Fab-Nanolipoprotein Conjugate Causes Vitreous Opacity and Cataracts Following a Single Intravitreal Administration in New Zealand White Rabbits.

One strategy employed to prolong the ocular half-life of large molecule therapeutics is via covalent attachment to a carrier, resulting in an increase in size thereby slowing their clearance from the eye. Rabbit antigen-binding fragment conjugated to nanolipoprotein (RabFab-NLP) is a novel conjugate intended to prolong ocular half-life through an increase in hydrodynamic radius compared to Fab alone (∼12 vs ∼3 nm). Nanolipoproteins are mimetics of endogenous high-density lipoproteins and consist of lipids and apolipoproteins (ApoE422k), both biologically derived materials. The objective of this study was to evaluate the ocular toxicity and toxicokinetics of RabFab-NLP after a single intravitreal administration in New Zealand White rabbits. Serum toxicokinetic data suggested a significant increase in ocular residence time of RabFab-NLP compared to RabFab alone. Ophthalmic examinations showed that RabFab-NLP caused vitreous and lens opacities as early as day 3 and day 8 postdose, respectively, which persisted for the entire study duration to day 30. The RabFab-NLP-related microscopic findings were present in the lens, vitreous cavity, and/or optic nerve head. Based on the observed ocular toxicity, a single intravitreal dose of 1.3 mg/eye RabFab-NLP was not tolerated and caused vitreous opacity and cataracts in rabbit eyes.

Nanolipoprotein particles for co-delivery of cystine-knot peptides and Fab-based therapeutics.

Nanolipoprotein particles (NLPs) have been evaluated as an in vivo delivery vehicle for a variety of molecules of therapeutic interest. However, delivery of peptide-like drugs in combination with therapeutic Fabs has not yet been evaluated. In this study, we describe the development and characterization of cystine-knot peptide (CKP)-containing NLPs and Fab-CKP-NLP conjugates. CKPs were incorporated into NLPs using a self-assembly strategy. The trypsin inhibitor EETI-II, a model CKP, was produced with a C16 fatty acyl chain to enable incorporation of the CKP into the lipid bilayer core during NLP assembly. The CKP-NLP retained trypsin inhibitory function although the overall activity was reduced by ∼5 fold compared to free CKP, which was presumably due to steric hindrance. The NLP platform was also shown to accommodate up to ∼60 CKP molecules. Moreover, the stability of the CKP-NLP was comparable to the NLP control, displaying a relatively short half-life (∼1 h) in 50% serum at 37 °C. Therapeutic Fabs were also loaded onto the CKP-NLP by introducing thiol-reactive lipids that would undergo a covalent reaction with the Fab. Using this strategy, Fab loading could be reliably controlled from 1-50 Fabs per CKP-NLP and was found to be independent of CKP density. Surprisingly, Fab incorporation into CKP-NLPs led to a substantial improvement in NLP stability (half-life > 24 h) at 37 °C; also, there was no reduction in CKP activity in the Fab-CKP-NLP conjugates compared to CKP-NLPs. Altogether, our data demonstrate the potential of NLPs as a promising platform for the targeted or multidrug delivery of peptide-based drug candidates in combination with Fabs.

Nanolipoprotein Particles as a Delivery Platform for Fab Based Therapeutics.

Nanolipoprotein particles (NLPs), a lipid bilayer-based nanoparticle platform, have recently been developed for in vivo delivery of a variety of molecules of therapeutic interest, but their potential to deliver Fabs with valencies that exceed those of current multivalent formats has not yet been evaluated. Here we describe the development, optimization, and characterization of Fab-NLP conjugates. NLPs were generated with maleimide reactive lipids for conjugation to a Fab with a C-terminal cysteine. Of note, maleimide reactive lipids were shown to conjugate to the apolipoprotein when the NLPs were assembled at pH 7.4. However, this undesirable reaction was not observed when assembled at pH 6. Site-specific Fab conjugation conditions were then optimized, and conjugation of up to 30 Fab per NLP was demonstrated. Interestingly, although conjugation of higher numbers of Fabs had a significant impact on NLP molecular weight, only a minimal impact on NLP hydrodynamic radius was observed, indicating that particle size is largely dictated by the discoidal shape of the NLP. Fab-NLP viscosity and its stability upon lyophilization were also evaluated as an assessment of the manufacturability of the Fab-NLP. Significantly higher Fab concentrations were achieved with the Fab-NLP conjugates relative to another multivalent format (Fab-PEG conjugates). Fab conjugation to the NLP was also not found to have an impact on Fab activity in both an inhibitory and agonist setting. Finally, the stability of the Fab-NLP conjugates was evaluated in 50% serum and Fab-NLPs demonstrated increased stability, with >63% of Fab-NLP remaining intact after 24 h at Fab per particle ratios of 7 or greater. Our findings suggest Fab-NLPs are a promising platform for the targeted delivery of Fabs in a multivalent format and are compatible with established manufacturing processes.

