CN109939126A - The application of CD3 × B7H3 bispecific antibody orientation killing adriamycin-resistant bladder cancer cell PUMC-91/ADM - Google Patents
The application of CD3 × B7H3 bispecific antibody orientation killing adriamycin-resistant bladder cancer cell PUMC-91/ADM Download PDFInfo
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Abstract
The purpose of the present invention is to provide the applications of coupling CD3 × B7H3 bispecific antibody orientation killing adriamycin-resistant Human transitional cell carcinoma of bladder PUMC-91/ADM a kind of, specially coupling CD3 × B7H3 bispecific antibody can enhance activating T cell (ATC) to the lethal effect of PUMC-91/ADM adriamycin-resistant bladder cancer cell, provide new immunotherapeutic targets for the drug resistant targeted therapy of bladder cancer.The present invention confirms B7H3 high expression in PUMC-91/ADM adriamycin-resistant bladder cancer cell by research；Compared with the ATC for not being coupled CD3 × B7H3 bispecific antibody, in conjunction with the ability enhancing of the ATC orientation killing PUMC-91/ADM adriamycin-resistant bladder cancer cell of CD3 × B7H3 bispecific antibody, there is significant cytotoxic activity to PUMC-91/ADM adriamycin-resistant bladder cancer cell.When the ATC and tumour cell for combining CD3 × B7H3 bispecific antibody are co-cultured under the conditions of imitating target ratio and being 10:1, fragmentation effect is obviously increased.Meanwhile in conjunction with the ATC of CD3 × B7H3 bispecific antibody secretion gamma interferon (IFN-γ) and the horizontal increase of α-tumornecrosisfactor (TNF-α).
Description
Technical field
Killing adriamycin-resistant people is oriented the purpose of the present invention is to provide a kind of coupling CD3 × B7H3 bispecific antibody to move
The application of row cell bladder cancer cell PUMC-91/ADM, specially coupling CD3 × B7H3 bispecific antibody can enhance activation T
Cell (ATC) is the drug resistant target of bladder cancer to the lethal effect of PUMC-91/ADM adriamycin-resistant Human transitional cell carcinoma of bladder
New immunotherapeutic targets are provided to treatment.
Background technique
Bladder cancer is the 4th common cancer in male, the 11st common cancer in women.It is estimated to be in the U.S. within 2017
79030 new hair bladder cancer cases and 19870 deaths, wherein male's morbidity and mortality are higher than 4 times of women.Most
Just, about 85% bladder cancer patients are diagnosed as superficial bladder cancer, and the superficial bladder cancer more than 50% can recur, also, only
There are 46% three phase patients and 15% 4 phase patients to can achieve five Nian Shengcun.Therefore, the form that bladder cancer patients are faced is very
It is severe.Despite the presence of multiple therapy methods, such as operation, radiotherapy, chemotherapy, the survival rates of bladder cancer are still paid no attention to very much
Think.These treatment methods can limit the development and growth of tumour, but they can not contain the recurrence and drug resistant generation of tumour.
Therefore, developing new therapy is vital to bladder cancer patients.
With immunologic progress, immunization therapy is acknowledged as the 4th in 21st century combined therapy of tumour mode
Kind treatment method.For example, targeting CTLA-4(cytotoxic T lymphocyte epitope) and PD-1/PD-L(programmed cell death
1/ PD-1 ligand) immunologic test point inhibitor promoted the treatment of cancer, another effective method is that targeting T is thin
The utilization of the bispecific antibody (BiAb) of extracellular antigen recipient T cells (TCR) and tumor associated antigen (TAA).
In past 2 years, bispecific antibody targets different tumor associated antigens, including EGFR, Her2, CD19,
CD20, CD30, CEA, CA125, PSA etc. obtain encouraging achievement in experiment and clinical research.B7H3, also referred to as
CD276 has up to 30% identical amino acid with B7 family.Its height expression has been demonstrated in a plurality of types of cancers
It can promote the progress of tumour and the transfer of cancer cell, including acute leukemia, glioma, hepatocellular carcinoma, lung cancer, breast cancer is preceding
Column gland cancer, osteosarcoma, cutaneous melanoma and cancer of pancreas.The gene transfer that Luo et al. demonstrates mouse B7H3 produces effectively
Anti tumor immune response, Lupu etc. injects the adenovirus containing B7H3 in the model of colon cancer of mouse original position, and discovery can inhibit swollen
The transfer of tumor, MJ18, a kind of anti-B7H3 mouse is anti-, is proved to can inhibit the growth of tumour in pancreatic cancer models.Result of study table
Bright B7H3 is expected to be used for based on immune antineoplaston.
