reduced to 10.7% and 6.8%.And it is anti-through anti-PD-1
After body processing, DC-CIK cells are not influenced by PD-L1, continue to secrete more IFN-γ, respectively from 20.8% and 12.5%,
Increase to 39.6.7% and 21.9%, 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 (2)
1.DC-CIK cells and anti-PD-1 antibody compositions are used to prepare treating cancer or delay in the immune drug of cancer progression
Purposes, it is characterised in that：The immune drug is by 1-1.5 × 1010A DC-CIK cells are formed with the anti-PD-1 antibody of 10mg；Institute
It is incubated 30 minutes in 37 DEG C of insulating boxs before stating immune drug input human body；The cancer is selected from：Liver cancer, kidney, colorectal cancer,
Lung cancer, carcinoma of urinary bladder, breast cancer, oophoroma or nasopharyngeal carcinoma；The cancer is in late stage.
2. a kind of combined immunization composition for being used for treating cancer or delaying cancer progression, it is characterised in that：The composition by
1-1.5×1010A DC-CIK cells are formed with the anti-PD-1 antibody of 10mg；After the DC-CIK cells and anti-PD-1 antibody combinations
37 DEG C of insulating boxs are incubated 30 minutes；The cancer is selected from：Liver cancer, kidney, colorectal cancer, lung cancer, carcinoma of urinary bladder, breast cancer, ovum
Nest cancer or nasopharyngeal carcinoma；The cancer is in late stage.
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