M. Jack Borrok

pH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

Background: FcRn controls the serum persistence of antibodies. Results: A panel of novel Fc mutations reveals sites controlling pH dependence and FcRn affinity. Conclusion: FcRn affinity thresholds determine IgG recycling efficiency. Significance: Knowledge of the relationship between FcRn binding and serum persistence can aid in designing better therapeutic antibodies. The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.

Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells.

Immunoglobulin E (IgE) plays a key role in allergic asthma and is a clinically validated target for monoclonal antibodies. Therapeutic anti-IgE antibodies block the interaction between IgE and the Fc epsilon (Fcε) receptor, which eliminates or minimizes the allergic phenotype but does not typically curtail the ongoing production of IgE by B cells. We generated high-affinity anti-IgE antibodies (MEDI4212) that have the potential to both neutralize soluble IgE and eliminate IgE-expressing B-cells through antibody-dependent cell-mediated cytotoxicity. MEDI4212 variants were generated that contain mutations in the Fc region of the antibody or alterations in fucosylation in order to enhance the antibodys affinity for FcγRIIIa. All MEDI4212 variants bound to human IgE with affinities comparable to the wild-type (WT) antibody. Each variant was shown to inhibit the interaction between IgE and FcεRI, which translated into potent inhibition of FcγRI-mediated function responses. Importantly, all variants bound similarly to IgE at the surface of membrane IgE expressing cells. However, MEDI4212 variants demonstrated enhanced affinity for FcγRIIIa including the polymorphic variants at position 158. The improvement in FcγRIIIa binding led to increased effector function in cell based assays using both engineered cell lines and class switched human IgE B cells. Through its superior suppression of IgE, we anticipate that effector function enhanced MEDI4212 may be able to neutralize high levels of soluble IgE and provide increased long-term benefit by eliminating the IgE expressing B cells before they differentiate and become IgE secreting plasma cells.

Monovalent IgG4 molecules: Immunoglobulin Fc mutations that result in a monomeric structure

Antibodies have become the fastest growing class of biological therapeutics, in part due to their exquisite specificity and ability to modulate protein-protein interactions with a high biological potency. The relatively large size and bivalency of antibodies, however, limits their use as therapeutics in certain circumstances. Antibody fragments, such as single-chain variable fragments and antigen binding-fragments, have emerged as viable alternatives, but without further modifications these monovalent formats have reduced terminal serum half-lives because of their small size and lack of an Fc domain, which is required for FcRn-mediated recycling. Using rational engineering of the IgG4 Fc domain to disrupt key interactions at the CH3-CH3 interface, we identified a number of point mutations that abolish Fc dimerization and created half-antibodies, a novel monovalent antibody format that retains a monomeric Fc domain. Introduction of these mutations into an IgG1 framework also led to the creation of half-antibodies. These half-antibodies were shown to be soluble, thermodynamically stable and monomeric, characteristics that are favorable for use as therapeutic proteins. Despite significantly reduced FcRn binding in vitro, which suggests that avidity gains in a dimeric Fc are critical to optimal FcRn binding, this format demonstrated an increased terminal serum half-life compared with that expected for most alternative antibody fragments.

Enhancement of Immune Effector Functions by Modulating IgG’s Intrinsic Affinity for Target Antigen

Antibody-mediated immune effector functions play an essential role in the anti-tumor efficacy of many therapeutic mAbs. While much of the effort to improve effector potency has focused on augmenting the interaction between the antibody-Fc and activating Fc-receptors expressed on immune cells, the role of antibody binding interactions with the target antigen remains poorly understood. We show that antibody intrinsic affinity to the target antigen clearly influences the extent and efficiency of Fc-mediated effector mechanisms, and report the pivotal role of antibody binding valence on the ability to regulate effector functions. More particularly, we used an array of affinity modulated variants of three different mAbs, anti-CD4, anti-EGFR and anti-HER2 against a panel of target cell lines expressing disparate levels of the target antigen. We found that at saturating antibody concentrations, IgG variants with moderate intrinsic affinities, similar to those generated by the natural humoral immune response, promoted superior effector functions compared to higher affinity antibodies. We hypothesize that at saturating concentrations, effector function correlates most directly with the amount of Fc bound to the cell surface. Thus, high affinity antibodies exhibiting slow off-rates are more likely to interact bivalently with the target cell, occupying two antigen sites with a single Fc. In contrast, antibodies with faster off-rates are likely to dissociate each binding arm more rapidly, resulting in a higher likelihood of monovalent binding. Monovalent binding may in turn increase target cell opsonization and lead to improved recruitment of effector cells. This unpredicted relationship between target affinity and effector function potency suggests a careful examination of antibody design and engineering for the development of next-generation immunotherapeutics.

