iLite® ADCP Bioassays 

A Biological Relevant and powerful way to assess CD32 / FcyRIIa induced Effects

iLite  ADCP Bioassays  - A biological relevant and powerful way to assess CD32 / FcyRIIa induced effects

iLite® ADCP BIOASSAYS

The iLite ADCP product portfolio is based on a reporter gene system and offers a convenient and powerful way of measuring the efficacy antibodies have to elicit ADCP through the CD32/ FcyRIIa receptor in vitro.

This is achieved by combining a cleverly engineered effector cell, that closely resembles the natural FcγRIIA signal transduction pathway, with homologous target cells with a controlled antigen expression (+) or depletion (-).

Taken together, our unique iLite ADCP portfolio offers unparalleled sensitivity for each target and a system that is truly greater than the sum of its parts.

ADCP-Effector-Positive-Target-Cell-May-2022

The FcγRIIa ADCP Reporter Bioassay is a biologically relevant MOA-based assay that can be used to measure the potency and stability of antibodies and other biologics that specifically bind and activate FcγRIIa. The product portfolio consists of cell lines provided in an “assay-ready” format for a rapid and convenient workflow and a further reduction in assay variability, enabling their use in antibody screening, characterization, stability, and potency studies.

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S-018-GB-White-paper-iLITE ADCC BIOASSAYS
In-vivo-ADCP-illustration

ADCP, Antibody-dependent cellular phagocytosis - in Vivo
A mechanism of cell-mediated immune defense

ADCP is a potent immune mechanism to eliminate pathogens.  Elimination happens by antibody-coating of foreign particles such as microbes or tumor cells.

This engages FcγRIIa and FcγRI expressed on macrophages and triggers a signaling cascade leading to the engulfment of the IgG-opsonised particle.

In vivo, ADCP can be mediated by monocytes, macrophages, neutrophils, and dendritic cells via FcγRIIa, FcγRI, and FcγRIIIa. While all three receptors can participate in ADCP, FcγRIIa is believed to be the predominant Fcγ receptor involved.

ILITE ADCC TECHNOLOGY - How does it work?

Antibody-dependent cell-mediated phagocytosis (ADCP) is an immune response where Fc receptor bearing Effector cells engulf antibody-coated target cells that express tumor or pathogen-specific antigens on their surface. Many antibody-based therapeutics rely, at least in part, on their ability to induce ADCP in patients. The iLite ADCP product line consists of Jurkat cells stably expressing human FcγRIIa-H (the high-affinity H131 variant) and NFAT-induced luciferase. This cell line offers a convenient and powerful way of measuring the efficacy of antibodies to elicit ADCP in vitro.  

Luminescence based on the crosslinking of Fc receptor, antibody, and target

The ADCP process is triggered when the effector cell interacts with a drug antibody bound to its target cell. A Fcγlla (CD32) receptor on the surface of the effector cell binds to the Fc region of the antibody, thus creating a bridge between the effector and target cells.

Following the formation of this crosslinking, the engineered effector cells will, instead of engulfing  the target cells as in the in vivo situation, produce luciferase through an intracellular pathway and generate luminescence exclusively from this cross-linking and signaling. The strength of the luminescence correlates to the ability of the drug to induce ADCP.

 

 

Intracellular Pathway Signaling

Upon binding to the antibody, the NFAT intra cellular pathway signaling is activated in the effector cell. The promoter controlling the firefly luciferase gene relays on the NFAT-responsive promoter. This process mimics  the in vivo FcγRIIA signal transduction pathway and the MOA of the therapeutic antibodies  eliciting this effect

 

A normalization gene for easy comparisons between samples

Like most iLite cell lines, the iLite ADCP Effector Cells also have a secondary luciferase readout, from a luciferase gene expressed under the control of a constitutive promotor. This enables normalization of each individual readout according to cell number and compensates for potential matrix effects.

 

APPLICATIONS for the iLite® ADCP Portfolio

Increasing evidence suggests that antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) plays a significant role in antibody-mediated protection and control of viral infection and several laboratory methods exist for determining the efficacy of antibodies or effector cells in eliciting ADCC and ADCP

The iLite ADCP Reporter Bioassay is designed for evaluating ADCP of therapeutic monoclonal antibodies (mAbs) and exhibits greatly reduced variability and offers an easier workflow than traditional ADCP assays.

It is an ideal assay for applications such as potency lot release and antibody screening, as well as for assessing comparability between innovator and biosimilars. Furthermore, the bioassay can be used to assess the impact of post-translational modifications, such as glycosylation and fucosylation, on the potency of monoclonal antibodies (mAbs).

Svar-Life-Science-hand-holding-iLite-cell-based-reporter-gene-assay-plate-placing-it-into-incubator

 

Determine Mechanism of Action for new drug candidates

Many therapeutic monoclonal antibodies (mAbs) include ADCP as part of their mechanism of action (MoA). This makes accurate determination of ADCP activity an essential part of the development and characterization of therapeutic antibodies.

