Immunology of gene and cell therapy
The outcome of gene and cell therapies is determined in large part by the immune system. On one hand, immune responses can be drivers for therapy-induced immunotoxicities as well as loss of therapy in instances when components of the vector, its gene product or cells producing the therapeutic protein are recognized by the immune system and eliminated. On the other hand, gene and cell-based therapies can be designed to engage the immune system leading to either induction or suppression of immune responses in order to combat cancer or autoimmune disorders, respectively.
At CLS, we have expert knowledge of the immune system and we are able to support gene and cell therapy customers wishing to assess the impact of their gene or cell therapy product on immune cells. We will work with viral constructs and cell products provided by you, using your preferred handling and transduction methods.
Below are some of the services we offer in the field of gene and cell therapy:
- Assessment of transduction efficiency using different immune cells or tumor cells; measurement of transgene expression by flow cytometry or the Incucyte Live-Cell Analysis System
- Measuring lytic potential of oncoviruses in tumor cell lines
- Optimization and generation of chimeric antigen receptor (CAR)-transduced immune cells e.g. T cells, Tregs and macrophages
- Design and performance of downstream experiments consistent with the immunomodulatory genes’ or cell products’ mode of action including cell proliferation, cytokine production, expression of activation markers, cytotoxicity towards tumor cells, phagocytosis
Mixed lymphocyte reaction (MLR)
Mixed lymphocyte reaction (MLR) assays can be used to understand the effects of biologics or small molecules on T cell activation, an important immune response in the tumor microenvironment (TME).
There are different MLR protocols available:
- “one-way” or “two-way” MLR systems using PBMCs
- Autologous MLR assay using monocyte-derived dendritic cells (Mo-DC) and CD4+ T cells from the same donor, and anti CD3/CD28 as the stimulator
- Allogenic MLR assay using Mo-DC and CD4+ T cells from HLA-mismatched donors (immature or mature)
We can advise on the most suitable MLR to understand your drug candidate mechanism of action (MOA) and tailor the assay accordingly.
We offer both custom and standard assay readouts, such as cytokine expression (e.g. IFN-γ), T cell activation (e.g. CD80), proliferation (e.g. CFSE) and cell cycle (e.g. Ki-67) measured by ELISA and/or flow cytometry.
PBMC and T Cell stimulation assays
Understanding how immune cells are activated, proliferate and are regulated is key to developing novel and effective immunotherapy cancer drugs.
We offer activated human peripheral mononuclear cell (Human PBMC) and T cell activation assays to help you understand the interaction between your compound or biologic and the immune system, taking you to the clinic faster.
As well as tailored assay readouts, we also offer standard analysis options, such as cytokine levels by ELISA (e.g. IFN-γ) or proliferation (e.g. CFSE) with multi-color flow cytometry. Our 96-well plate format allows for moderate-throughput screening of multiple compounds, helping you achieve fast, precise results.
Exhausted T cell assays
We have developed both a murine and human exhausted CD4+ T cell assay, an important tool to test the efficacy of compounds in reversing T cell exhaustion and restoring normal effector function.
Exhaustion of T cells in the tumor microenvironment (TME) results in a reduced capacity to mount an effective immune response to the proliferating tumor.
Our moderate-throughput assay is ideal for evaluating the efficacy of compounds able to modulate checkpoint targets on immune cells. Your compounds can be tested against our validated reference controls. Several readouts are available to suit your requirements, including proliferation (e.g. Ki-67, CFSE) and cytokine analysis (e.g. IFN-γ).
The basis of our proprietary mouse assay centers around our TCR transgenic CD4+ T cells, which can be stimulated with WT-MBP to induce a fully responsive effector T cell, or with an altered ligand MBP (APL-MBP), which acts as a superagonist to generate exhausted T cells. These cells effectively mimic the exhausted state of T cells in the tumor microenvironment so are highly relevant to immuno-oncology research.
Our validated human exhausted T cell assay has been validated and demonstrated to phenotypically and functionally recapitulate the exhausted T cells in the tumor microenvironment. To find out more you can listen to our video blog below about our poster describing this work.
Understanding whether a test biologic or small molecule can enhance tumor cell killing could well be a key component of your IO drug discovery program.
We offer a range of in vitro assays to evaluate the various mechanisms by which the immune system drives targeted cell death. We use Sartorius’ IncuCyte Live-Cell Analysis System to capture real-time quantitative live-cell imaging and analysis to investigate the kinetics and mechanisms of apoptosis.
