Bivalent Antibody Research Articles

High-resolution structure of the vWF A1 domain in complex with caplacizumab, the first nanobody-based medicine for treating acquired TTP

von Willebrand factor (vWF) is a huge oligomeric glycoprotein involved in blood homeostasis. However, this protein is also implicated in acquired thrombotic thrombocytopenic purpura (TTP). The blocking of its binding with platelets has been recognized as an attractive therapeutic strategy for treating acquired TTP. Caplacizumab, a bivalent single-domain antibody (VHH), is the first FDA-approved nanobody drug against vWF for the treatment of acquired TTP. Here, we describe the crystal structure of the A1 domain of vWF in complex with the caplacizumab nanobody at the resolution of 1.60 Å. This structure elucidates the precise epitope and binding mode of caplacizumab. Unexpectedly, caplacizumab binds to the bottom face of the vWF A1 domain and does not create any steric clash with platelet-receptor glycoprotein Ib (GPIb) bound to vWF. However, its binding can stabilize the different conformation within the N-terminus and α1β2 loop from the GPIb bound structure, suggesting that the mechanisms of caplacizumab would not be the direct competition of GPIb binding to vWF A1 domain but the conformational arrestment of vWF in an inappropriate state to platelet adhesion. This high-resolution structure would provide helpful information for the design of improved anti-vWF therapeutics for the treatment of acquired TTP.

Nanoscale spatial dependence of avidity in an IgG1 antibody

Antibodies are secreted proteins that are crucial to recognition of pathogens by the immune system and are also efficient pharmaceuticals. The affinity and specificity of target recognition can increase remarkably through avidity effects, when the antibody can bind a multivalent antigen through more than one epitope simultaneously. A key goal of antibody engineering is thus to optimize avidity, but little is known about the nanoscale spatial dependence of avidity in antibodies. Here, we develop a set of anti-parallel coiled-coils spanning from 7 to 20 nm and validate their structure using biophysical techniques. We use the coiled-coils to control the spacing between two epitopes, and measure how antigen spacing affects the stability of the bivalent antibody:antigen complex. We find a maximal avidity enhancement at a spacing of 13 nm. In contrast to recent studies, we find the avidity to be relatively insensitive to epitope spacing near the avidity maximum as long as it is within the spatial tolerance of the antibody. We thus only see a ~ twofold variation of avidity in the range from 7 to 20 nm. The coiled-coil systems developed here may prove a useful protein nanocaliper for profiling the spatial tolerance and avidity profile of bispecific antibodies.

Interphase epichromatin: last refuge for the 30-nm chromatin fiber?

"Interphase epichromatin" describes the surface of chromatin located adjacent to the interphase nuclear envelope. It was discovered in 2011 using a bivalent anti-nucleosome antibody (mAb PL2-6), now known to be directed against the nucleosome acidic patch. The molecular structure of interphase epichromatin is unknown, but is thought to be heterochromatic with a high density of "exposed" acidic patches. In the 1960s, transmission electron microscopy of fixed, dehydrated, sectioned, and stained inactive chromatin revealed "unit threads," frequently organized into parallel arrays at the nuclear envelope, which were interpreted as regular helices with ~ 30-nm center-to-center distance. Also observed in certain cell types, the nuclear envelope forms a "sandwich" around a layer of closely packed unit threads (ELCS, envelope-limited chromatin sheets). Discovery of the nucleosome in 1974 led to revised helical models of chromatin. But these models became very controversial and the existence of in situ 30-nm chromatin fibers has been challenged. Development of cryo-electron microscopy (Cryo-EM) gave hope that in situ chromatin fibers, devoid of artifacts, could be structurally defined. Combining a contrast-enhancing phase plate and cryo-electron tomography (Cryo-ET), it is now possible to visualize chromatin in a "close-to-native" situation. ELCS are particularly interesting to study by Cryo-ET. The chromatin sheet appears to have two layers of ~ 30-nm chromatin fibers arranged in a criss-crossed pattern. The chromatin in ELCS is continuous with adjacent interphase epichromatin. It appears that hydrated ~ 30-nm chromatin fibers are quite rare in most cells, possibly confined to interphase epichromatin at the nuclear envelope.

Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments

The elution and adsorptive behavior of a bivalent bispecific antibody (BiSAb), comprising an IgG1 framework with a scFv domain genetically fused to each heavy chain C-terminus via flexible linkers, and of two associated fragments were studied on two cation exchange chromatography media – ProPac WCX-10, which is pellicular and suitable for analytical use, and Nuvia HR-S, which is macroporous and suitable for preparative and process scale uses. Both fragments were identified by MS as missing one of the two scFv domains and its flexible linker, but one of them also contains an additional C-terminal lysine. The separation of these fragments on both resins occurs as a result of differences in non-specific ligand-protein interactions that are modulated by the salt concentration. For the ProPac WCX-10 column, complex, multipeak elution behaviors are observed, since, as a result of the linker flexibility, both the intact molecule and the fragments appear to exist in multiple binding configurations with each scFv domains either collapsed onto the IgG framework or extended away from it. With a residence time of 2.5 min and at 21 °C, two peak elution is observed for the fragments which contain a single linked scFv and three peak elution for the intact molecule which contains two linked scFvs. This behavior is affected by residence time, temperature, and hold time. Increasing the residence time to 25 min or increasing temperature to 40°C results in elution of a single, merged peak for each of the protein species. For Nuvia HR-S, the broader peaks, obtained as a result of mass transfer limitations, tend to obscure the multipeak elution behavior. Nevertheless, even for this resin, the effects of configurational flexibility are still manifested at the single-particle scale and affect the evolution of the patterns of protein binding within individual resin particles as evident from confocal microscopy observations.

Efficacy and safety of orally administered heat-killed Lactobacillus paracasei LCW23 in patients with allergic rhinitis: A randomized controlled clinical trial

Objective: In this study, we primarily evaluated the effects of IMMUPHYLA LCW23 on symptoms of allergic rhinitis. IMMUPHYLA LCW23 is a commercial probiotic product containing the Lactobacillus paracasei LCW23 strain. Materials and Methods: Sixty 5–18-year-old children with allergic rhinitis positive for the dust mite-specific bivalent antibody and meeting the inclusion criteria were enrolled in this double-blind, randomized, placebo-controlled trial. They were administered 2–4 g of the probiotic product containing L. paracasei LCW23 (2.5 × 109 cells/g; n = 28) or a placebo supplement (n = 32) according to their body weights for 12 weeks. After the treatment period, a self-assessment of allergic rhinitis symptoms in the nose and eyes was performed. Results: This study results revealed that 12-week supplementation with IMMUPHYLA LCW23 is safe, with no side effects. In addition, the Pediatric Rhinoconjunctivitis Quality of Life Questionnaire (PRQLQ) assessment revealed that the probiotic significantly alleviated general discomfort (P = 0.003) and common symptoms of allergic rhinitis, such as nasal congestion (P = 0.033), runny nose (P = 0.001), and blowing nose (P = 0.008). Conclusion: Administration of IMMUPHYLA LCW23 was safe in the long-term follow-up study. The probiotic likely reduced the risk of allergy prevalence, without any major side effects.

Anti-NKG2D single domain-based antibodies for the modulation of anti-tumor immune response

