Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Target arm affinities determine preclinical efficacy and safety of anti-HER2/CD3 bispecific antibody.

Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.

Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation.

The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid ß (Aß) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aß pathology in mice producing both human Aß and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aß deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aß pathology and may represent a selective approach to treat AD.

Relative Target Affinities of T-Cell-Dependent Bispecific Antibodies Determine Biodistribution in a Solid Tumor Mouse Model.

Anti-HER2/CD3, a T-cell-dependent bispecific antibody (TDB) construct, induces T-cell-mediated cell death in cancer cells expressing HER2 by cross-linking tumor HER2 with CD3 on cytotoxic T cells, thereby creating a functional cytolytic synapse. TDB design is a very challenging process that requires consideration of multiple parameters. Although therapeutic antibody design strategy is commonly driven by striving for the highest attainable antigen-binding affinity, little is known about how the affinity of each TDB arm can affect the targeting ability of the other arm and the consequent distribution and efficacy. To our knowledge, no distribution studies have been published using preclinical models wherein the T-cell-targeting arm of the TDB is actively bound to T cells. We used a combined approach involving radiochemistry, invasive biodistribution, and noninvasive single-photon emission tomographic (SPECT) imaging to measure TDB distribution and catabolism in transgenic mice with human CD3ε expression on T cells. Using CD3 affinity variants, we assessed the impact of CD3 affinity on short-term pharmacokinetics, tissue distribution, and cellular uptake. Our experimental approach determined the relative effects of (i) CD3 targeting to normal tissues, (ii) HER2 targeting to HER2-expressing tumors, and (iii) relative HER2/CD3 affinity, all as critical drivers for TDB distribution. We observed a strong correlation between CD3 affinity and distribution to T-cell-rich tissues, with higher CD3 affinity reducing systemic exposure and shifting TDB distribution away from tumor to T-cell-containing tissues. These observations have important implications for clinical translation of bispecific antibodies for cancer immunotherapy. Mol Cancer Ther; 17(4); 776-85. ©2018 AACR.

An Anti-GDNF Family Receptor Alpha 1 (GFRA1) Antibody-Drug Conjugate for the Treatment of Hormone Receptor-Positive Breast Cancer.

Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.

An anti-CD3/anti-CLL-1 bispecific antibody for the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a major unmet medical need. Most patients have poor long-term survival, and treatment has not significantly changed in 40 years. Recently, bispecific antibodies that redirect the cytotoxic activity of effector T cells by binding to CD3, the signaling component of the T-cell receptor, and a tumor target have shown clinical activity. Notably, blinatumomab is approved to treat relapsed/refractory acute lymphoid leukemia. Here we describe the design, discovery, pharmacologic activity, pharmacokinetics, and safety of a CD3 T cell-dependent bispecific (TDB) full-length human IgG1 therapeutic antibody targeting CLL-1 that could potentially be used in humans to treat AML. CLL-1 is prevalent in AML and, unlike other targets such as CD33 and CD123, is not expressed on hematopoietic stem cells providing potential hematopoietic recovery. We selected a high-affinity monkey cross-reactive anti-CLL-1 arm and tested several anti-CD3 arms that varied in affinity, and determined that the high-affinity CD3 arms were up to 100-fold more potent in vitro. However, in mouse models, the efficacy differences were less pronounced, probably because of prolonged exposure to TDB found with lower-affinity CD3 TDBs. In monkeys, assessment of safety and target cell depletion by the high- and low-affinity TDBs revealed that only the low-affinity CD3/CLL1 TDB was well tolerated and able to deplete target cells. Our data suggest that an appropriately engineered CLL-1 TDB could be effective in the treatment of AML.

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.

An antimesothelin-monomethyl auristatin e conjugate with potent antitumor activity in ovarian, pancreatic, and mesothelioma models.

Mesothelin (MSLN) is an attractive target for antibody-drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine-citrulline linker. The humanized (h7D9.v3) version, αMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of αMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose-dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of αMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial.

Antitumor efficacy of a bispecific antibody that targets HER2 and activates T cells.

Clinical results from the latest strategies for T-cell activation in cancer have fired interest in combination immunotherapies that can fully engage T-cell immunity. In this study, we describe a trastuzumab-based bispecific antibody, HER2-TDB, which targets HER2 and conditionally activates T cells. HER2-TDB specifically killed HER2-expressing cancer cells at low picomolar concentrations. Because of its unique mechanism of action, which is independent of HER2 signaling or chemotherapeutic sensitivity, HER2-TDB eliminated cells refractory to currently approved HER2 therapies. HER2-TDB exhibited potent antitumor activity in four preclinical model systems, including MMTV-huHER2 and huCD3 transgenic mice. PD-L1 expression in tumors limited HER2-TDB activity, but this resistance could be reversed by anti-PD-L1 treatment. Thus, combining HER2-TDB with anti-PD-L1 yielded a combination immunotherapy that enhanced tumor growth inhibition, increasing the rates and durability of therapeutic response.

Anti-EGFL7 antibodies enhance stress-induced endothelial cell death and anti-VEGF efficacy.

Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix-associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF-mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non-small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.

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.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent.

Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF ß-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not ß-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4.

Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.

Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Armed antibodies targeting the mucin repeats of the ovarian cancer antigen, MUC16, are highly efficacious in animal tumor models.

