Rapid assessment of CAR T-cell strategies for multiple myeloma with Cell Avidity analysis

Identifying effective immunotherapeutic treatment strategies for multiple myeloma that also mitigate relapse often requires tedious and time-consuming validations, such as cell-killing assays and in vivo engraftments. In this 2020 collaboration with Dr. Eric Smith (Dana-Farber Cancer Institute) and Prof. Carlos de Larrea (Hospital Clinic Barcelona), we show that intercellular binding strength (Cell Avidity), quickly predicts CAR T-cell efficacies that correlate with treatment outcomes in vivo.

Cell Avidity in Multiple Myeloma

The continuous development of treatment options for multiple myeloma has consistently improved the clinical outcomes during the past decades. In recent years, several research groups have reported promising results from early clinical trials of CAR-engineered T cells targeting B-cell maturation antigen (BCMA). While BCMAspecific CAR T cells have yielded high response rates, most patients relapse shortly after treatment due to BCMA antigen escape [1].

Expression of G protein-coupled receptor class C group 5 member D (GPRC5D) was recently identified on bone marrow-localized multiple myeloma cells and proposed as a promising target candidate [2]. Hence, targeting both BCMA and GPRC5D is an attractive approach to treat multiple myeloma while avoiding BCMA antigen escape. However, confirming the added benefits of dual-targeting CAR T cells against BCMA and GPRC5D and testing the most effective combination strategy can be time-consuming through conventional assays.

In this review, we introduce you to a unique analysis method using the z-Movi Cell Avidity Analyzer that reliably predicts CAR T-cell functionality by assessing intercellular binding strength (cell avidity). We describe cell-avidity measurements of mono-targeting and dual-targeting CAR T cells against BCMA and GPRC5D and compare the results with in vivo outcomes from a recent publication by de Larrea et al. [3].

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CAR T-cell strategies to simultaneously target two antigens

This review evaluates two immunotherapeutic strategies that simultaneously target the two multiple myeloma-specific antigens BCMA and GPRC5D. In a pooled mono-targeting CAR T cell approach (mtCAR), the antigenspecific CARs (BCMA or GPRC5D) are expressed in separate T-cell populations and subsequently pooled in the relevant ratios.

Dual-targeting CAR T cells (dtCAR) express both antigen-specific CARs (BCMA and GPRC5D) in parallel, which can be generated in different ways:

• A bicistronic form where the respective CARs are delivered to the T cells as a single vector.
• A so-called “dual single-stalk” approach where both CARs are introduced to the T cells as a fused single-chain variable fragment (scFv) vector.

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How to measure Cell Avidity in CAR-T cells

Avidity analyses can serve as a facilitating tool to distinguish between low and high efficacy CAR T cells. Measuring cell avidity through applied acoustic forces can predict CAR T-cell functionality and outcome. We measure cell avidity by first allowing the tracked CAR T cells to sediment on top of a monolayer of target cells. Next, we apply a force ramp of increasing force magnitudes to pull the tracked cells. The force required to separate the tracked cells from the monolayer indicates the specific cellular avidity of the assessed CAR T-cell population.

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Measuring CAR T-cell avidity on BCMA/GPRC5Dexpressing cells to compare functionalities between different CAR configurations

How do dtCARs compare against populations of mtCARs targeting BCMA or GPRC5D?

Researchers evaluated cell–cell interactions between CAR T cells and artificial antigen-presenting 3T3 cells expressing both BCMA and GPRC5D. To validate the effectiveness of clinically relevant strategies, they compared cellular avidity of mtCARs (targeting BCMA or GPRC5D) and two types of dtCARs, a bicistronic (dtCAR1) and single stalk (dtCAR2), upon exposure to an acoustic force ramp.

Separate cell avidity measurements revealed that dtCARs required significantly higher separation forces than both mtCAR populations (Figure 3). At 200 pN, which was the force required to detach non-transduced T cells, percentages of bound mtCARs and dtCARs ranged between 18% and 27% (p < 0.05; mtCAR-BCMA: 18%, mtCAR-GPRC5D: 21%, dtCAR1: 25%, and dtCAR2: 27%). The results suggest that dtCARs exhibit superior binding interactions to target cells expressing both antigens compared with mtCARs.