ABSTRACT The natural killer group 2 member D (NKG2D) receptor is a C-type lectin-like activating receptor mainly expressed by cytotoxic immune cells including NK, CD8+ T, γδ T and NKT cells and in some pathological conditions by a subset of CD4+ T cells. It binds a variety of ligands (NKG2DL) whose expressions is finely regulated by stress-related conditions. The NKG2DL/NKG2D axis plays a central and complex role in the regulation of immune responses against diverse cellular threats such as oncogene-mediated transformations or infections. We generated a panel of seven highly specific anti-human NKG2D single-domain antibodies targeting various epitopes. These single-domain antibodies were integrated into bivalent and bispecific antibodies using a versatile plug-and-play Fab-like format. Depending on the context, these Fab-like antibodies exhibited activating or inhibitory effects on the immune response mediated by the NKG2DL/NKG2D axis. In solution, the bivalent anti-NKG2D antibodies that compete with NKG2DL potently blocked the activation of NK cells seeded on immobilized MICA, thus constituting antagonizing candidates. Bispecific anti-NKG2DxHER2 antibodies that concomitantly engage HER2 on tumor cells and NKG2D on NK cells elicited cytotoxicity of unstimulated NK in a tumor-specific manner, regardless of their apparent affinities and epitopes. Importantly, the bispecific antibodies that do not compete with ligands binding retained their full cytotoxic activity in the presence of ligands, a valuable property to circumvent immunosuppressive effects induced by soluble ligands in the microenvironment.

A reporter cell system for the triggering receptor expressed on myeloid cells 2 reveals differential effects of disease-associated variants on receptor signaling and activation by antibodies against the stalk region.

The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on myeloid-derived cell types. The extracellular immunoglobulin-like domain of TREM2 binds anionic ligands including Apolipoprotein E and Amyloid-β. The transmembrane domain interacts with its adaptor protein DAP12/TYROBP that is responsible for propagation of downstream signaling upon ligand interaction. Several sequence variants of TREM2 have been linked to different neurodegenerative diseases including Alzheimer's disease. Here, we generated HEK 293 Flp-In cell lines stably expressing human TREM2 and DAP12 using a bicistronic construct with a T2A linker sequence allowing initial expression of both proteins in stoichiometric amounts. Cell biological and biochemical analyses revealed transport of TREM2 to the cell surface, and canonical sequential proteolytic processing and shedding of TREM2 (sTREM2). The functionality of this cell system was demonstrated by detection of phosphorylated spleen tyrosine kinase (SYK) upon stimulation of TREM2 with the anionic membrane lipid phosphatidylserine or anti-TREM2 antibodies. Using this cell model, we demonstrated impaired signaling of disease associated TREM2 variants. We also identified a monoclonal antibody against the stalk region of TREM2 with agonistic activity. Activation of TREM2-DAP12 signaling with the monoclonal antibody and the partial loss of function of disease associated variants were recapitulated in induced pluripotent stem cell derived microglia. Thus, this reporter cell model represents a suitable experimental system to investigate signaling of TREM2 variants, and for the identification of ligands and compounds that modulate TREM2-DAP12 signaling. MAIN POINTS: Disease associated variants impair the signaling activity of TREM2 by distinct mechanisms. Targeting the stalk region of TREM2 with bivalent antibodies activates TREM2 signaling.

Bivalent antibody pliers inhibit \u03b2-tryptase by an allosteric mechanism dependent on the IgG hinge

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of allergic inflammatory responses in asthma. Antibodies generally inhibit proteases by blocking substrate access by binding to active sites or exosites or by allosteric modulation. The bivalency of IgG antibodies can increase potency via avidity, but has never been described as essential for activity. Here we report an inhibitory anti-tryptase IgG antibody with a bivalency-driven mechanism of action. Using biochemical and structural data, we determine that four Fabs simultaneously occupy four exosites on the β-tryptase tetramer, inducing allosteric changes at the small interface. In the presence of heparin, the monovalent Fab shows essentially no inhibition, whereas the bivalent IgG fully inhibits β-tryptase activity in a hinge-dependent manner. Our results suggest a model where the bivalent IgG acts akin to molecular pliers, pulling the tetramer apart into inactive β-tryptase monomers, and may provide an alternative strategy for antibody engineering.

Site-Specific Linking of an Oligonucleotide to Mono- and Bivalent Recombinant Antibodies with SpyCatcher-SpyTag System for Immuno-PCR.

