ASSAY DEVELOPMENT - Case Study: How Custom Assay Development Helped Spur Precision Medicine Research in Multiple Myeloma

INTRODUCTION

From big pharma to emerging biotech companies, a robust assay is an integral part of any pharmaceutical company’s drug discovery and development process. Yet many organizations may not want to devote the substantial time or resources required to develop custom, reproducible assays on their own. For these companies, having a trusted laboratory partner with the necessary expertise and technology can make all the difference – both in creating assays for specific biomarkers and in successfully integrating these assays into clinical trials in a more cost-effective manner.

Starting in 2010, the Menarini Silicon Biosystems, Inc. (MSB) R&D team (then part of Veridex, a Johnson & Johnson company) led the process of developing their first assay to isolate and enumerate circulating multiple myeloma cells (CMMC) from peripheral blood. Our primary goal for the assay was to determine if CMMCs could be used to better understand the biology of multiple myeloma and monitor clinical progression of the disease. The assay was developed in collaboration with Janssen Oncology Pharmaceuticals as part of a research agreement.

The following provides a “behind-the-scenes” look at the CMMC assay development process (from 2010 through today) to show how pharmaceutical companies can effectively partner with a laboratory to design customized assays that complement their drug discovery and development programs.

TECHNOLOGY & EXPERTISE: THE POWER OF CELLSEARCH

When selecting a partner for assay development, both the technology and expertise of those in the lab are important factors to consider. Our team developed the circulating multiple myeloma cells (CMMC) assay with the CELLSEARCH^® platform, which is considered the gold standard of liquid biopsy technology for detecting circulating tumor cells (CTCs). It is the first and only clinically validated, FDA-cleared system for identification, isolation, and enumeration of CTCs in metastatic breast, metastatic prostate, and metastatic colon cancer.

The CELLSEARCH system uses automated sample capture and analysis on a four-color semi-automated fluorescence microscope. It separates rare CTCs from blood using ferrofluid nanoparticles coated with antibodies to target the epithelial cell adhesion molecule (EpCAM), a protein expressed on the surface of epithelial cells. EpCAM is expressed at a high level by solid tumors and has allowed researchers to develop assays for cancers like prostate and breast cancer. The cells are then stained with detection antibodies and an antibody to exclude leukocytes, and image analysis is carried out to identify tumor cell candidates. The technology is not limited to EpCAM-expressing cells, however. We can develop custom ferrofluids to capture non-EpCAM-expressing cells, and MSB has additional kits targeting circulating endothelial cells (CEC kit) and circulating melanoma cells (CMC kit).

With a fourth detection channel available for additional screening of cells, the system has built-in flexibility to move beyond just counting cells and incorporate other markers such as PD-L1, HER2, and EGFR. As we did with Janssen, pharmaceutical companies could use a primary detection marker for CTCs, while using the open channel to look for the expression of a specific target marker as inclusion criteria for a clinical trial, or to monitor the therapeutic effect of a drug.

By working with our R&D team, the Janssen group also had the benefit of working with some of the same people who created the CELLSEARCH platform and pioneered the development of custom assays with the technology. Many of us were part of a group of researchers, led by Massimo Cristofanilli, MD, that helped validate the CELLSEARCH technology in the early 2000s. That research established that the number of CTCs before treatment was an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer.¹ When it comes to rare cell or CTC assay biomarker development, this is a level of expertise not available at an average reference laboratory.

THE CMMC ASSAY DEVELOPMENT PROCESS

Multiple myeloma is the second most common hematological cancer in the US, with an estimated incidence of over 20,000 new cases per year. Despite advances in treatment, the disease remains incurable with a 5-year survival rate of only 45%.

In 2010, Janssen’s hematology group asked our team to develop an assay for the detection and enumeration of CMMCs from peripheral blood samples. The desire for the assay was spurred by a growing body of research that showed the presence of CMMCs in patients with the disease, as well as those with two precursor diseases: monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma (SMM). It was recognized that those circulating cells might be usable as a liquid biopsy to learn more about the progression of the disease and identify actionable mutations for targeted therapy without an invasive bone marrow biopsy. In addition, the ability to access CMMCs through a simple blood draw means we can capture these cells more easily than with bone marrow biopsies, which could potentially make it possible to monitor the disease in real time.