MUC16 is a well-validated cell surface marker for serous adenocarcinomas of the ovary and other gynecologic malignancies that is distinguished by highly repetitive sequences ("mucin repeats") in the extracellular domain (ECD). We produced and compared two monoclonal antibodies: one (11D10) recognizing a unique, nonrepeating epitope in the ECD and another (3A5) that recognizes the repeats and binds multiple sites on each MUC16 protein. 3A5 conjugated to cytotoxic drugs exhibited superior toxicity against tumor cells in vitro and in tumor xenograft models compared with antibody-drug conjugates of 11D10. Importantly, drug conjugates of 3A5 were well tolerated in primates at levels in excess of therapeutic doses. Additionally, the presence of circulating CA125 in a rat model did not exacerbate the toxicity of 3A5 drug conjugates. We conclude that targeting the repeat MUC16 domains, thereby increasing cell-associated levels of drug-conjugated antibody, provides superior efficacy in vitro and in vivo without compromising safety.

Imaging tumors with an albumin-binding Fab, a novel tumor-targeting agent.

Association with albumin as a means to improve biodistribution and tumor deposition of a Fab was investigated using AB.Fab4D5, a bifunctional molecule derived from trastuzumab (HERCEPTIN) capable of binding albumin and tumor antigen HER2 (erbB2) simultaneously. AB.Fab4D5 was compared with trastuzumab and a trastuzumab-derived Fab (Fab4D5) for the ability to target tumors overexpressing HER2 in mouse mammary tumor virus/HER2 allograft models. Biodistribution was monitored using intravital microscopy, histology, and integrated single-photon emission computed tomography/computed tomography analysis. Fab4D5 tumor deposition was characterized by rapid but transient appearance in tumor at 2 h with little retention, followed by rapid accumulation in kidney by 6 h. Trastuzumab was slow to accumulate in tumors and slow to clear from normal tissues, although significant tumor deposition was achieved by 24 h. In contrast, AB.Fab4D5 was observed at 2 h in tumor and its presence was sustained beyond 24 h similar to trastuzumab. Intravital microscopy revealed that at peak tumor accumulation, tumor cell staining by AB.Fab4D5 was more uniform than for Fab4D5 or trastuzumab. Similar tumor deposition was achieved for both AB.Fab4D5 and trastuzumab at 48 h (35.9 +/- 1.8% and 38.2 +/- 3.1% injected dose/g); however, AB.Fab4D5 targeted tumors more rapidly and quickly cleared from blood, leading to a lower overall normal tissue exposure. Importantly, unlike Fab4D5, AB.Fab4D5 did not accumulate in kidney, suggesting that association with albumin leads to an altered route of clearance and metabolism. Rapid targeting, excellent tumor deposition and retention, coupled with high tumor to blood ratios may make AB.Fab an exceptional molecule for imaging and cancer therapy.

Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library.

Non-immune (naïve) antibody phage libraries have become an important source of human antibodies. The synthetic phage antibody library described here utilizes a single human framework with a template containing human consensus complementarity-determining regions (CDRs). Diversity of the libraries was introduced at select CDR positions using tailored degenerate and trinucleotide codons that mimic natural human antibodies. Neuropilin-1 (NRP1), a cell-surface receptor for both vascular endothelial growth factor (VEGF) and class 3 semaphorins, is expressed on endothelial cells and neurons. NRP1 is required for vascular development and is expressed widely in the developing vasculature. To investigate the possibility of function blocking antibodies to NRP1 as potential therapeutics, and study the consequence of targeting NRP1 in murine tumor models, panels of antibodies that cross-react with human and murine NRP1 were generated from a designed antibody phage library. Antibody (YW64.3) binds to the CUB domains (a1a2) of NRP1 and completely blocks Sema3A induced neuron collapse; antibody (YW107.4.87) binds to the coagulation factor V/VIII domains (b1b2) of NRP1 and blocks VEGF binding and VEGF induced cell migration. YW107.4.87 inhibits tumor growth in animal xenograft models. These antibodies have provided valuable tools to study the roles of NRP1 in vascular and tumor biology.

Fusion of two distinct peptide exosite inhibitors of Factor VIIa.

Highly potent bifunctional inhibitors of Factor VIIa (FVIIa) were generated by linking two distinct peptides, recently shown to bind to two discrete exosites on the FVIIa protease domain [Dennis, Eigenbrot, Skelton, Ultsch, Santell, Dwyer, O'Connell and Lazarus (2000) Nature (London) 404, 465-470; Dennis, Roberge, Quan and Lazarus (2001) Biochemistry 40, 9513-9521; Roberge, Santell, Dennis, Eigenbrot, Dwyer and Lazarus (2001) Biochemistry 40, 9522-9531]. Fusion peptides consisting of an N-terminal A-series peptide followed by flexible linkers, an E-series peptide, and the Z-domain of protein A were expressed in Escherichia coli and purified using IgG-Sepharose affinity chromatography. The fusion peptides were potent anticoagulants and had steep concentration dependence curves in tissue factor-dependent prothrombin time (PT) assays in comparison to the individual peptides or their noncovalent combination. This phenomenon was dependent on the length of the linker joining the A- and E-peptides. The fusion of the peptides increased the extent of inhibition of Factor X (FX) activation to 100% at saturating peptide concentrations, but did not improve the binding affinity for Factor VIIa (FVIIa) at the A- and E- binding sites or the IC(50) for the inhibition of FX activation. Differences between the peptides in the PT fold prolongation in normal and FVII-deficient plasma, in conjunction with the inhibition of (125)I-FVII activation, suggest that the enhanced effects of the fusion peptides involve the inhibition of FVII autoactivation.