Immuno-Oncology

Immuno-Oncology

Source: http://www.cancerresearchuk.org/sites/default/files/cs_report_world.pdf

Our immune system is an extraordinary sentinel that protects our body from harmful pathogens such us bacteria and viruses (the innate immunity), produce antibodies against foreign substances and destroy abnormal cells that may evolve into cancer cells (the adaptive immunity).

A balanced immune system can perfectly target and remove cancer cells before its evolution to a detectable tumour. However, an imbalanced immune system response could contribute to tumorigenesis or induce inflammation stimulating cancer cell proliferation and metastasis, leaving these cells growing effectively avoiding our immune system [1]. The very beginning of a therapeutic regimen supporting the activation of our immune system to fight disease can be traced back to the China’s Qin dynasty period, around the third century BC. This was associated with purposeful inoculation with variola minor virus to prevent smallpox infections [2].  Nowadays, we have acquired a strong understanding of how we can help our immune system play an effective role against cancer (or other diseases). It was in 2010 that the FDA approved the first immune therapy against prostate cancer, launching de facto the modern immunotherapy [3].

Cancer immunotherapy or immuno-oncology, by definition, represents a specific targeted therapy by using our immune system to fight and destroy cancer cells. As this does not involve surgery or use of radiation or chemotherapy, it is relatively safe to the healthy cells of the body. Importantly, it could be applicable at all stages of the disease, with higher efficiency.

Currently cancer immunotherapy is divided into several areas depending on how the immune system can be improved or supported. For example, the immune cell therapy called CAR-T (Chimeric antigen receptor T-cell therapy), consists in collecting part of the patient’s T cells (a type of immune cells), re-engineer them in the lab to produce surface receptors that are capable of identifying and attacking specific tumour cells and infusing them back into the patient via the bloodstream. By remaining active in the body for a period, these CAR-T cells can ward off cancer recurrence, resulting in long-term remission. Novartis’ targeted therapy is based on this technology to treat acute lymphoblastic leukaemia, a type of blood cancer, in children.

Source: http://www.cancerresearchuk.org/sites/default/files/cs_report_world.pdf

The whole objective of Immunotherapy is to enable our immune system understand differences between healthy and cancer cells and then specifically target the cancer cells. We now have the tools to produce specific substances that stimulate our immune system to recognise and fight specific cancer cells. Monoclonal antibodies, checkpoint inhibitors and cytokines represent a class of immuno-oncology therapies that trigger an immune response to destroy cancer cells. As a fact, we have now several such therapies available to successfully treat several cancers including lung, kidney, bladder, head and neck, melanoma and some autoimmune diseases such as rheumatoid arthritis, psoriasis and alopecia.

Finally, we have a separate class of immunotherapy, represented by cancer vaccines. They do not work in the same way as regular vaccines. Once injected into the patient’s bloodstream, they will stimulate an immune response within the body to attack existing cancer cells. The objective here is to trigger a sort of immune memory, preventing cancer recurrence.

In conclusion, immunotherapy (or immuno-oncology) has opened a new era in healthcare and indeed we can soon expect some ground-breaking discoveries that will completely erase our current notion of cancer as a life-threatening disease. Having said that, modelling the immune system in vitro to stimulate an effective response to cancer sets a particularly difficult challenge. Potential roles of multiple immune cells, the heterogeneity of tumours and the molecular mechanisms involved mean that multiple advanced assay models are required before moving the most promising immunotherapeutic approaches into clinical trials.

References:

  1. Hanahan D and Weinberg RA. Cell. 2011
  2. Decker WK et al. Front Immunol. 2017
  3. Handy CE et al. Future Oncol. 2018

Assays Offered

1. ADCC

Raji cells were treated with Anti-CD20 and T cells for 6 hours and 24 hours respectively and the Relative Luminescence Units (RLU) were measured 6 hours [A] and 24 hours [B] post incubation. The higher concentration of the Anti-CD20 resulted in an increase in the activation of the NFAT pathway. This corresponds directly to an increase in the target cell death.

ADC assay was performed using Human Mast Cells 1.2 (HMC1.2). CD13 and CD33, respectively markers for solid tumors and acute myeloid leukaemia, are targeted using secondary antibody MMAE-conjugated.

