9 pathway-specific screening assays in Immunotherapy

The immune system is a system of cells and organs whose function is to defend an organism from foreign pathogens. With the ability to mount a response against virtually any foreign material and return to a quiescent state following neutralization of the threat, this fascinating organ system displays remarkable specificity and plasticity. To achieve this, there is a multifaceted balancing act between the many activators and suppressors which maintains homeostasis of the body’s perhaps most complex organ system.

[Read more…]

Immunotherapy Screening – IDO pathway

In previous blogs, I invited you to join me in exploring the relevance of the following pathways:

PD1 - PD-L1 - PD-L2

 

Today I’d like to focus on the IDO pathway. [Read more…]

Cancer immunotherapy and PBMCs

Patients who receive cancer immunotherapy treatments in clinical trials must have their peripheral blood mononuclear cell (PBMC) samples collected at baseline and at later time points. Immune monitoring facilities provide laboratory testing of these PBMC samples in order to gauge the response of the patient’s immune system to the test treatment.

A primary focus of immune monitoring facilities is therefore to develop cutting-edge technologies whilst also standardizing and validating immune assays with rigorous quality-control standards to ensure data reliability. After all, the weight of clinical trial results depends on the accuracy, precision, and reliability of data generated from these PBMC samples.

The fragile nature of biological samples makes standardization of laboratory procedures an especially important focus. Parameters that can affect data include the anticoagulant used for preserving PBMC samples, the time frame between sampling and processing, storage/shipping temperature en route to the central processing lab, the cryopreservation and thawing process, as well as the cell culture media.[1]

[Read more…]

Immunotherapy Screening – CD137:CD137L pathway

In previous blogs, I invited you to read about the relevance of the B7-1 : CD28, B7-1 : CTLA4, the BLTA:HVEM, CD47:SIRPα , the GITR:GITRL, the CD40:CD40L and the  PD-1/PD-L1/PD-L2 pathway for immunotherapy screenings and discussed the products available to work on these pathways. Today, I will focus on the CD137:CD137L pathway.

CD137 is another co-stimulatory protein that is expressed on activated T cells. Unlike CD40:CD40L signaling, which primarily involves helper T-cells, CD137 has a crucial role in the development of cytotoxic T-cells and anti-tumor immunity. Its ligand, CD137L, is mainly expressed on antigen-presenting cells, such as activated B cells, macrophages, and dendritic cells, as well as on human tumor cells.

Co-stimulation through CD137:CD137L enhances T-cell activation, promotes the rejection of cardiac allografts and skin transplants, and eradicates experimentally induced tumors in animal models. Several clinical studies are on-going that use agonistic anti-CD137 antibodies to induce an anti-cancer response to solid tumors. [Read more…]

Immunotherapy Screening – CD40:CD40L pathway

In my previous blogs, I invited you to read about the relevance of the B7-1 : CD28, B7-1 : CTLA4, the BLTA:HVEM, CD47:SIRPα , the GITR:GITRL and the PD-1/PD-L1/PD-L2 pathway for immunotherapy screenings and discussed the research products available to study these pathways. Today, I’d like to focus on the CD40:CD40L pathway.

[Read more…]

Immunotherapy Screening – GITR:GITRL pathway

In previous blogs, I invited you to read about the relevance of the B7-1 : CD28, B7-1 : CTLA4, the BLTA:HVEM, CD47:SIRPα , and the  PD-1/PD-L1/PD-L2 pathway for immunotherapy screenings and discussed the products available to work on these pathways. Today, let’s focus on the GITR:GITRL pathway (glucocorticoid-induced tumor-necrosis-factor-receptor-related protein).

The GITR:GITRL pathway is a very important target for immunotherapy drug discovery — in fact, the National Cancer Institute (USA) identified GITR modulation as one of the top 25 most promising research areas! GITR is expressed on most immune cells, including T-cells, B-cells, macrophages, and NK cells. Binding of GITR by the GITR ligand (found on antigen-presenting cells and many tumors) activates the immune response by stimulating macrophages and triggering T-cells to expand, proliferate, and differentiate. Using anti-GITR mAb to trigger GITR signaling is effective in treating viral, bacterial, and parasitic infections, as well in boosting immune response against tumors. Not surprisingly, clinical trials testing anti-GITR mAb in melanoma patients are already in progress (1).GITR - GITRL Figure

Of course, it’s never that simple. Stimulating GITR signaling in NK cells has the opposite effect compared to T-cells and macrophages—GITR signaling inhibits NK cell activity (2). Likewise, stimulation of T-suppressor cells by GITR signaling also down-regulates the immune response.   Researchers are using this to their advantage; down-regulation of the immune response can be used to treat autoimmune and inflammatory diseases, including arthritis, allergy, inflammatory bowel diseases, and graft rejection. Therefore, GITR-Fc fusion protein and mAb that block GITR signaling instead of stimulating the pathway are also being studied as potential drug candidates.

