Bivalent Constructs Research Articles

206 Evaluation of the effect of oral administration of bivalent VHH constructs in weaned piglets challenged with enterotoxigenic Escherichia coli F4+

Abstract Following the European restriction on the use of antibiotics and zinc oxide (ZnO) in livestock, the search for novel and sustainable strategies to reduce the incidence of enteric infections in piglets caused by Escherichia coli (colibacillosis) has increased. Among them, the ongoing development of bivalent VHH constructs designed to bind toxins and/or virulence factors of pathogens, appears to hold potential. The aim of this study was to evaluate the effect of in-feed administration of bivalent VHH constructs designed to bind to heat-labile enterotoxin (LT) and F4 fimbria of enterotoxigenic E. coli (ETEC) in orally challenged piglets. A total of 96, 21-d old, weaned piglets were assigned to three experimental diets: control diet (CTR); diet supplemented with 3,100 ppm ZnO (ZnO); or diet supplemented with 1.71 kg/MT bivalent VHH constructs (VHH). Animals on the VHH treatment also received an oral booster dose (15 mgּ pig-1ּ d-1) of the same product just before the challenge and one d later. Pigs were orally challenged with ETEC F4+ (4.62×109 colony forming unit/animal), 1 wk after weaning (d 28). Data on growth, feed intake and clinical health signs of the animals were recorded throughout the 15 d of the study. On d 4 and 8 post inoculation (PI), fecal samples, digestive contents, and ileal scrapings were taken for microbiological plate counts of lactobacilli and ETEC in selective media. Ileal tissue samples were also taken for histomorphology. After the challenge, animals in the supplemented groups (ZnO and VHH) showed statistically greater growth rates than the CTR (28, 112 and 106 g/d (0-4 PI period) for CTR, ZnO and VHH, respectively, P = 0.018; Table 1). The experimental ZnO and VHH groups showed numerical reductions in the incidence of diarrhea at d 2 PI (60, 29 and 40 % for CTR, ZnO and VHH, P = 0.107), in agreement with reduced fecal ETEC counts also registered at d 8 PI in the VHH group compared with CTR (5.77 vs. 7.21 cfu/g, P = 0.046), and also greater lactobacilli/ETEC ratio (d 8 PI: 1.52 vs. 1.20, P = 0.071). Histomorphology of ileal samples showed significantly greater numbers of intraepithelial lymphocytes cells (IEL) with VHH supplementation compared with ZnO, with CTR showing intermediate values (8.06, 5.80 and 9.46 cell nº/villi for CTR, ZnO and VHH respectively, P = 0.045). The results from this study suggest that bivalent VHH constructs can reduce the negative effects of an oral ETEC F4+ challenge through reducing their growth in the gut, improving intestinal mucosa architecture and function, and increasing piglet growth rate. With results similar to those observed in pigs supplemented with ZnO, bivalent VHH constructs appear as a promising alternative technology for the management of post-weaning colibacillosis.

Abstract 3109: Development of novel mono and bivalent nanobodies against EGFR for targeted cancer therapy

Abstract Introduction: The epidermal growth factor receptor (EGFR) plays a central role in the tumorigenesis of various solid tumors, rendering it a noteworthy target for cancer therapy. Nanobodies (Nbs) offer an appealing therapeutic approach due to their low immunogenicity, rapid clearance, and high targeting specificity. The aim of this study is to isolate, design, and characterize Nbs directed against EGFR. Methods: The Nb library was constructed following llama immunization with cells overexpressing EGFR. After three rounds of panning the phage-display library (both in solid phase and solution), individual clones were screened for EGFR reactivity using ELISA against Virus-Like Particles. Positive clones were sequenced, cloned into an expression vector, and then purified by immobilized metal affinity chromatography (IMAC). The Nbs were characterized through immunofluorescence (IF), flow cytometry (FACS), and surface plasmon resonance (SPR). The most promising candidates were used to design bivalent constructs, which were subsequently characterized by SPR and in a cell proliferation assay. Results: We successfully constructed an EGFR-targeted Nb library, which allowed the selection of multiple EGFR-specific Nbs following the optimization of the panning strategy. Two of the candidates exhibit strong binding to native EGFR in cells and outperform the gold standard anti-EGFR Nb 7D12. They demonstrate enhanced binding parameters in surface plasmon resonance (SPR) assays, with fast association and slow dissociation rates. The homo/heterodimeric bivalent constructs show improved affinity and the ability to inhibit cancer cell proliferation. Conclusion: We were able to develop novel mono and bivalent nanobodies with enhanced binding properties which can be used for targeted cancer therapy. Citation Format: Diana C. Aguilar Cortes, Mauro O. Heitrich, Marisa M. Fernandez, Santiago Werbajh, Gabriela Canziani, Vanesa Zylberman, Emilio L. Malchiod, Osvaldo L. Podhajcer, Sabrina E. Vinzón. Development of novel mono and bivalent nanobodies against EGFR for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3109.