How does Cell Avidity correlate with CAR T-cell functionality?

De Larrea et al. compared the overall survival of mtCAR- or dtCAR-treated xenograft mouse models injected with OPM2 myeloma cells expressing both antigens. At low (subtherapeutic) doses of CAR T cells (2.5 × 105), dtCAR treatment resulted in an enhanced response, observed by a lower tumor burden and improved overall survival. The results support the cell-avidity measurements, indicating that dtCARs effectively bind and kill cells expressing both BCMA and GPRC5D.

Measuring the effectiveness of dtCARs on BCMA-deficient cells to study response to antigen escape

Multiple myeloma relapses often occur due to BCMA antigen escape, resulting in a positive selection and expansion of cells with reduced or absent expression of BCMA. Validating the functionality of dtCARs on BCMA-knockout target cells can reveal their efficacy upon antigen-escape in vivo.

Here, researchers validated the cell avidity between CAR T cells and BCMAnegative 3T3 target cells expressing GPRC5D. They compared mtCARs against BCMA or GPRC5D and dtCARs that simultaneously target both antigens (dtCAR1 and dtCAR2).

Avidity measurements recorded the highest avidity in the two dtCAR populations (41% and 57% bound dtCARs at 200 pN) and the GPRC5Dtargeting mtCAR (54% bound mtCAR-GPRC5D at 200 pN). By contrast, BCMA-targeting CAR T cells established significantly weaker interactions with the target cells (20% bound mtCAR-BCMA at 200 pN; p < 0.01; Figure 4). As expected, cell avidity was negligible between any of the CAR T-cell populations on 3T3 cells negative for BCMA and GPRC5D.

How does Cell Avidity correlate with CAR T-cell functionality?

De Larrea et al. rechallenged myeloma-engrafted mice that had survived an initial CAR T-cell treatment (with mtCARs or dtCARs) by injecting them with BCMA-deficient OPM2 cells. Mice that had previously been treated with mtCARs targeting GPRC5D only or dtCARs at the initial treatment point were protected from the OPM2 rechallenge. Notably, the mice previously treated with BCMA-specific mtCARs developed progressive disease and had a comparatively shorter median survival than their counterparts.

Supporting our cell avidity data, these in vivo results demonstrate that both GPRC5D-specific CAR T cells and dtCARs effectively bind and eradicate BCMA-deficient cells.

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Conclusions

With 100s of ongoing clinical trials [4], CAR T-cell therapies are rapidly emerging as promising treatment strategies for multiple myeloma. These fastpaced advancements require an accelerated identification of target candidates that can improve CAR T-cell effectiveness and mitigate relapses.

We have showcased the use of cell avidity analysis to explore and validate CAR T-cell functionality in the context of multiple myeloma. Specifically, we demonstrate that these measurements correlate with treatment outcomes in mouse models, suggesting that cell avidity measurements are reliable predictors of CAR T-cell efficacy.Researchers have traditionally relied on assays that are either inconsistent, non-correlating with the immune-cell response, or time-consuming.

High-throughput cell avidity analyses have correlated with immune-cell response, producing accurate results within hours. This technology offers a predictive, reproducible, and rapid results at the single-cell level without compromising cell viability.

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References

1. Brudno JN, Maric I, Hartman SD, Rose JJ, Wang M, Lam N, et al. T cells genetically modified to express an anti–B-Cell maturation antigen chimeric antigen receptor cause remissions of poor-prognosis relapsed multiple myeloma. J Clin Oncol. 2018
2. Smith EL, Harrington K, Staehr M, Masakayan R, Jones J, Long TJ, et al. GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Sci Transl Med. 2019
3. Fernández de Larrea C, Staehr M, Lopez A V., Ng KY, Chen Y, Godfrey WD, et al. Defining an optimal dual-targeted CAR T-cell therapy approach simultaneously targeting BCMA and GPRC5D to prevent BCMA escape–driven relapse in multiple myeloma. Blood Cancer Discov. 2020;
4. https://clinicaltrials.gov/ (September 2025)