Similar results were observed for EGFR degradation, with no major proteome-wide changes occurring and EGFR being virtually the only proteins significantly down regulated in CXCL12- Ctx treatment compared to control in both the surface-enriched and whole cell proteomics . Interestingly, a previously published proteomics dataset of LYTAC-mediated EGFR degradation identified additional proteins significantly up- or down-regulated following LYTAC treatment.Comparing to our experiment in the same cell line suggests that KineTACs are more selective in degrading EGFR. As there is large overlap in peptide IDs observed between the two datasets, the observed greater selectivity is not due to lack of sensitivity of the KineTAC proteomics experiment . CXCR4 and CXCR7 peptide IDs were not altered in the surface-enriched sample, and CXCR4 IDs were also unchanged in the whole cell sample, indicating that treatment with KineTAC does not significantly impact CXCR4 or CXCR7 levels. Furthermore, protein levels of GRB2 and SHC1, which are known interacting partners of EGFR4 , were also not significantly changed. Together, these data demonstrate the exquisite selectivity of KineTACs for degrading only the target protein and not inducing unwanted, off-target proteome wide changes. To elucidate whether KineTAC-mediated degradation could impart functional cellular consequences, cell viability of HER2 expressing cells was measured following treatment with CXCL12-Tras. MDA-MB-175VII breast cancer cells are reported to be sensitive to trastuzumab treatment, and as such serve as an ideal model to test the functional consequence of degrading HER2 compared to inhibition with trastuzumab IgG.To this end, cells were treated with either CXCL12-Tras or trastuzumab IgG for 5 days,vertical tower planter after which the cell viability was determined using a modified MTT assay. Reduction in cell viability was observed at higher concentrations of CXCL12-Tras and was significantly greater than trastuzumab IgG alone .
These data demonstrate that KineTAC-mediated degradation has functional consequences in reducing cancer cell viability in vitro and highlights that KineTACs could provide advantages over traditional antibody therapeutics which bind but do not degrade. Finally, we asked whether KineTACs would have similar antibody clearance to IgGs in vivo. To this end, male nude mice were injected intravenously with 5, 10, or 15 mg/kg CXCL12- Tras, which is a typical dose range for antibody xenograft studies. Western blotting analysis of plasma antibody levels revealed that the KineTAC remained in plasma up to 10 days post-injection with a half-life of 8.7 days, which is comparable to the reported half-life of IgGs in mice .Given the high homology between human and mouse CXCL12, we tested whether human CXCL12 isotype could be cross-reactive. Human CXCL12 isotype binding to mouse cell lines MC38 and CT26, which endogenously express mouse CXCR7, was confirmed . Together, these results demonstrate that KineTACs have favorable stability and are not being rapidly cleared despite cross-reactivity with mouse CXCR7 receptors. Since atezolizumab is also known to be cross-reactive, CXCL12-Atz ability to degrade mouse PD-L1 was tested in both MC38 and CT26. Indeed, CXCL12-Atz mediates near complete degradation of mouse PD-L1 in both cell lines . Thus, PD-L1 degradation may serve as an ideal mouse model to assay the efficacy of KineTACs in vivo. Having demonstrated the ability of KineTACs to mediate cell surface protein degradation, we next asked whether KineTACs could also be applied towards the degradation of soluble extracellular proteins. Soluble ligands, such as inflammatory cytokines and growth factors, have been recognized as an increasingly important therapeutic class.
