We demonstrate proof-of-concept that conjugation of an adenosine 2a receptor agonist to an RNF targeting antibody leads to efficient degradation of A2AR in vitro. Furthermore, we find that A2AR degradation is dependent on site of conjugation on the antibody scaffold and linker length between the small molecule and antibody. Overall, this strategy has the potential to convert any GPCR-directed small molecule into an effective degradation-based antagonist. Importantly, ADC-TACs can be generally applied to the targeted degradation of other multi-pass membrane protein classes. To determine whether it is possible to degrade GPCRs using ADC-TACs, A2AR was first targeted as a proof-of-concept. A2AR is expressed on the surface of natural killer and CD8+ T cells and is primarily involved in responding to levels of adenosine in the extracellular environment. Binding of adenosine agonizes A2AR to activate a downstream signaling cascade, resulting in an increase in intracellular cyclic AMP levels and overall immunosuppression. This is especially evident in the tumor micro-environment,vertical grow shelf where high levels of extracellular ATP decompose to generate excess adenosine.Along with the immunosuppressive role, A2AR agonism is thought to contribute to cell proliferation via the MAPK/ERK/JNK signaling pathways.
Given this role in cell proliferation, there has been an effort to develop antagonists to counteract agonism of the receptor in both the immune and cancer cell context. As such, A2AR is an appealing first target for applying our ADC-TAC approach towards the targeted degradation of GPCRs. To this end, A2AR agonist CGS21680 was chosen for conjugation as it had previously been conjugated onto an Fc domain, and the crystal structure indicated that our proposed conjugation site off the solvent exposed carboxylic acid was unlikely to interfere with small molecule binding .Three analogs of CGS21680 with DBCO- n-NH2 linkers of varying lengths were synthesized . We hypothesized that conjugation of CGS21680 onto the variable domain of an antibody with the indicated linker lengths would be capable of spanning the interface between A2AR and RNF43. Final compounds were confirmed by LC/MS and purified by HPLC before conjugation onto previously described anti-RNF43 Fabs. To enable site-specific labeling, methionine conjugation chemistry involving oxaziridine labeling of methionines was chosen due to its high selectivity. Sites were chosen that have been previously demonstrated to have both high labeling percentage and stability to hydrolysis of the sulfimide for methionine mutations.To prevent off-target labeling, a methionine that was present in the H1 complementarity determining region of the anti-RNF Fab was mutated to leucine. Two additional methionines that are buried in the Fab scaffold were also removed to limit unwanted labeling.
As seen by multi-point biolayer interferometry , removing the endogenous methionine had little effect on binding to RNF Fc fusion . Five sites of methionine mutation were then each incorporated into the scaffold . Sites were chosen in the variable domain under the assumption that conjugation closer to the CDRs would require shorter linker lengths to the small molecule to allow simultaneous binding of E3 ligase and target protein. These mutants were expressed as Fabs in the periplasm of the C43 E. coli strain. Multi-point BLI confirmed that introduction of methionine at the five sites had little to no effect on binding to RNF43 Fc fusion . Next, we sought to determine whether the anti-RNF43 Fab mutants could be selectively labeled with the azide derivative of the piperidine-derived oxaziridine 8 and the CGS21680-DBCO conjugates . All mutants showed robust and rapid labeling with the oxaziridine reagent after 30 min incubation, with little unlabeled antibody remaining in solution . Furthermore, all mutants showed robust click chemistry labeling after overnight incubation with CGS21680-DBCO, with near complete conversion to agonist-labeled product . Ability of oxaziridine- and agonist-labeled Fabs to bind RNF43 Fc fusion was confirmed with multi-point BLI as compared to unlabeled Fabs . Furthermore, we find that the conjugation sites are highly stable at both room temperature and 37ºC after 3 days . These conjugates were used for testing A2aR degradation in vitro. To determine if an ADC-TACs could degrade endogenous A2AR, a MOLT-4 derived line that co-expresses A2AR and RNF43 was used for degradation experiments.