Antibody-oligonucleotide conjugates (AOCs) are a versatile class of chimeric biomolecules for therapeutics and biotechnological applications. Most widely employed chemical labeling methods for proteins are based on targeting of Lys or Cys residues that leads to mixed stoichiometry in the degree of conjugation and may interfere with antigen binding, thus, compromising the function of the antibody. A site-specific oligonucleotide conjugation technology providing full control over valency in mild reaction conditions would be an advancement to the state-of-the-art in bioconjugation. Herein, we demonstrate the production of single-chain variable fragment antibodies with fused SpyCatcher (scFv-SpyCatcher, monovalent) and alkaline phosphatase-SpyCatcher (scFv-AP-SpyCatcher, bivalent) on C-terminus and their conjugation to SpyTag002-oligonucleotide in phosphate-buffered saline (PBS). The formation of a covalent isopeptide bond between the protein and SpyTag002-oligonucleotide was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the functionality of the obtained AOCs was confirmed in immuno-polymerase chain reaction (PCR) assays for the detection of microcystin-LR and 17β-estradiol. Based on time-resolved fluorescence immunoassays with scFv-AP fusion constructs, we observed that the SpyCatcher and SpyCatcher-SpyTag002-oligonucleotide part lowered the absolute signal obtained from the assay by 27.6 and 48.4% at 2 nM and by 26.2 and 27.6% at 100 pM microcystin-LR and 17β-estradiol concentrations, respectively. Nevertheless, the overall sensitivity of the immuno-PCR assays was similar to the time-resolved fluorescence immunoassays performed with the same components. In this study, vectors for SpyCatcher-fusion construction were created for directional cloning with SfiI sites enabling the rapid generation of AOC constructs for site-specific SpyTag-oligonucleotide conjugation.

Anti-Insulin Receptor Antibodies Improve Hyperglycemia in a Mouse Model of Human Insulin Receptoropathy.

Loss-of-function mutations in both alleles of the human insulin receptor gene (INSR) cause extreme insulin resistance (IR) and usually death in childhood, with few effective therapeutic options. Bivalent antireceptor antibodies can elicit insulin-like signaling by mutant INSR in cultured cells, but whether this translates into meaningful metabolic benefits in vivo, wherein the dynamics of insulin signaling and receptor recycling are more complex, is unknown. To address this, we adopted a strategy to model human insulin receptoropathy in mice, using Cre recombinase delivered by adeno-associated virus to knockout endogenous hepatic Insr acutely in floxed Insr mice (liver insulin receptor knockout [L-IRKO] + GFP), before adenovirus-mediated add back of wild-type (WT) or mutant human INSR. Two murine anti-INSR monoclonal antibodies, previously shown to be surrogate agonists for mutant INSR, were then tested by intraperitoneal injections. As expected, L-IRKO + GFP mice showed glucose intolerance and severe hyperinsulinemia. This was fully corrected by add back of WT but not with either D734A or S350L mutant INSR. Antibody injection improved glucose tolerance in D734A INSR-expressing mice and reduced hyperinsulinemia in both S350L and D734A INSR-expressing animals. It did not cause hypoglycemia in WT INSR-expressing mice. Antibody treatment also downregulated both WT and mutant INSR protein, attenuating its beneficial metabolic effects. Anti-INSR antibodies thus improve IR in an acute model of insulin receptoropathy, but these findings imply a narrow therapeutic window determined by competing effects of antibodies to stimulate receptors and induce their downregulation.

