Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.

Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action.

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.

Generation of a Porcine Antibody Fab Fragment Using Protein Engineering to Facilitate the Evaluation of Ocular Sustained Delivery Technology.

Treatment of age-related macular degeneration (AMD) with anti-vascular endothelial growth factor (VEGF) biologic agents has been shown to restore and maintain visual acuity for many patients afflicted with wet AMD. These agents are usually administered via intravitreal injection at a dosing interval of 4-8 weeks. Employment of long-acting delivery (LAD) technologies could improve the therapeutic outcome, ensure timely treatment, and reduce burden on patients, caregivers, and the health care system. Development of LAD approaches requires thorough testing in pre-clinical species; however, therapeutic proteins of human origin may not be well tolerated during testing in non-human species due to immunogenicity. Here, we have engineered a surrogate porcine antibody Fab fragment (pigG6.31) from a human antibody for testing ocular LAD technologies in a porcine model. The engineered Fab retains the VEGF-A-binding and inhibition properties of the parental human Fab and has stability properties suitable for LAD evaluation. Upon intravitreal injection in minipigs, pigG6.31 showed first-order clearance from the ocular compartments with vitreal elimination rates consistent with other molecules of this size. Application of the surrogate molecule in an in vivo evaluation in minipigs of a prototype of the port delivery (PD) platform indicated continuous ocular delivery from the implant, with release kinetics consistent with both the results from in vitro release studies and the efficacy observed in human clinical studies of the PD system with ranibizumab (PDS). Anti-drug antibodies in the serum against pigG6.31 were not detected over exposure durations up to 16 weeks, suggesting that this molecule has low porcine immunogenicity.

Development of an ultra-sensitive human IL-33 biomarker assay for age-related macular degeneration and asthma drug development.

BACKGROUND: Over the past decade, human Interleukin 33 (hIL-33) has emerged as a key contributor to the pathogenesis of numerous inflammatory diseases. Despite the existence of several commercial hIL-33 assays spanning multiple platform technologies, their ability to provide accurate hIL-33 concentration measurements and to differentiate between active (reduced) and inactive (oxidized) hIL-33 in various matrices remains uncertain. This is especially true for lower sample volumes, matrices with low hIL-33 concentrations, and matrices with elevated levels of soluble Interleukin 1 Receptor-Like 1 (sST2), an inactive form of ST2 that competes with membrane bound ST2 for hIL-33 binding. RESULTS: We tested the performance of several commercially available hIL-33 detection assays in various human matrices and found that most of these assays lacked the sensitivity to accurately detect reduced hIL-33 at biologically relevant levels (sub-to-low pg/mL), especially in the presence of human sST2 (hsST2), and/or lacked sufficient target specificity. To address this, we developed and validated a sensitive and specific enzyme-linked immunosorbent assay (ELISA) capable of detecting reduced and total hIL-33 levels even in the presence of high concentrations of sST2. By incorporating the immuno-polymerase chain reaction (iPCR) platform, we further increased the sensitivity of this assay for the reduced form of hIL-33 by ~ 52-fold. Using this hIL-33 iPCR assay, we detected hIL-33 in postmortem human vitreous humor (VH) samples from donors with age-related macular degeneration (AMD) and found significantly increased hIL-33 levels when compared to control individuals. No statistically significant difference was observed in aqueous humor (AH) from AMD donors nor in plasma and nasosorption fluid (NF) from asthma patients compared to control individuals. CONCLUSIONS: Unlike existing commercial hIL-33 assays, our hIL-33 bioassays are highly sensitive and specific and can accurately quantify hIL-33 in various human clinical matrices, including those with high levels of hsST2. Our results provide a proof of concept of the utility of these assays in clinical trials targeting the hIL-33/hST2 pathway.

A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants.

Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.

Hyaluronic Acid-Antibody Fragment Bioconjugates for Extended Ocular Pharmacokinetics.

Treatment of ocular diseases associated with neovascularization currently requires frequent intravitreal injections of antivascular endothelial growth factor (anti-VEGF) therapies. Reducing the required frequency of anti-VEGF injections and associated clinical visits may improve patient adherence to the prescribed treatment regimen and improve outcomes. Herein, we explore conjugation of rabbit and fragment antibodies (Fab) to the biopolymer hyaluronic acid (HA) as a half-life modifying strategy, and assess the impact on Fab biophysical properties and vitreal pharmacokinetics. HA-Fab conjugates of three distinct molecular weights and hydrodynamic radii (RH) were assessed for in vivo pharmacokinetic performance relative to unconjugated Fab after intravitreal injection in rabbits. Covalent conjugation to HA did not significantly alter the thermal stability or secondary or tertiary structure, or diminish the potency of the Fab, thereby preserving its pharmacological properties. Conjugation to HA did significantly slow the in vivo clearance of Fab from the rabbit vitreous in an RH-dependent manner. Compared to free Fab (observed vitreal half-life of 2.8 days), HA-Fab conjugates cleared with observed half-lives of 7.6, 10.2, and 18.3 days for 40 kDa, 200 kDa, and 600 kDa HA conjugates, respectively. This work elucidates a possible strategy for long-acting delivery of proteins intended for the treatment of chronic posterior ocular diseases.

Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109.

Cytomegalovirus (CMV) is a widespread opportunistic pathogen that causes birth defects when transmitted transplacentally and severe systemic illness in immunocompromised individuals. MSL-109, a human monoclonal IgG isolated from a CMV seropositive individual, binds to the essential CMV entry glycoprotein H (gH) and prevents infection of cells. Here, we suggest a mechanism for neutralization activity by MSL-109. We define a genetic basis for resistance to MSL-109 and have generated a structural model of gH that reveals the epitope of this neutralizing antibody. Using surface-based, time-resolved FRET, we demonstrate that gH/gL interacts with glycoprotein B (gB). Additionally, we detect homodimers of soluble gH/gL heterodimers and confirm this novel oligomeric assembly on full-length gH/gL expressed on the cell surface. We show that MSL-109 perturbs the dimerization of gH/gL:gH/gL, suggesting that dimerization of gH/gL may be required for infectivity. gH/gL homodimerization may be conserved between alpha- and betaherpesviruses, because both CMV and HSV gH/gL demonstrate self-association in the FRET system. This study provides evidence for a novel mechanism of action for MSL-109 and reveals a previously undescribed aspect of viral entry that may be susceptible to therapeutic intervention.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor ßKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and ßKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.

The oligomeric state sets GABA(B) receptor signalling efficacy.

G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.

Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering.

Nectins (nectin1-4) and Necls [nectin-like (Necl1-5)] are Ig superfamily cell adhesion molecules that regulate cell differentiation and tissue morphogenesis. Adherens junction formation and subsequent cell-cell signaling is initiated by the assembly of higher-order receptor clusters of cognate molecules on juxtaposed cells. However, the structural and mechanistic details of signaling cluster formation remain unclear. Here, we report the crystal structure of poliovirus receptor (PVR)/Nectin-like-5/CD155) in complex with its cognate immunoreceptor ligand T-cell-Ig-and-ITIM-domain (TIGIT). The TIGIT/PVR interface reveals a conserved specific "lock-and-key" interaction. Notably, two TIGIT/PVR dimers assemble into a heterotetramer with a core TIGIT/TIGIT cis-homodimer, each TIGIT molecule binding one PVR molecule. Structure-guided mutations that disrupt the TIGIT/TIGIT interface limit both TIGIT/PVR-mediated cell adhesion and TIGIT-induced PVR phosphorylation in primary dendritic cells. Our data suggest a cis-trans receptor clustering mechanism for cell adhesion and signaling by the TIGIT/PVR complex and provide structural insights into how the PVR family of immunoregulators function.

Up-regulation of GABA(B) receptor signaling by constitutive assembly with the K+ channel tetramerization domain-containing protein 12 (KCTD12).

GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.

HPK1 citron homology domain regulates phosphorylation of SLP76 and modulates kinase domain interaction dynamics.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell receptor signaling and as such is an attractive target for cancer immunotherapy. Although the role of the HPK1 kinase domain (KD) has been extensively characterized, the function of its citron homology domain (CHD) remains elusive. Through a combination of structural, biochemical, and mechanistic studies, we characterize the structure-function of CHD in relationship to KD. Crystallography and hydrogen-deuterium exchange mass spectrometry reveal that CHD adopts a seven-bladed ß-propellor fold that binds to KD. Mutagenesis associated with binding and functional studies show a direct correlation between domain-domain interaction and negative regulation of kinase activity. We further demonstrate that the CHD provides stability to HPK1 protein in cells as well as contributes to the docking of its substrate SLP76. Altogether, this study highlights the importance of the CHD in the direct and indirect regulation of HPK1 function.

Stability of GABAB receptor oligomers revealed by dual TR-FRET and drug-induced cell surface targeting.

The function of cell surface proteins likely involves the formation and dissociation of oligomeric complexes. However, the dynamics of this process are unknown. Here we examined this process for the GABA(B) receptors that assemble into oligomers of heterodimers through the association of their GABA(B1) subunit. We report a method to study oligomer dynamics based on a drug-controlled cell surface targeting of intracellularly retained receptors and a parallel measurement of two FRET signals in HEK293 cells. GABA(B1) subunits at the cell surface (4.0 ± 0.6 a.u.) are labeled with a pair of fluorophores (donor and red acceptor). New receptors are then targeted to the cell surface during 3h treatment with AP21967 such that the number of receptors is doubled (9.1 ± 0.7 a.u.). After labeling these new receptors with a second acceptor (green), the red FRET remained unchanged (5189 ± 36 vs. 4783 ± 32 cps), supporting the stability of the preformed oligomers. However, new oligomers are detected by the green FRET signal indicating both receptor populations are in the same microdomains. As a control, we confirmed the strict stability of the GABA(B) heterodimer itself. Herein, using a novel method to monitor the dynamics of cell surface complexes, we provide evidence for the stability of GABA(B) oligomers.