Cells were dosed with either PBS control, 100 nM CGS21680 alone, or varying concentrations of ADC-TAC and A2AR levels analyzed by western blotting. Excitingly, dose-dependent degradation of endogenous A2AR was observed for multiple antibody-small molecule conjugates tested, with maximal degradation of 50-60% observed . Interestingly, a “hook effect”, characteristic of over saturation by bispecific molecules,was observed in which higher concentrations of conjugate led to decreased degradation of A2AR. We also observed a dependence on the site of antibody conjugation as well as linker length. Specifically, LC S7M and R66M conjugation sites show dose-dependent degradation while other labeling sites do not. Furthermore, the PEG4 linker length conjugates at these sites show successful degradation while the slightly longer PEG6 and 9 conjugates do not. Going forward, conjugates that show efficient degradation in preliminary experiments will be triaged for further validation and understanding of degradation mechanism using pathway inhibitors. Future studies will also focus on expanding the ADC-TAC target scope to other therapeutically relevant GPCRs,vertical hydroponic such as chemokine receptors CXCR4 and CCR5. For conjugation with oxaziridine, 50 µM Fab was incubated with 5 molar equivalents of oxaziridine azide for 30 min at room temperature in phosphate-buffer saline . The reaction was quenched with 10 molar equivalents methionine. The antibody was buffer exchanged into PBS and desalted using a 0.5-mL Zeba 7-kDa desalting column . Then, 10 molar equivalents of DBCO-CGS21680 was added and incubated at room temperature overnight. The agonist-labeled conjugate was desalted using the 0.5-mL Zeba 7-kDa desalting column to remove excess DBCO-CGS21680. Full conjugation at each step was monitored by intact mass spectrometry using a Xevo G2-XS Mass Spectrometer .
Cells at 1 million cells/mL were treated with antibody-drug conjugate, agonist, or antagonist in complete growth medium. After 24 hrs, cells were pelleted by centrifugation . Cell pellets were lysed with RIPA buffer containing complete mini protease inhibitor cocktail on ice for 40 min. Lysates were spun at 16,000xg for 10 min at 4ºC and protein concentrations were normalized using BCA assay. 4x NuPAGE LDS sample buffer and 2-mercaptoethanol was added to the lysates. Equal amounts of lysates were 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 then transferred onto a polyvinylidene difluoride membrane. The membrane was blocked in PBS with 0.1% Tween20 + 5% bovine serum albumin for 30 min at room temperature with gentle shaking. Membranes were co-incubated overnight with rabbit-anti-A2aR and mouse-anti-tubulin at 4ºC with gentle shaking in PBS + 0.2% Tween20 + 5% BSA. Membranes were washed four times with tris-buffered saline + 0.1% Tween20 and then co-incubated with HRP-anti-rabbit IgG and 680RD goat anti-mouse IgG in PBS + 0.2% Tween20 + 5% BSA for 1 hr at room temperature. Membranes were washed four times with TBS + 0.1% Tween20, then washed with PBS. Membranes were first imaged using an OdysseyCLxImager . Super Signal West Pico PLUS Chemiluminescent Substrate was then added and image using a ChemiDoc Imager . Band intensities were quantified using Image Studio Software .SARS-CoV-2 has emerged as a global health concern and effective therapeutics are necessary to curb the COVID-19 pandemic. Many potential therapeutic options for treating COVID-19 have been explored, from small molecules,convalescent patient sera,decoy receptors,neutralizing antibodies,and other protein scaffolds.In particular, antibodies are advantageous due to their specific and potent binding, demonstrated pharmacokinetics, and ability to be recombinantly produced and manufactured at scale. SARS-CoV-2 antibodies have been derived from several sources, including B-cells of convalescent patients,animal immunization,prior coronavirus infections,and synthetic libraries or de novo design.Most of the antibodies reported to date potently target the receptor binding domain in the trimeric Spike protein on the surface of SARS-CoV-2,which is highly immunogenic and is the key protein that mediates cellular entry via interaction with the host angiotensin-converting enzyme II receptor.However, given the widespread global impact of this pandemic and limitations in biologic manufacturing capacities, means to further increase the potency of these antibodies and thereby decrease the dose required will be critical in meeting the global demand for therapeutics.