BPS Biosciences recently released a set of products linked to immunotherapeutically relevant targets, amongst them the human glucocorticoid-induced TNF-related ligand (GITRL), also known as AITRL, CD357L, or tumor necrosis factor ligand superfamily member 18 (TNFSF18).

If you are interested in this specific ligand or any other of the topics and reagents covered in previous blogs (there are links to them at the beginning of this post), don’t hesitate to get in touch (you can use the form below).

References:

(1) Nocentini, G., et al., Br J Pharmacol. 165(7): 2089-99 (2012)

(2) Barao, I., Front Immunol. 3: 402 (2012)

Immunotherapy – BLTA:HVEM, CD47:SIRPα in drug discovery

The treatment of diseases by inducing, enhancing, or surpressing an immune response is referred to as Immunotherapy. T-cell activation and inactivation requires the coordination of various co-inhibitory and co-stimulatory signals and most immunotherapies modulate these signals.

Therapeutic manipulation of immunopathways has lead to promising clinical results for the treatment of a number of diseases such as cancer, autoimmune diseases and inflammatory diseases. Research in this field is rapidly evolving as scientists seek to identify the next generation of therapies.

In the third and last article in this series,  I’ll describe the BLTA:HVEM and CD47:SIRPα pathways and summarize the portfolio of tools to investigate this pathway produced by our partner BPS Biosciences. In the first article, I focused on B7-1 : CD28 and B7-1 : CTLA4 pathways, and in the second article the emphasis was on the PD-1/PD-L1/PD-L2 pathway.

After a brief summary of the impact of these pathways in immune response, we’ll be looking at the screening tools I’ve selected from our partner BPS Biosciences to investigate these pathways in drug discovery activities, and especially in inhibitor screenings. In fact, some of these tools are the first ready-to-use assays to screen for inhibitors of the respective pathways.

Why study the BLTA:HVEM pathway in immunotherapeutics?Untitled

HVEM, also known as herpesvirus entry mediator, is the receptor for the HSVglycoprotein D involved in viral entry. It’s also the receptor for several ligands, including BTLA or CD160, which deliver a co-inhibitory signal to down-regulate T cell response. For example, BTLA activation inhibits the function of human CD8+ cancer-specific T cells. Conversely, the binding of two other ligands, LIGHT or lymphotoxin-α, deliver a costimulatory signal.   Thus, HVEM is a bidirectional switch, with the outcome depending on which ligand is engaged. A cysteine-rich domain (CRD1) of HVEM is required for the binding of inhibitory ligands CD160 and BTLA but not stimulatory ligand LIGHT. Therapies targeting the CRD1 of HVEM to block BTLA and CD160 binding are being developed to enhance immune responses and vaccination.

BTLA can also bind to another receptor, B7-H4. Like BLTA binding to HVEM, BTLA engagement of B7-H4 results in downregulation of T-cell activation, and mice deficient in BTLA show increased incidence and severity of autoimmune disorders.

The most advanced BLTA:HVEM screening reagents

These new therapeutical advances highlight the importance of reliable R&D tools in Drug discovery approaches and translation medicine programs. Access to robust research reagents covering the needs of Drug discoverers from target validation to screening (HTS or secondary) or even DMPK studies is crucial. I have made a selection of some of the latest releases for scientists interested in the BLTA:HVEM pathway.

       #1 – BTLA – a ligand for HVEM and B7-H4

BTLA (CD272) (Id. nr 71141)  is a ligand for nHVEM and B7-H4, a mediator of co-inhibitory signals to down-regulate the T cell response.

       #2 – HVEM – a receptor for BTLA

HVEM is one of the receptors for BTLA. Furthermore it serves as the receptor for the HSVglycoprotein D involved in viral entry.

HVEM is available as a Fc fusion (Id. nr 71142) and as a biotinylated Fc fusion (Id. nr 71143).

       #3 – B7-H4 – a receptor for BTLA

B7-H4 is another receptor for BTLA. Complex formation also results in downregulation of T-cell activation.

