A 10-minute assay to prioritize and rank constructs with direct mechanistic insight

The measure of avidity provides one simple assay to assess the efficacy and safety of your cell therapy or cell engager with results that correlate to in vivo outcomes better than killing assays or cytokine secretion

Marcela Maus, MD, PhD, Associate Professor at Harvard Medical School and Director of Cellular Immunotherapy at Massachusetts General Hospital recently published in Nature and Cancer Cell, showcasing the necessity of measuring cell avidity to pick the best CAR candidates. In her webinar with our team, she mentions:

“It’s been challenging to identify either in vitro models or in vivo models that will be predictive… we think that with measuring avidity, which was sort of lucky testing on our part, is the assay that seems to predict the best for in vivo activity… We don’t have enough data to say whether it predicts clinical activity, but at least in terms of in vivo functionality it seems like avidity is quite an exciting new assay to add to the repertoire of picking the best CAR.”  

From Dr. Maus’ groundbreaking work in Cancer Cell, Non-cleavable hinge enhances avidity and expansion of CAR T cells for acute myeloid leukemia, Leick et al. tested various CAR variants with different in-vitro methods and mouse models, looking for the best functioning CAR to develop further.

They found that cytokine secretion and cytotoxicity assays, even serial re-stimulation assays, couldn’t differentiate between the different CAR constructs. However, when her group used avidity to evaluate their constructs it very clearly indicated a lead candidate.

In fact, avidity correlated with in vivo tumor control (R^2: 0.91) significantly better than killing assays (R^2: 0.91) 28-day serial re-stim followed by killing assay) or cytokine secretion (R^2: 0.55).

The missing correlation in existing assays

The open secret in the field is that we’re relying on the same sets of assays to select candidates for further study when they have very poor correlations to in-vivo outcomes. From 2011-2020, the likelihood of a therapeutic approval from a Phase I clinical trial in Immuno-Oncology was 12.4%.

With the vast majority of clinical trials failing, at tremendous cost, do we really have the best information available to select candidates for further study?

Killing assays, serial killing or restimulation, and even EC50’s have tremendous variability in their results. The same applies to IFNg release and other cytokine secretion assays. We’re desperate for a better understanding with our in-vitro assays, but we’re all stuck comparing amongst the same sets of variable assays and hoping for some collective confidence to distill out… and it often doesn’t.

So we end up taking our best guess on a lead candidate, because that’s all the information we’ve had, until recently.

Avidity: where function begins

We’re accustomed to thinking about the affinity of receptor-ligand interactions (Kd, Ka, etc) because that’s what has been readily measurable by SPR. However, this measurement is isolated within a 3d, forceless environment… and it’s missing all other interactions happening within the immune synapse. CD2-CD58, ICAM-1 – LFA-1, amongst hundreds of others, are vital dynamic interactions happening within the immune synapse that are missed with SPR.

So how can a 10-minute avidity assay correlate so well to in vivo function?

It seems that the binding strength between cells communicates a proportionate signal strength that determines an effector function.

We can think of analogous situation with thymic selection. The right amount of binding communicates through signal strength some functional outcome. If a T cell binds too tightly or too weakly, it’s not selected. The ‘right’ strength of binding results in the desired function.

So we can think of avidity, or the strength of all these interactions, as the initiating event for downstream function. This binding strength is the ‘input’ while most of our other functional assays measure some variable output.

The data seems to indicate that activation, expansion, cytokine secretion, killing and long-term persistence are all ‘outputs’ – the downstream result of initial effector-tumor interactions. The assays we’ve had can be thought of as ability to indirectly infer something about the effector-tumor interaction… because we haven’t had the ability to directly quantify that interaction.

Until now…

The strength or weakness of binding initiates a proportionate signal amplification which in large part determines the efficacy of a functional response.

The z-Movi Cell Avidity Analyzer provides the ONLY simple, direct solution to measure cell avidity

The easiest way to think about setting up assays with the z-Movi is that we’re setting up a killing assay with an effector-target ratio of 1:1… but we don’t let them kill.

You incubate your fluorescently stained effectors with targets for an average of 5 minutes and then use acoustic force to ‘pull’ on the effectors. Since we’re tracking each effector as we gradually increase the force, we can quantify the exact force required to break the synapse. This allows you tremendously flexibility in your workflows so you can very rapidly get compelling data… often before your other functional assays can even be set up.

What's next?