Data on charge separation of bispecific and mispaired IgGs using native charge-variant mass spectrometry.

The data supplied in this work are related to the research article entitled "Characterization of Bispecific and Mispaired IgGs by Native Charge-Variant Mass Spectrometry" (Phung et al., 2019). This data article describes a powerful analytical platform using native weak cation exchange chromatography coupled to a high-resolution mass spectrometer, charge variant mass spectrometry (CV-MS), to characterize bispecific and mispaired antibody species. Elution order is investigated through analytical methods and molecular modeling in an effort to understand the intrinsic charge, size and shape differences of these molecules.

Influence of Charge, Hydrophobicity, and Size on Vitreous Pharmacokinetics of Large Molecules.

PURPOSE: Development of therapeutics for retinal disease with improved durability is hampered by inadequate understanding of pharmacokinetic (PK) drivers following intravitreal injection. Previous work shows that hydrodynamic radius is correlated with vitreal half-life over the range of 3 to 7 nm, and that charge and hydrophobicity influence systemic clearance. Better understanding the molecular attributes affecting vitreal elimination half-life enables improved design of therapeutics and enhances clinical translatability. METHODS: Impacts of charge and hydrophobicity on vitreal PK in the rabbit were systematically assessed using antibody and antibody fragment (Fab) variant series, including ranibizumab, altered through amino acid changes in hypervariable regions of the light chain. The impact of molecule size on vitreal PK was assessed in the rabbit, nonhuman primate, and human for a range of molecules (1-45 nm, net charge -1324 to +22.9 in rabbit), including published and internal data. RESULTS: No correlation was observed between vitreal PK and charge or hydrophobicity. Equivalent rabbit vitreal PK was observed for ranibizumab and its variants with isoelectric points (pI) in the range of 6.8 to 10.2, and hydrophobicities of the variable domain unit (FvHI) between 1009 and 1296; additional variant series had vitreal PK similarly unaffected by pI (5.4-10.2) and FvHI (1004-1358). Strong correlations were observed between vitreal half-life and hydrodynamic radius for preclinical species (R 2 = 0.8794-0.9366). CONCLUSIONS: Diffusive properties of soluble large molecules, as quantified by hydrodynamic radius, make a key contribution to vitreal elimination, whereas differences in charge or hydrophobicity make minor or negligible contributions. TRANSLATIONAL RELEVANCE: These results support estimation of vitreal elimination rates based on molecular size in relevant preclinical species and humans.

Identification and characterization of an octameric PEG-protein conjugate system for intravitreal long-acting delivery to the back of the eye.

Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives <10 days in humans, requiring frequent administration. Since IVT injection is burdensome to patients, there exists a strong need to develop therapeutics with prolonged residence time in the eye. We report here a strategy to increase retention of a therapeutic fragment antibody (Fab) in the eye, using an anti-complement factor D Fab previously optimized for ocular delivery. Polyethylene glycol structures, varying in length, geometry and degree of branching, were coupled to the Fab via maleimide-activated termini. A screening strategy was developed to allow for key determinants of ocular half-life to be measured in vitro. After compound selection, a scalable process was established to enable tolerability and pharmacokinetic studies in cynomolgus monkeys, demonstrating an increase in vitreal half-life with no associated adverse events. Further, we show that the technique for compound selection, analytical characterization, and scalable production is general for a range of antibody fragments. The application of the technology has broad impact in across many therapeutic areas with the first major advancement in the treatment of an important ocular disease.

Strategies for modifying drug residence time and ocular bioavailability to decrease treatment frequency for back of the eye diseases.

INTRODUCTION: Treating posterior eye diseases has become a major area of focus for pharmaceutical and biotechnology companies. Current standard of care for treating posterior eye diseases relies on administration via intravitreal injection. Although effective, this is not without complications and there is great incentive to develop longer-acting therapeutics and/or sustained release delivery systems. Here, we present an overview of emerging technologies for delivery of biologics to the back of the eye. AREAS COVERED: Posterior eye diseases, intravitreal injection, age-related macular degeneration, anti-VEGF, ocular pharmacokinetics, novel technologies to extend half-life, in vivo models, translation to the clinic, and hurdles to effective patient care. EXPERT OPINION: Posterior eye diseases are a worldwide public health issue. Although anti-VEGF molecules represent a major advance for treating diseases involving choroidal neovascularization, frequent injection can be burdensome for patients and clinicians. There is a need for effective and patient-friendly treatments for posterior eye diseases. Many technologies that enable long-acting delivery to the back of the eye are being evaluated. However, successful development of novel therapies and delivery technologies is hampered by a multitude of factors, including patient education, translatability of in vitro/in vivo preclinical data to the clinic, and regulatory challenges associated with novel technologies.

