Broad Epitope Coverage of Therapeutic Multi-Antibody Combinations Targeting SARS-CoV-2 Boosts In Vivo Protection and Neutralization Potency to Corner an Immune-Evading Virus.

Therapeutic antibodies (Abs) which act on a broader range of epitopes may provide more durable protection against the genetic drift of a target, typical of viruses or tumors. When these Abs exist concurrently on the targeted antigen, several mechanisms of action (MoAs) can be engaged, boosting therapeutic potency. This study selected combinations of four and five Abs with non- or partially overlapping epitopes to the SARS-CoV-2 spike glycoprotein, on or outside the crucial receptor binding domain (RBD), to offer resilience to emerging variants and trigger multiple MoAs.

Development of [Zr]Zr-hCD103.Fab01A and [Ga]Ga-hCD103.Fab01A for PET imaging to noninvasively assess cancer reactive T cell infiltration: Fab-based CD103 immunoPET.

Background: CD103 is an integrin specifically expressed on the surface of cancer-reactive T cells. The number of CD103+ T cells significantly increases during successful immunotherapy and might therefore be an attractive biomarker for noninvasive PET imaging of immunotherapy response. Since the long half-life of antibodies preclude repeat imaging of CD103+ T cell dynamics early in therapy, we therefore here explored PET imaging with CD103 Fab fragments radiolabeled with a longer (Zr) and shorter-lived radionuclide (Ga).

Intratumoral injection of IL-12-encoding mRNA targeted to CSFR1 and PD-L1 exerts potent anti-tumor effects without substantial systemic exposure.

IL-12 is a potent cytokine for cancer immunotherapy. However, its systemic delivery as a recombinant protein has shown unacceptable toxicity in the clinic. Currently, the intratumoral injection of IL-12-encoding mRNA or DNA to avoid such side effects is being evaluated in clinical trials.

Development of Zr-anti-CD103 PET imaging for non-invasive assessment of cancer reactive T cell infiltration.

Purpose: CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses.

Experimental Design: First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an Zr-anti-murine CD103 PET tracer.

Generation and characterization of novel co-stimulatory anti-mouse TNFR2 antibodies.

Tumor necrosis factor receptor 2 (TNFR2) has gained much research interest in recent years because of its potential pivotal role in autoimmune disease and cancer. However, its function in regulating different immune cells is not well understood. There is a need for well-characterized reagents to selectively modulate TNFR2 function, thereby enabling definition of TNFR2-dependent biology in human and mouse surrogate models.

Determinants of Ligand-Functionalized DNA Nanostructure-Cell Interactions.

Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnology has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential.

Purification of murine immunoglobulin E (IgE) by thiophilic interaction chromatography (TIC).

In addition to their known implication in allergy studies, IgE antibodies are becoming an increasingly interesting antibody class in cancer research. However, large-scale purification of IgE antibodies still poses substantial challenges, as they cannot be purified using techniques commonly used for other immunoglobulins such as protein A or protein G chromatography. Here, we have developed and optimised a gentle and simple IgE purification method based on thiophilic interaction chromatography (TIC).

Bispecific antibodies targeting dual tumor-associated antigens in cancer therapy.

Purpose: Bispecific antibodies (BsAbs) have emerged as a leading drug class for cancer therapy and are becoming increasingly of interest for therapeutic applications. As of April 2020, over 123 BsAbs are under clinical evaluation for use in oncology (including the two marketed BsAbs Blinatumomab and Catumaxomab). The majority (82 of 123) of BsAbs under clinical evaluation can be categorized as bispecific immune cell engager whereas a second less well-discussed subclass of BsAbs targets two tumor-associated antigens (TAAs).

A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing.

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly.

Functional characterization of the selective pan-allele anti-SIRPα antibody ADU-1805 that blocks the SIRPα-CD47 innate immune checkpoint.

Background: Accumulating preclinical data indicate that targeting the SIRPα/CD47 axis alone or in combination with existing targeted therapies or immune checkpoint inhibitors enhances tumor rejection. Although several CD47-targeting agents are currently in phase I clinical trials and demonstrate activity in combination therapy, high and frequent dosing was required and safety signals (acute anemia, thrombocytopenia) were recorded frequently as adverse events. Based on the restricted expression pattern of SIRPα we hypothesized that antibodies targeting SIRPα might avoid some of the concerns noted for CD47-targeting agents.

The effect of subcellular localization on the efficiency of EGFR-targeted VHH photosensitizer conjugates.

Photodynamic therapy (PDT) is an emerging method to treat light-accessible malignancies. To increase specificity and allow dose reduction, conjugates of photosensitizers (PS) with antibodies against tumor-associated antigens have been developed for photoimmunotherapy (PIT). However, so far it is unclear whether cellular internalization of these conjugates after binding affects PIT efficacy.

A Conjugate of an Anti-Epidermal Growth Factor Receptor (EGFR) VHH and a Cell-Penetrating Peptide Drives Receptor Internalization and Blocks EGFR Activation.

Overexpression of (mutated) receptor tyrosine kinases is a characteristic of many aggressive tumors, and induction of receptor uptake has long been recognized as a therapeutic modality. A conjugate of a synthetically produced cell-penetrating peptide (CPP), corresponding to amino acids 38-59 of human lactoferrin, and the recombinant llama single-domain antibody (VHH) 7D12, which binds the human epidermal growth factor receptor (EGFR), was generated by sortase A mediated transpeptidation. The conjugate blocks EGF-mediated EGFR activation with higher efficacy than that of both modalities alone; a phenomenon that is caused by both effective receptor blockade and internalization.

