RE:RE:RE:Answer about the linker I remember having this conversation on TH's linker vs zoptarelin here a while back, and my idea about the 2 added methyl groups, classically bringing chemical stability, was the same. I like even more your analysis about the esterases' pockets.
On another angle, my idea is there's more to the zoptarelin failure than just the linker. We have to consider the LHRH receptor is only accidentally mediating endocytosis, as a function of managing/recycling receptors on the cell surface. Whereas SORT1 is by design a scavenging receptor. Internalization is going to happen a lot more on the latter than the former.
See e.g. https://d1wqtxts1xzle7.cloudfront.net/48203874/0303-7207_2883_2990065-520160820-21764-1wsmtn2.pdf
On page 6, fraction of diseapearing ligand-bound LHRH receptors is ~20%, with ~70% of those (see page 9) being attributed to internalization.
Also later on p.10: "In the pituitary, it has recently been shown that neither formation of large-scale clusters nor internalization of LHRH receptors is required for LH release [..]. Thus internalization of LHRH receptors may simply be related to degradation of the hormone and/or receptors"
Therefore lack of specificity of zoptarelin-doxorubicin could result from the conjugate largely not being internalized in time and ending up being cleaved early by the TME's more acidic nature (this point I have read somewhere but didn't kept the reference, sorry) and having thus broadly the same properties as doxorubicin alone.
jfm1330 wrote: Just to make things clearer about the linkers. This is the one used in zoptarelin (glutaryl)
https://spectrabase.com/compound/2hThxt1mJSH
And this is the one used in TH1902 (dimethyl glutaryl)
https://spectrabase.com/compound/6JqpwoZbYZK
These are the diacid forms (not linked). So the two added methyl groups in the middle of the molecule (the V shape up), makes it harder for the catalytic pocket of esterases enzymes to access the carbobyl groups (C=O), hence, the slower catalytic rate. I think these images make it easier to understand for non biochemists.
jfm1330 wrote: I spent a few hours today doing internet research to try to understand what is the linker used in TH1902 and TH1904. What I knew is the fact that it was supposed to be pH sensitive and since the intracellular pH of cancer cells is more acidic than the pH of the serum in the bloodstream, the linker is almost not cleaved in the bloodstream, but totally cleaved once inside the cancer cells.
That being said, as much as I said here that Lutathera was an example of an approved PDC that was giving me confidence, there are significant differences between Lutathera and TH1902. In Lutathera, the cytotoxic agent is a Lu177, a radioistope that does not need to be free inside the cancer cell to be active, so no need for a linker that is selectively cleavable inside the cancer cell. That lead me to look closer to another PDC that is very similar to TH1902 and especially TH1904. This PDC is AEZS 108, also called Zoptarelin. This PDC, a LHRH analog carrying one molecule of doxorubicin, went into phase III and the results released in 2017 showed Zoptarelin provided no improvment over treatment with doxorubicin alone.
I read in a scientific article that the reason for the failure in phase III of Zoptarelin may be due to the fact that its linker (glutaryl linker) was not stable enough outside of the cancer cells. Read this:
Unfortunately, AEZS-108 could not achieve its primary endpoint in clinical phase III studies on endometrial cancer, which was caused by the lack of a significant difference in the median period of overall survival of patients treated with Zoptrex™ as compared to patients treated with doxorubicin [26]. The main reason for this might be the poor enzymatic stability of the conjugate in circulation. It has been shown that the ester bond can be hydrolyzed rapidly by carboxylesterases in presence of mouse (t1/2 = 19 min) and human serum (t1/2 = 126 min) [27]. Taking this into account, drug-linkers with higher enzymatic stability under physiological conditions might help to overcome this weakness and ensure antitumor activity without toxic side effects.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320914/
So, reading that, worried me a bit. That's whay I wanted to understand what TH1902 linker is, the mechanism by which it is cleaved and if it allowed for a cleavage that will really happen inside the cancer cells in real humans with real tumors. Remember, Zoptarelin linker passed all the tests in the pre-clinical phase. Then, even in human trials, in phase I and phase II, no obvious problem was detected. The reason for that is that it is impossible to directly monitor the fate of the PDC in the human body, so it's impossible to really monitor the behavior of the linker in the bloodstream, just outside of the cancer cells in the tumor environement, and inside the cancer cells.
To vizualize Zoptarelin and its glutaryl receptor, look at this link:
https://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5942387_Beilstein_J_Org_Chem-14-930-g007.jpg
Now, if you want to see the linker of TH1902 look at page 62 of the Investor Presentation at this link:
https://www.theratech.com/wp-content/uploads/2019/10/Investor_Day_Nasdaq_Oct_23-1.pdf
You will see that the linkers are different, but also similar. In the case of TH1902, insted of a simple glutaryl linker, it uses a dimethyl glutaryl linker. What needs to be understood here, is that both linkers are not cleaved by the higher acidity inside the cancer cells, as I thought previously, but by esterases enzymes that have a higher activity under mild acidity, like it's the case inside a cancer cell (pH 6.0-6.8), instead of pH 7.4 in the bloodstream. So in the bloodstream, the cleavage by esterases is very slow, but much faster inside the cancer cells. That being said, from my understanding, with my backgound in biochemistry, (I found no reference for that explaination), the dimethyl glutaryl linker used in TH1902 would be more stable because the two added methyl groups on the central carbon atom make it sterically hindered. That means that these added groups, from a 3-D standpoint, partially block the ideal access of the enzyme to the ester bond between docetaxel or doxorubycin and the linker, that the enzyme would cleave. Since the 3-D access is not ideal, the rate of cleavage is slower, hence the ester bond more stable.
This is my understanding not having access to a reference with an explaination. If I am right it means that the worry about the linker not being stable enough outside of cancer cells is not warranted. But being more stable also means a slower cleavage rate inside the cancer cells. About that we know that in vitro and in vivo, in xenograft animal model, docetaxel is active, so cleaved inside the cancer cells. Now, we have to hope it will be the same inside human cancer cells in real tumors in real humans. That being said, it would be a good idea for the company to explain clearly how their linker work and why they are confident that it will work as needed in real cancer patients. A linker that will work as needed is critical to a successful jump from mice to humans.