Abstract 2270: RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells

Abstract Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific or bispecific antibodies. Up-regulation of TIM-3 has been described as an adaptive CPI resistance mechanism, and internal prevalence data on archival samples of CPI-naïve and -experienced patients showed co-expression of PD-1 and TIM-3 in various tumor types, consistent with literature reports. Here, we describe RG7769 (PD1-TIM3), a novel avidity driven heterodimeric PD-1/TIM-3 1+1 bispecific CrossMabVH-VL intentionally designed as high affinity PD-1 (KD 250 pM, 37°C) and low affinity TIM-3 (KD 130 nM, 37°C) Fab-moieties to specifically target PD-1+ and PD-1+ TIM-3+ T cells through avidity gain, while bypassing PD-1- TIM3+ myeloid and NK cells. In contrast to IgG4-based PD-1 antibodies and conventional IgG1-based TIM-3 Fc-effector function competent antibodies, RG7769 harbors a PG LALA containing heterodimeric KiH IgG1 Fc-region rendering the BsAb refractory to drug shaving by FcgR-expressing macrophages in the TME, while retaining IgG-pharmacokinetics. RG7769 binds to PD-1 with higher affinity than pembrolizumab and nivolumab. X-ray crystallography demonstrated that the humanized PD-1 binding Fab recognizes a unique glycosylated epitope on PD-1, and potently blocks the PD-1/PD-L1 and PD-1/PD-L2 interactions in both biochemical and reporter cell line assays. The humanized TIM-3 binding arm was identified for maximal functional activity using mixed lymphocyte reaction (MLR) assays. Compared with bivalent TIM-3 antibodies, RG7769 shows reduced binding to TIM-3+ myeloid and NK cells, but binds preferentially to dysfunctional T cells expressing PD-1 or both PD-1 and TIM-3, like tumor infiltrating lymphocytes (TILs) in the tumor microenvironment. By virtue of its monovalency, RG7769 induced low antibody internalization on activated T cells when compared with bivalent TIM-3 antibodies, overcoming a major cellular sink for TIM-3 antibodies. In functional assays, RG7769 showed increased IFN-γ secretion by in vitro generated tumor-specific T-cells, increased ex vivo tumor-specific effector functions of T cells from PBMCs of melanoma patients, and enhanced the anti-tumor-activity of TILs from melanoma patients when compared to the monospecific parental PD-1 antibody. Finally, RG7769 showed superior efficacy in controlling s.c. MC38 tumor growth in huPD-1/huTIM-3 transgenic C57/BL6 mice compared to the parental PD-1 antibody. In summary, these preclinical data support the use of RG7769 as a monotherapy and as combination partner for the treatment of patients with solid/hematological tumors. A phase I study is currently ongoing in patients with advanced metastatic solid tumors (NCT03708328). Citation Format: Laura Laura Codarri Deak, Stefan Seeber, Mario Perro, Patrick Weber, Laura Lauener, Standford Chen, Sonja Offner, Stefan Dengl, Friederike Hesse, Adrian Zwick, Marco Boettger, Alexander Bujotzek, Jörg Benz, Guy Georges, Georg Fertig, Valeria Lifke, Jens Fischer, Stephane Leclair, Victor Levitsky, Marta Canamero, Juha Lindner, Sara Colombetti, Stefanie Bendels, Christophe Boetsch, Matthias Fueth, Merlind Muecke, Henry Kao, Pablo Umana, Christian Klein. RG7769 (PD1-TIM3), a novel heterodimeric avidity-driven T cell specific PD-1/TIM-3 bispecific antibody lacking Fc-mediated effector functions for dual checkpoint inhibition to reactivate dysfunctional T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2270.