In Vivo Stability Profiles of Anti-factor D Molecules Support Long-Acting Delivery Approaches.

The collection of aqueous humor (phase 1 b/2 Mahalo study) from patients dosed intravitreally with anti-factor D (AFD; FCFD4514S, lampalizumab), a humanized antibody fragment previously under investigation to treat geographic atrophy (GA) secondary to age-related macular degeneration, presented a unique opportunity to examine AFD properties in clinical samples. We investigated AFD stability and target-binding characteristics to set up strategies for engineering and evaluating optimized molecules that enable less frequent dosing. Two variants, AFD.v8 and AFD.v14, were evaluated as alternatives to AFD for longer-acting treatments. Mass spectrometry, surface plasmon resonance, and immunoassay were used to assess AFD stability and binding activity in aqueous humor samples from Mahalo patients. In vitro stability and binding activity of AFD, AFD.v8, and AFD.v14 were assessed in human vitreous humor versus buffer at 37 °C over 16 weeks and in vivo in rabbits over 28 days along with pharmacokinetic determinations. In human aqueous humor, AFD specific binding was >85% through 30 days, and deamidation was <3% through 60 days, consistent with the AFD stability and binding activity in vitreous humor from humans in vitro and rabbits in vivo. Target binding, stability, and rabbit pharmacokinetic parameters of AFD.v8 and AFD.v14 were similar to those of AFD. Physiological stability and activity of AFD translated across in vitro and in vivo studies in humans and rabbits. The two variants AFD.v8 and AFD.v14 demonstrated comparable potency and pharmacokinetics. These findings, along with previously demonstrated improved solubility of AFD.v8 and AFD.v14, provide proof-of-concept for developing other similar long-acting therapeutic variants.

Impact of polymer geometry on the interactions of protein-PEG conjugates.

The conjugation of high molecular weight polyethylene glycol (PEG) to an active pharmaceutical ingredient (API) is an attractive strategy for the modification of biophysical and biodistribution properties of the API. Indeed, several therapeutic proteins conjugated to PEG have been safely administered in the clinic. While there have been studies on the configuration of these conjugates in solution, investigations on the impact of PEG geometry on protein-PEG conjugate interactions is limited. In this study, we use dynamic light scattering (DLS), rheology, and small-angle neutron scattering (SANS) to investigate the biophysical solution and interaction behavior of a 50kDa Fab protein attached to either a linear or tetrameric (branched) 40kDa PEG molecule. The hydrodynamic radii, diffusivity, viscosity and pair distance distribution function (PDDF) were obtained for the protein-PEG conjugates in solution. An analysis revealed that interactions between unconjugated proteins were quite attractive, however linear PEG-protein conjugates exhibited net repulsive interactions, similar to that of the unconjugated polymer. Tetramer PEG-protein conjugates on the other hand, exhibited a net weak attractive interaction, indicating a more balanced distribution of repulsive and attractive interaction states. Further analysis of the SANS data using geometric models consistent with the PDDF elucidated the conjugates' equilibrium configuration in solution. Insights gained from measurements and analysis used here can also be useful in predicting how conjugate geometries affect viscosity and aggregation behavior, which are important in determining suitable protein-polymer drug formulations.

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye.

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.

An efficient route to bispecific antibody production using single-reactor mammalian co-culture.

Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho ß that is being developed for type 2 diabetes and other obesity-linked disorders.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Design and Pharmacokinetic Characterization of Novel Antibody Formats for Ocular Therapeutics.

PURPOSE: To design and select the next generation of ocular therapeutics, we performed a comprehensive ocular and systemic pharmacokinetic (PK) analysis of a variety of antibodies and antibody fragments, including a novel-designed bispecific antibody. METHODS: Molecules were administrated via intravitreal (IVT) or intravenous (IV) injections in rabbits, and antibody concentrations in each tissue were determined by ELISA. A novel mathematical model was developed to quantitate the structure-PK relationship. RESULTS: After IVT injection, differences in vitreal half-life observed across all molecules ranged between 3.2 and 5.2 days. Modification or elimination of the fragment crystallizable (Fc) region reduced serum half-life from 9 days for the IgG to 5 days for the neonatal Fc receptor (FcRn) null mAb, to 3.1 to 3.4 days for the other formats. The F(ab')2 was the optimal format for ocular therapeutics with comparable vitreal half-life to full-length antibodies, but with minimized systemic exposure. Concomitantly, the consistency among mathematical model predictions and observed data validated the model for future PK predictions. In addition, we showed a novel design to develop bispecific antibodies, here with activity targeting multiple angiogenesis pathways. CONCLUSIONS: We demonstrated that protein molecular weight and Fc region do not play a critical role in ocular PK, as they do systemically. Moreover, the mathematical model supports the selection of the "ideal therapeutic" by predicting ocular and systemic PK of any antibody format for any dose regimen. These findings have important implications for the design and selection of ocular therapeutics according to treatment needs, such as maximizing ocular half-life and minimizing systemic exposure.