Additionally, testing different scaffolds and targeting mechanisms against coronavirus could lead to a better understanding of the most effective modalities and ultimately lead to a more resilient therapeutic arsenal against viral infections. Following identification of an initial candidate antibody, various methods for improving antibody affinity and potency are typically employed, each with their advantages and drawbacks. Affinity maturation using mutagenesis or library display is a powerful tool to improve candidate antibodies and can screen large sequence spaces.However, this process is labor intensive and may result in an antibody sequence with altered biophysical or pharmacokinetic properties that requires additional optimization. A parallel strategy to improve potency is to target multiple epitopes, either by engineering bi-specific or multi-specific molecules or by combining multiple antibodies into a cocktail.Targeting multiple epitopes has the added benefit of decreasing the likelihood of viral escape and resistance,and has shown promise as a powerful viral immunotherapy against viruses such as influenza and HIV.Indeed, several cocktails and engineered multi-specific binders have been shown to be effective against SARS-CoV-2. Recently, our lab demonstrated the benefits of linking multiple neutralizing epitopes on the SARSCoV-2 Spike using bi-paratopic binders derived from variable heavy domains.By linking multiple neutralizing VH together in tandem, we were able to improve antibody potency through avidity. Here we explored whether linking non-neutralizing binders to neutralizing binders in a bispecific scaffold could be used as a means to rapidly improve neutralization potency. Using phage display, we identified Fabs that bind RBD but do not block ACE2 binding, and then assembled them in a knob-in-hole bi-specific IgG scaffold with VH binders that block ACE2. These VH/Fab bi-specifics have the additional advantage of avoiding the light-chain mispairing problem common to bi-specific IgGs that utilize Fabs on both arms.Remarkably, the resulting VH/Fab bi-specifics are ~20 to 25-fold more potent in neutralizing both pseudotyped and authentic SARS-CoV-2 virus than the mono-specific bi-valent VH-Fc or IgG alone or as a cocktail. This effect is epitope dependent, illustrating the unique geometry that bi-specific VH/Fab IgGs could capture on the trimeric Spike protein. Our findings highlight how targeting multiple epitopes within a single therapeutic molecule, both neutralizing and non-neutralizing, can confer significant gains in efficacy, and could potentially be generalized to other therapeutic targets to rapidly enhance antibody potency. Recently, we reported the identification and engineering of human variable heavy chain binders against SARS-CoV-2 Spike from an in-house VH-phage library, using a masked phage selection strategy to enrich for binders to Spike-RBD that compete with ACE2. From this process, we identified VH domains against two epitopes that bind within the ACE2 binding site of SARS-CoV-2 Spike. In the bi-valent VH-Fc format, both site A and site B binders blockbinding of ACE2 to Spike and neutralize pseudotyped and authentic SARS-CoV-2. VH domains that bind outside of the ACE2 binding site were not identified with this selection campaign.Here, we utilized an in-house Fab-phage library to identify unbiased Fab binders that recognize Spike-RBD. Briefly, for each round of selection, the Fab-phage pool was pre-cleared with biotinylated Fc immobilized on streptavidin -coated magnetic beads before incubating with SA-beads conjugated with biotinylated Spike-RBD-Fc . After 3-4 rounds of selection, significant enrichment was observed for Fab-phage that bound Spike-RBD-Fc over Fc alone. Individual phage clones were isolated and phage ELISA was used to characterize binding to Spike-RBD-Fc alone and in complex with ACE2-Fc. We hypothesized that Fab-phage that can bind similarly to Spike-RBD-Fc alone or when masked with ACE2-Fc would bind an epitope outside of the ACE2 binding site and would therefore occupy a unique epitope from the VH. From here, we identified over 200 unique Fab-phage sequences that bound Spike-RBD-Fc, a majority of which did not bind at the Spike-ACE2 interface . We characterized a subset of these and identified two lead Fabs, C01 and D01, which bound Spike-RBD-Fc and the trimeric Spike full ectodomain with high affinity . Conversion of these Fabs into a traditional bi-valent IgG scaffold further improved affinity to Secto to single-digit nanomolar KD . The increased affinity of the IgG compared to the Fab is driven by the avidity of the two binding arms. Due to the challenges of modeling the interaction between a bi-valent binder and a conformationally dynamic, trimeric Spike, we have reported affinities as apparent KDs derived from a 1:1 binding model of the data.