Our killing assays can be adapted to a range of tumor cell lines, immune cell populations, such as PBMCs, NK cells, macrophages and neutrophils as well as complement activated responses and combinations of your drug candidates. We offer custom and standard readouts, from cytokine expression by ELISA, multiplex or MSD, proliferation and cell surface markers by multi-color flow cytometry, to gene expression by qPCR or NGS. Our 96-well plate format allows for moderate-throughput screening of multiple compounds, helping you achieve fast, precise results.
Our antibody-dependent cell-mediated cytotoxicity (ADCC) assays can help you understand if your biologic engages target antigen, activating immune cells through their Fc receptors initiating cell death or alternatively determine if Fc-silencing approaches are effective.
We can optimize specific targets using non-radioactive labeling techniques, and the assay can be adapted to a variety of immune cells, from mixed cell populations (PBMCs) to isolated cells (e.g. NK cells).
Complement-dependent cytotoxicity (CDC) assays allow the evaluation of antibody-based therapeutics to activate the complement immune system, an innate response that also results in target cell death.
Cancer cells typically express high levels of complement inhibitor molecules, protecting them from this innate immune mechanism. We can support your understanding of the interaction between your biologic and the immune system, taking you to the clinic faster.
Understanding whether your novel therapeutic can modulate or trigger phagocytosis could be key to the discovery phase of your immunotherapy drug. Phagocytosis is one of the main mechanisms of the innate immune response and the phagocytic cells most commonly involved are neutrophils, macrophages, monocytes and DCs.
We have a range of phagocytosis-based assays to help you understand the interaction between your compound or biologic and the phagocytic cells of the immune system, taking you to clinic faster.
Our phagocytosis assays, which can be tailored to your requirements, include:
- Clearance of dead or apoptotic cells (efferocytosis)
- Antibody-dependent cellular cytotoxicity (ADCP)
- Blocking of phagocytosis signals (e.g. CD47)
- Uptake of bacteria or yeast bioparticles
We work closely with you to select the most suitable approach based on the immune cells or disease of interest. Cargo for phagocytosis include bacteria, beads or tumor cells. Our standard readout uses the IncuCyte® Live-Cell Analysis System and customized readouts can include cytokine expression by ELISA, multiplex or MSD, proliferation and cell surface markers by multi-color flow cytometry, or gene expression by qPCR or NGS.
As antibody-dependent cell-mediated phagocytosis (ADCP) is an important MOA for many monoclonal antibody therapeutics. Our ADCP assays use monocytes or macrophages as effector cells for phagocytosis of target cells, to understand the effectiveness of antibody engagement with specific antigens through their Fc receptors.
Our strong expertise in macrophage differentiation in vitro, combined with our bespoke approach and readout options, ensures a phagocytosis assay design tailored to you.
Myeloid cell assays
Myeloid cells are an important potential target immune cell for immunotherapy cancer treatment. Screening molecules or biologics for their ability to drive phenotypic and functional immune cell changes in the tumour microenvironment (TME) could be key to your immuno-oncology discovery program.
We provide custom assays to investigate the effect of client compounds on a variety of myeloid cells. Whatever your myeloid cell of interest (monocyte, macrophage, dendritic cell (DC) or granulocyte), we can tailor our myeloid cell assays to meet your needs. We offer both monoculture assays, to examine the effect of your compound on a single cell type, as well as mixed cell assays.
Whatever your scientific question, our immunology expertise and myeloid cell experience can support you in your IO discovery programs. Our suite of myeloid cell assays includes:
- M1 and M2 macrophage differentiation assay
- M1 and M2 macrophage polarisation assay
- DC activation and maturation
- Mixed lymphocyte reaction (MLR)
- Antigen-specific T cell responses
- Phagocytosis assays
- M1-mediated T cell activation assay
- M2 suppression assay
- MDSC suppression assay
We can advise on the most suitable assay set up to understand your drug candidate mechanism of action (MOA), and tailor the assay accordingly. We offer both custom and standard assay readouts such as cytokine expression (ELISA, multiplex or MSD), proliferation, cell activation and cell surface markers (flow cytometry).
We offer a suite of assays for screening molecules or biologics for their ability to reverse the suppression of T cells mediated by different immune cell populations such as M2 macrophages, MDSC and Tregs, an important mechanism of action (MOA) for any potential immunotherapy cancer treatment.
Macrophage suppression assay
The tumor microenvironment (TME) can drive differentiation and accumulation of immune cells, which support tumor growth e.g. tumor-associated macrophages (TAMs) and M2 macrophages. We have a range of customizable assays to screen compounds for their potential to block these mechanisms, reducing the suppression of T cells and boosting anti-tumor responses. Reversal of T cell suppression can be measured by cytokine release (ELISA, multiplex or MSD), gene expression changes (qPCR) or by proliferation and activation markers using flow cytometry.
