Scrutiny of chimeric antigen receptor activation by the extracellular domain: experience with single domain antibodies targeting multiple myeloma cells highlights the need for case-by-case optimization.

Introduction: Multiple myeloma (MM) remains incurable, despite the advent of chimeric antigen receptor (CAR)-T cell therapy. This unfulfilled potential can be attributed to two untackled issues: the lack of suitable CAR targets and formats. In relation to the former, the target should be highly expressed and reluctant to shedding; two characteristics that are attributed to the CS1-antigen. Furthermore, conventional CARs rely on scFvs for antigen recognition, yet this withholds disadvantages, mainly caused by the intrinsic instability of this format. VHHs have been proposed as valid scFv alternatives. We therefore intended to develop VHH-based CAR-T cells, targeting CS1, and to identify VHHs that induce optimal CAR-T cell activation together with the VHH parameters required to achieve this. Methods: CS1-specific VHHs were generated, identified and fully characterized, in vitro and in vivo. Next, they were incorporated into second-generation CARs that only differ in their antigen-binding moiety. Reporter T-cell lines were lentivirally transduced with the different VHH-CARs and CAR-T cell activation kinetics were evaluated side-by-side. Affinity, cell-binding capacity, epitope location, in vivo behavior, binding distance, and orientation of the CAR-T:MM cell interaction pair were investigated as predictive parameters for CAR-T cell activation. Results: Our data show that the VHHs affinity for its target antigen is relatively predictive for its in vivo tumor-tracing capacity, as tumor uptake generally decreased with decreasing affinity in an in vivo model of MM. This does not hold true for their CAR-T cell activation potential, as some intermediate affinity-binding VHHs proved surprisingly potent, while some higher affinity VHHs failed to induce equal levels of T-cell activation. This could not be attributed to cell-binding capacity, in vivo VHH behavior, epitope location, cell-to-cell distance or binding orientation. Hence, none of the investigated parameters proved to have significant predictive value for the extent of CAR-T cell activation. Conclusions: We gained insight into the predictive parameters of VHHs in the CAR-context using a VHH library against CS1, a highly relevant MM antigen. As none of the studied VHH parameters had predictive value, defining VHHs for optimal CAR-T cell activation remains bound to serendipity. These findings highlight the importance of screening multiple candidates.

Nanobody-based pannexin1 channel inhibitors reduce inflammation in acute liver injury.

BACKGROUND: The opening of pannexin1 channels is considered as a key event in inflammation. Pannexin1 channel-mediated release of adenosine triphosphate triggers inflammasome signaling and activation of immune cells. By doing so, pannexin1 channels play an important role in several inflammatory diseases. Although pannexin1 channel inhibition could represent a novel clinical strategy for treatment of inflammatory disorders, therapeutic pannexin1 channel targeting is impeded by the lack of specific, potent and/or in vivo-applicable inhibitors. The goal of this study is to generate nanobody-based inhibitors of pannexin1 channels. RESULTS: Pannexin1-targeting nanobodies were developed as potential new pannexin1 channel inhibitors. We identified 3 cross-reactive nanobodies that showed affinity for both murine and human pannexin1 proteins. Flow cytometry experiments revealed binding capacities in the nanomolar range. Moreover, the pannexin1-targeting nanobodies were found to block pannexin1 channel-mediated release of adenosine triphosphate. The pannexin1-targeting nanobodies were also demonstrated to display anti-inflammatory effects in vitro through reduction of interleukin 1 beta amounts. This anti-inflammatory outcome was reproduced in vivo using a human-relevant mouse model of acute liver disease relying on acetaminophen overdosing. More specifically, the pannexin1-targeting nanobodies lowered serum levels of inflammatory cytokines and diminished liver damage. These effects were linked with alteration of the expression of several NLRP3 inflammasome components. CONCLUSIONS: This study introduced for the first time specific, potent and in vivo-applicable nanobody-based inhibitors of pannexin1 channels. As demonstrated for the case of liver disease, the pannexin1-targeting nanobodies hold great promise as anti-inflammatory agents, yet this should be further tested for extrahepatic inflammatory disorders. Moreover, the pannexin1-targeting nanobodies represent novel tools for fundamental research regarding the role of pannexin1 channels in pathological and physiological processes.