Knobs-into-holes antibody production in mammalian cell lines reveals that asymmetric afucosylation is sufficient for full antibody-dependent cellular cytotoxicity.

Knobs-into-holes is a well-validated heterodimerization technology for the third constant domain of an antibody. This technology has been used to produce a monovalent IgG for clinical development (onartuzumab) and multiple bispecific antibodies. The most advanced uses of this approach, however, have been limited to E. coli as an expression host to produce non-glycosylated antibodies. Here, we applied the technology to mammalian host expression systems to produce glycosylated, effector-function competent heterodimeric antibodies. In our mammalian host system, each arm is secreted as a heavy chain-light chain (H-L) fragment with either the knob or hole mutations to allow for preferential heterodimer formation in vitro with low levels of homodimer contaminants. Like full antibodies, the secreted H-L fragments undergo Fc glycosylation in the endoplasmic reticulum. Using a monospecific anti-CD20 antibody, we show that full antibody-dependent cell-mediated cytotoxicity (ADCC) activity can be retained in the context of a knobs-into-holes heterodimer. Because the knobs-into-holes mutations convert the Fc into an asymmetric heterodimer, this technology was further used to systematically explore asymmetric recognition of the Fc. Our results indicate that afucosylation of half the heterodimer is sufficient to produce ADCC-enhancement similar to that observed for a fully afucosylated antibody with wild-type Fc. However, the most dramatic effect on ADCC activity is observed when two carbohydrate chains are present rather than one, regardless of afucosylation state.

Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies.

By enabling the simultaneous engagement of two distinct targets, bispecific antibodies broaden the potential utility of antibody-based therapies. However, bispecific-antibody design and production remain challenging, owing to the need to incorporate two distinct heavy and light chain pairs while maintaining natural nonimmunogenic antibody architecture. Here we present a bispecific-antibody production strategy that relies on co-culture of two bacterial strains, each expressing a half-antibody. Using this approach, we produce 28 unique bispecific antibodies. A bispecific antibody against the receptor tyrosine kinases MET and EGFR binds both targets monovalently, inhibits their signaling, and suppresses MET and EGFR-driven cell and tumor growth. Our strategy allows rapid generation of bispecific antibodies from any two existing antibodies and yields milligram to gram quantities of bispecific antibodies sufficient for a wide range of discovery and preclinical applications.

Development of a human IgG4 bispecific antibody for dual targeting of interleukin-4 (IL-4) and interleukin-13 (IL-13) cytokines.

Human bispecific antibodies have great potential for the treatment of human diseases. Although human IgG1 bispecific antibodies have been generated, few attempts have been reported in the scientific literature that extend bispecific antibodies to other human antibody isotypes. In this paper, we report our work expanding the knobs-into-holes bispecific antibody technology to the human IgG4 isotype. We apply this approach to generate a bispecific antibody that targets IL-4 and IL-13, two cytokines that play roles in type 2 inflammation. We show that IgG4 bispecific antibodies can be generated in large quantities with equivalent efficiency and quality and have comparable pharmacokinetic properties and lung partitioning, compared with the IgG1 isotype. This work broadens the range of published therapeutic bispecific antibodies with natural surface architecture and provides additional options for the generation of bispecific antibodies with differing effector functions through the use of different antibody isotypes.

Long-range structural and dynamical changes induced by cofactor binding in DNA methyltransferase M.HhaI.

The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNA in an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (GCGC) into an extrahelical position, with a concomitant large movement of an active site loop involving residues 80-99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly 80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural and dynamical characterization. We examined details of the previously unknown effects of the cofactor binding with M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout the protein, including those decorating the cofactor binding site, and distal residues more than 30 A away. The active site loop is involved in motions both on a picosecond to nanosecond time scale and on a microsecond to millisecond time scale and is not significantly affected by cofactor binding except for a few N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a role for the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closely related to the significant amount of dynamics and dynamical changes in response to ligand binding detected in the protein.