advances in research & design of car-t therapy for solid

Advances in Research & Design of CAR-T Therapy for Solid Tumors

Page 3CAR-T Cell Therapy Faces a Solid Challenge

Page 4Analyzing and Validating CAR-T Data

Page 5The Underestimated Impact of R&D Basics

Page 6Happy Cells, Better Data: Easy Steps for Reliable CAR-T Cell Therapy Data

Smarter Technologies for Advancing the Next Generation of CAR-T Therapies

For immunotherapy researchers, Sartorius in vitro assay solutions reduce complexity through deeper insights, streamlined workflows via the collapse of various assays, validation of results by complementary technologies, and robust multiparametric software analysis.

Learn about our Smarter Solutions at www.sartorius.com/car-t-research

© 2020 Sartorius. All rights reserved. IncuCyte®, iQue® and all names of Essen BioScience products are registered trademarks and the property of Essen Bioscience unless otherwise specified. Essen Bioscience is a Sartorius company.

Sartorius 2020 sartorius.com/car-t-research3

ADVANCES IN RESEARCH & DESIGN OF CAR-T THERAPY FOR SOLID TUMORS

CAR-T Cell Therapy Faces a Solid Challenge Chimeric antigen receptor T-cell (CAR-T) therapy is a novel approach to cancer treatment that uses the body’s natural defense, the immune system, to specifically target and kill cancer cells. In CAR-T therapy, researchers extract T cells from the blood of cancer patients or donors and genetically modify them to express cell surface receptors that will recognize antigens on the surface of tumor cells. The modified T-cells are then infused back into patients where they facilitate an immune system wide response to eradicate tumor cells. Since its inception, CAR-T therapy has revolutionized the field of cancer treatment resulting in hundreds of clinical trials and several government-approved cell therapy treatments.1 The Challenge

CAR-T therapy offers unapparelled potential to specifically and efficiently kill tumor cells while avoiding damage to healthy tissue. However, many CAR-T therapies have only found success in liquid or hematological tumors, largely due to cell type homogeneity.2 Solid tumors, on the other hand, are comprised of many different cell types, including malignant cells, stromal cells for scaffolding, tumor-associated immune cells, and vascular cells for oxygen and nutrient supply.3 In addition to these broad cell types, there can be subpopulations of malignant cells with stochastic mutations or that have developed drug resistance.4 This significant cellular heterogeneity results in vastly different protein expression profiles, which makes finding an ideal broad-acting tumor antigen difficult. Furthermore, solid tumor cells derive from healthy cells with which they share similar protein expression profiles. CAR-T cells built to recognize tumor cells may also target and kill a portion of healthy cells in a toxic off-tumor side effect.2

The microenvironment of solid tumors also poses a significant challenge. CAR-T cells that have been infused through the vascular system may not be able to penetrate unorganized

tumor vessels to infiltrate the tumor tissue. If they infiltrate the tissue, the dense fibrotic matrix and low chemokine expression within solid tumors can significantly inhibit CAR-T cell migration to target sites.2

Screening to the Rescue

Large-scale screening is an essential aspect of CAR-T cell research and design and a real asset for facing the unique challenges of solid tumors. Researchers may need to screen hundreds of novel tumor target proteins on different subpopulations of tumor cells from different cancer types. Classical flow cytometry for detecting protein targets or secretions involves multiple washing steps which can significantly slow down sample processing. The Intellicyt® iQue advanced high throughput flow cytometry platform from Sartorius is the fastest flow cytometry platform on the market. It eliminates time-consuming washing steps and can process multiple cell populations within a single run, using a minimal amount of sample volume. This allows for large-scale sample multiplexing to study a variety of cell population attributes in an efficient, timely, and cost-effective manner.5,6 Other automated scalable platforms, such as Sartorius’s Incucyte® Live-Cell Analysis System, allow researchers to observe their cells in real time while recording automated and direct measurements of interactions between immune cells and cancer cells.7 Using this platform, researchers can get an unmatched look at CAR-T cell migration within an in vitro microenvironment, as well as detect live changes in protein expression and track cell number and viability.

See references on page 7.



Research and design are the cornerstone of CAR-T therapy success. Before potential CAR-T therapies can make it to patients, they must be designed and thoroughly vetted in research labs. Researchers generate genes for chimeric antigen receptors and then transfer them into T cells using a variety of approaches, including viral vector transduction, transposon systems, or direct mRNA transduction. They then expand the modified T-cells in culture and evaluate the functionality of CAR-T cells in cell lines or animal models.1 Screening at each of these steps is critical to CAR-T research and design.