Abstract 1076: Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies

Abstract TNF Receptor Superfamily members (TNF-R-SF), including CD40, are key regulators of the immune system and have been immunotherapeutic targets for over 20 years. CD40 signaling serves as an important co-stimulatory signal for antigen-presenting cells (APC). For the induction of a CD40 driven anti-tumor immune response multiple approaches, - most of them antibody based - are currently under investigation. However, the structural necessity of trimerization within the TNF-R-SF, defines bivalent antibodies generally as unfeasible inducers of signaling events within this protein family. To overcome the known inadequacies of antibodies, we developed HERA-CD40L, composed of two trivalent CD40L-receptor-binding domains, fused to a silenced human IgG1 Fc-domain. This hexavalent CD40 agonist mimics the natural ligand and enables efficient receptor clustering and superior signaling. HERA-CD40L treatment increased the pro-inflammatory state of all CD40-expressing cells examined. It promoted the licensing of dendritic cells (DC), macrophages, B cells and other APC. Comparison to benchmark antibodies revealed that HERA-CD40L elicited a stronger and more rapid activation of NFκB signaling in primary B cells. HERA-CD40L treatment, but not clinical benchmark antibodies triggered immediate NFκB, MAPK, PI3K and STAT-1 signaling in primary monocyte-derived immature DC. As a result, HERA-CD40L induced upregulation of activation markers and co-stimulatory molecules in B cells and DC. Using SEC fractionation followed by a CD40 reporter assay, we could furthermore demonstrate that the activity of a clinical benchmark antibody derived exclusively from antibody aggregates. In vitro HERA-CD40L treatment converted immature phagocytic macrophages into mature/professional APC and induced repolarization of M2- to M1-like macrophages. These findings were confirmed in vivo using a mouse surrogate (mmHERA-CD40L). Upon treatment of MC38-CEA and CT26wt syngeneic mouse models, we observed single agent anti-tumor efficacy. In the CT26wt model, mmHERA-CD40L treatment converted cold into hot tumors by increasing T cell infiltration. Furthermore, mmHERA-CD40L induced a strong dose dependent decrease of tumor associated pro-tumorigenic M2-macrophages indicating a profound reorganization of the tumor microenvironment. Both in vitro (human) and in vivo (mouse), HERA-CD40L increased antigen-specific immune system activation without affecting the non-specific immune cells. These data, together with pilot PD/safety results, demonstrate that the activity of HERA-CD40L is both potent and safe. In conclusion, HERA-CD40L is a potent agonist able to show single agent anti-tumor activity. The biological activity is distinct from and superior to clinical benchmark “agonistic” antibodies. HERA-CD40L has a well-defined mechanism of action, does not depend on Fc gamma receptor-mediated crosslinking and hence functions as a true agonist. Citation Format: Christian Gieffers, David M. Richards, Jaromir Sykora, Christian Merz, Julian P. Sefrin, Katharina Billian-Frey, Karl Heinonen, Mauricio Redondo Müller, Matthias Schröder, Meinolf Thiemann, Oliver Hill. Hexavalent HERA-CD40L induces a productive T cell-mediated anti-tumor immune response and shows superior activity in comparison to benchmark CD40 agonistic antibodies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1076.

Recognition of a highly conserved glycoprotein B epitope by a bivalent antibody neutralizing HCMV at a post-attachment step.

Human cytomegalovirus (HCMV) is one of the main causative agents of congenital viral infection in neonates. HCMV infection also causes serious morbidity and mortality among organ transplant patients. Glycoprotein B (gB) is a major target for HCMV neutralizing antibodies, yet the underlying neutralization mechanisms remain largely unknown. Here we report that 3-25, a gB-specific monoclonal antibody previously isolated from a healthy HCMV-positive donor, efficiently neutralized 14 HCMV strains in both ARPE-19 cells and MRC-5 cells. The core epitope of 3-25 was mapped to a highly conserved linear epitope on antigenic domain 2 (AD-2) of gB. A 1.8 Å crystal structure of 3-25 Fab in complex with the peptide epitope revealed the molecular determinants of 3-25 binding to gB at atomic resolution. Negative-staining electron microscopy (EM) 3D reconstruction of 3-25 Fab in complex with de-glycosylated postfusion gB showed that 3-25 Fab fully occupied the gB trimer at the N-terminus with flexible binding angles. Functionally, 3-25 efficiently inhibited HCMV infection at a post-attachment step by interfering with viral membrane fusion, and restricted post-infection viral spreading in ARPE-19 cells. Interestingly, bivalency was required for HCMV neutralization by AD-2 specific antibody 3-25 but not the AD-4 specific antibody LJP538. In contrast, bivalency was not required for HCMV binding by both antibodies. Taken together, our results reveal the structural basis of gB recognition by 3-25 and demonstrate that inhibition of viral membrane fusion and a requirement of bivalency may be common for gB AD-2 specific neutralizing antibody.

