During the last few years, significant advancement has been made in the clinical application of cancer immunotherapies. Molecules directed against immune checkpoints and other agonists show great promise for treatment of a variety of malignancies. Next to CTLA-4 and PD-1 blockade, a wide range of therapeutics with the potential to reverse the tumor-induced suppression are under development. Despite the first successes, only a relative small portion of patients benefit from these treatments and it has been shown that combination therapies can improve the outcome. Today, there are over a thousand trials of immune checkpoints running resulting in some critical questions like: Are we running too fast and testing combinations that might never work? Are we doubling some of the work and wasting some efforts and chances of patients to participate in the most promising trials?
Early evaluation of the effectiveness of candidate therapeutics and combination therapies can be done using mouse models and in vitro bioassays with primary mouse or human immune cells.
Also, a better understanding of the tumor micro environment and the steps needed to generate an anti-tumor response by the immune system will help design clinical trials and ultimately discover relevant biomarkers.
Finally, it is also important to deal with the potential unwanted immunogenicity of oncology drugs, as the development of anti-drugantibodies might lead to an altered efficacy and potency and auto immune responses.
The development and application of in vitro assays using human or mouse immune cells to accelerate the design and development of therapeutics drugs is becoming a major research subject. Implementing these assays early in the research supports the selection of those candidates and combinations that show a functional response and allows the pharmaceutical companies to focus on the most promising candidates.
Screening Candidate Checkpoint Inhibitors
There are multiple assays used for the functional screening of checkpoint inhibitors or check point inhibitor combinations. In the Human Mixed Lymphocyte Assay, cells from one donor are ‘mixed’ with cells from another donor, resulting in an allogenic response. Upon addition of checkpoint inhibiting candidates, this immune response is elevated. Several parameters such as cytokine production and proliferation can be used to measure this ‘check point blocking’ effect. The use of multicolor flow cytometry allows the identification of the specific responding population, which, in combination with multiplex cytokine analysis, can help to reveal the mechanism of action of certain newer generation checkpoints such as VISTA and TIM3. As not all checkpoints and/or ligands are expressed on all immune cells, it is important to screen this expression prior to selecting the specific format of this assay. Multiple combinations of for example immature and mature dendritic cells with either CD3+, CD4+ or CD8+ T cells can be evaluated and adapted in function of the specific test molecule, target and/or ligand expression. The format allows testing of small molecules, classical antibodies, new antibody formats and scaffolds and can also be used for the evaluation of combination therapies, either as a mixture of antibodies or for example as bispecific format. Examples of applications of this assay are to have a fast answer on whether candidate molecules or combinations are functional, or to evaluate the potency of candidates by comparison with benchmark molecules or among a series of variants. In cases of comparison with benchmark molecules, a dose-titration series is evaluated and compared.
There is also a ‘mouse’ version available of the Mixed Lymphocyte Reaction assay, especially designed to pre-test the potency and functionality of mouse targeting checkpoints. Using an in vitro assay as the first step of selection allows the reduction of the number of laboratory animals used in extended in vivo models and provides information of the functionality in weeks, thereby avoiding testing of non- functional candidates in animal experiments and saving time and costs in the long run.
Another in vitro assay used for the functional screening of checkpoint inhibiting candidates is a CMV-reactivation assay or a superantigen activation assay. In the latter, PBMC of healthy donors are stimulated with staphylococcal enterotoxin B in the presence of the checkpoint candidates and the immune response is quantified using multicolor flow cytometry and cytokine measurement in the supernatant. Again, the functionality of the test samples can be measured and compared and given the short incubation period of the assay, certain specific conditions such as the potency of test samples at lower pH can be evaluated. For the CMV assay, CMV responding pre-screened donors are used and stimulated in vitro with CMV peptide mixes or lysate in the presence of the test samples and relevant controls. The potency of the checkpoint blocking candidates is evaluated by the analysis of proliferation and cytokine production.
