Potency AssaysI’ve read the recent FDA guidance documents on...

Potency Assays

I’ve read the recent FDA guidance documents on GvHD and potency tests in conjunction. This was helpful. Thank you@irenekwshiu for suggesting it.

I find this area difficult as I’m no scientist and IMO the specifics re. Ryoncil's potency assay would be proprietary knowledge.

Also, it’s hard to know where the truth lies because I find a strong indication cellular medicine is way more advanced than is in the public domain simply because:

• the supporting technology can be outsourced or has become a commodity.

• AI and machine-learning have enabled great advances in simulation. Computational Biology (one of the areas Dr. Jessica Rose is in) is an actual discipline!

I don’t know how much of this is useful but I’ll throw some things out with the intention to learn. Happy to be corrected.

Having read the FDA document, the clearest takeaway for me is that a potency assay is the cornerstone of a biological product and something to be sorted out at the earliest stage using a “progressive” and “incremental” approach.

Biological and non biological assays are covered and specific and helpful examples are given.

With reference to the recent FDA GvHD document:

“For drugs developed in a population selected on the basis of a biomarker of disease activity, an in vitro companion diagnostic device (referred to as a "companion diagnostic" herein) may be needed. A companion diagnostic is an in vitro diagnostic (IVD) that provides information that is essential for the safe and effective use of the drug”.

Am I right in thinking these have been available as kits for some time? If so, surely that speaks to standardization.

The FDA guidelines for potency assays make it impossible for me to believe that any issue with a potency assay for a biological product could not have been anticipated:

“When possible, you should retain samples of each lot of in house reference material for comparison with newly manufactured reference material and prepare in advance for depletion or expiration of reference materials”.

“You should also show that the assay can discriminate between active product and an inactive or degraded form of the product”

Back in 2013, Bravery et al provided a detailed overview of challenges and expectations, stressing that “aggressive early investment in a solid potency evaluation strategy can greatly enhance eventual CTP deployment because it can mitigate the risk of costly product failure in late-stage development”.

The FDA document states it may be difficult to determine which product attributes are most relevant to measuring potency; however, back in 2012, Lehman et al. reported on a model for the development of an in vitro surrogate potency assay for Athersys’ matrix. The aim was to identify the factors for the desired effect of angiogenesis and thereby set “pass/fail criteria based on minimum levels of these factors required for activity”.

Removal of any one of the factors lessened the effect and researchers were also able to test the minimum level required by adding back increasing amounts of these cytokines into the depleted serum-free conditioned media. Authors describe the process as “quantitative”.

FDA:

Ideally, the potency assay will represent the product's mechanism of action (i.e., relevant therapeutic activity or intended biological effect).

The FDA gives an example of gene therapy assays “which should incorporate both a measure of gene transfer and the biological effect of the transferred gene”.

I don’t know exactly what the desired effect would be in the case of Ryoncil but I guess it could be the effect on cytokines, the shift from M1 to M2, demonstrated by increased expression of IL-10 or ability to remove fluid (Note Rux’s lack of effect on edema in the photo of the foot in their real-world study) and Dr. Matthay’s presentation on MSCs in ARDS 5 years ago showed very specifically that when VEGF was removed the cells couldn’t achieve this effect, which speaks to mucosal barrier integrity, applicable also to the gut because of the well-known gut/lung axis.

CBP would require a different assay:

Brachyury dysregulation is implicated in disc degeneration causing lower back pain and has a downstream effect on nerve growth factor (which is what Fasinumab and other mabs trialled in CBP target).

For personal reasons, I’ve been interested in learning more about a rare brain cancer called chordoma, which in a late stage looks like a weed growing through the brain. These occur where the spine meets the skull.

Since reading about brachyury implicated in disc degeneration, my layperson’s question is if brachyury dysregulation is telling the notochord to be a spine in the wrong place (brain)?

I asked around a year ago but no one got back to me; however, this is what I came across last week from the “least bad pharma”:

This Anti-Brachyury Antibody, clone 3E4.2 is validated for use in WB(western blotting) for the detection of Brachyury.

In addition, current findings also indicate that the dysregulation of brachyury may also be linked to the formation of chordomas, which are malignant tumors that develop along the spine

Am I right in thinking the anti-brachyury antibody can be supplied?

What about transcription factors? I know a company called Cell Mogrify (Monash connection?) was supplying them at least 5 years ago. Just how advanced is the area of cellular medicine that transcription factors (perhaps also brachyury) can be supplied as a commodity?

Importantly, If brachyury is key, it speaks to regeneration (more than just reducing inflammation) of the disc being a possibility, provided it’s caught early.

Organoids

Organoids have been studied intensively in cancers and the gut, where there’s considerable heterogeneity, as is the case with GvHD.

Clevers at al. (2016) say there’s been a revival in organoids since around 15 years ago in that the term came to refer to 3D structures grown from stem cells which are organ-specific.