Development and evaluation of nanobody tracers for noninvasive nuclear imaging of the immune-checkpoint TIGIT.

Introduction: T cell Ig and ITIM domain receptor (TIGIT) is a next-generation immune checkpoint predominantly expressed on activated T cells and NK cells, exhibiting an unfavorable prognostic association with various malignancies. Despite the emergence of multiple TIGIT-blocking agents entering clinical trials, only a fraction of patients responded positively to anti-TIGIT therapy. Consequently, an urgent demand arises for noninvasive techniques to quantify and monitor TIGIT expression, facilitating patient stratification and enhancing therapeutic outcomes. Small antigen binding moieties such as nanobodies, are promising candidates for such tracer development. Methods: We generated a panel of anti-human or anti-mouse TIGIT nanobodies from immunized llamas. In addition, we designed a single-chain variable fragment derived from the clinically tested monoclonal antibody Vibostolimab targeting TIGIT, and assessed its performance alongside the nanobodies. In vitro characterization studies were performed, including binding ability and affinity to cell expressed or recombinant TIGIT. After Technetium-99m labeling, the nanobodies and the single-chain variable fragment were evaluated in vivo for their ability to detect TIGIT expression using SPECT/CT imaging, followed by ex vivo biodistribution analysis. Results: Nine nanobodies were selected for binding to recombinant and cell expressed TIGIT with low sub-nanomolar affinities and are thermostable. A six-fold higher uptake in TIGIT-overexpressing tumor was demonstrated one hour post- injection with Technetium-99m labeled nanobodies compared to an irrelevant control nanobody. Though the single-chain variable fragment exhibited superior binding to TIGIT-expressing peripheral blood mononuclear cells in vitro, its in vivo behavior yielded lower tumor-to-background ratios at one hour post- injection, indicating that nanobodies are better suited for in vivo imaging than the single-chain variable fragment. Despite the good affinity, high specificity and on-target uptake in mice in this setting, imaging of TIGIT expression on tumor- infiltrating lymphocytes within MC38 tumors remained elusive. This is likely due to the low expression levels of TIGIT in this model. Discussion: The excellent affinity, high specificity and rapid on-target uptake in mice bearing TIGIT- overexpressing tumors showed the promising diagnostic potential of nanobodies to noninvasively image high TIGIT expression within the tumor. These findings hold promise for clinical translation to aid patient selection and improve therapy response.

Current status and future expectations of nanobodies in oncology trials.

INTRODUCTION: Monoclonal antibodies have revolutionized personalized medicine for cancer in recent decades. Despite their broad application in oncology, their large size and complexity may interfere with successful tumor targeting for certain applications of cancer diagnosis and therapy. Nanobodies have unique structural and pharmacological features compared to monoclonal antibodies and have successfully been used as complementary anti-cancer diagnostic and/or therapeutic tools. AREAS COVERED: Here, an overview is given of the nanobody-based diagnostics and therapeutics that have been or are currently being tested in oncological clinical trials. Furthermore, preclinical developments, which are likely to be translated into the clinic in the near future, are highlighted. EXPERT OPINION: Overall, the presented studies show the application potential of nanobodies in the field of oncology, making it likely that more nanobodies will be clinically approved in the upcoming future.