Screening Regulatory T Cell Modulating Candidates
The role of regulatory T cells in relation to cancer development and progression has shown to be of great importance. A suppressive regulatory T cell assay can be used for screening molecules that have a potential impact on regulatory T cell functionality. In this assay, purified regulatory T cells are co-cultured with autologous T cells that are either stimulated with anti-CD3 or with allogenic dendritic cells. After an optimized period of in vitro co-culture, the ability of the test molecules to (partly) block the suppressive function of the regulatory T cells can be evaluated by analysis of proliferation and cytokine production.
Screening the Effects of Test Molecules on Macrophage Polarization And Function
Macrophages possess important active and regulatory functions in both innate and adaptive immune responses. Classical activated macrophages, also named M1 macrophages, comprise immune effector cells with an acute inflammatory phenotype while the alternatively activated M2 macrophages have suppressive capacities. Tumor associated macrophages (TAMs) are present at high densities in solid tumors and share many characteristics with so called M2 macrophages.
Macrophages display extraordinary plasticity in response to exogenous and endogenous stimuli which can convert M2- polarized macrophages towards the M1-activated status. Using in vitro polarization and functional macrophage assays, one can screen molecules with the potential to influence M1 and M2 like macrophage generation and polarization. Although distinguished classification and in vitro generation and polarization of M1- and M2- like macrophages is challenging, in vitro assays can be a first step to screen the effect of the test molecules on the phenotype and function of the macrophages. For that, monocytes can be cultured in vitro with M1 and M2 polarizing cytokines and test samples, or test samples can be added after generation to evaluate the effect on re-polarization. Using multicolor flow cytometry, a series of membrane markers in combination with the production of cytokines and enzymes like iNOS and arginase can be used to analyze the phenotype of the macrophage population. Additionally, their suppressive function can be evaluated in a suppressive T cell activation assay.
Screening of Unwanted Immunogenicity of Test Samples
A large proportion of oncology drugs are monoclonal antibodies or other antibody formats that come with an intrinsic risk of unwanted immunogenicity. Especially with combination therapies, it was observed that using a combination of two immune checkpoint blocking antibodies resulted in a higher percentage of patients developing antidrug antibodies (ADA) compared to the sum of the percentages of the ADA positive patients treated with the single antibodies.
Often used as a first step for early immunogenicity assessment are in silico T cell epitope prediction algorithms such as NetMHCIIpan which can be used to assess and compare the immunogenic potential of the lead candidates and guide de-immunization strategies.
Further evaluation of the immunogenic risk can be performed using in vitro T cell proliferation assays to determine and rank the immunogenic risk of the test proteins or identify specific regions of concern. MHC Class II Associated Peptide Proteomics (MAPPs) is becoming increasingly popular to allow a relative ranking of the immunogenic potential based on the peptides that are bound, processed, and presented by dendritic cells.
For assessment of early immune responses, cytokine releaseassociated responses can be evaluated using in vitro whole blood or cellular assays can be used. Analysis of the cytokine signature via Luminex technology allows the evaluation of the effect of drug attributes such as subvisible proteinaceous particles, host cell proteins and other contaminants on the innate immune system.
Finally, the compilation of the different datasets and translation of the results into a comprehensive risk management plan allows further derisking of test candidates and the selection of the best candidates to move forward into humans.
Critical Reagents and Customization
Performing in vitro tests with primary cells can be quite challenging in terms of reproducibility and standardization. For this reason, we prefer to control the entire process from collecting blood or tissues from donors and isolating primary cells to data interpretation and analysis. Almost all cell preparations used in our assays are isolated and prepared in our own labs and undergo a stringent quality control.
For most assays, donor cells or donor pairs are pretested to allow optimal performance in the specific assays. Also, candidate specific adaptations and optimizations of our assays are preferred as not all targets are expressed by all cells and are at different levels for different donors and often, small adaptations are required to reach the most optimal assay window.