Because much of assay development can be a trial-and-error process, we used a phased approach to minimize risk for Janssen. For each of the three phases (feasibility, verification, and final development), we submitted reports and obtained approval from Janssen before moving on to the next phase.

THE CHALLENGE: IDENTIFYING DETECTION & CAPTURE ANTIBODIES

The initial phase of the CMMC assay development process began in February 2010. Developing the first assay for a hematological cancer presented a distinct challenge for our team. Capture and detection of tumors that typically express EpCAM is relatively straightforward on the CELLSEARCH platform. Most blood cancers, on the other hand, do not have unique markers that differentiate them from normal white blood cells in circulation.

In order to develop the assay, our CTC experts collaborated with Janssen’s multiple myeloma experts to identify antibodies that would allow for the detection and capture of the CMMCs. Fortunately, multiple myeloma is one of the few hematologic cancers that have a marker unique enough to identify the cell: CD138. We chose to use CD138 as the capture antibody and CD38 for detection, with CD19 and CD45 as exclusion antibodies.

THE STRUGGLE: UTILIZING THE OPEN CHANNEL

Next, a great deal of discussion went into selecting a marker for identification in the open channel. While the available literature offered a wide variety of markers used in multiple myeloma, we wanted to select the most common ones in order to pick up the highest percentage of patients with the disease.

Ten years ago, most diagnostic testing for multiple myeloma was done in bone marrow, but finding those same cells in circulation can be a difficult process. Some markers may not be ideal for testing cells in peripheral blood. Though our team finally decided on a marker for the open channel, it turned out that marker was not found frequently on CMMCs in the circulation. While the cells were positive for that marker in the bone marrow, when they moved into circulation, we discovered they tended to lose that marker. That part of the assay was subsequently dropped from development.

THE RESULT: A REPRODUCIBLE CMMC ASSAY

By March 2011, our team succeeded in developing an automated assay capable of reproducibly isolating CMMCs from the peripheral blood of patients with multiple myeloma. The assay provided valuable information regarding how the number of CMMCs changed with progression of the disease, or in response to treatment.

In the final phase of development, our group transferred the assay to the clinical development team. Both groups carried out side-by-side testing to make sure the results matched, and then trained team members on the assay so they could run it independently.

In 2018, we published results of a study based on the CMMC assay in The British Journal of Hematology.² Our research indicated the assay could be used to investigate the biology of the disease and potentially monitor progression, especially in early stage asymptomatic disease.

In the study, we counted CMMCs from over 1,000 patient samples, including those with newly diagnosed MM and SMM. In the newly diagnosed patients, CMMC counts correlated with other clinical measures of disease burden, including percentage of bone marrow plasma cells, serum M protein, and International Staging System stage. Patients with CMMC counts equal to or greater than 100 in 4 ml of blood during remission had worse progression free survival compared with patients who had less than 100. Those with undetectable CMMCs had even greater overall survival benefits. Patients with SMM showed a trend toward higher-risk myeloma states with higher CMMC counts.

ASSAY & EXPERTISE TRANSFER TO MENARINI LAB SERVICES

In 2017, Menarini Silicon Biosystems acquired CELLSEARCH from Janssen. This marked the transfer of the CMMC assay to Menarini’s Laboratory Services, which provides global laboratory testing with the CELLSEARCH platform, from Phase 1 to Phase 3 clinical trials. With a CLIA-certified and ISO15189-accredited lab in the US and a mirror lab in Italy, as well as its proprietary Cell-Save tube, which stabilizes samples for 96 hours to enable international shipping, MSB partners with clinical investigators and pharmaceutical companies throughout the world to perform global clinical trials.

A majority of the R&D team that originally developed the CMMC assay also became part of Menarini. With nearly two decades of pharma services development and clinical trial testing experience, our Assay Development and Lab Services teams have created close to a dozen custom assays and participated in testing for nearly 100 clinical trials.

We continue to use our expertise with the CELLSEARCH platform to develop custom assays, helping our partners make the best use of the technology in order to develop companion diagnostics that can accelerate the validation of personalized therapies. By using the only FDA-cleared CTC platform, our partners who use their data for FDA submissions are confident in the quality and reproducibility of their assays.

CUSTOM ASSAY DEVELOPMENT

The CMMC assay is now available as a laboratory service, part of a broad menu of assays available to Menarini’s pharmaceutical and academic partners. Other CLIA-regulated tests on the menu include tests for enumeration of epithelial cells, melanoma cells, and breast cancer cells expressing PDL-1.