Preclinical and Clinical Development of Therapeutic Antibodies Targeting Functions of CD47 in the Tumor Microenvironment.

CD47 is a ubiquitously expressed cell surface glycoprotein that functions as a signaling receptor for thrombospondin-1 and as the counter-receptor for signal regulatory protein-α (SIRPα). Engaging SIRPα on macrophages inhibits phagocytosis, and CD47 thereby serves as a physiological marker of self. However, elevated CD47 expression on some cancer cells also protects tumors from innate immune surveillance and limits adaptive antitumor immunity via inhibitory SIRPα signaling in antigen presenting cells. CD47 also mediates inhibitory thrombospondin-1 signaling in vascular cells, T cells, and NK cells, and blocking inhibitory CD47 signaling on cytotoxic T cells directly increases tumor cell killing. Therefore, CD47 functions as an innate and adaptive immune checkpoint. These findings have led to the development of antibodies and other therapeutic approaches to block CD47 functions in the tumor microenvironment. Preclinical studies in mice demonstrated that blocking CD47 can limit the growth of hematologic malignancies and solid tumors and enhance the efficacy of conventional chemotherapy, radiation therapy, and some targeted cancer therapies. Humanized CD47 antibodies are showing promise in early clinical trials, but side effects related to enhanced phagocytic clearance of circulating blood cells remain a concern. Approaches to circumvent these include antibody preloading strategies, development of antibodies that recognize tumor-specific epitopes of CD47, SIRPα antibodies, and bivalent antibodies that restrict CD47 blockade to specific tumor cells. Preclinical and clinical development of antibodies and related biologics that inhibit CD47/SIRPα signaling are reviewed, including strategies to combine these agents with various conventional and targeted therapeutics to improve patient outcome for various cancers.

Design and Synthesis of Megamolecule Mimics of a Therapeutic Antibody.

This communication describes the design, synthesis, and biological activity of a megamolecule mimic of an anti-HER2 antibody. The antibody mimic was prepared by linking two Fabs from the therapeutic antibody trastuzumab, which are fused through the heavy chain variable domain to either cutinase or SnapTag, with a linker terminated in an irreversible inhibitor for each enzyme. This mimic binds HER2 with comparable avidity to trastuzumab, has similar activity in a cell-based assay, and can arrest tumor growth in a mouse xenograft BT474 tumor model. A panel of 16 bivalent anti-HER2 antibodies were prepared wherein each varied in the orientation of the fusion domain on the Fabs. The analogs displayed a range of cytotoxic activity, and surprisingly, the most active mimic binds to cells with a 10-fold lower avidity than the least active variant suggesting that structure plays a large role in their efficacy. This work suggests that the megamolecule approach can be used to prepare antibody mimics having a broad structural diversity.

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization.

Fibroblast growth factor receptor 1 (FGFR1) transmits signals through the plasma membrane regulating essential cellular processes like division, motility, metabolism, and death. Overexpression of FGFR1 is observed in numerous tumors and thus constitutes an attractive molecular target for selective cancer treatment. Targeted anti‐cancer therapies aim for the precise delivery of drugs into cancer cells, sparing the healthy ones and thus limiting unwanted side effects. One of the key steps in targeted drug delivery is receptor‐mediated endocytosis. Here, we show that the efficiency and the mechanism of FGFR1 internalization are governed by the spatial distribution of the receptor in the plasma membrane. Using engineered antibodies of different valency, we demonstrate that dimerization of FGFR1 with bivalent antibody triggers clathrin‐mediated endocytosis (CME) of the receptor. Clustering of FGFR1 into larger oligomers with tetravalent antibody stimulates fast and highly efficient uptake of the receptor that occurs via two distinct mechanisms: CME and dynamin‐dependent clathrin‐independent endocytic routes. Furthermore, we show that all endocytic pathways engaged in FGFR1 internalization do not require receptor activation. Our data provide novel insights into the mechanisms of intracellular trafficking of FGFR1 and constitute guidelines for development of highly internalizing antibody‐based drug carriers for targeted therapy of FGFR1‐overproducing cancers.

