A Novel Bispecific Antibody Targeting EGFR and cMet Is Effective against EGFR Inhibitor-Resistant Lung Tumors.

Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR.

An efficient process of generating bispecific antibodies via controlled Fab-arm exchange using culture supernatants.

Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.

An anti-PCSK9 antibody reduces LDL-cholesterol on top of a statin and suppresses hepatocyte SREBP-regulated genes.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.

A rate-limiting role for Dickkopf-1 in bone formation and the remediation of bone loss in mouse and primate models of postmenopausal osteoporosis by an experimental therapeutic antibody.

Genetic studies have linked both osteoporotic and high bone mass phenotypes to low-density lipoprotein receptor-related proteins (LRP4, LRP5, and LRP6). LRPs are receptors for inhibitory Dickkopf-1 (DKK1) protein, and treatment modalities that modulate LRP/DKK1 binding therefore may act as stimulators of bone mass accrual. Here, we report that RH2-18, a fully human monoclonal anti-DKK1 antibody elicits systemic pharmacologic bone efficacy and new bone formation at endosteal bone surfaces in vivo in a mouse model of estrogen-deficiency-induced osteopenia. This was paralleled by partial-to-complete resolution of osteopenia (bone mineral density) at all of the skeletal sites investigated in femur and lumbar-vertebral bodies and the restoration of trabecular bone microarchitecture. More importantly, testing of RH2-18 in adult, osteopenic rhesus macaques demonstrated a rate-limiting role of DKK1 at multiple skeletal sites and responsiveness to treatment. In conclusion, this study provides pharmacologic evidence for the modulation of DKK1 bioactivity in the adult osteopenic skeleton as a viable approach to resolve osteopenia in animal models. Thus, data described here suggest that targeting DKK1 through means such as a fully human anti-DKK1-antibody provides a potential bone-anabolic treatment for postmenopausal osteoporosis.

A PCSK9-binding antibody that structurally mimics the EGF(A) domain of LDL-receptor reduces LDL cholesterol in vivo.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.

Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo.

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.

Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.

Affinity maturation and characterization of a human monoclonal antibody against HIV-1 gp41.

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.

IgG2m4, an engineered antibody isotype with reduced Fc function.

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.

Improving consistency of cell-based assays by using division-arrested cells.

In this article we describe the use of division-arrested cells for cell-based assays designed for high-throughput screening. Cells are the most critical and variable reagent for cell-based high-throughput screening. The robustness of robotic screening depends on the quality and consistency of cell reagents. We demonstrate that for most cell types commonly used for high-throughput screening, cells can be irreversibly division-arrested by mitomycin C treatment at doses that cause no apparent toxicity or obvious change to the cell signaling properties we measured. Our data also suggest that division-arrested cells perform favorably compared to regular growing cells in reporter and calcium flux assays, two platforms most commonly used in robotic screening. Division arrest technology effectively uncouples the process of cell production from robotic screening and brings the convenience of having quality-approved cell reagent on demand for cell-based high-throughput screening.