Tumor uptake of pegylated diabodies: Balancing systemic clearance and vascular transport

ABSTRACT The accumulation, dissemination and clearance of monoclonal antibody‐based therapeutics or imaging reagents targeting tumor associated antigens is governed by several factors including affinity, size, charge, and valency. Tumor targeting antibody fragments have distinct advantages over intact monoclonal antibodies such as enhanced penetration within the tumor and rapid accumulation but are subject to rapid clearance. Polyethylene glycol (PEG)‐modified antibody fragments can provide a way to balance tumor penetration and accumulation with improved serum persistence. In this study, we use a diabody, the dimeric antibody fragment, targeting the 5T4 antigen to assess the impact of PEGs of distinct size and shape on tumor accumulation and pharmacokinetics (PK). We show that PEG‐modified diabodies improved the PK of the parental diabody from a half‐life of 40min to over 40h for the higher molecular weight PEG conjugated diabodies. This improvement correlates with the increasing hydrodynamic size of pegylated diabodies, and can serve as a better predictor of the PK behavior of pegylated molecules than molecular weight alone. Tumor uptake profiles determined by quantitative PET imaging differed significantly based on PEG size and shape with diabody‐PEG5K showing peak accumulation early on, but with the larger diabody‐PEG20K showing better sustained tumor uptake at later time points. In addition, we demonstrate that a diabody‐PEG20K‐B with a hydrodynamic radius (Rh) of 6nm had superior tumor uptake than the larger diabody‐PEG40K‐B with Rh of 12nm, indicating that beyond 6nm, larger pegylated diabodies have a slower tumor uptake rate while having comparable clearance kinetics. Our data demonstrate that pegylated diabodies with Rh of ˜6nm have an optimal size and PK profile for tumor uptake. Understanding the impact of pegylation on PK and tumor uptake could facilitate the development of pegylated diabodies as therapeutics.

Enhancing IgG distribution to lung mucosal tissue improves protective effect of anti–Pseudomonas aeruginosa antibodies

IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti-Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.

Abstract LB-295: MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies

MEDI7247 is a first in class ADC consisting of a human anti-ASCT2 monoclonal antibody site specifically conjugated to DNA cross-linking pyrrolobenzodiazepine (PBD) dimers. ASCT2 (SLC1A5) is a multi-pass, Na+-dependent neutral amino acid transporter that mediates the uptake of amino acids required for tumor growth and progression. ASCT2 is highly overexpressed in many hematologic cancers, most notably Multiple Myeloma (MM - 100% positive), Acute Myeloid Leukemia (AML - 100% positive) and Diffuse Large B cell lymphoma (DLBCL - 95% positive). ASCT2 expression is low in normal tissues. MEDI7247 (Q1Wx4) demonstrated a significant survival advantage in 3 disseminated AML cell line models, TF1α(ASCT2-High), MOLM-13(ASCT2-low) and M.V.411(ASCT2-High), when compared to the untreated control at the lowest dose levels examined: 0.05, 0.1 and 0.1 mg/kg, respectively. Further exemplifying the activity of MEDI7247, both the TF1α and MOLM-13 models did not reach 50% survival by the end of the study, with 80% survival at >200 days for TF1α and 70% survival at >180 days for MOLM-13. Similarly, a single dose of MEDI7247 in the TF1α model resulted in a 60% survival at >200 days at 0.05 mg/kg. MEDI7247 was also tested in a disseminated AML PDX(ASCT2-low) model at 0.05, 0.1 and 0.4 mg/kg. A significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by >80 days. MEDI7247 activity was further confirmed by monitoring peripheral blood CD33+ve cells, which initially receded, with the timing of reappearance preempting survival. Multiple Myeloma is another indication that exhibits a high level of ASCT2 expression. MEDI7247 (Q1Wx4) efficacy was examined in 3 disseminated MM cell line models, NCI-H929(ASCT2-High), MM.1S(ASCT2-medium) and OPM2(ASCT2-Medium), with a significant improvement in survival from control at the lowest dose levels examined: 0.1, 0.1 and 0.05 mg/kg, respectively. The activity of MEDI7247 (Q1Wx4) was also examined in the subcutaneous DLBCL model KARPAS 422(ASCT2-High). Tumor regressions were observed at all dose levels tested (0.1, 0.2, 0.3 and 0.4 mg/kg), with the higher two dose levels resulting in complete tumor regression without regrowth beyond 150 days. Additionally, MEDI7247 (Q1Wx4) is efficacious against the disseminated 697(ASCT2-Low) (Acute Lymphoblastic Leukemia - ALL) and RAJI(ASCT2-High) (Burkitt9s lymphoma) models. A significant survival advantage was seen in both tumor models at the lowest dose examined of 0.05 mg/kg. In conclusion, MEDI7247 demonstrates antitumor efficacy across all tumor indications tested and varying levels of ASCT2 expression. These data support the use of MEDI7247 in ASCT2 positive hematological malignancies. MEDI7247 is currently in Phase 1 clinical trials. Citation Format: Noel R. Monks, Kevin P. Schifferli, Ravinder Tammali, M. Jack Borrok, Steven R. Coats, Ronald Herbst, David A. Tice, Nabendu Pore. MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-295.

