Intrinsic physicochemical profile of marketed antibody-based biotherapeutics.

Feeding biopharma pipelines with biotherapeutic candidates that possess desirable developability profiles can help improve the productivity of biologic drug discovery and development. Here, we have derived an in silico profile by analyzing computed physicochemical descriptors for the variable regions (Fv) found in 77 marketed antibody-based biotherapeutics. Fv regions of these biotherapeutics demonstrate significant diversities in their germlines, complementarity determining region loop lengths, hydrophobicity, and charge distributions. Furthermore, an analysis of 24 physicochemical descriptors, calculated using homology-based molecular models, has yielded five nonredundant descriptors whose distributions represent stability, isoelectric point, and molecular surface characteristics of their Fv regions. Fv regions of candidates from our internal discovery campaigns, human next-generation sequencing repertoires, and those in clinical-stages (CST) were assessed for similarity with the physicochemical profile derived here. The Fv regions in 33% of CST antibodies show physicochemical properties that are dissimilar to currently marketed biotherapeutics. In comparison, physicochemical characteristics of ∼29% of the Fv regions in human antibodies and ∼27% of our internal hits deviated significantly from those of marketed biotherapeutics. The early availability of this information can help guide hit selection, lead identification, and optimization of biotherapeutic candidates. Insights from this work can also help support portfolio risk assessment, in-licensing, and biopharma collaborations.

Preclinical efficacy of humanized, non-FcγR-binding anti-CD3 antibodies in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy that accounts for ∼20% of ALL cases. Intensive chemotherapy regimens result in cure rates >85% in children and <50% in adults, warranting a search of novel therapeutic strategies. Although immune-based therapies have tremendously improved the treatment of B-ALL and other B-cell malignancies, they are not yet available for T-ALL. We report here that humanized, non-Fcγ receptor (FcγR)-binding monoclonal antibodies (mAbs) to CD3 have antileukemic properties in xenograft (PDX) models of CD3+ T-ALL, resulting in prolonged host survival. We also report that these antibodies cooperate with chemotherapy to enhance antileukemic effects and host survival. Because these antibodies show only minor, manageable adverse effects in humans, they offer a new therapeutic option for the treatment of T-ALL. Our results also show that the antileukemic properties of anti-CD3 mAbs are largely independent of FcγR-mediated pathways in T-ALL PDXs.

Antibodies with Weakly Basic Isoelectric Points Minimize Trade-offs between Formulation and Physiological Colloidal Properties.

The widespread interest in antibody therapeutics has led to much focus on identifying antibody candidates with favorable developability properties. In particular, there is broad interest in identifying antibody candidates with highly repulsive self-interactions in standard formulations (e.g., low ionic strength buffers at pH 5-6) for high solubility and low viscosity. Likewise, there is also broad interest in identifying antibody candidates with low levels of non-specific interactions in physiological solution conditions (PBS, pH 7.4) to promote favorable pharmacokinetic properties. To what extent antibodies that possess both highly repulsive self-interactions in standard formulations and weak non-specific interactions in physiological solution conditions can be systematically identified remains unclear and is a potential impediment to successful therapeutic drug development. Here, we evaluate these two properties for 42 IgG1 variants based on the variable fragments (Fvs) from four clinical-stage antibodies and complementarity-determining regions from 10 clinical-stage antibodies. Interestingly, we find that antibodies with the strongest repulsive self-interactions in a standard formulation (pH 6 and 10 mM histidine) display the strongest non-specific interactions in physiological solution conditions. Conversely, antibodies with the weakest non-specific interactions under physiological conditions display the least repulsive self-interactions in standard formulations. This behavior can be largely explained by the antibody isoelectric point, as highly basic antibodies that are highly positively charged under standard formulation conditions (pH 5-6) promote repulsive self-interactions that mediate high colloidal stability but also mediate strong non-specific interactions with negatively charged biomolecules at physiological pH and vice versa for antibodies with negatively charged Fv regions. Therefore, IgG1s with weakly basic isoelectric points between 8 and 8.5 and Fv isoelectric points between 7.5 and 9 typically display the best combinations of strong repulsive self-interactions and weak non-specific interactions. We expect that these findings will improve the identification and engineering of antibody candidates with drug-like biophysical properties.

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.

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.

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.

Geometric Antibody Engineering Reveals the Spatial Factor on the Efficacy of Bispecific T Cell Engagers.