B7-H4 is available as a His tagged version (Id. nr 71144) and as a biotinylated version (Id. nr 71129).

Ready-to-use BLTA:HVEM4 screening assay kits

BTLA-HVEM interaction

Typical results with BPS’ ready-to-use BTLA:HVEM [Biotinylated] Inhibitor Screening Assay Kit

Recently, BPS Biosciences have released a unique ready-to-use assay kits to screen and profile inhibitors for the BLTA:HVEM4 signaling pathways (BTLA:HVEM[Biotinylated] Inhibitor Screening Assay Kit [Id. nr 72008]). This 96-well format assay kits come with biotinylated HVEM, purified BTLA,  streptavidin labeled HRP and assay buffer sufficient for 100 binding reactions.

 

Why study the CD47:SIRPα pathway in immunotherapeutics?

CD47 is an anti-phagocytic signal found on many cancer cells that signals macrophages not to “eat” the cancer cell. It binds to the SIRP-α receptor on the surface of macrophages and dendritic cells. Anti-CD47 antibody therapy inhibited tumor growth and prevented metastasis in mice, suggesting CD47 blockade may be an important immunotherapeutic target for human cancer.

Products available

       #1 – CD47 – a ligand for SIRP-alpha receptor

CD47 (Id. nr 71127) binds to the SIRP-α receptor on the surface of macrophages and dendritic cells.

       #2 – SIRP-alpha  (CD172a)

SIRP-α (Id. nr 71145) is located on the surface of macrophages and dendritic cells and serves as the receptor for CD47.

 

Looking for BLTA:HVEM or CD47:SIRPαtestings?

Choosing the right reagents or screening kits to raise the value of your compounds of interest by looking to BLTA:HVEM or CD47:SIRPα pathways can be challenging.

Research tools reviewed in this final post (of a series of 3 posts dedicated to Immunotherapy screening) will definitely help you there. Otherwise, you can simply contact me with the form below.

Recently released Immunotherapy Screening posts on the blog “being bio-reactive”:

Immunotherapy Screening – A focus on PD-1/PD-L1/PD-L2 Pathway in drug discovery

The treatment of diseases by inducing, enhancing, or surpressing an immune response is referred to as Immunotherapy. T-cell activation and inactivation requires the coordination of various co-inhibitory and co-stimulatory signals which are modulated by most immunotherapeutic approaches.

Therapeutic manipulation of immunopathways has lead to promising clinical results for the treatment of a number of diseases (ex. cancer, autoimmune diseases and inflammatory diseases…). Research projects in this field are rapidly evolving as scientists seek to identify the next generation of therapies.

In an earlier post on this blog, I introduced the latest advances regarding the the B7-1 : CD28 and B7-1 : CTLA4 pathways for immunotherapy screenings. Today, I’d like to focus on the PD-1/PD-L1/PD-L2 pathway.

After a brief review of the role of this pathway in immune response, let’s take a look at some of the screening tools I’ve selected (by our partner BPS Biosciences) to investigate this pathway for drug discovery applications. Interestingly, some of these tools are the first ready-to-use assay kits made available for drug discoverers to screen for inhibitors of the PD-1/PD-L1/PD-L2pathway.

Why look at PD-1/PD-L1/PD-L2 pathways in immunotherapeutics?

PD-1 is a receptor present on the surface of activated T-cells and other immune cells.  PD-1 has two ligands, PD-L1 and PD-L2, which are overexpressed in most human cancers.  Binding of PD-L1 to PD-1 negatively regulates T cell signaling and inhibits cytotoxic T-cell activity.

PD1 - PD-L1 - PD-L2

Similarly, engagement of PD-1 by PD-L2 dramatically inhibits T-cell proliferation, especially in CD4+ (T helper) cells. Therefore, this upregulation of PD-L1 and PD-L2 may allow cancers to evade the host immune system.

Neutralizing antibodies can prevent PD-L1 from binding to PD-1 or to B7-1. Blockade of PD-L1 enables the activation of T-cells, restoring their ability to detect and attack tumor cells. Therefore a number of pharmaceutical companies are actively developing monoclonal antibodies targeting PD-1 and PD-L to boost the immune system for the treatment of non-small-cell lung cancer, melanoma, and bladder, renal, and triple-negative breast cancers.