A few good reasons to use nanobodies for cancer treatment.

mAbs have been instrumental for targeted cancer therapies. However, their relatively large size and physicochemical properties result in a heterogenous distribution in the tumor microenvironment, usually restricted to the first cell layers surrounding blood vessels, and a limited ability to penetrate the brain. Nanobodies are tenfold smaller, resulting in a deeper tumor penetration and the ability to reach cells in poorly perfused tumor areas. Nanobodies are rapidly cleared from the circulation, which generates a fast target-to-background contrast that is ideally suited for molecular imaging purposes but may be less optimal for therapy. To circumvent this problem, nanobodies have been formatted to noncovalently bind albumin, increasing their serum half-life without majorly increasing their size. Finally, nanobodies have shown superior qualities to infiltrate brain tumors as compared to mAbs. In this review, we discuss why these features make nanobodies prime candidates for targeted therapy of cancer.

Optimizing the Safety and Efficacy of Bio-Radiopharmaceuticals for Cancer Therapy.

The precise delivery of cytotoxic radiation to cancer cells through the combination of a specific targeting vector with a radionuclide for targeted radionuclide therapy (TRT) has proven valuable for cancer care. TRT is increasingly being considered a relevant treatment method in fighting micro-metastases in the case of relapsed and disseminated disease. While antibodies were the first vectors applied in TRT, increasing research data has cited antibody fragments and peptides with superior properties and thus a growing interest in application. As further studies are completed and the need for novel radiopharmaceuticals nurtures, rigorous considerations in the design, laboratory analysis, pre-clinical evaluation, and clinical translation must be considered to ensure improved safety and effectiveness. Here, we assess the status and recent development of biological-based radiopharmaceuticals, with a focus on peptides and antibody fragments. Challenges in radiopharmaceutical design range from target selection, vector design, choice of radionuclides and associated radiochemistry. Dosimetry estimation, and the assessment of mechanisms to increase tumor uptake while reducing off-target exposure are discussed.

Emerging applications of nanobodies in cancer therapy.

Cancer is a heterogeneous disease, requiring treatment tailored to the unique phenotype of the patient's tumor. Monoclonal antibodies (mAbs) and variants thereof have enabled targeted therapies to selectively target cancer cells. Cancer cell-specific mAbs have been used for image-guided surgery and targeted delivery of radionuclides or toxic agents, improving classical treatment strategies. Cancer cell-specific mAbs can further inhibit tumor cell growth or can stimulate immune-mediated destruction of cancer cells, a feature that has also been achieved through mAb-mediated manipulation of immune cells and pathways. Drawbacks of mAbs and their variants, together with the discovery of camelid heavy chain-only antibodies and the many advantageous features of their variable domains, referred to as VHHs, single domain antibodies or nanobodies (Nbs), resulted in the exploration of Nbs as an alternative targeting moiety. We therefore review the state-of-the-art as well as novel exploitation strategies of Nbs for targeted cancer therapy.

In Vitro Assessment of Binding Affinity, Selectivity, Uptake, Intracellular Degradation, and Toxicity of Nanobody-Photosensitizer Conjugates.

Photosensitizers have recently been conjugated to nanobodies for targeted photodynamic therapy (PDT) to selectively kill cancer cells. The success of this approach relies on nanobody-photosensitizer conjugates that bind specifically to their targets with very high affinities (kD in low nM range). Subsequently, upon illumination, these conjugates are very toxic and selective to cells overexpressing the target of interest (EC50 in low nM range). In this chapter, protocols are described to determine the binding affinity of the nanobody-photosensitizer conjugates and assess the toxicity and selectivity of the conjugates when performing in vitro PDT studies. In addition, and because the efficacy of PDT also depends on the (subcellular) localization of the conjugates at the time of illumination, assays are described to investigate the uptake and the intracellular degradation of the nanobody-photosensitizer conjugates.

Imaging and therapeutic targeting of the tumor immune microenvironment with biologics.