Bispecific antibodies (BsAbs) represent an emerging class of biologics that can recognize two different antigens or epitopes. T-cell engagers (TcEs) bind two targets in trans on the cell surface of the effector and target cell to induce proximal immune effects, opening exciting windows for immunotherapies. To date, the engineering of BsAbs has been mainly focused on tuning the molecular weight and valency. However, the effects of spatial factors on the biological functions of BsAbs have been less explored due to the lack of biochemical methods to precisely manipulate protein geometry. Here, we studied the geometric effects of the TcEs. First, by genetically inserting rigidly designed ankyrin repeat proteins into TcEs, we revealed that the efficacy progressively decreased as the spacer distance of the two binding domains increased. Then, we constructed 26 pairs of TcEs with the same size but varying orientations using click chemistry-mediated conjugation at different mutation sites. We found that linear ligation sites play a minor role in modulating cell-killing efficacy. Next, we rendered the TcEs' advanced topology by cyclization chemistry using the SpyTag/SpyCatcher pair or sortase ligation approaches. Cyclized TcEs were generally more potent than their linear counterparts. Particularly, sortase A cyclized TcEs, bearing a minimal tagging motif, exhibited better cell-killing efficacy in vitro and improved stability both in vitro and in vivo compared to the linear TcE. This work combines modern bioconjugation chemistry and protein engineering tools for antibody engineering, shedding light on the elusive spatial factors of BsAbs functionality.

An adapted consensus protein design strategy for identifying globally optimal biotherapeutics.

Biotherapeutic optimization, whether to improve general properties or to engineer specific attributes, is a time-consuming process with uncertain outcomes. Conversely, Consensus Protein Design has been shown to be a viable approach to enhance protein stability while retaining function. In adapting this method for a more limited number of protein sequences, we studied 21 consensus single-point variants from eight publicly available CD3 binding sequences with high similarity but diverse biophysical and pharmacological properties. All single-point consensus variants retained CD3 binding and performed similarly in cell-based functional assays. Using Ridge regression analysis, we identified the variants and sequence positions with overall beneficial effects on developability attributes of the CD3 binders. A second round of sequence generation that combined these substitutions into a single molecule yielded a unique CD3 binder with globally optimized developability attributes. In this first application to therapeutic antibodies, adapted Consensus Protein Design was found to be highly beneficial within lead optimization, conserving resources and minimizing iterations. Future implementations of this general strategy may help accelerate drug discovery and improve success rates in bringing novel biotherapeutics to market.

Enhanced delivery of antibodies across the blood-brain barrier via TEMs with inherent receptor-mediated phagocytosis.

BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.

Development of a two-part strategy to identify a therapeutic human bispecific antibody that inhibits IgE receptor signaling.

The development of bispecific antibodies as therapeutic agents for human diseases has great clinical potential, but broad application has been hindered by the difficulty of identifying bispecific antibody formats that exhibit favorable pharmacokinetic properties and ease of large-scale manufacturing. Previously, the development of an antibody technology utilizing heavy chain knobs-into-holes mutations and a single common light chain enabled the small-scale generation of human full-length bispecific antibodies. Here we have extended the technology by developing a two-part bispecific antibody discovery strategy that facilitates proof-of-concept studies and clinical candidate antibody generation. Our scheme consists of the efficient small-scale generation of bispecific antibodies lacking a common light chain and the hinge disulfides for proof-of-concept studies coupled with the identification of a common light chain bispecific antibody for large-scale production with high purity and yield. We have applied this technology to generate a bispecific antibody suitable for development as a human therapeutic. This antibody directly inhibits the activation of the high affinity IgE receptor FcepsilonRI on mast cells and basophils by cross-linking FcepsilonRI with the inhibitory receptor FcgammaRIIb, an approach that has strong therapeutic potential for asthma and other allergic diseases. Our approach for producing human bispecific full-length antibodies enables the clinical application of bispecific antibodies to a validated therapeutic pathway in asthma.

X chromosome-linked inhibitor of apoptosis regulates cell death induction by proapoptotic receptor agonists.

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-alpha signaling. By contrast, blockade of tumor necrosis factor-alpha does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.

Discovery Strategies to Maximize the Clinical Potential of T-Cell Engaging Antibodies for the Treatment of Solid Tumors.