Jacob et al. (2020) published their research in Cell, which has one of the highest impact factors. They developed a biobank for patient-derived glioblastoma organoids (GBO). Authors say this “live” bank can be used for optimal testing of personalized therapies by correlating GBO mutational profiles with responses to specific drugs…”

(Note the references to Car T therapy which MSB became involved in through Cartherics. Prior to this, a friend of mine attended a talk on this therapy where an insider said the problem with this therapy is that it was uncontrollable - the cells became a rogue army - and I suspect MSB’s platform was required for targeted delivery. I’m not sure exactly how but it would be good to have some elucidation from the experts on this forum.)

Vlachogiannis et al. (2018) Found patient-derived organoids (PDOs) of gastrointestinal cancers had 88% accuracy for predicting whether patients would respond to some drugs and 100% accuracy for predicting whether they would not.

Yang et al. (2018) say PDOs can also be expanded long-term in vitro and used for high-throughput drug screening.

Zhao et al. (Nature, 2022) say organ cultures have been used as personalized companion diagnostics (which is what the FDA recommended). They speak as if they’re routinely used (a “trend”) and that they don’t even need to be that complex:

“There is an optimism to creating highly complex models and expecting them to accurately mimic the in vivo organ of origin. For the majority of users, simpler models — such as a model with one or two cells in monolayer or 3D culture — are more robust for mechanistic studies and applications than more complex models”.

Kumari et al. (2022) say “PDOs are also genetically-stable, can be cultured long-term and cryopreserved (Sachs et al., 2018; Weeber et al., 2015), and are highly predictive of clinical treatment out-comes, as demonstrated in recent “co-clinical trials” of in vitro PDO and matched patients in the clinic (Ganesh et al., 2019; Ooft et al., 2019; Vlachogiannis et al., 2018; Yao et al., 2020”.

@Bazsa (apologises for the delay in replying) has asked if organoids could be used to validate the potency assay. Bazsa also suggested this could be extended to IBD. I didn't consider that but IMO this is most likely as Ryoncil is most effective in GI inflammation and intestinal organoids have been intensively studied.

I wonder if MSB also has a bank of cryopreserved samples from patients on GvHD001 and could randomly select some of those and “put the patient in a dish” as researchers say is possible? Or could they have grown organoids from those same samples and still have them?

Could a representation of a living subject in organoid form enable using the same potency assay to test the commercial batch to see if the same intended biological effect is achieved and/or the process recapitulated according to the patients’ clinical records?

Perhaps even this is not necessary and it’s possible to grow organoids matched according to biomarkers, even including some adult samples?

Human intestinal organoids have been shown to carry out key functions of the gastrointestinal tract including establishment and maintenance of an appropriate epithelial barrier, mucus production, absorption, and secretion of biomolecules.

Fair et al (2018) say intestinal organoids can be used to model epithelial permeability and changes can be recorded in real time. Alterations to intestinal permeability can be studied under chronic inflammatory conditions and during bacterial invasions.

Corruption or a High Bar?

I believe Ryoncil is a victim (and by extension the children and adults who have not had access to it) of its potency (and likely consistency) and the time MSB is trying to commercialise.

Drugs tested in RCTs are failing real-world patients and there’s a clear move to personalized and precision medicine.

FDA says “Baseline disease severity is a prognostic factor for aGVHD” and sponsors should “provide objective criteria for categorizing aGVHD severity”. Problem is there’s no standardized criteria. Yet. I infer here a call perhaps for a standardized panel of biomarkers, that the FDA may be on the same page as Malard et al. Coincidentally.

I can prove that MSB not doing a randomized controlled study in GvHD being an issue was a lie - unscientific nonsense - from the get-go but not the apparent “issue” about the potency assay:

“FDA regulations allow for considerable flexibility in determining the appropriate measurement(s) of potency for each product”.

The FDA could exercise flexibility and move the goalposts. What they deem “appropriate” or “suitable” is subjective and subject to temporal factors.

If you don't go with corruption, a positive take is that whatever force is in control could have a very long-term view. There are several reasons why allo-BMTs' days could be numbered.

There’s obvious application for Ryoncil in IBD and cytokine storms in children (i.e HLH in juvenile arthritis) and young adults, which is a huge market. Not only that, but this is a cellular medicine platform and if you’ve been following the parallel world of science that can’t be questioned, you may know about the controversy over another class of biological product.

Jessica Rose says the precautionary principle should be exercised. She isn’t saying anything is definitely the case but that it’s important to prove it’s NOT the case and for that you need the “right assay".

I acknowledge it’s important to get the assay right and that it’s SEEN to be right, to send a clear message to insurers and end-users; however, Ryoncil could have been approved for children under certain conditions and I view the delay as heinous and not the fault of MSB.

https://www.fda.gov/files/vaccines,%20blood%20%26%20biologics/published/Final-Guidance-for-Industry--Potency-Tests-for-Cellular-and-Gene-Therapy-Products.pdf

https://www.fda.gov/media/172524/download

https://pubmed.ncbi.nlm.nih.gov/22687190/

https://pubmed.ncbi.nlm.nih.gov/23260082/

https://www.sciencedirect.com/science/article/pii/S0092867416307292

https://www.cell.com/cell/pdf/S0092-8674(19)31321-2.pdf

https://pubmed.ncbi.nlm.nih.gov/29472484/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225812/

https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.431

https://www.nature.com/articles/s43586-022-00174-y

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974440/