CD13+ and CD33+ HMC1.2 cells were exposed to primary antibodies CD13 and CD33 specific, followed by to scalar concentrations of secondary antibody MMAE-conjugated. Cytotoxicity was assessed by ATP release assay. Percentage of mortality was calculated versus cells treated with primary antibodies only. Each point is the average of 4 replicates. Bars represent the Standard Error Median (SEM)

2. Immune Cell Killing

T cell-based tumour killing assay

Summary of T-cell Cytotoxicity Assay

  1. Quantification of green fluorescent cells relative to A549 cells (black), using the IncuCyte rapid green alive cells dye (Sartorius).
  2. Quantification of Annexin V red fluorescent cells, relative to A549 cells (grey), IncuCyte Annexin V red (Sartorius). Incubation of A549 cells with activated pan-T cells determined a significant increase of the number of apoptotic cells.

Data are expressed as mean ± SEM, **p<0.05 ANOVA one-way.

3. Immune Checkpoints Involved In Cancer Development - xMAP Technology

  • 4-1BB (CD137/TNFRSF9)
  • AKT1, ARG1,
  • B7-1 (CD80), Bp50 (CD40), BTLA (CD272)
  • CCL2, CCL3, CCL5, CCL7, CX3CL1, CD27, CD28, CD70, CSF1, CTLA4, CX3CL1, CX3CR1, CXCL10, CXCL13, CXCL9
  • ICAM1, ICOS, IDO, IFNα, IFNβ1, IFNγ, IL-10, IL-12A, IL-12B, IL-13, IL-17A, IL-18, IL-1B, IL-2
  • IL-20, IL-21, IL-33, IL-4, IL-6, IL-7
  • LAG3
  • MICA, MICB
  • PD-1 (CD279/PDCD1), PD-2 (PDCD2), PD-L1 (CD274/PDCD1L1), PD-L2 (CD273/PDCD1L2),
  • PTGDS
  • TGFβ1, TIM3 (HAVCR2), TNF-a

xMAP – proteins multiplexing

BTLA; GITR; HVEM; IDO; LAG-3; PD-1; PD-L1; PD-L2; TIM-3; CD28; CD80; CD137; CD27; CD152

4. NK cell-based Cytotoxicity Assay

  • Evaluation of NK cells-mediated ability to kill target tumour cells

    -Quantification of the pro-apoptotic effects on tumour cells over time.

Quantification of Caspase 3/7 and LDH activity in a co-culture model of K562 cells incubated with unstimulated/stimulated primary CD56+ NK cells. Data is expressed as Relative Luminescence Units (RLUs), mean ± SEM.

Staurosporine = positive control

5. PBMCs and PD-1 blockade

PBMCs proliferate in response to anti-CD3 stimulation

PBMCs from 2 different healthy volunteers were stimulated with increasing concentrations of anti-CD3 antibody (0µM, 0.25µM, 1µM and 5µM) over the period of 144 hours and the cell proliferation was recorded. A dose dependent increase in PBMC cell counts was observed after 48 hours of stimulation

PBMCs proliferate in response to anti-CD3 stimulation

PBMCs were treated for 96 hours with increasing concentrations of anti-CD3 antibody (0µM, 0.25µM, 1µM and 5µM; red line) or with Isotype control (blue line) or with Nivolumab (black line). Supernatants were collected and IFN-γ release was quantified by ELISA.

Results demonstrate a dose dependent increase in IFNγ levels which are further potentiated by PD-1 blockade as suggested by Nivolumab treatment.

6. PD-1/PD-L1 immune checkpoint bioassay

The PD-1/PD-L1 luciferase bioassay enables detection of the inhibitory activity of anti-PD-1 and anti-PD-L1 blocking antibodies as quantified by increases in luminescence. In the assay workflow, PD-L1 expressing aAPC/CHO-K1 cells target cells are plated and incubated for 16–20 hours prior to the addition of increasing concentrations of test antibodies and the PD-1 expressing effector cells. Luminescence is detected after 6 hours using Bio-Glo™ Reagent and the GloMax® Discover System. Data is fitted to a 4PL curve using GraphPad Prism. Validation of the assay has been performed using Nivolumab and Pembrolizumab. EC50 for Nivolumab and Pembrolizumab is 0.28µg/ml and 0.11µg/ml respectively (a). The assay is specific for PD-1 blockade as confirmed by incubation with Ipilumamb, a CTLA-4 inhibitory antibody. A high degree of intra-plate reproducibility in Nivolumab inhibitory response is demonstrated (b).

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