Screening: A Shared CAR-T Problem

Each step in CAR-T research and development involves screening a large number of samples and replicates. Automation streamlines the research and discovery process by facilitating the screening of numerous samples in a timely and efficient manner, increasing data confidence, reliability, and accuracy. For example, automation can be used to efficiently assess and compare the degree of tumor death or cytolytic activity caused by CAR-T cells that were generated using different transduction methods.1

iQue® software can help alleviate bottlenecks associated with processing mountains of data. By combining data visualization with rapid interpretation, the iQue® system saves users from individually sifting and evaluating samples for days or weeks at a time.2 Other platforms, such as the Sartorius Incucyte®, allow for multiple users and applications at a time, so that researchers do not have to wait for colleagues to finish their day-long live-cell imaging before they can process their data.3

Combating High Throughput Error

High throughput sample processing and multiplexing are prone to bias and false positive or negative results. It is important that such screens are accompanied by robust analysis solutions and candidate validation. Large-scale multiplexing platforms come equipped with software tools to assist researchers in interrogating their data. The iQue® Forecyt® software automatically generates profile maps from flow cytometry runs and has built in data reduction tools that can help decrease the bias associated with multiplexed sample processing. With the iQue® system´s advanced analysis feature, Panorama, researchers can analyze multiple flow sorted plates, even plates that were processed on different days, a feature that is unheard of in standard flow sorting technology. The iQue® platform also utilizes automated vortex mixing, no-wash assays, and a 48-hour run time that decreases the possibility for user associated sample variability.4

Error and bias can also easily creep into live-cell imaging. Cell identification, size, and characterization can be inadvertently influenced by visualization and measurement. Advances in live-cell imaging, such as those seen with the Incucyte® software, allow researchers to define cell characteristics so as to harmonize data and aid researchers in objective decision making. Such platforms are also equipped with guided interfaces to help researchers to unbiasedly and accurately process data from thousands of wells on a sample plate. Researchers can also observe their entire sample to get a holistic view of the interactions and activities of many cells at once. However, this “birds-eye” view can be overwhelming. The Incucyte® software automatically identifies trends within experiments and helps researchers to quickly identify potential outliers and validate metric overlays.3


Analyzing and Validating CAR-T Data



As busy researchers, it is easy to forget the impact of the supportive things that contribute to endpoint data. The materials used in CAR-T research and design have direct impact on the quality and function of CAR-T cells. It is important for researchers to consistently evaluate and choose the best materials for their research. It is also important to have clear and established lab protocols that everyone in the lab can easily follow.1

Quality Control & Consistency

New researchers often assume that culture media is the same regardless of the supplier. However, there can be minute differences in culture media between suppliers and even between batches from the same supplier. These small differences can cause significant biological changes.It is important to perform regular batch testing on culture medium, serum, cytokines, enzymes, antibodies, antibiotics, magnetic beads, and any other substance that is used in the research and production of CAR-T cells.1 This highlights the importance of established quality testing protocols that are regularly performed to ensure that the materials used are comparable and of the best quality. Consistently using the same donor cell lines for CAR transduction or tumor cell lines for CAR-T cell characterization aids in reducing significant data variability. It is also important to assess the health of these cells throughout the research and design process by routinely screening for possible culture contaminants that may affect cell viability and function.1

Screening for contaminants can be a lengthy and time-consuming process. Sartorius’s Microsart® ATMP kits cut down on that time by rapidly screening for bacteria, fungi, and mycoplasma in only three hours, using existing real-time PCR machinery already present in many labs. Microsart® ATMP is also the only kit on market that does not use infectious bacterial or fungal validation standards, enabling researchers to be confident that they are using the safest possible methods to assess their sample quality.2

Small Volumes, Big Impact

It is easy to underestimate the impact that the simple task of pipetting can have on sample accuracy, sterility, and function. Slight variations in the push of the plunger or in the angle of a pipette can change dispense volumes and introduce an unexpected and sometimes cumulative effect on sample variability. Novel changes to the traditional pipette are helping researchers to guard against pipetting error and ensure accuracy. Sartorius’s Picus® and Picus NxT®

electronic pipettes come equipped with an electronic piston control and brake that helps guarantee precise pipetting results regardless of if the user is a trained lab veteran or new undergraduate student.

For multiplex CAR-T cell research, pipetting a microwell plate can be particularly challenging. Select electronic pipettes such as the Picus® have a built-in tracking function that aids researchers in reliably dispensing into microwell plates.3 New advances in mechanical pipette design can also aid in better data collection. Sartorius’s mechanical Tacta® pipette has a specially calibrated function for pipetting various viscosity types.4 New pipettes such as these are also equipped with a safe-cone filter that helps to safeguard precious samples from possible contamination.


The Underestimated Impact of R&D Basics

Happy Cells, Better Data: Easy Steps for Reliable CAR-T Cell Therapy Data

With the right basics— careful and accurate pipetting, batch reagent testing, regular cell health assessment, contamination testing, and established culture protocols—cells are happier and healthier. This ensures consistent sample output and optimal CAR-T cell functionality.

