A deep dive in CAR+ T-cell therapy

Most patients receiving CAR+ T-cell therapy treatment are pre-treated with daratumumab (anti-CD38), as this has been shown to have a demonstrable effect on the expression of CD38.

Image Credit: Nemes Laszlo/Shutterstock.com

Alternative markers such as CD229 and CD319 can also be introduced into a panel, meaning that it is advisable to check whether or not a targeted antigen is expressed on the malignant plasma cells prior to commencing CAR+ T-cell treatment or a different immunotherapeutic treatment.

A range of other antigens are currently being investigated for their potential in developing CAR+ T-cell therapies. These antigens include CD229, CD44, Lewis, among others. Several of these antigens are now under investigation and undergoing clinical trials.

Effects of immunotherapeutic drugs on plasma cell phenotype in current treatments

This article examines the impact of two popular treatments: daratumumab and BCMA-targeted CAR+ T-cells.

Treatment with daratumumab can directly interfere with a drug’s next-generation flow by impacting CD38 receptor occupancy. This leads to the plasma cells halting their expression of CD38.

This issue, however, can be overcome using a special CD38 multi-epitope antibody that binds to a different epitope than daratumumab. Another alternative approach involves the use of intra cytoplast staining for CD38 that is bound to the endoplasmatic reticulum.

The use of different plasma cell-defining antibodies is also possible, for example, CD229 and CD319.

Treatment with daratumumab may also lead to CD38 antigen loss, lasting up to six months after the last infusion of the drug. This may be due to the temporary downregulation of the CD38 antigen in the plasma cells, or another transient phenomenon.

It is important to note that low or lost CD38 expression can result from the genetic selection of CD38-negative plasma cell clones as a means to escape daratumumab therapy. CD38 might also be lost due to trogocytosis by monocytes and granulocytes, where these cells consume parts of the plasma cells and other antigens in the CD38 complex neighborhood.

This phenomenon has been described for CD56 and CD44. To retrieve the plasma cells, another plasma cell-defining antibody must be used. The most popular antigens for this purpose are CD229 and CD319.

Frequently targeted antigens for CAR+ T-cell therapy

The most commonly targeted antigen for CAR+ T cell therapy continues to be B cell maturation antigen (BCMA) - also referred to as CD269.

Plasma cells in multiple myeloma and other patients see the antigen expressed at very high levels, but this is not found in B cell precursor cells. It is also important to note that gamma-secretase removes the BCMA from plasma cells, allowing these to be retrieved in patients’ serum as soluble BCMA.

BCMA expression has been shown to correlate positively with disease progression. For example, it is higher in patients with multiple myeloma but lower in patients with MGUS.

A reversible down-regulation of BCMA on plasma cells is expected following treatment with anti-BCMA CAR+ T-cells. Clonal selection of BCMA negative or BCMA low expressing plasma cells may occur as an escape mechanism. This expresses multiple myeloma cells, which will proliferate.

Molecular aberration of chromosome 16 can result in BCMA antigen loss, or this antigen loss may be prompted by the removal of BCMA from the plasma cell surface by gamma-secretase.

All of these phenomena will ultimately lead to relapse with plasma cells that are only weakly positive for BCMA or are entirely BCMA negative.

Trogocytosis could occur, but in these cases, the trogocytosis will be prompted by the CAR+ T-cells, resulting in the expression of BCMA on the CAR+ T-cells themselves. This scenario leads to a phenomenon termed ‘fratricide,’ whereby certain CAR+ T-cells kill other CAR+ T-cells.

Using next-generation flow cytometry (NGF) to identify rare plasma cells in patient bone marrow

NGF is a popular and useful tool when evaluating treated patients’ bone marrow for the presence of rare plasma cells. The International Myeloma Working Group (IMWG) has developed a series of criteria for treatment response in multiple myeloma patients.

Studies have shown that 50 % of the treated multiple myeloma patients will reach a complete response - defined as the presence of less than 5 % plasma cells in the patient’s bone marrow, as well as the disappearance of soft tissue plasmacytoma and confirmation of negative unification in serum and urine.

A significant portion of these patients are likely to relapse, however, even in cases considered to be in stringent complete remission. Highly sensitive analytical techniques are, therefore, required to detect persistent disease below the levels typically associated with complete remission.

The IMWG has added to this its list of complete remission criteria, requiring confirmation of minimal residual disease (MRD) negativity via flow cytometric, molecular, or imaging techniques.

How else does MRD relate to clinical outcomes?

MRD is considered one of the most relevant predictors of clinical outcomes - defined as progression-free survival and overall survival. As an indicator, MRD is wholly independent of the risk profile of the patient, the disease stage at diagnosis, and the effect of whether or not the patient received any form of autologous stem cell transplantation.

