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In the last decade, researchers have studied biological mechanisms that initiate and control cancer cell growth. This research includes an effort to understand the tumor microenvironment and the influence of checkpoint inhibitors, facilitating the development of therapies that are personalized for the individual patient and their particular type of cancer. The following are among the more promising immunotherapies:

CAR-T, a novel immunotherapy
CAR-T therapies offer a new approach for the treatment of solid tumors. A patient’s own immune cells are collected, genetically altered, multiplied, and finally returned to the patient. This process is known as adoptive T-cell transfer and the alteration occurs via retroviral gene transfer of the CAR-T cell receptors. The re-engineered CAR-T cells possess cytokine-producing and cytolytic activity targeted at specific antigens on the tumor cell surface.

Checkpoint inhibitors as targeted therapy
The T-cell, one of the white blood cells, plays an important role in many immunotherapies. In healthy people, T-cells identify and kill infected or abnormal cells including cancer. To activate T-cells to become antigen-specific, CARs combine the Fv portion of monoclonal antibodies with the cellular signaling from the CD3 molecule.

T-cell regulation is vital for tumor development since T-cells can eliminate cancer cells if they are functioning properly. By regulating T-cell response, some proteins act as immune checkpoints providing information about tumor behavior. Immune checkpoint inhibitors attempt to restore the immune response by targeting programmed death protein (PD-1), its ligand PD-L1, or cytotoxic T-lymphocyte associated protein (CTLA-4).

Using malignant cells to target the immune system
An innovative technology called BiTE, was developed to engage the body’s endogenous T-cells to target malignant cells. Ordinarily activated T-cells will use various mechanisms to eliminate target cells causing cytotoxic components to be released. Malignant cells are capable of evading destruction by cytotoxic T-cells and BiTE technology is designed to overcome malignant cell evasion by binding polyclonal cytotoxic T-cells and targeted malignant cells.

Solutions for immunotherapy development
Developing a successful immunotherapy requires complex and challenging research. Often animal models are unreliable and patient samples are limited. To increase the likelihood of success, technologies utilized during the process need to be precise, sensitive, and absolute.

Foundational research
In this early phase of discovery, research is focused on understanding the tumor, its microenvironment, the patient’s immune system, and how they interact with each other. Researchers reveal insights that allow potential targets to be identified, a tumor cell related target or an immune cell. Potential therapies such as proteins, antibodies, antibody-drug conjugates, modified immune cells or vaccines are developed and tested.

Understanding the tumor microenvironment requires analysis at the single cell level to truly reveal cellular heterogeneity. Bio-Rad’s ddSEQ Single Cell Isolator enables single cell isolation prior to sequencing. In addition single cell isolation can be performed using the S3e Cell Sorter to provide cells for downstream cellular, proteomic, or genomic assays. These assays typically involve screening large numbers of parameters. For genomic analysis, the automated CFX Real-Time PCR Detection System provides gene expression data from multiple sample types.

Translational research
In translational research, insights are validated through multiple models as the potential therapy is further optimized for human safety and efficacy. Development of the most pertinent model system ensures success when translating the potential therapy from pre-clinical to clinical trials. Optimal purification processes and formulations for the therapy are investigated concurrently. The discovery of potential biomarkers can also be validated at this stage. In addition, applications may be submitted to regulatory agencies requesting approval to test potential therapies in humans.

For cell based assays, the automated Ze5 Cell Analyzer can examine up to 30 parameters simultaneously allowing rare or transient cell types to be easily analyzed in high throughput. Additionally, screening for the expression of key immune or inflammation related proteins is performed to track potential biomarkers during preclinical models. The Bio-Plex Multiplex Immunoassay platform measures up to 500 proteins simultaneously, automating the analysis of 96-well plates and yielding up to 9,600 data points in about 35 minutes.

Clinical research
Clinical trials put the potential therapeutic through rigorous, regulated tests in humans. The patient’s tumor type or subtypes will be profiled, and the patient will be matched to the most appropriate potential therapy. Safety of the selected therapy is determined during early phases of clinical research. In later phases, efficacy of the therapeutic as well as its associated side effects will be monitored and measured. Concurrently, manufacturers will expand production in preparation for approval.

Patient safety can be assessed through biomarker tracking using technologies like Bio-Plex Multiplex Immunoassays to measure up to 500 analytes simultaneously, including important immune and inflammation biomarkers. The ddPCR System provides a simple, sensitive method to identify and measure genetic mutations in the tumor or from liquid biopsies. It can also be used to monitor residual disease in response to the potential therapy. Sometimes when developing clinical assays, like PK assays, it may not be possible to generate reagents in-house. Bio-Rad’s HuCAL Custom Antibody service can provide key assay components in a continuous, reliable supply within 6 weeks using an animal free platform. Copy number variance in CAR T cell preparations can be QC-verified using ddPCR, also important for monitoring residual disease in cancer patients via liquid biopsy.

Manufacturing, QC, and patient monitoring
When regulatory agencies provide approval for the therapy, commercial scale manufacturing begins and includes rigorous QC testing. Continuous patient monitoring ensures patient safety and the dissemination of appropriate treatment plans.

Bio-Rad’s extensive resin portfolio covers a range of chromatographic techniques and offers unique resin chemistries that improve the purification process. Measuring and monitoring nucleic acid impurities in your final product can be achieved using ddPCR residual DNA solutions which provide sensitivity for nucleic acid detection without the need for a standard curve. Our host cell protein workflow validates host cell detection ELISA antibody coverage and ensures the integrity of host cell protein ELISA. In addition, ddPCR technology is used to determine CAR gene copy number in cell therapy preparations prior to patient administration and to monitor cell levels within the patient during treatment.