A novel, label-free, pre-equilibrium assay to determine the association and dissociation rate constants of therapeutic antibodies on living cells.

BACKGROUND AND PURPOSE: Monoclonal antibodies (Ab) represent the fastest growing drug class. Knowledge of the biophysical parameters (kon , koff and KD ) that dictate Ab:receptor interaction is critical during the drug discovery process. However, with the increasing complexity of Ab formats and their targets, it became apparent that existing technologies present limitations and are not always suitable to determine these parameters. Therefore, novel affinity determination methods represent an unmet assay need. EXPERIMENTAL APPROACH: We developed a pre-equilibrium kinetic exclusion assay using recent mathematical advances to determine the kon , koff and KD of monoclonal Ab:receptor interactions on living cells. The assay is amenable to all human IgG1 and rabbit Abs. KEY RESULTS: Using our novel assay, we demonstrated for several monoclonal Ab:receptor pairs that the calculated kinetic rate constants were comparable with orthogonal methods that were lower throughput or more resource consuming. We ran simulations to predict the critical conditions to improve the performance of the assays. We further showed that this method could successfully be applied to both suspension and adherent cells. Finally, we demonstrated that kon and koff , but not KD , correlate with in vitro potency for a panel of monoclonal Abs. CONCLUSIONS AND IMPLICATIONS: Our novel assay has the potential to systematically probe binding kinetics of monoclonal Abs to cells and can be incorporated in a screening cascade to identify new therapeutic candidates. Wide-spread adoption of pre-equilibrium assays using physiologically relevant systems will lead to a more holistic understanding of how Ab binding kinetics influence their potency.

Structural basis of antibody inhibition and chemokine activation of the human CC chemokine receptor 8.

The C-C motif chemokine receptor 8 (CCR8) is a class A G-protein coupled receptor that has emerged as a promising therapeutic target in cancer. Targeting CCR8 with an antibody has appeared to be an attractive therapeutic approach, but the molecular basis for chemokine-mediated activation and antibody-mediated inhibition of CCR8 are not fully elucidated. Here, we obtain an antagonist antibody against human CCR8 and determine structures of CCR8 in complex with either the antibody or the endogenous agonist ligand CCL1. Our studies reveal characteristic antibody features allowing recognition of the CCR8 extracellular loops and CCL1-CCR8 interaction modes that are distinct from other chemokine receptor - ligand pairs. Informed by these structural insights, we demonstrate that CCL1 follows a two-step, two-site binding sequence to CCR8 and that antibody-mediated inhibition of CCL1 signaling can occur by preventing the second binding event. Together, our results provide a detailed structural and mechanistic framework of CCR8 activation and inhibition that expands our molecular understanding of chemokine - receptor interactions and offers insight into the development of therapeutic antibodies targeting chemokine GPCRs.

Structural basis for HCMV Pentamer receptor recognition and antibody neutralization.

Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.

Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization.

Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput-compatible format. Using this approach, we examined whether G protein-coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers--a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA(B) receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Long-Term Stability of Anti-Vascular Endothelial Growth Factor (a-VEGF) Biologics Under Physiologically Relevant Conditions and Its Impact on the Development of Long-Acting Delivery Systems.

The port delivery system with ranibizumab (PDS) is an investigational long-acting drug delivery system for the continuous release of ranibizumab, an anti-VEGF biologic, in the vitreous humor. The efficacy of the PDS implant relies on the maintenance of long-term drug stability under physiological conditions. Herein, the long-term stability of three anti-VEGF biologics - ranibizumab, bevacizumab and aflibercept - was investigated in phosphate buffered saline (PBS) at 37 °C for several months. Comparison of stability profiles shows that bevacizumab and aflibercept are increasingly prone to aggregation whereas ranibizumab undergoes minimal aggregation. Ranibizumab also shows the smallest loss in antigen binding capacity after long-term incubation in PBS. Even though the aggregated forms of bevacizumab and aflibercept bind to VEGF, the consequences of aggregation on immunogenicity, implant function and efficacy are unknown. These results highlight the importance of maintaining long-term drug stability under physiologically relevant conditions which is necessary for achieving efficacy with an in vivo continuous drug delivery device such as the PDS implant.