Using the iLite® ADCP Solution enables the researchers to use only one platform for screening a large pool of compounds to establish a mechanism of action/ effect on target.

iLite ADCP cell lines can successfully be used:

  • In high throughput formats, such as 384 or 1536 plates
  • In automated plate handlers
  • To get assay results within a workday
  • To compare structurally different candidates

In-vivo-ADCC-illustration-2

 

Potency for therapeutic antibodies

When investigating potential drug candidates, it is essential to determine the presence of ADCP activity and if found, quantify the activity. This is an important step in identifying the most potent antibody and in lot release testing. If ADCP has been determined to be the mechanism of action of a biological drug this should be reflected in the potency assay.

iLite cell lines can be successfully used to:

  • Measure potency in a biologically relevant setting
  • Get a reproducible, sensitive, and specific cell-based potency assay

ADCC-Target-cells-CD20

 

Glycosylated Antibodies

To increase the ADCP activity of therapeutic antibodies, the pharmaceutical industry has structurally improved the Fc region of the antibodies. Point mutations or modifications into the glycosylation profiles of Fc regions have been shown to increase their affinity towards Fc receptors on a range of effector cells.

BILD!

Proving Biosimilarity

With many of the existing patents of monoclonal antibody blockbuster drugs set to expire in the next few years, the development of biologic therapeutics similar to the original drug (biosimilars) has become increasingly important.

However, extensive requirements for analytical characterization are needed to show comparability between innovator and biosimilar and it must be proven that “the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components” and that “there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency”. This is the biosimilar development approach from the EMA, FDA, and WHO.



The most critical evaluation is that of biological function, through assays that replicate the likely mechanism of action in vivo, with ADCC/ADCP assessments forming an important component.

There are strict requirements to show comparability between innovator drugs and biosimilars. ADCC adn ADCP evaluation studies are an essential part of the comparability profile. It is generally accepted that the innovator and biosimilar will be different due to the complex nature of the production process. However, the regulatory approach is that the compounds must be shown to be sufficiently similar to provide the same clinical outcome when used to treat disease.

Increasing evidence suggests that antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) plays a significant role in antibody-mediated protection and control of viral infection and several laboratory methods exist for determining the efficacy of antibodies or effector cells in eliciting ADCC and ADCP

The iLite ADCP Reporter Bioassay is designed for evaluating ADCP of therapeutic monoclonal antibodies (mAbs) and exhibits greatly reduced variability and offers an easier workflow than traditional ADCP assays.

It is an ideal assay for applications such as potency lot release and antibody screening, as well as for assessing comparability between innovator and biosimilars. Furthermore, the bioassay can be used to assess the impact of post-translational modifications, such as glycosylation and fucosylation, on the potency of monoclonal antibodies (mAbs).

Svar-Life-Science-hand-holding-iLite-cell-based-reporter-gene-assay-plate-placing-it-into-incubator

 

Determine Mechanism of Action for new drug candidates

Many therapeutic monoclonal antibodies (mAbs) include ADCP as part of their mechanism of action (MoA). This makes accurate determination of ADCP activity an essential part of the development and characterization of therapeutic antibodies.

Using the iLite® ADCP Solution enables the researchers to use only one platform for screening a large pool of compounds to establish a mechanism of action/ effect on target.

iLite ADCP cell lines can successfully be used:

  • In high throughput formats, such as 384 or 1536 plates
  • In automated plate handlers
  • To get assay results within a workday
  • To compare structurally different candidates

In-vivo-ADCC-illustration-2

 

Potency for therapeutic antibodies

When investigating potential drug candidates, it is essential to determine the presence of ADCP activity and if found, quantify the activity. This is an important step in identifying the most potent antibody and in lot release testing. If ADCP has been determined to be the mechanism of action of a biological drug this should be reflected in the potency assay.

iLite cell lines can be successfully used to:

  • Measure potency in a biologically relevant setting
  • Get a reproducible, sensitive, and specific cell-based potency assay

ADCC-Target-cells-CD20

 

Glycosylated Antibodies

To increase the ADCP activity of therapeutic antibodies, the pharmaceutical industry has structurally improved the Fc region of the antibodies. Point mutations or modifications into the glycosylation profiles of Fc regions have been shown to increase their affinity towards Fc receptors on a range of effector cells.

BILD!

Proving Biosimilarity

With many of the existing patents of monoclonal antibody blockbuster drugs set to expire in the next few years, the development of biologic therapeutics similar to the original drug (biosimilars) has become increasingly important.

However, extensive requirements for analytical characterization are needed to show comparability between innovator and biosimilar and it must be proven that “the biological product is highly similar to the reference product notwithstanding minor differences in clinically inactive components” and that “there are no clinically meaningful differences between the biological product and the reference product in terms of the safety, purity, and potency”. This is the biosimilar development approach from the EMA, FDA, and WHO.



The most critical evaluation is that of biological function, through assays that replicate the likely mechanism of action in vivo, with ADCC/ADCP assessments forming an important component.

There are strict requirements to show comparability between innovator drugs and biosimilars. ADCC adn ADCP evaluation studies are an essential part of the comparability profile. It is generally accepted that the innovator and biosimilar will be different due to the complex nature of the production process. However, the regulatory approach is that the compounds must be shown to be sufficiently similar to provide the same clinical outcome when used to treat disease.

TARGET CELL PORTFOLIO

Engineered, homologous, and specific target cells that enable screening for antibody induced effector functions

Can´t find a perfect match? Let us develop it for you!

In case you haven't got an ideal target cell line and can't find what you’re looking for in our off the shelf solution, you can rely on our extensive expertise to develop the most appropriate one and we have vast experience in designing cell lines ideally fit for purpose!

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