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Summary: 

The study by Crook et al. falls in the promising area of targeted protein degradation (TPD) in modern drug discovery. The authors make use of the endolysosomal pathway for degradation of proteins of interest (POI), here the epidermal growth factor receptor (EGFR) and the programmed death-ligand 1 (PD-L1). The authors address the transferrin receptor (TfR), a recycling receptor, via engineered nanobodies mainly based on Fc domains, as core structures decorated with cystine-dense peptide (CDP) miniproteins as specific targeting units. The highlight of this study is the connection of a pH-sensitive POI-binding moiety with a pH-insensitive TfR-binding moiety. This is where the platform gets its name from: CYpHER (CatalYtic pH-dependent Endolysosomal delivery with Recycling). The platform makes use of the occurring acidification in the late endosomes leading to a release of the POI and subsequent endosomal degradation while the CYpHER remains bound to the TfR which recycles back to the cell surface. This fact makes the systems (to a certain extent) working catalytically opposed to stoichiometric limitation of previous examples in the field. As evidence of the success of the concept, in vitro EGFR and PD-L1 were degraded, as well as EGFR in EGFR-driven non-small cell lung cancer in vivo. 

The authors chose the TfR, as it naturally works by the same principle, i.e., binding transferrin which releases its cargo, iron, upon acidification while remaining bound to TfR returning to the surface together with its receptor. On the other hand, CDP miniproteins or EGF variants (in case of EGFR) are used to flexibly design specific targeting units. The central core structure is formed by Fc domains which have the intrinsic property to extend the in vivo half-life in serum. This platform is advertised as adjustable to any POI or recycling receptor making it an attractive way to reach versatility in the production of such extracellular degradation technologies. 

General comments: 

As a general remark, if the authors wish to address their preprint to an interdisciplinary readership the manuscript would benefit from some more information on the rationale and selection of the experiments and conditions, as well as the conclusion which can be made based on the data. The subdivision of the Results section is appropriate but when comparing the sections writing styles differ which lets the manuscript appear a bit fragmentary.  

Information about the statistics applied on each experiment will surely be highly appreciated by the readership to assess the reliability of the data. 

For contextualisation, the preprint reviewing was done by two scientists with backgrounds in chemistry, biophysics and cell biology, and particular interest in plasma membrane/endocytosis, drug targeting and protein degradation. 

Section- and figure-specific comments:  

Introduction: 

• Instead of the general paragraph about the receptor tyrosine kinases (RTKs), even though the EGFR is one of them, we would suggest focussing on information relevant to both the proteins degraded in the present study, namely EGFR and PD-L1. 

• Biological motivation for PD-L1 is missing. 

• The third paragraph about intracellular TPD, e.g., molecular glue, PROTACs and LYTACs, is written in an inconvenient way which makes this section not easy to follow without deep knowledge of the TPD concepts. This paragraph has more potential to serve as a basis for emphasising the novelty of the developed CYpHER platform. 

• The paragraphs about the CYpHER concept and TfR as relevant uptake receptor could benefit from a more concise formulation. 

Results: 

The CYpHER concept 

• The description of the CYpHER concept is easy to follow. 

• Please highlight the rationale why initially “PD-L1-binding CDP”–“(flexible Gly-Ser linker)”–“TfR-binding CDP” was used, and which observations/results led to the motivation to change the platform to the incorporation of Fc domains and linkers. 

• Figure 1B: From the design point of view, not the whole process is pictured logically: 

• e.g., TfR is on the right, next to it CYpHER and far left the Target à maybe place TfR and CYpHER on the very left side and the Target between “(Recycled CYpHER + TfR) and “Ternary Complex; and adding the corresponding arrows. 

• Figure 1E: The caption for E is unclear, specifically the rationale behind the use of sample enrichment instead of single clones 

Engineering CYpHER candidates 

• Page 4: Figure 2A and first paragraph: Why is the design for CT-4212-1 and CT-4212-3 like it was done? Which properties would change when comparing both nanobodies? 

• Page 4: The paragraph about the EGFR and its (cancer) biological function interferes with the reading flow in this subchapter as it does not serve the design but the motivation (maybe in better hand in the Introduction)  

• Page 4: “caused by better cooperative CYpHER accumulation when more target is present, by saturation of the recycling pathway, or both.” 

• Have you doublechecked this hypothesis and what kind of consequences would this observation have for your system? 

• Page 5: “In one method, a pool of variants with His substitutions was screened in mammalian surface display (45, 46) through four “rounds of enrichment””. Please explain the rationale behind the number of rounds of enrichment.  

Adapting a native ligand for CYpHER 

• Page 5: It would be good to have more information about why engineered EGF would be better than engineered (“standard”) CDRs in your case or in general as it seems to be a rather elaborated procedure to engineered EGF (like EGFd1.5.36)? 

EGFR CYpHER induces EGFR surface clearance and elimination. 

• N/A 

CYpHERs with any of the three engineered EGFR binders clear surface EGFR 

• N/A 

CYpHER-driven EGFR intracellular sequestration 

• Page 8/9: What is the motivation to test the different variants? – What conclusions could you make from the experiments? 

CYpHER catalytic target uptake 

• Page 10: Why soluble targets were used for the catalytic uptake assay only? What was the motivation?  

• Figure 6 (page10): Errors in title of (C) EGFRvIII instead of EGFR, same for (D). In (D) CYpHER instead of CYPHER.   

• We strongly recommend presenting a proven measure of the expression levels of TfR and PD-L1 in all cell lines in order to understand the variability in performance of the cell lines. 

Pharmacodynamic effects of CYpHER in vitro 

• Page 12: “The potency against cancer vs the potency against keratinocytes was higher for CT-1212-1 than for any of the three clinical drugs in the five cell lines, …” – any idea why? 

• Figure 7. If WB of all samples were run in different membranes, please state this fact. If not, the grid generated around each individual band appears confusing to us.  

In vivo CYpHER pharmacokinetics and pharmacodynamics 

• Page 12: please rationalise the doses chosen for the CYpHER. 

• Page 12: “The nanobody does not cross-react with murine EGFR, so only the murine cross-reactive TfR-binding CDP would be expected to influence PK apart from the Fc domain.” - Please, provide some more information about the importance of this conclusion for your study. 

Discussion:  

• The discussion section is mainly written as an outlook regarding what kind of potential impact the presented work could have in the future. However, the discussion of the results from the many experiments, which have been conducted, come up short. Readers would certainly appreciate, if there would be more explanations about the rationale of each step in the series of experiments. Also, it would be advantageous for the reader if the interpretation of the results of the individual experiments would be brought into context with each other. This may include also the evaluation of which engineered nanobodies work best under which circumstances, etc.  

• A Discussion about the degradation efficiency in comparison with other eTPG platforms (as far as possible) would add value regarding comparability as EGFR is used in almost all literature studies as kind of a benchmark. 

Competing interests

The authors declare that they have no competing interests.