Microsart® ATMP Sterile Release

• Rapid bacteria, mycoplasma, and fungi detection

• Results in just 3 hours• Only kit with non-infectious validation

standards

Intellicyt iQue® Advanced Flow Cytometry System

• Flow-cytometry analysis of secreted proteins or beads

• Conserves sample by using minimum sample volumes

• Comprehensive analysis and visualization tools

• Data reduction tools• Fastest plate sampling on market• Scalable multiplexed analysis• Multi-user walkaway automation

Incucyte® Live-Cell Analysis System

• Automated, real-time continuous live-cell profiling

• Observe and analyze dynamic cell interactions

• Offers valuable morphological and spatial information

• Sample multiplexing• Built-in direct image-based analysis

Picus® Electronic Pipette

• Ergonomic, electronic pipette• Electronic piston control and

break for accuracy• Tracking functionality for microwell

plates• Safe-cone filters to prevent sample

contamination



Article 1

1. Y. Li et. al., “Quality Control and Nonclinical Research on CAR-T Cell Products: General Principles and Key Issues,” Engineering, 5(1):122-131, 2019.

2. Q. Zhang et. al., “CAR-T Cell Therapy in Cancer: Tribulations and Road Ahead.” Sato T, ed. J Immunol Res, 2020:1924379, 2020.

3. H.M. Levitin et. al., “Single-Cell Transcriptomic Analysis of Tumor Heterogeneity.” Trends Cancer, 4(4):264-268, 2018.

4. M.L. Suvà, I. Tirosh, “Single-Cell RNA Sequencing in Cancer: Lessons Learned and Emerging Challenges.” Mol Cell. 75(1):7-12. 2019.

5. Sartorius, “Intellicyt® iQue® | Suspension Cell Screening |,” Sartorius, https://www.sartorius.com/en/products/cell-analysis/intellicyt-ique-screener-suspension-cell-analysis.

6. Sartorius, “Car-T Therapies Solid Tumors,” Sartorius, https://www.sartorius.com/en/pr/car-t/car-t-therapies-solid-tumors.

7. Sartorius, “Incucyte® Immuno-oncology: Monitoring Immune and Cancer cells,” Sartorius, https://www.essenbioscience.com/en/communications/immuno-oncology/?utm_source=sartorius&utm_medium=extbanner&utm_campaign=car-t&utm_term=sartorius_referral&utm_content=overall.

Article 2

1. Y. Li et. al., “Quality Control and Nonclinical Research on CAR-T Cell Products: General Principles and Key Issues,” Engineering, 5(1):122-131. 2019.

2. Sartorius, "Intellicyt® iQue® | Suspension Cell Screening," Sartorius, https://www.sartorius.com/en/products/cell-analysis/intellicyt-ique-screener-suspension-cell-analysis.

3. Sartorius, "Incucyte® Analysis Software - Software," Sartorius, https://www.essenbioscience.com/en/products/software/incucyte-base-software/.

4. Sartorius, "iQue® | Advanced High Throughput Flow Cytometry Instrument," Sartorius, https://intellicyt.com/products/instrumentation/.

Article 3

1. Y. Li et. al., “Quality Control and Nonclinical Research on CAR-T Cell Products: General Principles and Key Issues,” Engineering, 5(1):122-131. 2019.

2. Sartorius, "Bacterial Detection," Sartorius, https://www.sartorius.com/en/applications/quality-control-testing/microbiological-quality-control/rapid-testing/bacterial-detection.

3. Sartorius, "Picus® Electronic Pipette | Ergonomic Pipette |," Sartorius, https://www.sartorius.com/en/products/pipetting-and-dispensing/pipettes/electronic-pipettes/picus.

4. Sartorius, "Tacta Mechanical Pipette | Laboratory Pipettes |," Sartorius, https://www.sartorius.com/en/products/pipetting-and-dispensing/pipettes/mechanical-pipettes/tacta.

Information-Rich Cell Analysis Solutions to Transform CAR-T Research and Discovery

Unmatched speed, depth and scale—Sartorius’ Incucyte® Live-Cell Analysis System and iQue® Advanced Flow Cytometry platforms offer unique, high capacity solutions for characterizing immune cell phenotype, activation and function. Other approaches require multiple assays or time points. Our comprehensive analysis and visualization capabilities reduce variability by delivering more data per time point, or multiple time points, in the same context, under the same conditions for faster, actionable results.

Learn about our Smarter Solutions at www.sartorius.com/car-t-research

© 2020 Sartorius. All rights reserved. IncuCyte®, iQue® and all names of Essen BioScience products are registered trademarks and the property of Essen Bioscience unless otherwise specified. Essen Bioscience is a Sartorius company.

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