Development of biochemical assays for immunotherapy
drug discovery and development
Andrew C. Newman, Aaron Snead, Stephen Edgcomb, and Henry Zhu
BPS Bioscience, Inc. San Diego, CA 92121
Immunotherapy has become an important approach for the treatment of
numerous diseases including cancer. A number of immunotherapies
target one or more co-stimulatory or co-inhibitory pathways regulating
immune activation such as cell surface receptors and enzymes like IDO1
and TDO. Reliable high throughput screening (HTS) methods are
needed to successfully screen and identify small molecules, antibodies,
or antibody fragments (Fab) that modulate these pathways. Here, we
report the development of a toolbox of HTS biochemical assays to
screen for inhibitors of immune receptor-ligand interaction and for
screening inhibitors of IDO1 and TDO activity. These assays have been
internally validated using known inhibitors. Future work will focus on
establishing novel biochemical and
cell-based assays for
immunoregulatory pathways.
INTRODUCTION
Recognition and clearance of cancerous cells by the immune system
requires a series of steps. Some of these steps involve signaling
cascades that are initiated by proteins expressed on the surface of
cancerous cells and/or immune cells that regulate immune activation.
Many tumors have evolved specific mechanisms to evade the immune
response. This often involves cancer cells expressing ligands that
directly inhibit T cell activation by activating co-inhibitory pathways (Fig.
1). A number of therapies targeting T cell co-inhibitory receptors have
demonstrated profound clinical benefit1,2,3.
Indoleamine 2,3 dioxygenase 1 (IDO1) and tryptophan 2,3 dixoygenase
(TDO) are enzymes that catalyze the rate-limiting step of tryptophan
degradation and indirectly inhibits T cell activation through tryptophan
depletion and generation of toxic metabolites. Importantly, both of these
enzymes have been shown to be upregulated by subsets of cancers4
(Fig. 4) and IDO1 inhibitors are currently being pursued in the clinic5,6.
Bioscience, Inc. San Diego, CA
RESULTS
A.
Wash
Strep HRP is added,
followed by
chemiluminescent or
colorimetric substrate
Biotin-labeled
ligand is added
with or without
inhibitor
Wash
Figure 2. Immunotherapy biochemical assay design. BPS’ ELISA-based immunotherapy
assays take advantage of our biotin-labeled proteins. One binding partner is allowed to adhere to a
plate overnight and the next day the cognate binding partner along with test inhibitor is added.
Binding is detected by addition of Strep-HRP along with chemiliuminescent or colorimetric HRP
substrate.
A.
Figure 4. The role of IDO1 & TDO in the tumor microenvironement. (A) PD-1 inhibitors have
shown clinical efficacy, but many tumors upregulate expression of IDO1 and/or TDO, resulting in
Trp depletion in the tumor microenvironment and increased levels of the Trp metabolite,
Kynunerinine (Kyn) which combine to induce T cell cycle arrest and apoptosis. (B) Inhibition of
IDO1 and TDO has the potential to halt T cell inhibition caused by Trp catabolism.
A.
C.
B.
B.
siWnt5a
D.
F.
E.
However, not all cancers have responded to current therapies, providing
rationale for targeting other co-regulatory pathways. BPS has developed
biochemical-based assays designed to screen for inhibitors of relevant
immunotherapy targets in an effort to accelerate the discovery and
development of novel immunotherapies.
Figure 3. Validation of BPS’ Immunotherapy Receptor
Inhibitor Screening Assay Kits. BPS Bioscience’s assay kits
are validated by inhibiting receptor-ligand interaction with known
inhibitors, when available, or with unlabeled ligand. Figure 3
depicts inhibition of (A) PD-1:PD-L1 (B) PD-1:PD-L2 (C)
CD137:CD137L (D) CD28:B7-1 (E) CTLA4:B7-1 (F) CTLA4:B72, and (G) BTLA-HVEM interaction by PD-1 neutralizing antibody
(BPS Cat. #71120), CTLA4 neutralizing antibody (BPS Cat.
#71212), or unlabeled ligand as indicated.