These assays are designed in collaboration with company scientists and can be customized for many different types of cells, based on the target of the trial. The system can be tailored to detect different types of tumor cells in the same way that it was tailored to capture and detect CMMCs.

After developing the assay for a particular biomarker of interest, we can also provide information beyond just the number of cells captured, depending on the specific parameters of the clinical trial. Although the available open channel on the CELLSEARCH platform ultimately wasn’t needed for the CMMC assay, it offers potential for even more selectivity for assay development. Using that channel, we can look for the expression of the marker to verify patient eligibility for the trial, or monitor how that expression changes before, during, and after therapy to determine the therapeutic effect of the drug being studied.

Similar to the development process for the CMMC assay, we approach custom assay development in phases. If at any point we conclude the assay won’t work, the company does not have to pay to proceed to the next phase of development. This gives our R&D team the opportunity to explore new, novel markers and develop innovative assays while minimizing the financial risk for our partners.

A MORE COMPLETE LIQUID BIOPSY

With the addition of Menarini’s DEPArray^TM technology to the assay workflow³, the possibilities for customized enumeration, isolation, and molecular characterization of rare cells are even greater. DEPArray is an automated image-based cell selection platform that can recover single cells of interest from heterogeneous samples for downstream molecular analysis. Combining this functionality with CELLSEARCH allows further identification, isolation, and characterization of cells of interest based on a wide range of parameters, including cell characteristics, expression analysis, mutations, next-generation sequencing, and more.

With the CMMC assay, the CELLSEARCH platform can separate the cells from a blood sample, producing an enriched population of CMMCs to be analyzed with the DEPArray technology, which enables molecular characterization of those individual cells in real-time. Not only does the workflow³ allow the researchers to monitor the number of CMMCs, it also enables them to observe the specific molecular changes that occur throughout the disease progression, which will likely have therapeutic implications for this rapidly moving, dynamic cancer.

PUSHING PRECISION MEDICINE FORWARD

In 2020, Menarini Silicon Biosystems will launch the CMMC assay as a research-use-only commercial kit. Like the development of the original assay, this process was not without its challenges, including the need to design a more stable capture reagent when it was discovered that CD138 alone was not stable enough for commercial use. To address this at the time, our team developed a modified capture reagent that combined both CD138 and CD38. The CMMC RUO kit will be used with a new research-use-only blood collection tube containing a new blood preservative that helps stabilize CD138. This eliminates the need for the CD138/38 hybrid capture reagent, which results in significantly lower background.

By offering the CMMC assay as a kit that researchers can run on their own instrumentation, the hope is that it will open new doors on further investigating these cells. As researchers learn more about the molecular dynamics of multiple myeloma, this could have a significant impact by helping to identify patients that will respond to an already existing drug, or lead to the development of new, more effective treatments, an important step forward for precision medicine research.

ACKNOWLEDGEMENT

The author would like to thank the members of the Menarini Silicon Biosystems R&D group, clinical group, and the commercial team for their contributions to the development of the CMMC assay.

REFERENCES

1. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. NEJM. 2004;351(8):781-791. doi: 10.1056/nejmoa040766.
2. Foulk B, Schaffer M, Gross S, et al. Enumeration and characterization of circulating multiple myeloma cells in patients with plasma cell disorders. Bri J Haematol. 2017;180(1):71-81. doi:10.1111/bjh.15003.
3. The workflow described is for research use only. Not for use in diagnostic procedures. The performance characteristics, safety, and effectiveness of the workflow have not been established and are not cleared or approved by the FDA. The CMMC Assay and the associated blood collection tube used for the detection of CMMCs is for research use only. Not for use in diagnostic procedures. The DEPArray technology is for research use only. Not for use in diagnostic procedures.

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Steven Gross is the Head of Assay Development for Menarini Silicon Biosystems. As a Scientist/Senior Scientist, he has been a part of the CELLSEARCH story from its founding at Immunicon Corporation, then with Johnson & Johnson, and now Menarini Silicon Biosystems. In addition to executing R&D research agreements and assay development, he works closely with both the clinical and commercial teams to supply reagents for clinical lab services and interact with research partners and customers. He earned his Master’s degree in Biology from Temple University in Philadelphia.