The PD-1/PD-L pathway is also a therapeutic target for chronic viral-infections (including HIV and HCV). This therapeutic appraoch is based on the observation that PD-1 expression is upregulated by virus-specific T cells and that the inhibition of this loop is hoped to decrease viral load by increasing  T cell function. Conversely, since the PD-1/PD-L interaction regulates self-tolerance and immunologic homeostasis, agonists of these proteins are promising drug targets for many autoimmune disorders including multiple sclerosis, arthritis, lupus, and type I diabetes.

The most advanced PD-1/PD-L1/PD-L2 screening reagents

These new therapeutical advances highlight the importance of reliable R&D tools in Drug discovery approaches and translation medicine programs. Access to robust research reagents covering the needs of Drug discoverers from target validation to screening (HTS or secondary) or even DMPK studies is crucial. Below, I’ve made a selection of some of the latest releases for scientists interested in the PD-1/PD-L1/PD-L2 pathway.

         #1 – PD-1  – a receptor for PD-L1

PD-1 is the receptor for PD-L1. Formation of the receptor-ligand complex leads to an inhibitory signal which reduces proliferation of CD8x T cells.

PD-1 is available as a Fc Fusion (Id. nr 71106), as a FLAG-tagged variant (Id. nr 71115), and as a Biotin labeled variant (Id. nr 71109)

        #2 – PD-L1 – a ligand for PD-1 receptor

PL-L1 is one of the ligands for PD-1, which plays a role in surpressing the immune response especially during pregnancy, tissue allografts, autoimmune diseases and diseases such as Hepatitis.

PL-L1 is available as a non labeled variant (Id. nr 71104) and a Biotin-labeled variant (Id. nr 71105).

        #3 – PD-L2 –  aligand for PD-2 receptor

PL-L2 is another ligand for PD-1 which, upon complex formation T cell proliferation, especially of CD4+ (T helper) cells, is dramatically decreased.

PL-L2 is available as a non labeled variant (Id. nr 71107) and a Biotin-labeled variant (Id. nr 71108).

        #4 – PD1-neutralizing antibody

This antibody (Id. nr 71120) can be used as a control inhibitor using the PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit (see below).

Ready-to-use PD-1/PD-L1/PD-L2 screening assay kits

Recently, BPS Biosciences have released 3 unique ready-to-use assay kits to screen and profile inhibitors for the PD-1/PD-L1/PD-L2 signaling pathways

PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit (Id. nr 72003)

PD-1:PD-L2[Biotinylated] Inhibitor Screening Assay Kit Id. nr 72004)

Both are 96-well format assay kits and come with biotinylated PD-L1 or PD-L2, purified PD-1,  streptavidin labeled HRP for 100 binding reactions.

PD-L1-PD-1 Interaction measured by Luminescence

Typical data obtained with the BPS’ Ready-to-use Inhibitor Screening assays.

PD-L2 Inhibitor Screening Assay Kit (Id. nr 72006).

This 96-well format assay kits comes with biotinylated PD-1, purified PD-L2,  streptavidin labeled HRP  for 100 binding reactions.

Looking for PD-1/PD-L1/PD-L2 testings?

Choosing the right reagents or screening kits to raise the value of your compounds of interest by looking to PD-1/PD-L1/PD-L2 pathway can be challenging.

Research tools reviewed in this second post (belonging to a series of 3 posts dedicated to  Immunotherapy screening) will definitely help you there. Otherwise, you can simply contact me with the form below.

Recently released Immunotherapy Screening posts on the blog “being bio-reactive”:

Upcoming Immunotherapy Screening posts on the blog “being bio-reactive”:

  • Immunotherapy Screening – Part 3:  BLTA:HVEM and CD47:SIRPalpha Pathways

Immunotherapy Screening – B7-1 / CD28 and B7-1 / CTLA4 Pathways in Drug Discovery

The treatment of diseases by inducing, enhancing, or surpressing an immune response is referred to as Immunotherapy. T-cell activation and inactivation requires the coordination of various co-inhibitory and co-stimulatory signals and most immunotherapies modulate these signals.

Therapeutic manipulation of immunopathways has lead to promising clinical results for the treatment of a number of diseases such as cancer, autoimmune diseases and inflammatory diseases. Research in this field is rapidly evolving as scientists seek to identify the next generation of therapies.

In the first post of a series of three,  I will describe the B7-1 : CD28 and B7-1 : CTLA4 pathways and summarize the portfolio of tools currently available to researchers to investigate these pathways. In fact, these are the first kits on the market to screen for inhibitors of these pathways. [Read more…]