G.
Figure 1. Immunotherapy targets. T cells are activated when the T cell
receptor engages with peptides displayed by MHC class I or II by antigen
presenting cells or cancer cells. A large repertoire of co-inhibitory and costimulatory receptors and ligands integrate a signaling network that
regulates T cell activation, differentiation, survival, and effector function.
B.
Wells are
coated with
receptor
Y Y Y Y
1 BPS
Y Y Y Y
ABSTRACT
Catalog #
Product Name
Catalog #
Product Name
72003
PD-1:PD-L1[Biotinylated]
72014
PD1:PDL1 Homogeneous Assay
72004
PD-1:PD-L2[Biotinylated]
72015
PD-1:PD-L2 Homogeneous Assay
72005
PD-1[Biotinylated]:PD-L1
72016
PD-1:PD-L1[Biotinylated]
72006
PD-1[Biotinylated]:PD-L2
72017
PD-1:PD-L2[Biotinylated]
72007
CD28:B7-1[Biotinylated]
72018
PD-1[Biotinylated]:PD-L1
72008
BTLA:HVEM[Biotinylated]
72019
PD-1[Biotinylated]:PD-L2
72009
CTLA4:B7-1[Biotinylated]
72024
CTLA4[Biotinylated]:B7-2
72012
PD-1:PD-L2 TR-FRET
72025
CD137[Biotinylated]:CD137L
Table 1. BPS Bioscience’s Immunotherapy receptor portfolio. This table represents a complete
list of BPS’ biochemical-based immunotherapy receptor assay kits as of April 2015. This portfolio is
continually expanding and will eventually include all the receptor-ligand pairs in Figure 1 and more.
Figure 5. Validation of BPS’ IDO1 and TDO inhibitor screening assay kit Both the IDO1 (BPS
Cat. #72021) and TDO (BPS Cat. #72023) Inhibitor Screening Assay Kits measure enzyme activity
by analyzing absorption of product (Kyn) at λ = 320-325 nm. (A) IDO1 activity in the presence of
the IDO1-specific inhibitor INCB024360, the IC50 was determined to be 60 nM, demonstrating the
ability of the kit to serve as a high throughput screening tool for IDO1 inhibitors (B) TDO activity in
the presence of the TDO-specific inhibitor 680C91, the IC50 was determined to be 338 nM,
demonstrating the ability of the kit to serve as a high throughput screening tool for TDO inhibitors.
SUMMARY
Immunotherapy is a rapidly growing field with a number of currently approved therapies that are
showing excellent efficacy in the clinic, especially in cancer. However, these success stories also
have their shortcomings as not all cancers have been responsive, emphasizing the need for novel
therapeutics.
In an effort to increase the rate of immunotherapy drug discovery and development, we have
developed and validated biochemical high throughput screens for established drug targets as well
as potential new targets. These assays are straightforward, easy-to-use, and come in different
formats such as chemiluminescent, colorimetric, TR-FRET, and AlphaLISATM. In addition to these
assay kits, BPS also provides screening services for its entire immunotherapy portfolio, allowing
researchers to take advantage of our assay expertise. Future work will focus on developing assays
for novel therapeutic targets in addition to cell-based assays that complement our portfolio of
biochemical-based assays.
REFERENCES/CONTACT INFO
1.
2.
3.
4.
5.
6.
Robert, C., et al., N. Engl. J. Med. 2011; 364: 2517-2526
Wolchok, J.D., et al., N. Engl. J. Med. 2013; 369: 122-133.
Sznol, M., et al., J. Clin. Oncol. 2014; 32: (Suppl.; abstr LBA9003).
Platten, M., et al., Front. Immunol. 2015; 5(63): 1-7.
Holmgaard, R.B. et al., J. Exp. Med. 2013; 210: 1389-1402
Gibney, G.T. et al., J. Clin. Oncol. 2014; 32: (Suppl.: abstr 301).
For more information visit bpsbioscience.com/immunotherapy or contact Andrew Newman at
[email protected] , for general information, email [email protected].