The majority of clinical degrader candidates utilize an immunomodulatory imide drug (IMiD)-based derivative, that directs their target substrate to the E3 ligase receptor Cereblon (CRBN), however, concomitant neo-substrate degradation by IMiDs often results in severe off-target effects. Further biophysical screening is needed to discover CRBN binders that might overcome these safety concerns, but the previously reported CRBN protein constructs suffer significant limitations that reduce their applicability as tools for biophysical assays making large-scale screening efforts a costly endeavor. This is due to the requirement of CRBN co-expression with DDB1 in insect cells to produce soluble protein that contains all the reported structural features necessary for proper compound interaction. Here, a near full-length human CRBN construct was designed that retains these required features, but for the first time allows the generation of highly homogenous and cost-efficient expression in E.coli , eliminating the need for DDB1 co-expression. We have extensively profiled the new construct against the existing CRBN constructs in binary and ternary degrader complexes, showing it to be the “best of both worlds” in terms of binding characteristics and ease of protein production. We then designed the Enamine CRBN focused IMiD library of 4480-compounds and demonstrated applicability of the new construct to high throughput screening by identifying novel CRBN binders with high potency and ligand efficiency. The new methods described here should serve as valuable tools for the development of the next generation IMiD-based degraders.### Competing Interest StatementThe authors have declared no competing interest.

The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative that directs their target to the E3 ligase receptor cereblon (CRBN); however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs, which retain all features for ternary complex formation, while allowing generation of homogenous and cost-efficient expression in E. coli. Extensive profiling of the construct shows it to be the “best of both worlds” in terms of binding activity and ease of production. We next designed the “Enamine focused IMiD library” and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency, and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues.

Herein, we describe a systematic SAR- and SPR-investigation of the peptidomimetic hydroxy-proline based VHL-ligand VH032, from which most to-date published VHL-targeting PROTACs have been derived. This study provides for the first time a consistent data set which allows for direct comparison of structural variations including those which were so far hidden in patent literature. The gained knowledge about improved VHL binders was used to design a small library of highly potent BRD4-degraders comprising different VHL exit vectors. Newly designed degraders showed favorable molecular properties and significantly improved degradation potency compared to MZ1.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen–antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera—as in cases of non-ARDS sera—combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen–antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

Many of the approved SARS-CoV-2 vaccines are based on a stabilized variant of the spike protein. This raises the question of whether the immune response against the stabilized spike is identical to the immune response that is elicited by the native spike in the case of a SARS-CoV-2 infection. Using a peptide array-based approach, we analysed the binding of antibodies from Comirnaty-elicited, convalescent, and control sera to the peptides covering the spike protein. A total of 37 linear epitopes were identified. A total of 26 of these epitopes were almost exclusively recognized by the convalescent sera. Mapping these epitopes to the spike structures revealed that most of these 26 epitopes are masked in the pre-fusion structure. In particular, in the conserved central helix, three epitopes that are only exposed in the post-fusion conformation were identified. This indicates a higher spike-specific antibody diversity in convalescent sera. These differences could be relevant for the breadth of spike-specific immune response.

SummaryBackgroundThe mRNA vaccine BNT162b2 (Comirnaty, BioNTech/Pfizer) and the vaccine candidate CVnCoV (Curevac) each encode a stabilized spike protein of SARS‐CoV2 as antigen but differ with respect to the nature of the mRNA (modified versus unmodified nucleotides) and the mRNA amount (30 μg versus 12 μg RNA). This study characterizes antisera elicited by these two vaccines in comparison to convalescent sera.MethodsSera from BNT162b2 vaccinated healthcare workers, and sera from participants of a phase I trial vaccinated with 2, 4, 6, 8, or 12 μg CVnCoV and convalescent sera from hospitalized patients were analyzed by ELISA, neutralization tests, surface plasmon resonance (SPR), and peptide arrays.ResultsBNT162b2‐elicited sera and convalescent sera have a higher titer of spike‐RBD‐specific antibodies and neutralizing antibodies as compared to the CVnCoV‐elicited sera. For all analyzed sera a reduction in binding and neutralizing antibodies was found for the lineage B.1.351 variant of concern. SPR analyses revealed that the CVnCoV‐elicited sera have a lower fraction of slow‐dissociating antibodies. Accordingly, the CVnCoV sera almost fail to compete with the spike‐ACE2 interaction. The significance of common VOC mutations K417N, E484K, or N501Y focused on linear epitopes was analyzed using a peptide array approach. The peptide arrays showed a strong difference between convalescent sera and vaccine‐elicited sera. Specifically, the linear epitope at position N501 was affected by the mutation and elucidates the escape of viral variants to antibodies against this linear epitope.ConclusionThese data reveal differences in titer, neutralizing capacity, and affinity of the antibodies between BNT162b2‐ and CVnCoV‐elicited sera, which could contribute to the apparent differences in vaccine efficacy.