Of these, vascular endothelial growth factor and tumor necrosis factor alpha represent the most targeted soluble ligands by antibody and small molecule drug candidates, highlighting their importance in disease.Thus, we chose VEGF and TNFa as ideal proof-of-concept targets to determine whether KineTACs could be expanded to degrading extracellular soluble ligands . First, we targeted VEGF by incorporating bevacizumab , an FDA approved VEGF inhibitor, into the KineTAC scaffold . Next, HeLa cells were incubated with VEGF-647 or VEGF-647 and CXCL12-Beva for 24 hr. Following treatment, flow cytometry analysis showed a robust increase in cellular fluorescence when VEGF-647 was co-incubated with CXCL12-Beva, but not bevacizumab isotype which lacks the CXCL12 arm . To ensure that the increased cellular fluorescence was due to intracellular uptake of VEGF-647 and not surface binding, we determined the effect of an acid wash which removes any cell surface binding after 24 hr incubation . We found that there was no significant difference in cellular fluorescence levels between acid and normal washed cells. This data suggests that KineTACs successfully mediate the intracellular uptake of extracellular VEGF. Similar to membrane protein degradation, KineTAC-mediated uptake of VEGF occurs in a time-dependent manner, with robust internalization occurring before 6 hrs and reaching steady state by 24 hrs . Furthermore, the levels of VEGF uptake are dependent on the KineTAC:ligand ratio and saturate at ratios greater than 1:1 . We next tested the ability of CXCL12-Beva to promote uptake on other cell lines and find that these cells also significantly uptake VEGF . Moreover, the extent of uptake is correlated with the transcript levels of CXCR7 in these cells . These data suggest that KineTACs directed against soluble ligands can promote broad tissue clearance of these targets as compared to glycan- or Fc-mediated clearance mechanisms. To demonstrate the generalizable nature of the KineTAC platform for targeting soluble ligands, we next targeted TNFa by incorporating adalimumab , an FDA approved TNFa inhibitor, into the KineTAC scaffold . Following 24 hr treatment of HeLa cells, significant increase in cellular fluorescence was observed when TNFa-647 was coincubated with CXCL12-Ada compared to adalimumab isotype .
Consistent with the VEGF uptake experiments, acid wash did not alter the level of cellular fluorescence increase observed, and uptake was dependent on the KineTAC:ligand ratio . Thus, KineTACs are generalizable in mediating the intracellular uptake of soluble ligands, significantly expanding the target scope of KineTAC-mediated targeted degradation.In summary, our data suggest that KineTACs are a versatile and modular targeted degradation platform that enable robust lysosomal degradation of both cell surface and extracellular proteins. We find that KineTAC-mediated degradation is driven by recruitment of both CXCR7 and target protein, and that factors such as binding affinity, epitope, and construct design can affect efficiency. Other factors, such as signaling competence and pH dependency for the protein of interest, did not impact degradation for CXCL12 bearing KineTACs. These results provide valuable insights into how to engineer effective KineTACs going forward. Furthermore, we show that KineTACs operate via time-, lysosome-, and CXCR7-dependence and are exquisitely selective in degrading target proteins with minimal off-target effects. Initial experiments with an alternative cytokine, CXCL11, highlight the versatility of the KineTAC platform and the exciting possibility of using various cytokines and cytokine receptors for targeted lysosomal degradation. KineTACs are built from simple genetically encoded parts that are readily accessible from the genome and published human antibody sequences. Given differences in selectivity and target scope that we and others have observed between degradation pathways,lettuce vertical farming there is an ongoing need to co-opt novel receptors for lysosomal degradation, such as CXCR7, that may offer advantages in terms of tissue selectivity or degradation efficiency. Thus, we anticipate ongoing work on the KineTAC platform to offer new insights into which receptors can be hijacked and to greatly expand targeted protein degradation to the extracellular proteome for both therapeutic and research applications.SILAC proteomics data were analyzed using PEAKSOnline . For all samples, searches were performed with a precursor mass error tolerance of 20 ppm and a fragment mass error tolerance of 0.03 Da. For whole cell proteome data, the reviewed SwissProt database for the human proteome was used. For surface enriched samples, a database composed of SwissProt proteins annotated “membrane” but not “nuclear” or “mitochondrial” was used to ensure accurate unique peptide identification for surface proteins, as previously described.Carbamidomethylation of cystine was used as a fixed modification, whereas the isotopic labels for arginine and lysine, acetylation of the N-terminus, oxidation of methionine, and deamidation of asparagine and glutamine were set as variable modifications. Only PSMs and protein groups with an FDR of less than 1% were considered for downstream analysis. SILAC analysis was performed using the forward and reverse samples, and at least 2 labels for the ID and features were required. Proteins showing a >2-fold change from PBS control with a significance of P<0.01 were considered to be significantly changed.Cell viability assays were performed using an MTT modified assay. In brief, on day 0 15,000 MDA-MB-175VII cells were plated in each well of a 96-well plate. On day 1, bispecifics or control antibodies were added in a dilution series. Cells were incubated at 37ºC under 5% CO2 for 5 days. On day 6, 40 µL of 2.5 mg/mL thiazolyl blue tetrazolium bromide was added to each well and incubated at 37ºC under 5% CO2 for 4 hrs. 100 µL of 10% SDS in 0.01M HCl was then added to lyse cells and release MTT product.
