Immuno-Oncology


Source: http://www.cancerresearchuk.org/sites/default/files/cs_report_world.pdf
  • A different approach to treat cancer.
  • It is a type of immunotherapy to specifically treat cancer.
  • Immunotherapy activates the body's immune response to fight the disease.

Source: http://www.cancerresearchuk.org/sites/default/files/cs_report_world.pdf
Cells Services MoA/ Principles Examples of Assays
Tumour cells
  • Proliferation
  • Cytotoxicity
    E.g. Antibody drug conjugate (ADC)
  • Blocking cell function
  • Complement dependent cytotoxicity (CDC)
ADC cytotoxicity- HEL92.1.7 cells ADC assay CDC- Ramos Burkitt's lymphoma cell line treated with anti-CD20 antibody + human serum
  • Tumour metastasis
Quantitative
  • Invasion/ migration
MCF7 with chemoattractant.
  • Indoleamine 2,3- dioxygenases (IDO)/ Tryptophan 2,3- dioxygenases (TDO) modulation
  • Overexpression of IDO/TDO in cancer inhibits Tryptophan catabolism essential for T-cell response.
Tumour cell lines treated with IDO/TDO inhibitors. Endpoint: Proliferation
Immune Cells PBMCs, T-Cells, B cells, Macrophage, NK cells
  • Immune cell separation
Magnetic bead separation followed by FACS
  • Immune cell clustering
Test compounds on isolated cells
  • Immune cell activation
T-cell, B-cell, Macrophage, NK cells
  • Mixed Lymphocyte Reaction (MLR)
  • Cytokine profiling
  • T cell activation by anti- CD3/Il-2 or anti-CD28
  • Luminex, ELISA
  • Proliferation
Test compounds on isolated cells (Cell Titer Glo assay)
  • Cytotoxicity
  • Blocking cell function
L-kynurenine inhibits T cell activation
  • Chemotaxis
  • Trans-endothelial migration
Activated T cell migration through HUVEC monolayer by SDF-1a (CXCR4 ligand)
Tumour and Immune Cell Co-culture
  • Immune checkpoints
Stimulatory (+);
Inhibitory (-)
  • Inhibitor assay
  • Checkpoint analysis
  • Immune cell killing
T-Cell, PBMCs, NK
mediated cell killing
  • T-cell mediated lysis
  • Antibody dependent cell mediated cytotoxicity (ADCC)
  • Complement dependent cell mediated cytotoxicity (CDCC)
  • PBMCs and cytotoxic T lymphocytes co cultured with adherent or suspension tumour cells. End point: Caspase 3/7 or LDH assays.
  • ADCC- Her2+ SKOV3 cells with PBMC in presence of Trastuzumab.
  • CDCC- Tumour cells + Serum + NK cells
  • Phagocytosis
Macrophages
  • Antibody dependent cell phagocytosis (ADCP)
  • Efferocytosis
  • ADCP- Anti-CD47 binds to CCRF-CEM on human leukemic lymphoblasts and engulfed by bone-marrow derived macrophages

Immuno-Oncology expertise

  • Immune checkpoints are molecules able to switch on or off the immune system signalling.
  • Immune checkpoints can be:
    • - stimulatory: CD27, CD40, OX40, GITR, CD137, CD28, ICOS, CD154, CD122
    • - inhibitory: A2AR, B7-H3, B7-H4, BTLA, CTLA, IDO, KIR, LAG3, PD-1, TIM-3, VISTA
  • Cancer cells are able to activate/inactivate checkpoints in order to create a microenvironment to support tumor growth and avoid the attack from T cell.

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
  • EGFR
  • 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

QuantiGene single or multiplex:

B cells BLK, CD19, TNFRSF17,

T cells CD2, CD3E, CD3G, BATF, CD28, ICOS, CD38, CSF2, IFNG, IL12RB2, LTA, CTLA4, STAT4, CXCR6, GATA3, IL26, LAIR2, PMCH, SMAD2, STAT6, IL17A, IL17RA, RORC, CXCL13, MAF, PDCD1, BCL6, FOXP3, ATM, DOCK9, NEFL, USP9Y, AKT3, CCR2, EWS, ENFATC4.

The T cells genes can be clusterised based on the specific T cell subtype.

Natural killer: BCL2, NCR1, FOXJ1, MPPED1, PLA2G6, GZMB, IL21R

Macrophages: APOE, CCL7, CD68, CXCL5, MARCO, MSR1, CMA1, CTSG, KIT, PRG2, TPSAB1, CSF3R, FPR2, MME, CCR3, IL5RA, PTGDR2, THBS1

Dendritic cells: CCL13, CCL17, CCL22, CD209, HSD11B1, CD1A, CD1B, CD1E, F13A1, CCL1, EBI3, IDO1, IDO2, LAMP3, OAS3, IL3RA


Case Study


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)


For a complete list of relevant biomarkers, please click on the pdf link below:
Immuno-Oncology