Affinity enhancement of polo-like kinase 1 polo box domain-binding ligands by a bivalent approach using a covalent kinase-binding component.

The polo-like kinase 1 (Plk1) is an important cell cycle regulator that is recognized as a target molecule for development of anti-cancer agents. Plk1 consists of a catalytic kinase domain (KD) and a polo-box domain (PBD), which engages in protein-protein interactions (PPIs) essential to proper Plk1 function. Recently, we developed extremely high-affinity PBD-binding inhibitors based on a bivalent approach using the Plk1 KD-binding inhibitor, BI2536, and a PBD-binding peptide. Certain of the resulting bivalent constructs exhibited more than 100-fold Plk1 affinity enhancement relative to the best monovalent PBD-binding ligands. Herein, we report an extensive investigation of bivalent ligands that utilize the non-selective kinase inhibitor Wortmannin as a Plk1 KD-binding component. We found that bivalent ligands incorporating Wortmannin demonstrated affinity enhancements that could be similar to what we had obtained with BI2536 and that they could tightly bind to the protein. This suggests that these tight binding ligands might be useful for structural analysis of full-length Plk1.

Abstract 2891: Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody

Abstract Intracellular tumor antigens presented as peptides on MHC (pMHC) class I molecules are attractive targets for more tumor-selective immunotherapeutic approaches with promising data already emerging from clinical trials. pMHCs have been targeted by TCR-engineered T cells or soluble recombinant T-cell receptors (TCRs) fused to an anti-CD3 fragment. Naturally occurring cancer-reactive TCRs have weak affinity and require substantial affinity enhancements for their cognate pMHC. However, the outcome of this process is difficult to predict and bears the risk for off-target cross reactivities in normal tissues, which may lead to severe adverse events in the clinic. Here, we describe a highly potent dual pMHC T-cell engager (TCE) antibody format with high specificity towards tumor-specific pMHCs utilizing an HLA-A*02:01 restricted MAGE-A4 epitope to validate the concept. A series of monovalent and bivalent antibody constructs composed of anti-MAGE-A4 binding arms, ranging in affinities from 30 nM to 100 pM, were fused to an anti-CD3 Fab fragment with lower affinity compared to that commonly used for TCR-fusions. The different antibody constructs were evaluated for selective killing of MAGE-A4/HLA-A*02 positive human U2OS osteosarcoma and A375 melanoma cancer cells versus a panel of different MAGE-A4-negative/HLA-A*02-positive human cell lines. Bivalent bispecific antibody variants mediated a 7-fold greater degree of cancer cell killing and similarly increased T cell activation compared to their monovalent bispecific counterparts. EC50 values ranged as low as single digit picomolar, while the overall cross reactivity against MAGE-A4-negative/HLA-A*02-positive cells was not substantially affected. These results prove that dual targeting of pMHCs on cancer cells provides selective and efficient T cell-mediated target cell killing and T cell activation, even at very low levels of pMHC on the cell surface, highlighting the pivotal roles played by the affinity of the individual arms, valency, and epitope densities. We further analyzed the dual pMHC TCE for potential off-target effects by recognition of similar and physiologically relevant non-MAGE-A4 peptides. T2 cells pulsed with similar peptides and co-cultured with PBMC effector cells showed no significant T cell activation or IFNg release in the presence of the dual pMHC TCE in comparison to MAGE-A4 peptide-pulsed T2 cells. Finally, we compared the dual pMHC TCE against a recombinant TCR fused to an anti-CD3 scFv, a construct that is currently in clinical development. Interestingly, the dual pMHC TCE resulted in a 3-fold more potent cancer cell killing while having significantly lower effect on cytokine production. In conclusion, dual pMHC targeting with TCE antibodies is an attractive alternative to soluble affinity-enhanced TCR-based cancer immunotherapies as they facilitate potent tumor targeting without the need for extensive affinity enhancements. Citation Format: Stephanie Jungmichel, Fabian Scheifele, Anna Sobieraj, Severin Wendelspiess, Thomas Schleier, Philip Knobel, Camilla Winnewisser, Melissa Vrohlings, Philipp Richle, Hannes Merten, Jacqueline Blunschi, Christian Leisner, Leonardo Borras. Enhanced anti-tumor responses with a novel dual pMHC T-cell engager bispecific antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2891.

Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis

Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two NaV1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade

Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.

Selective targeting of HER2-overexpressing solid tumors with a next-generation CAR-T cell therapy.

3041 Background: Conventional chimeric antigen receptor T cell (CAR-T) therapies have achieved limited clinical success in the treatment of solid tumors, in part due to the challenges of identifying tumor antigen(s) that are uniquely expressed on tumor cells. The dearth of such targets requires that current CAR-T therapies be re-engineered to preferentially target tumor cells thereby mitigating potential on-target off-tumor toxicity to normal cells. Herein we describe a novel cell therapy platform comprising Antigen Receptor Complex T (ARC-T) cells that are readily activated, silenced, and reprogrammed in vivo by administration of a novel tumor-targeting soluble protein antigen-receptor X-linker (sparX). The formation of the ARC-T, sparX, and tumor complex is required for the ARC-T to kill the tumor. Because ARC-T activity is entirely dependent on the dose of sparX administered, therapeutic doses of sparX may be defined that preferentially target cells over-expressing a target antigen and thus limit coincident kill of normal cells expressing lower levels of target antigen. Methods: We have created a library of sparX that bind different cell surface antigens, including HER2. The HER2 sparX was tested as both monovalent and bivalent constructs in vitro by assessing ARC-T cell activation, cytokine release and target cell cytotoxicity. In vivo efficacy models utilized NSG mice and incorporated tumor volume measurements and histopathologic assessments to evaluate tumor clearance. Results: In vitro studies demonstrate that co-culture of ARC-T cells, sparX-HER2 and HER2-expressing target cells drives T cell activation, expansion, cytokine secretion and cytotoxicity of target cells in a dose-dependent manner. Furthermore, by affinity tuning the HER2 binding domain and bivalent formatting of sparX-HER2, we achieved selective killing of HER2-overexpressing breast cancer cells with minimal effect on cells expressing HER2 levels representative of normal tissues. In vivo proof-of-principal studies with ARC-T/sparX-HER2 similarly demonstrate complete eradication of HER2-overexpressing solid tumor cells. Conclusions: These results demonstrate that a single intravenous dose of ARC-T cells can traffic to a solid tumor site and induce tumor eradication upon systemic administration and co-localization of tumor-targeting sparX in a mouse model. Bivalent formatting of sparX-HER2 further enabled ARC-T sensitivity to target antigen density to avoid the on-target off-tumor toxicity that has hindered conventional monovalent CAR-T treatments.

Identification of New DR5 Agonistic Nanobodies and Generation of Multivalent Nanobody Constructs for Cancer Treatment.

Current cancer therapeutics suffer from a lack of specificity in targeting tumor cells and cause severe side effects. Therefore, the design of highly specialized drugs comprising antibody derivatives inducing apoptosis in targeted cancer cells is considered to be a promising strategy. Drugs acting on death receptor 5 (DR5) such as DR5 agonist antibodies replacing “TNF-related apoptosis-inducing ligand” (TRAIL) offer feasible opportunities in this direction. Although such agonists provided good antitumor activity in preclinical studies, they were less effective in clinical studies, possibly due to a disturbed Fc interaction with Fc-γ receptors. Thus, multimerized antigen binding fragments without Fc have been proposed to increase their efficacy. We generated nanobodies (Nbs), recombinant variable domains of heavy chain-only antibodies of camelids, against the DR5 ectodomain. Nb24 and Nb28 had an affinity in the nM and sub-nM range, but only Nb28 competes with TRAIL for binding to DR5. Bivalent, trivalent, and tetravalent constructs were generated, as well as an innovative pentameric Nb complex, to provoke avidity effects. In our cellular assays, these trimeric, tetrameric, and pentameric Nbs have a higher apoptotic capacity than monomeric Nbs and seem to mimic the activity of the natural TRAIL ligand on various cancer cells.

Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22

Despite high remission rates following CAR-T cell therapy in B-ALL, relapse due to loss of the targeted antigen is increasingly recognized as a mechanism of immune escape. We hypothesized that simultaneous targeting of CD19 and CD22 may reduce the likelihood of antigen loss, thus improving sustained remission rates. A systematic approach to the generation of CAR constructs incorporating two target-binding domains led to several novel CD19/CD22 bivalent CAR constructs. Importantly, we demonstrate the challenges associated with the construction of a bivalent CAR format that preserves bifunctionality against both CD19 and CD22. Using the most active bivalent CAR constructs, we found similar transduction efficiency compared to that of either CD19 or CD22 single CARs alone. When expressed on human T cells, the optimized CD19/CD22 CAR construct induced comparable interferon γ and interleukin-2 in vitro compared to single CARs against dual-antigen-expressing as well as single-antigen-expressing cell lines. Finally, the T cells expressing CD19/CD22 CAR eradicated ALL cell line xenografts and patient-derived xenografts (PDX), including a PDX generated from a patient with CD19− relapse following CD19-directed CAR therapy. The CD19/CD22 bivalent CAR provides an opportunity to test whether simultaneous targeting may reduce risk of antigen loss.

Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions.

The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™) were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3) were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

High affinity targeting of CD23 inhibits IgE synthesis in human B cells.

The low-affinity IgE receptor FcϵRII (CD23) is part of the regulatory system controlling IgE synthesis in human B cells and exists in membrane and soluble forms. Binding of IgE to CD23 has been described to have stabilizing effects and to prevent cleavage of CD23. Previous experiments using anti-CD23 antibodies reduced IgE synthesis but were difficult to interpret as the antibody Fc part might also mediate feedback mechanisms. The purpose of this study was to investigate the regulatory role of CD23, by using designed ankyrin repeat proteins (DARPins) that specifically recognize CD23. Anti-CD23 DARPins were isolated by ribosome display and were produced as monovalent and bivalent constructs. Affinities to CD23 were measured by surface plasmon resonance. IgE synthesis and up-regulation of CD23 in human peripheral B cells were induced by IL-4 and anti-CD40 antibody. We assessed CD23 expression and its stabilization by FACS and used an ELISA for detecting soluble CD23. IgE synthesis was measured by ELISA and real-time PCR. Surface plasmon resonance revealed affinities of the DARPins to CD23 in the pico-molar range. Anti-CD23 DARPins strongly inhibited binding of IgE to CD23 and share thus a similar binding epitope as IgE. The DARPins stabilized membrane CD23 and reduced IgE synthesis in an isotype specific manner. Furthermore, the anti-CD23 DARPins decreased IgE transcript through inhibition of mature Cϵ RNA synthesis suggesting a posttranscriptional control mechanism. This study demonstrates that targeting CD23 alone is sufficient to inhibit IgE synthesis and suggests that a negative signaling occurs directly through the CD23 molecule.

Protective effect of different anti-rabies virus VHH constructs against rabies disease in mice.

Rabies virus causes lethal brain infection in about 61000 people per year. Each year, tens of thousands of people receive anti-rabies prophylaxis with plasma-derived immunoglobulins and vaccine soon after exposure. Anti-rabies immunoglobulins are however expensive and have limited availability. VHH are the smallest antigen-binding functional fragments of camelid heavy chain antibodies, also called Nanobodies. The therapeutic potential of anti-rabies VHH was examined in a mouse model using intranasal challenge with a lethal dose of rabies virus. Anti-rabies VHH were administered directly into the brain or systemically, by intraperitoneal injection, 24 hours after virus challenge. Anti-rabies VHH were able to significantly prolong survival or even completely rescue mice from disease. The therapeutic effect depended on the dose, affinity and brain and plasma half-life of the VHH construct. Increasing the affinity by combining two VHH with a glycine-serine linker into bivalent or biparatopic constructs, increased the neutralizing potency to the picomolar range. Upon direct intracerebral administration, a dose as low as 33 µg of the biparatopic Rab-E8/H7 was still able to establish an anti-rabies effect. The effect of systemic treatment was significantly improved by increasing the half-life of Rab-E8/H7 through linkage with a third VHH targeted against albumin. Intraperitoneal treatment with 1.5 mg (2505 IU, 1 ml) of anti-albumin Rab-E8/H7 prolonged the median survival time from 9 to 15 days and completely rescued 43% of mice. For comparison, intraperitoneal treatment with the highest available dose of human anti-rabies immunoglobulins (65 mg, 111 IU, 1 ml) only prolonged survival by 2 days, without rescue. Overall, the therapeutic benefit seemed well correlated with the time of brain exposure and the plasma half-life of the used VHH construct. These results, together with the ease-of-production and superior thermal stability, render anti-rabies VHH into valuable candidates for development of alternative post exposure treatment drugs against rabies.

Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25±10Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH2 was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-c[Asp-His-2′-D-Nal-Arg-Trp-Lys]-NH2 and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)3 units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.

Patent foramen ovale and cryptogenic stroke: many unanswered questions.

Patent foramen ovale (PFO) is associated with cryptogenic stroke, but uncertainty remains about the exact relationship and the best management. Percutaneous closure of PFO is safe and effective, but this procedure has yet to be definitely proven to be better than medical therapy. The scenario of PFO and cryptogenic stroke poses unique challenges to primary care physicians and subspecialists and requires an understanding of the relationship between cryptogenic stroke and PFO, and of current data on the safety, efficacy, and comparative effectiveness of management options.

Multivalent cell specific therapeutics for treatment of metabolic disorders (1108.1)

Metabolic Homeostasis is mediated via multi‐organ endocrine signaling. Because many systems are involved, therapeutics often have wide‐ranging side‐effects. We have shown therapeutic specificity can be achieved with agents that simultaneously bind two different receptors that, as a combination, are relatively unique to a given cell type. Bivalent ligands, that target cells within the homeostatic axis, were produced; melanocyte stimulating hormone linked to CCK (MSH/CCK), and Glucagon Like Peptide 1 (GLP‐1) linked to Glibenclamide (Glb, SUR1 antagonist). Binding analyses, assessed with cells expressing the complementary receptor pair, indicate an apparent Kd of ~5 nM for MSH/CCK, ~10nM for GLP‐1/Glb. Constants for the individual domains within the bivalent constructs were 30 nM or greater, and neither ligand bound with high affinity to cells expressing only one of the complementary receptors, indicating that high affinity binding requires both receptors. Once bound, the ligand/ receptor complex is rapidly endocytosed. Both ligands maintain signaling capacity, though the magnitude, sensitivity of 2nd messenger and physiological effects, differ from the constituent monomers. Uniquely, GLP‐1/Glb was found to potentiate insulin secretion at stimulatory glucose concentrations, but inhibit, rather than activate, at non‐stimulatory glucose. Thus, these bivalent ligands exhibit enhanced affinity, cell type specificity and maintain functional efficacy, and thereby hold promise as cell specific therapeutic agents.Grant Funding Source: Supported by: Juvenile Diabetes Research Foundation, National Institutes of Health, NCI, CA123547

The Effect of Molecular Weight, PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)2 (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)2versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)2 with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.

SNAP-tag based Agents for Preclinical In Vitro Imaging in Malignant Diseases

Although current cancer treatment strategies are highly aggressive, they are often not effective enough to destroy the collectivity of malignant cells. The residual tumor cells that survived the first-line treatment may continue to proliferate or even metastasize. Therefore, the development of novel more effective strategies to specifically eliminate also single cancer cells is urgently needed. In this respect, the development of antibody-based therapeutics, in particular example immunotoxins, has attracted broad interest. Since the internalization of immunotoxins is essential for their cytotoxic effectivity, it is of crucial importance to study their internalization behavior to assess the potential for their therapeutic use. In this study, we determined the internalization behavior of four different single-chain fragments variable (scFv) when binding to the corresponding target antigen as expressed on solid or non-solid tumor cell lines. The scFvs were recombinantly fused to the SNAP-tag, an engineered variant of the human repair enzyme O(6)-alkylguanine-DNA alkyltransferase that covalently reacts with benzylguanine derivatives. Since a large number of highly sensitive organic fluorescent dyes are already available or can easily be derivatized to react with the self-labeling SNAP-tag, this system provides versatile applications for imaging of intraand extracellular compartments of living cells. The fusion proteins were coupled to SNAP-surface(®) Alexa Fluor(®) 488 or SNAP-surface(®) Alexa Fluor(®) 647 and binding as well as internalization was monitored by flow cytometry and confocal microscopy, respectively. Depending on the respective target antigen, we could distinguish between slow and rapid internalization behavior. Moreover, we detected increased internalization rate for bivalent scFv constructs. Our approach allows for rapid and early stage evaluation of the internalization characteristics of new antibodies designated for further therapeutic development.

Llama-Derived Single Domain Antibodies to Build Multivalent, Superpotent and Broadened Neutralizing Anti-Viral Molecules

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5.The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve “best-in-class” and broader neutralization capacity.

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.