Initial thoughts on CD44v6 CAR-T (MolMed)

Originally written April 15th, 2015


CD44v6 CAR-T

MolMed today exercised an option on a CD44v6 CAR-T:
http://www.molmed.com/sites/default/files/uploads/press-releases/2638/2638_1428894007.pdf

I’ll primarily be referring to this paper describing the CAR construct:
http://www.bloodjournal.org/content/122/20/3461.long?sso-checked=true

This CAR targets a non-mutated tumor antigen, CD44v6. Non-mutated self antigens have previously been a dangerous class of antigens to target (such as Her2). Mesothelin is another one, and we will see some of the data from this target at the upcoming AACR, which I would expect will discuss trying to mitigate toxicity. As a guess, perhaps the Mesothelin mRNA CAR-Ts last a short enough time that you don’t get intolerable toxicity.

On to CD44v6 – this is an antigen expressed rarely in normal cells, mainly keratinocytes and monocytes, but seems to be expressed in a range of tumor types, such as AML, MM, pancreatic, and head & neck cancer. CD44v6 was found to be expressed in a high percentage of AML & MM samples. Importantly, they corroborated previous work suggesting expression is important for tumor growth, as knockdown of CD44v6 reduced tumor seeding and progression. This suggests that it might be more difficult for the tumor to lose CD44v6 expression as a way of becoming resistant to the CAR.

They made the CAR construct based off of a previously used antibody in clinical trials that is specific for CD44v6, called bivatuzumab. Injection of the CAR-T cells successfully suppressed growth of human AML & MM xenografts, although it looks like the first time point for evaluation was 5 weeks after T-cell injection. However, this doesn’t model potential on-target off-tumor effects of the CAR, which is the big concern for this type, and really any type, of CAR.

They looked at expression of CD44v6 in normal tissue and found it expressed, as expected, primarily in keratinocytes & monocytes, both at seemingly similar levels (maybe keratinocytes slightly less), and both considerably less than expression in AML samples.

First they tested skin (keratinocyte) toxicity, and surprisingly found the CAR-T cells did not react against keratinocytes. I was surprised the CAR didn’t have skin toxicity, although this was not tested in vivo, only in co-culture of CAR-T cells with keratinocytes. The antibody used to make the CD44v6 recognition portion (scFv) of the CAR construct comes from bivatuzumab. Previously, this antibody had been conjugated to radionuclides to deliver radiation to patients' head & neck tumors. Some responses were seen, toxicity was myelosuppression (presumed to be due to the circulating radionuclide) & grade 2 oral mucositis (potentially on-target toxicity). A second trial in head & neck cancer tested bivatuzumab conjugated to mertanzine (an anti-mitotic drug). Preclinical work suggested reversible skin toxicities, presumed to be due to expression of CD44v6 in skin, and reversible ones were seen in the first patients enrolled. There was some evidence of efficacy, some stable disease and minor tumor responses. However, at the highest dose, the first patient developed a fatal skin toxicity – toxic epidermal necrolysis, even though this patient had only a slightly higher dose than the previous patients, and a lower cumulative dose. There wasn’t evidence of deconjugation of the toxin from the antibody, as the free toxin has other toxicities that weren’t observed.

The authors of the CAR-T study propose that for somewhat unknown reasons the CAR-T may have a wider therapeutic index than the antibody conjugates. This would be somewhat surprising to me, as it generally seems that CAR-T cells can recognize very few molecules of the target on cells, and that they have one of the narrowest therapeutic indices of antibody targeted therapeutics. Perhaps playing with the affinity of the scFv could allow for broader therapeutic index, but this is the same antibody used here as in the previous clinical studies. Maybe there is something structural about the scFv on the surface of T-cell versus floating & conjugated to toxin, or perhaps keratinocytes can suppress T-cells, but it is surprising there seems to be little to no reactivity of the CAR-T cells against keratinocytes.

The CAR-T cells did react against monocytes, and when injected in mice reconstituted with a human hematopoietic system, the CD14+ monocytes appeared to be depleted relatively rapidly, in ~10 days. While the accompanying preview by Richard Morgan suggested that short term monocytopenia is tolerable, long term it is not tolerable: http://www.bloodjournal.org/content/122/20/3392.long?sso-checked=true

The authors dealt with this toxicity by introducing suicide gene constructs to be able to deplete the T-cells after a period of time, to allow reversibility of the monocytopenia. This seemed to work well, however I’m not sure at what duration monocytopenia become intolerable, and if the CAR-T cells will have significant enough activity by that point. From what I can tell, based on the xenograft, at 5 weeks there was some clear activity, but it only took 10 days for the CD14+ cells to be depleted, so it might be difficult to separate activity from toxicity. Although for the CD19 CAR-T cells in humans, significant anti-tumor activity appears to occur very rapidly, so CD44v6 CAR anti-tumor activity could occur more rapidly in humans.

Some of the most impressive data was with the suicide gene, iC9 (I believe this is the same as Bellicum’s suicide gene construct) – which was much better than expression of the other suicide construct thymidine kinase. iC9 killed both activated & resting T-cells and did so in only a few hours.

So with all these concerns, I would take a wait & see attitude about the application of this CAR-T in the clinic. It is differentiated, which is nice, considering the paucity of targets, however, I am concerned about the therapeutic index, specifically if skin toxicity shows up, and even the monocyte toxicity might be too difficult to separate from the anti-tumor activity. If everything works like in the paper, it could be a useful CAR in the clinic, however, for me, the above are some reasons to be cautious.

Let me know any feedback or questions/comments you have, happy to continue discussion.

Further reading:
Phase I Bivatuzumab Mertanzine trial: http://www.ncbi.nlm.nih.gov/pubmed/17062682
CD44(v6) as therapeutic target: http://www.ncbi.nlm.nih.gov/pubmed/20303742