Cancer Immunotherapy: Combination Therapy

Vivian Cao

The traditional oncology pharmacopia of small molecules is rapidly being supplemented with biologics such as checkpoint inhibitors, and will soon also include cellular therapies (CAR-T cells, etc.). With this expanding repertoire comes the possibility of boosting cancer killing efficacy by combining different modalities. The optimization of combination therapies would benefit from an assay platform that, by maintaining high sensitivity under physiologically relevant conditions, yields in vitro data that is predictive of in vivo behavior. Other desirable characteristics include an easy workflow and a high throughput format, enabling diverse permutations of combination therapies to be analyzed simultaneously. xCELLigence Real-Time Cell Analysis (RTCA) meets all of the above criteria.

In the below example xCELLigence RTCA was used to monitor the impact that a combination of PD-1 and CTLA-4 checkpoint inhibitors have on the killing of prostate cancer PC3 cells by PBMCs. Target PC3 cells were seeded in ACEA’s patented biosensor plate (E-Plate) and allowed to attach and proliferate. Frozen PBMCs were thawed, activated by incubation with SEB superantigen, and then added on top of the PC3 cells in the presence or absence of 38 nM anti-PD-1 antibody and two different concentrations of anti-CTLA-4 antibodies. The effector:target ratio was 5:1. The killing efficacy of PBMCs varies dramatically from donor to donor, and for this particular batch of cells adding 38 nM anti-PD-1 did not enhance the killing of target cells. However, adding anti-CTLA-4 along with anti-PD-1 promoted target cell killing in a dose dependent manner (green and pink traces).

By analyzing cancer cell killing with high sensitivity and without the need for labels/modifications, the xCELLigence Real-Time Cell Analysis (RTCA) instruments allow effector and target cells to be studied under conditions that approximate human physiology more closely than other in vitro techniques. By monitoring combination therapy-induced target cell killing continuously, these instruments also do away with laborious end points and thereby readily yield cell killing data under many different conditions simultaneously.

Key Benefits of Using xCELLigence for Combination Therapy Studies::

  • Label-Free: Because 51Cr, luciferace, dyes, etc. aren’t needed, target cancer cells and immune effector cells can be used directly…maximizing physiological relevance.
  • Exquisite Sensitivity: Analyze combination therapies at low, physiologically relevant effector:target ratios.
  • Unlimited Assay Window: The impact of combination therapies on target cell killing can be monitored over assay windows spanning anywhere from minutes to days, elucidating both fast and slow killing kinetics. Differences in long term killing behavior are now observable.
  • Astonishingly Simply Workflow: Plate target cells, add effector cells and molecules, and start monitoring. High throughput comparisons of different combination therapies are easy to setup, run, and analyze.
Combination Therapy – Supporting Information:
Dual Purpose Single Plate Multi Plate High Throughput
RTCA DP RTCA SP RTCA MP RTCA HT
3×16 wells 1×96 wells 6×96 wells Up to 4×384 wells
  • Combination Therapy Publications:
  1. Oncolytic Adenoviral Delivery of an EGFR-Targeting T-cell Engager Improves Antitumor Efficacy.  Fajardo CA, Guedan S, Rojas LA, Moreno R, Arias-Badia M, de Sostoa J, June CH, Alemany R.  Cancer Res. 2017 Apr 15;77(8):2052-2063.
  2. The MDM2-inhibitor Nutlin-3 synergizes with cisplatin to induce p53 dependent tumor cell apoptosis in non-small cell lung cancer. Deben C, Wouters A, Op de Beeck K, van Den Bossche J, Jacobs J, Zwaenepoel K, Peeters M, Van Meerbeeck J, Lardon F, Rolfo C, Deschoolmeester V, Pauwels P. Oncotarget. 2015 Sep 8;6(26):22666-79.
  3. Cotylenin A and arsenic trioxide cooperatively suppress cell proliferation and cell invasion activity in human breast cancer cells. Kasukabe T, Okabe-Kado J, Kato N, Honma Y, Kumakura S.  Int J Oncol. 2015 Feb;46(2):841-8.
  4. Combination therapy targeting ectopic ATP synthase and 26S proteasome induces ER stress in breast cancer cells.  Chang HY, Huang TC, Chen NN, Huang HC, Juan HF.  Cell Death Dis. 2014 Nov 27;5:e1540.
  5. Dianthin-EGF is an effective tumor targeted toxin in combination with saponins in a xenograft model for colon carcinoma. von Mallinckrodt B, Thakur M, Weng A, Gilabert-Oriol R, Dürkop H, Brenner W, Lukas M, Beindorff N, Melzig MF, Fuchs H.  Future Oncol. 2014 Nov;10(14):2161-75.
  6. Combination treatment for glioblastoma cells with tumor necrosis factor-related apoptosis-inducing ligand and oncolytic adenovirus delta-24. Tsamis KI, Alexiou GA, Vartholomatos E, Kyritsis AP.  Cancer Invest. 2013 Nov;31(9):630-8.