After 4 hrs at room temperature, absorbance at 600 nm was quantified using an Infinite M200 PRO plate reader . Data was plotted using GraphPad Prism software and curves were generated using non-linear regression with sigmoidal 4PL parameters. Male nude nu/nu mice were treated with 5, 10, or 15 mg/kg CXCL12-Tras via intravenous injection . Blood was collected from the lateral saphenous vein using EDTA capillary tubes at day 0 prior to intravenous injection and at days 3, 5, 7, and 10 post injection. Plasma was separated after centrifugation at 700xg at 4ºC for 15 min. To determine the levels of CXCL12-Tras, 1 µL of plasma was diluted into 30 µL of NuPAGE LDS sample buffer and loaded onto a 4-12% Bis-Tris gel and ran at 200V for 37 min. The gel was incubated in 20% ethanol for 10 min and transferred onto a polyvinylidene difluoride membrane. The membrane was washed with water followed by incubation for 5 min with REVERT 700 Total Protein Stain . The blot was then washed twice with REVERT 700 Wash Solution and imaged using an OdysseyCLxImager . The membrane was then blocked in PBS with 0.1% Tween-20 + 5% bovine serum albumin for 30 min at room temperature with gentle shaking. Membranes were incubated overnight with 800 CW goat anti-human IgG at 4ºC with gentle shaking in PBS + 0.2% Tween- 20 + 5% BSA. Membranes were washed four times with tris-buffered saline + 0.1% Tween-20 and then washed with PBS. Membranes were imaged using an OdysseyCLxImager . Band intensities were quantified using Image Studio Software .The concept of targeted degradation has emerged in the last two decades as an attractive alternative to conventional inhibition. Small molecule inhibitors primarily work through occupancy-driven pharmacology, resulting in temporary inhibition in which the therapeutic effect is largely dependent on high potency. On the other hand, PROteolysis TArgeting Chimeras utilize event-driven pharmacology to degrade proteins in a catalytic manner.Traditionally, PROTACs are heterobifunctional small molecules composed of a ligand binding a protein of interest chemically linked to a ligand binding an E3 ligase. The recruitment of an E3 ligase enables the transfer of ubiquitin onto the protein of interest, which is subsequently polyubiquitinated and recognized by the proteasome for degradation . In many cases, PROTACs have proven efficacious over the small molecule inhibitors alone, and several candidate PROTACs have progressed to clinical trials for treating human cancers and other diseases. Despite these successes, small molecule PROTACs are largely limited to targeting intracellular proteins. Given this challenge, there is a need for novel technologies that expand the scope of targeted degradation to membrane proteins. Recently, our lab has developed a method termed antibody-based PROTACs which utilize bispecific antibody scaffolds to bring membrane-bound E3 ligases in proximity to a membrane protein of interest for targeted degradation.Thus far, AbTACs have shown success in using bispecific IgGs to recruit E3 ligase RNF4 to programmed death ligand 1 for efficient lysosomal degradation. This data suggests that it is possible to use bispecific antibodies to degrade membrane proteins for which antibodies already exist or that have characteristics amenable to recombinant antibody selection strategies.However, the ability to degrade multipass membrane proteins, such as GPCRs, remains challenging due to few extracellular-binding antibodies existing for this target class. Here, we describe a novel approach to expand the scope of AbTACs to targeting multi-pass membrane proteins. This approach, termed antibody-drug conjugate PROTACs , comprises of an antibody targeting a cell surface E3 ligase chemically conjugated to a small molecule that specifically binds the protein of interest .