Membrane Association Transforms an Inert Anti-TCRβ Fab’ Ligand into a Potent T Cell Receptor Agonist

The natural peptide-major histocompatibility complex (pMHC) ligand for T cell receptors (TCRs) is inactive from solution yet capable of activating T cells at single-molecule levels when membrane-associated. This distinctive feature stems from the mechanism of TCR activation, which is thought to involve steric phosphatase exclusion as well as direct mechanical forces. It is possible to defeat this mechanism and activate T cells with solution ligands by cross-linking pMHC or using multivalent antibodies to TCR. However, these widely used strategies activate TCRs through a nonphysiological mechanism and can produce different activation profiles than natural, monovalent, membrane-associated pMHC. Here, we introduce a strictly monovalent anti-TCRβ H57 Fab’ ligand that, when coupled to a supported lipid bilayer via DNA complementation, triggers TCRs and activates nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) with a similar potency to pMHC in primary murine T cells. Importantly, like monovalent pMHC and unlike bivalent antibodies, monovalent Fab’-DNA triggers TCRs only when physically coupled to the membrane, and only around 100 individual Fab’:TCR interactions are necessary to stimulate early T cell activation.

Preclinical Efficacy of a Trivalent Human FcγRI-Targeted Adjuvant-Free Subunit Mucosal Vaccine against Pulmonary Pneumococcal Infection.

Lack of safe and effective mucosal adjuvants has severely hampered the development of mucosal subunit vaccines. In this regard, we have previously shown that immunogenicity of vaccine antigens can be improved by targeting the antigens to the antigen-presenting cells. Specifically, groups of mice immunized intranasally with a fusion protein (Bivalent-FP) containing a fragment of pneumococcal-surface-protein-A (PspA) as antigen and a single-chain bivalent antibody raised against the anti-human Fc-gamma-receptor-I (hFcγRI) elicited protective immunity to pulmonary Streptococcus pneumoniae infection. In order to further enhance the immunogenicity, an additional hFcγRI-binding moiety of the single chain antibody was incorporated. The modified vaccine (Trivalent-FP) induced significantly improved protection against lethal pulmonary S. pneumoniae challenge compared to Bivalent-FP. In addition, the modified vaccine exhibited over 85% protection with only two immunizations. Trivalent-FP also induced S. pneumoniae-specific systemic and mucosal antibodies. Moreover, Trivalent-FP also induced IL-17- and IL-22-producing CD4+ T cells. Furthermore, it was found that the hFcγRI facilitated uptake and presentation of Trivalent-FP. In addition, Trivalent-FP also induced IL-1α, MIP-1α, and TNF-α; modulated recruitment of dendritic cells and macrophages; and induced CD80/86 and MHC-II expression on antigen presenting cells.

A bispecific IgG format containing four independent antigen binding sites

Bispecific antibodies come in many different formats, including the particularly interesting two-in-one antibodies, where one conventional IgG binds two different antigens. The IgG format allows these antibodies to mediate Fc-related functionality, and their wild-type structure ensures low immunogenicity and enables standard methods to be used for development. It is however difficult, time-consuming and costly to generate two-in-one antibodies. Herein we demonstrate a new approach to create a similar type of antibody by combining two different variable heavy (VH) domains in each Fab arm of an IgG, a tetra-VH IgG format. The VHs are used as building blocks, where one VH is placed at its usual position, and the second VH replaces the variable light (VL) domain in a conventional IgG. VH domains, binding several different types of antigens, were discovered and could be rearranged in any combination, offering a convenient “plug and play” format. The tetra-VH IgGs were found to be functionally tetravalent, binding two antigens on each arm of the IgG molecule simultaneously. This offers a new strategy to also create monospecific, tetravalent IgGs that, depending on antigen architecture and mode-of-action, may have enhanced efficacy compared to traditional bivalent antibodies.

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.