The TGF-β inhibitory activity of antibody 37E1B5 depends on its H-CDR2 glycan

ABSTRACT Excessive transforming growth factor (TGF)-β is associated with pro-fibrotic responses in lung disease, yet it also plays essential roles in tissue homeostasis and autoimmunity. Therefore, selective inhibition of excessive and aberrant integrin-mediated TGF-β activation via targeting the α-v family of integrins is being pursued as a therapeutic strategy for chronic lung diseases, to mitigate any potential safety concerns with global TGF-β inhibition. In this work, we reveal a novel mechanism of inhibiting TGF-β activation utilized by an αvβ8 targeting antibody, 37E1B5. This antibody blocks TGF-β activation while not inhibiting cell adhesion. We show that an N-linked complex-type Fab glycan in H-CDR2 of 37E1B5 is directly involved in the inhibition of latent TGF-β activation. Removal of the Fab N-glycosylation site by single amino acid substitution, or removal of N-linked glycans by enzymatic digestion, drastically reduced the antibodys ability to inhibit latency-associated peptide (LAP) and αvβ8 association, and TGF-β activation in an αvβ8-mediated TGF-β signaling reporter assay. Our results indicate a non-competitive, allosteric inhibition of 37E1B5 on αvβ8-mediated TGF-β activation. This unique, H-CDR2 glycan-mediated mechanism may account for the potent but tolerable TGF-b activation inhibition and lack of an effect on cellular adhesion by the antibody.

An antidote approach to reduce risk and broaden utility of antibody-based therapeutics

Antibody therapeutics offer effective treatment options for a broad range of diseases. One of the greatest benefits of antibody therapeutics is their extraordinarily long serum half-life, allowing infrequent dosing with long-lasting effects. A characteristic of antibodies that drives long half-life is the ability to interact with the recycling receptor, FcRn, in a pH-dependent manner. The benefit of long half-life, however, carries with it liabilities. Although the positive effects of antibody therapeutics are long-lasting, any acute adverse events or chronic negative impacts, such as immunosuppression in the face of an infection, are also long-lasting. Therefore, we sought to develop antibodies with a chemical handle that alone would enjoy the long half-life of normal antibodies but, upon addition of a small-molecule antidote, would interact with the chemical handle and inhibit the antibody recycling mechanism, thus leading to rapid degradation and shortened half-life in vivo. Here we present a proof of concept study where we identify sites to incorporate a non-natural amino acid that can be chemically modified to modulate FcRn interaction in vitro and antibody half-life in vivo. This is an important first step in developing safer therapeutics, and the next step will be development of technology that can perform the modifying chemistry in vivo.