Established therapeutic strategies remain in place, irrespective of the patient’s MRD status, but a number of clinical trials are ongoing by which the most ideal time for autologous stem cell transplantation is being assessed, as well as the type of consolidation therapy and its duration.

MRD represents a robust predictive factor, both in terms of overall survival and progression-free survival.

MRD negativity could also be used as a surrogate endpoint in terms of the accelerated release of newly developed drugs, though this approach has yet to be considered acceptable by the FDA and EMA.

MRD assessment is also gaining importance in multi-center clinical trials, in cases where these have been FDA and EMA approved. In these cases, MRD can be used as a surrogate marker for progression-free survival.

The challenges of MRD assessment

There are a number of outstanding issues that require resolution before MRD assessment can be more widely used in the clinical space.

For example, the FDA recommends that MRD assessment only be used in patients exhibiting complete and stringent responses, though the EMA expands these criteria to also include patients exhibiting a very good partial response.

Good, usable definitions have yet to be developed in relation to the timing points for the initial MRD assessment, or the duration of ongoing MRD monitoring.

It is, however, anticipated that both agencies will agree by implementing the IMWG criteria of sustained one-year MRD negativity. Both the FDA and EMA seem to have agreed to employ a cut-off of 10-5.

It should also be noted that appropriate methods and imaging techniques are necessary to exclude the presence of extramedullary disease. Liquid biopsies have offered one potential option, but NGF results have not been encouraging until very recently. The determination of cell-free DNA holds significant potential in terms of its effectiveness.

Further risk stratification is required to better account for patients that are proven to be MRD-positive when clinical trials are performed.

Just two techniques currently offer sufficient sensitivity to properly assess MRD: NGF and next-generation sequencing (NGS). Each of these techniques offers a range of advantages and disadvantages which must be considered.

NGS (next-generation sequencing) versus NGF (next-generation flow)

NGS makes delayed analysis possible because this technique does not require the use of fresh samples. It is also important to note that NGF can be performed in the absence of a diagnostic sample, while NGS cannot.

Other cells being present alongside plasma cells is a useful indicator of both sample quality and its possible hemodilution.

Both NGS and NGF offer users complementary information. For example, molecular characterization via NGS provides useful information on clonal evolution and pharmacogenomics, while NGF offers useful information on the number of normal and abnormal plasma cells, cell characteristics, and the level of expression of specific antigens.

A further noteworthy feature of NGS is its capacity to yield usable results from the analysis of fewer cells compared to NGF.

Two standardized panels are commonly employed in MRD assessment: the two times eight-color Euroflow panel and the 10-color MSKCC panel. Both of these panels offer very high sensitivity, often to the minus six.

A number of other panels have been developed by French and German flow cytometry groups. These panels are useful alternatives, but they are not currently utilized as standardized panels.

Cerba Research’s MRD assessment solutions

Cerba Research’s current MRD assessment solution leverages the Euroflow panel on the FACSLyrics instrument.

The company continues to focus on developing larger and more informative panels, expanding the capacity of currently available NGF panels while working to include additional plasma cell defining or prognostic markers.

The primary goal of this development work lies in overcoming the impact of antigen changes on targetable antibodies and therapy-related phenotypical changes of plasma cells.

Should MRD and CAR+ T-cell persistence be simultaneously monitored following CAR+ T-cell infusion?

Clear guidelines regarding the appropriate monitoring of CAR+ T-cell therapy do not currently exist. One emerging approach involves the examination of both MRD and CAR+ T-cell persistence simultaneously, considering both the residual immune system and the functionality and persistence of CAR+ T-cells.

A patient may become MRD-positive for a number of reasons. In 50 % of cases, the primary reason for this is the absence of persistence, but the impact of the disappearance of antigens on plasma cells may also be a contributing factor.

It is, therefore, important to ensure that antigens are still present before reinfusing CAR+ T-cells. It is also important to examine the phenotype of the plasma cells themselves using an appropriate tool; for example, flow cytometric assays, which can detect very low events.

Are NGS assays able to identify more MRD-positive patients than flow cytometric assays?

On the other hand, finding data on comparisons between NGS and flow cytometric assays with similar sensitivities is not quite as easy. The Euroflow group has compared their two eight-color tube NGF method with the LymphoTrack NGS assay, both of which have a sensitivity of 10^-5.

They investigated 105 cases and found ten discordant cases, all of which were NGS-positive. Each of these cases had an MRD below 10^-5, and only three of these patients relapsed. NGS may produce some false-positive results at these very low MRD levels.

In most studies, there may also be an impact due to the fact that both assays are performed on different samples. Typically, there are two samplings: one for NGS and another for NGF. Discordant results can also occur if one of these samples is hemodiluted.

Acknowledgments

Produced from materials originally authored by Ans De Beuckelear, Ph.D., and Rowan Claeys from Cerba Research.

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