Legomedicine-A Versatile Chemo-Enzymatic Approach for the Preparation of Targeted Dual-Labeled Llama Antibody-Nanoparticle Conjugates.

Conjugation of llama single domain antibody fragments (Variable Heavy chain domains of Heavy chain antibodies, VHHs) to diagnostic or therapeutic nanoparticles, peptides, proteins, or drugs offers many opportunities for optimized targeted cancer treatment. Currently, mostly nonspecific conjugation strategies or genetic fusions are used that may compromise VHH functionality. In this paper we present a versatile modular approach for bioorthogonal VHH modification and conjugation.

Targeting the extracellular matrix of ovarian cancer using functionalized, drug loaded lyophilisomes.

Epithelial ovarian cancer is characterized by a high mortality rate and is in need for novel therapeutic avenues to improve patient outcome. The tumor's extracellular matrix ("stroma") offers new possibilities for targeted drug-delivery. Recently we identified highly sulfated chondroitin sulfate (CS-E) as a component abundantly present in the ovarian cancer extracellular matrix, and as a novel target for anti-cancer therapy.

Triggered Drug Release from an Antibody-Drug Conjugate Using Fast "Click-to-Release" Chemistry in Mice.

The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice.

Diabody Pretargeting with Click Chemistry In Vivo.

Unlabelled: Radioimmunotherapy and nuclear imaging (immuno-PET/SPECT) of cancer with radiometal-labeled antibody fragments or peptides is hampered by low tumor-to-kidney ratios because of high renal radiometal retention. Therefore, we developed and evaluated a pretargeting strategy using click chemistry in vivo to reduce kidney uptake and avoid unwanted radiation toxicity. We focused on the bioorthogonal reaction between a trans-cyclooctene (TCO)-functionalized TAG72 targeting diabody, AVP04-07, and a low-molecular-weight radiolabeled tetrazine probe that was previously shown to have low kidney retention and relatively fast renal clearance.

Development of Radiolabeled Membrane Type-1 Matrix Metalloproteinase Activatable Cell Penetrating Peptide Imaging Probes.

Membrane type-1 matrix metalloproteinase (MT1-MMP or MMP-14) plays an important role in adverse cardiac remodelling. Here, we aimed to develop radiolabeled activatable cell penetrating peptides (ACPP) sensitive to MT1-MMP for the detection of elevated MT1-MMP levels in adverse cardiac remodelling. Three ACPP analogs were synthesized and the most potent ACPP analog was selected using MT1-MMP sensitivity and enzyme specificity assays.

Bioresponsive probes for molecular imaging: concepts and in vivo applications.

Molecular imaging is a powerful tool to visualize and characterize biological processes at the cellular and molecular level in vivo. In most molecular imaging approaches, probes are used to bind to disease-specific biomarkers highlighting disease target sites. In recent years, a new subset of molecular imaging probes, known as bioresponsive molecular probes, has been developed.

Identification of a novel MET mutation in high-grade glioma resulting in an auto-active intracellular protein.

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas.

Trans-cyclooctene tag with improved properties for tumor pretargeting with the diels-alder reaction.

Radioimmunotherapy (RIT) of solid tumors is hampered by low tumor-to-nontumor (T/NT) ratios of the radiolabeled monoclonal antibodies resulting in low tumor doses in patients. Pretargeting technologies can improve the effectiveness of RIT in cancer therapy by increasing this ratio. We showed that a pretargeting strategy employing in vivo chemistry in combination with clearing agents, proceeds efficiently in tumor-bearing mice resulting in high T/NT ratios.

In vivo biodistribution of radiolabeled MMP-2/9 activatable cell-penetrating peptide probes in tumor-bearing mice.

Matrix metalloproteinases (MMPs) play a pivotal role in cancer progression and present therefore an interesting biomarker for early diagnosis, staging and therapy evaluation. Consequently, MMP-specific molecular imaging probes have been proposed for noninvasive visualization and quantification of MMP activity. An interesting approach is MMP-2/9 activatable cell-penetrating peptides (ACPP) that accumulate in the tumor tissue after activation.

Imaging of MMP activity in postischemic cardiac remodeling using radiolabeled MMP-2/9 activatable peptide probes.

The noninvasive imaging of matrix metalloproteinases (MMPs) activity in postischemic myocardial tissue holds great promise to predict cardiac function post-myocardial infarction. Consequently, development of MMP specific molecular imaging probes for noninvasive visualization and quantification of MMP activity is of great interest. A novel MMP imaging strategy is based on activatable cell-penetrating peptide probes (ACPP) that are sensitive to the proteolytic activity of MMP-2 and -9.

Evaluation of 111In-labeled EPep and FibPep as tracers for fibrin SPECT imaging.

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging.

SPECT imaging of fibrin using fibrin-binding peptides.

Noninvasive detection of fibrin in vivo using diagnostic imaging modalities may improve clinical decision-making on possible therapeutic options in atherosclerosis, cancer and thrombus-related pathologies such as pulmonary embolism and deep venous thrombosis. The aim of this study was to assess the potential of a novel (111)In-labeled fibrin-binding peptide (FibPep) to visualize thrombi in mice noninvasively using single-photon emission computed tomography (SPECT). FibPep and a negative control peptide (NCFibPep) were synthesized and their fibrin-binding properties were assessed in vitro.