Summary of the invention
Killing adriamycin-resistant people is oriented the purpose of the present invention is to provide a kind of coupling CD3 × B7H3 bispecific antibody to move
The application of row cell bladder cancer cell PUMC-91/ADM.
Preferably, the bladder cancer mdr cell is PUMC-91/ADM adriamycin-resistant Human transitional cell carcinoma of bladder.
Preferably, structure such as Fig. 1 of coupling CD3 × B7H3 bispecific antibody.
Preferably, the bispecific antibody can enhance activating T cell to PUMC-91/ADM adriamycin-resistant people's migratory cell
The lethal effect of bladder cancer cell.
Preferably, the activating T cell secretion gamma interferon and α-tumornecrosisfactor level increase.
Preferably, the activating T cell of combination CD3 × B7H3 bispecific antibody and tumour cell are in effect target ratio
When 10:1, lethal effect is obviously increased.
Inventor cultivates adriamycin-resistant people's transitional cell bladder cancer PUMC-91/ADM cell strain first.It is close using Ficoll
The peripheral blood that degree gradient centrifugation separates the healthy contributor of Beijing blood bank offer obtains peripheral blood mononuclear cells (PBMCs).?
It is supplemented with 10%FBS and 5ug/ml AntiCD3 McAb mAb(eBioscience, San Diego, CA, USA) and 100IU/ml recombined human IL-
With 1 × 10 in 2 RPMI-1640 culture medium6/ ml cultivates PBMC.Make anti-B7-H3 monoclonal antibody (R & D System,
Minneapolis, MN, USA) it is reacted with sulfo group-SMCC, AntiCD3 McAb (OKT3, eBioscience) is reacted with Traut's reagent.
The ATC of defrosting freezen protective, and in conjunction with B7-H3Bi-Ab, calculate Conjugate ratio.Utilize lactic acid dehydrogenase activity kit measurement
Cytotoxicity, ELISA detect cell factor, Beckman flow cytomery cell cycle, and CCK-8 detects cell Proliferation.Knot
Compared with fruit shows with independent ATC and be not coupled the ATC of bispecific antibody, dual anti-ATC is coupled to mankind's adriamycin-resistant bladder
Cancer cell has significant cytotoxic activity, also, is coupled dual anti-ATC and secretes higher levels of IFN-γ, TNF-α.As a result
The cytotoxic activity for showing T cell is the adriamycin-resistant bladder cancer cell that the B7H3 positive is targeted by the bridge of CD3-B7H3, from
And play lethal effect.
The present invention confirms that CD3XB7H3 bispecific antibody can enhance ATC and kill adriamycin-resistant bladder cancer cell by research
Ability, new immunotherapeutic targets may be provided for the drug resistant targeted therapy of bladder cancer.
The present invention will provide new strategy and experimental basis for the development of the drug resistant immunization therapy of bladder cancer and accurate medicine.
For above and other objects of the present invention, feature and advantage can be clearer and more comprehensible, preferred embodiment is cited below particularly,
And cooperate attached drawing, it is described in detail below.
Detailed description of the invention
Fig. 1 is the structure chart for being coupled CD3XB3H7 bispecific antibody.
Fig. 2 is B7H3 high expression in adriamycin-resistant people's transitional cell bladder cancer PUMC-91/ADM cell.
Fig. 3 is the expression of CD3 and ATC vegetative map in the ATC of amplification.
Fig. 4 is lethal effect of the ATC to adriamycin-resistant bladder cancer cell that bispecific antibody is coupled using LDH detection.
Fig. 5 is the ATC secrete cytokines increase for being coupled bispecific antibody.
Specific embodiment
Embodiment 1Cell culture
Adriamycin-resistant Human transitional cell carcinoma of bladder strain PUMC-91/ADM is gradually increased by its parental cell strain PUMC-91
The dosage of adriamycin is established, and the ultimate density of adriamycin is 1.0 μ g/ ml.15% fetal calf serum culture.Cell contains at 37 DEG C
It is cultivated in the incubator of 5% carbon dioxide.
Embodiment 2The separation of peripheral blood mononuclear cells and the T lymphocyte (ATC) for preparing freezen protective activation
Peripheral blood mononuclear is obtained using the peripheral blood that Ficoll density gradient centrifugation separates the healthy contributor of Beijing blood bank offer
Cell (PBMCs).It is being supplemented with 10%FBS and 5 μ g/ ml AntiCD3 McAb mAb(eBioscience, San Diego, CA, USA) and
With 1 × 10 in the RPMI-1640 culture medium of 100IU/ml recombinant human il-26/ ml cultivates PBMC.It is new needed for cell culture
Fresh culture medium contains 100IU/ml recombinant human il-2.13rd day, averagely there is ATC expression CD3+(CD4+and % of amplification
CD8+), freezen protective is to further use.