T-cell Engaging bispecific antibodies (TcEs) that can re-direct cytotoxic T-cells to kill cancer cells have been validated in clinical studies. To date, the clinical success with these agents has mainly been seen in hematologic tumor indications. However, an increasing number of TcEs are currently being developed to exploit the potent mode-of-action to treat solid tumor indications, which is more challenging in terms of tumor-cell accessibility and the complexity of the tumor microenvironment (TME). Of particular interest is the potential of TcEs as an immunotherapeutic approach for the treatment of non-immunogenic (often referred to as cold) tumors that do not respond to checkpoint inhibitors such as programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) antibodies. This has led to considerable discovery efforts for, firstly, the identification of tumor selective targeting approaches that can safely re-direct cytotoxic T-cells to cancer cells, and, secondly, bispecific antibodies and their derivatives with drug-like properties that promote a potent cytolytic synapse between T-cells and tumor cells, and in the most advanced TcEs, have IgG-like pharmacokinetics for dosing convenience. Based on encouraging pre-clinical data, a growing number of TcEs against a broad range of targets, and using an array of different molecular structures have entered clinical studies for solid tumor indications, and the first clinical data is beginning to emerge. This review outlines the different approaches that have been taken to date in addressing the challenges of exploiting the TcE mode-of-action for a broad range of solid indications, as well as opportunities for future discovery potential.

A Bispecific DLL3/CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer.

PURPOSE: Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy-resistant recurrence in the majority of patients. To effectively treat SCLC, we have developed a unique and novel IgG-like T-cell engaging bispecific antibody (ITE) that potently redirects T-cells to specifically lyse SCLC cells expressing Delta-like ligand 3 (DLL3), an antigen that is frequently expressed on the cell surface of SCLC cells, with no to very little detectable expression in normal tissues. EXPERIMENTAL DESIGN: The antitumor activity and mode of action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell lines and primary human effector cells and in vivo in an SCLC xenograft model reconstituted with human CD3+ T-cells. RESULTS: Selective binding of DLL3/CD3 ITE to DLL3-positive tumor cells and T-cells induces formation of an immunological synapse resulting in tumor cell lysis and activation of T-cells. In a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads to an increase in infiltration of T-cells into the tumor tissue resulting in apoptosis of the tumor cells and tumor regression. Consistent with the mode of action, the DLL3/CD3 ITE treatment led to upregulation of PD-1, PD-L1, and LAG-3. CONCLUSIONS: This study highlights the ability of the DLL3/CD3 ITE to induce strictly DLL3-dependent T-cell redirected lysis of tumor cells and recruitment of T-cells into noninflamed tumor tissues leading to tumor regression in a preclinical in vivo model. These data support clinical testing of the DLL3/CD3 ITE in patients with SCLC.

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.

bisFabs: Tools for rapidly screening hybridoma IgGs for their activities as bispecific antibodies.

Bispecific antibodies are a growing class of therapeutic molecules. Many of the current bispecific formats require DNA engineering to convert the parental monoclonal antibodies into the final bispecific molecules. We describe here a method to generate bispecific molecules from hybridoma IgGs in 3-4 d using chemical conjugation of antigen-binding fragments (Fabs) (bisFabs). Proteolytic digestion conditions for each IgG isotype were analyzed to optimize the yield and quality of the final conjugates. The resulting bisFabs showed no significant amounts of homodimers or aggregates. The predictive value of murine bisFabs was tested by comparing the T-cell redirected cytotoxic activity of a panel of antibodies in either the bisFab or full-length IgG formats. A variety of antigens with different structures and expression levels was used to extend the comparison to a wide range of binding geometries and antigen densities. The activity observed for different murine bisFabs correlated with those observed for the full-length IgG format across multiple different antigen targets, supporting the use of bisFabs as a screening tool. Our method may also be used for the screening of bispecific antibodies with other mechanisms of action, allowing for a more rapid selection of lead therapeutic candidates.

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.

Crystal structures of a ligand-free and malonate-bound human caspase-1: implications for the mechanism of substrate binding.

Caspase-1, a mediator of the posttranslational processing of IL-1beta and IL-18, requires an aspartic acid in the P1 position of its substrates. The mechanisms of caspase-1 activation remain poorly understood despite numerous structures of the enzyme complexed with aspartate-based inhibitors. Here we report a crystal structure of ligand-free caspase-1 that displays dramatic rearrangements of loops defining the active site to generate a closed conformation that is incompatible with substrate binding. A structure of the enzyme complexed with malonate shows the protein in its open (active-site ligand-bound) conformation in which malonate reproduces the hydrogen bonding network observed in structures with covalent inhibitors. These results illustrate the essential function of the obligatory aspartate recognition element that opens the active site of caspase-1 to substrates and may be the determinant responsible for the conformational changes between ligand-free and -bound forms of the enzyme, and suggest a new approach for identifying novel aspartic acid mimetics.

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.

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.

Anti-CD20/CD3 T cell-dependent bispecific antibody for the treatment of B cell malignancies.

Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.