We are developing ex vivo protocols, utilizing tumor conditioned media (tumor cell lines and ascites samples) as well isolating TAMs, for use in our macrophage suppression assays as a translational step-up in complexity from our customizable in vitro assay systems.
MDSC suppression assay
Myeloid-derived suppressor cells (MDSC) are key players in the TME. Our MDSC-mediated T cell suppression assay can help you determine if your compound or biologic can block MDSC function and relieve T cell activation.
Treg suppression assays
Regulatory T cells (Tregs) can inhibit unwanted immunity e.g. in autoimmune diseases, or conversely, they can suppress desirable immunity in cancer. We have a customizable Treg suppression assay to study your drug candidate’s ability to either boost or reduce Treg activity. We work closely with you to tailor key assay readouts to your requirements.
Our Treg suppression assays can incorporate in vitro generated inducible FoxP3+ Tregs (iTregs) or natural Tregs (nTregs) can be isolated for use in this assay, more closely reflecting the FoxP3-induction potentially driven by some tumor types and highly relevant to immuno-oncology research.
IO in vivo models
Immunotherapies for cancer work with a patient’s immune system to increase anti-tumor responses. Syngeneic mouse models allow the study of novel cancer immunotherapies in the presence of a functional immune system compared to other in vivo models e.g. xenograft models.
We know using in vivo mouse models is an important translational step in your immuno-oncology pre-clinical phases.
We offer several syngeneic tumor models e.g. B16.OVA and Lewis Lung models that can be coupled with many of our custom in vitro assay readouts to allow detailed MOA interrogation within the TME.
Our immunology and specialist histology expertise ensures robust analysis and reporting of your in vivo model. We can perform histological analysis from routine H&E up to 9-plex multiplex immunofluorescent staining on a single section. Our flow experts can design customized panels where required and we can also support with ELISA, multiplex, qPCR and NGS readouts.
We work closely with you to optimize existing or validate new in vivo models based on your requirements to accelerate your best route to clinic.
Oncology drug discovery
Our extensive expertise and consultative approach to oncology drug discovery ensures a customized, robust program for our clients. We can deliver a tailored, focused study or a fully integrated program to clinical candidate by being an extension of your team. We deliver bespoke programs of work to support a precision medicine approach through to direct tumor targeting and combination therapy investigations.
We provide whole project teams comprising medicinal & computational chemistry, biology and DMPK support for a range of mechanistic approaches including DNA damage repair processes, metabolic, immuno-oncology and precision medicine strategies.
At Concept, we tailor our team and approach to each client project and can support your program from identification of a small molecule hit, bespoke screening cascade design and execution, medicinal chemistry optimization through to API GMP manufacture, bioanalysis and patient biomarker assessment in clinic. We also work on alternative modalities such as ADCs, PROTACs and non-small molecule drug discovery approaches.
Our Oncology services include:
- Bespoke assay and screening cascade development
- High content phenotypic profiling
- Biophysical expertise including novel MOAs
- TME studies and translational assays including in vitro, in vivo and ex vivo screening of compounds for their ability to drive phenotypic and functional immune cell changes
- Target gene and protein expression in healthy and diseased tissue
- Biomarker/patient selection strategies
- PROTACs, ADCs and small molecule drug discovery
- Medicinal chemistry, synthetic chemistry and computational chemistry
- ADMET screening, bioanalysis and PK assessments
- Process research and development, API GMP manufacture and supporting analytical services
A simple, highly sensitive and precise solution for multiplex analysis of up to 800 RNA, DNA or protein targets. NanoString’s nCounter® technology is a variation on the DNA microarray, providing a digital count of transcript numbers within each sample using colour coded molecular barcodes.
We work closely with you to provide rational and stepwise discovery programs, which will provide insight into the signaling pathways, regulation of downstream genes and the target cell types of your drug candidate. Our NanoString and NGS services can help inform you on your best route to clinic.
Key advantages of NanoString:
- Profile up to 800 RNA (mRNA, microRNAs, SNVs, CNVs), DNA or protein targets
- Off-the-shelf and custom panels for Oncology, Immunology and Neuroscience
- Less precious sample material required
- Optimized for difficult sample types e.g. FFPE, tissue, crude-cell lysates and biofluids
- Human and mouse
- Accurate & faster than qPCR (no amplification steps), simpler than NGS
NanoString is available as a readout to our custom in vitro, in vivo and ex vivo assay systems. Alternatively, send us your samples for RNA extraction or as pre-extracted RNA, we tailor to you.