Embodiment 3Synthesis AntiCD3 McAb × anti-B7-H3 bispecific antibody (B7-H3Bi-Ab) and be coupled to activation T it is thin
Born of the same parents
Anti- B7-H3 monoclonal antibody (R & D System, Minneapolis, MN, USA) is reacted, AntiCD3 McAb with sulfo group-SMCC
(OKT3, eBioscience) is reacted with Traut's reagent.The ATC of defrosting freezen protective, and and B7-H3Bi-Ab with 50ng/
106The concentration of cell 30 minutes at room temperature, wash cell then to eliminate unbonded antibody.Using ImagJ to western
Blot band carries out gray analysis and calculates Conjugate ratio.
Embodiment 4The detection of cytotoxicity
Target cell is seeded in the 96 hole bottom U microplates, is separately added into and is coupled dual anti-ATC, is not coupled dual anti-ATC or individual
ATC.Every kind is done 3 multiple holes.Effector cell and tumour cell effect target ratio are and 10:1, after 24 hours that interact at 37 DEG C
Collect supernatant.Cytotoxicity mainly be measured by lactic acid dehydrogenase activity kit (Sigma-Aldrich, St.Louis,
MO, USA)
Embodiment 5ELISA detects cell factor
Target cell is inoculated in the 96 hole bottom U microwell plates, and concentration is 1 × 104/ hole, 37 DEG C overnight.Then added with E/T ratio of 10:1
Enter to be coupled dual anti-ATC, is not coupled dual anti-ATC or individual ATC and is incubated for 24 hours.Cell supernatant is collected, and according to system
The explanation for making quotient measures IFN-γ, TNF-α using specific mankind's ELISA kit (eBioscience).
Embodiment 6Flow cytometry
Anti human B 7-H 3-PE mAb and mouse IgG 1-PE isotpye antibody, anti-human CD3-FITC, anti-mouse IgG1-FITC, and
Anti-mouse IgG2a-FITC secondary antibody is purchased from eBioscience.Raji cell assay Raji Beckman flow cytomery, is used in combination
CytExpert software carries out data analysis.
Although the present invention has been disclosed in the preferred embodiments as above, however, it is not to limit the invention, any affiliated technology
The technical staff in field, without departing from the spirit and scope of the present invention, when can make a little change and improve, therefore the present invention
Protection scope when view as defined in claim.
Claims (6)
1. being coupled the application of CD3 × B7H3 bispecific antibody orientation killing human bladder cancer's mdr cell.
2. application according to claim 1, which is characterized in that the bladder cancer mdr cell be PUMC-91/ADM resistance to Ah
Mycin Human transitional cell carcinoma of bladder.
3. application according to claim 1, which is characterized in that the structure of coupling CD3 × B7H3 bispecific antibody is as schemed
1。
4. being applied according to shown in claim 3, which is characterized in that bispecific antibody can enhance activating T cell to PUMC-
The lethal effect of 91/ADM adriamycin-resistant Human transitional cell carcinoma of bladder.
5. being applied according to shown in claim 4, which is characterized in that activating T cell secretes gamma interferon and α-tumor necrosis factor
Sub horizontal increase.
6. being applied according to shown in claim 4, which is characterized in that in conjunction with the activating T cell of CD3 × B7H3 bispecific antibody
With tumour cell when imitating target ratio is 10:1, lethal effect is remarkably reinforced.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017030926A1 (en) * | 2015-08-17 | 2017-02-23 | Macrogenics, Inc. | Bispecific monovalent diabodies that are capable of binding b7-h3 and cd3, and uses thereof |
| CN106715469A (en) * | 2014-08-27 | 2017-05-24 | 纪念斯隆-凯特琳癌症中心 | Antibodies, compositions, and uses |
-
2017
- 2017-12-21 CN CN201711397309.9A patent/CN109939126A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106715469A (en) * | 2014-08-27 | 2017-05-24 | 纪念斯隆-凯特琳癌症中心 | Antibodies, compositions, and uses |
| WO2017030926A1 (en) * | 2015-08-17 | 2017-02-23 | Macrogenics, Inc. | Bispecific monovalent diabodies that are capable of binding b7-h3 and cd3, and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| PAUL MOORE等: "MGD009, a B7-H3 x CD3 Bispecific Dual-Affinity Re-Targeting (DART®) Molecule Directing T Cells to Solid Tumors", 《MACRO GENICS》 * |
| QINGZHONG HE等: "Purified anti-CD3 × anti-HER2 bispecific antibody potentiates cytokine-induced killer cells of poor spontaneous cytotoxicity against breast cancer cells", 《CELL & BIOSCIENCE》 * |
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Application publication date: 20190628 |