The important role of tumor microenvironmental elements in determining tumor progression and metastasis has been firmly established. In particular, the presence and activity profile of tumor-infiltrating immune cells may be associated with the outcome of the disease and may predict responsiveness to (immuno)therapy. Indeed, while some immune cell types, such as macrophages, support cancer cell outgrowth and mediate therapy resistance, the presence of activated CD8+ T cells is usually indicative of a better prognosis. It is therefore of the utmost interest to obtain a full picture of the immune infiltrate in tumors, either as a prognostic test, as a way to stratify patients to maximize therapeutic success, or as therapy follow-up. Hence, the non-invasive imaging of these cells is highly warranted, with biologics being prime candidates to achieve this goal.

Selective targeting of ligand-dependent and -independent signaling by GPCR conformation-specific anti-US28 intrabodies.

While various GPCRs, including US28, display constitutive, ligand-independent activity, it remains to be established whether ligand-dependent and -independent active conformations differ and can be selectively modulated. Previously, the agonist-bound conformation of US28 was stabilized and its structure was solved using the anti-US28 nanobody Nb7. Here we report the recognition of the constitutively active, apo-conformation of US28 by another nanobody VUN103. While the Nb7 intrabody selectively inhibits ligand-induced signaling, the VUN103 intrabody blocks constitutive signaling, indicating the existence of distinct US28 conformational states. By displacing Gαq protein, VUN103 prevents US28 signaling and reduces tumor spheroids growth. Overall, nanobodies specific for distinct GPCR conformational states, i.e. apo- and agonist-bound, can selectively target and discern functional consequences of ligand-dependent versus independent signaling.

Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies.

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

Mechanisms Underlying Connexin Hemichannel Activation in Disease.

Gap junctions and connexin hemichannels mediate intercellular and extracellular communication, respectively. While gap junctions are seen as the "good guys" by controlling homeostasis, connexin hemichannels are considered as the "bad guys", as their activation is associated with the onset and dissemination of disease. Open connexin hemichannels indeed mediate the transport of messengers between the cytosol and extracellular environment and, by doing so, fuel inflammation and cell death in a plethora of diseases. The present mini-review discusses the mechanisms involved in the activation of connexin hemichannels during pathology.

Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.

Herpesviruses are ubiquitous pathogens that establish lifelong, latent infections in their host. Spontaneous reactivation of herpesviruses is often asymptomatic or clinically manageable in healthy individuals, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with multiple proliferative cardiovascular and post-transplant diseases. Herpesviruses encode viral G protein-coupled receptors (vGPCRs) that alter the host cell by hijacking cellular pathways and play important roles in the viral life cycle and these different disease settings. In this review, we discuss the pharmacological and signaling properties of these vGPCRs, their role in the viral life cycle, and their contribution in different diseases. Because of their prominent role, vGPCRs have emerged as promising drug targets, and the potential of vGPCR-targeting therapeutics is being explored. Overall, these vGPCRs can be considered as attractive targets moving forward in the development of antiviral, cancer, and/or cardiovascular disease treatments. SIGNIFICANCE STATEMENT: In the last decade, herpesvirus-encoded G protein-coupled receptors (GPCRs) have emerged as interesting drug targets with the growing understanding of their critical role in the viral life cycle and in different disease settings. This review presents the pharmacological properties of these viral receptors, their role in the viral life cycle and different diseases, and the emergence of therapeutics targeting viral GPCRs.

Therapeutic Nanobodies Targeting Cell Plasma Membrane Transport Proteins: A High-Risk/High-Gain Endeavor.

Cell plasma membrane proteins are considered as gatekeepers of the cell and play a major role in regulating various processes. Transport proteins constitute a subclass of cell plasma membrane proteins enabling the exchange of molecules and ions between the extracellular environment and the cytosol. A plethora of human pathologies are associated with the altered expression or dysfunction of cell plasma membrane transport proteins, making them interesting therapeutic drug targets. However, the search for therapeutics is challenging, since many drug candidates targeting cell plasma membrane proteins fail in (pre)clinical testing due to inadequate selectivity, specificity, potency or stability. These latter characteristics are met by nanobodies, which potentially renders them eligible therapeutics targeting cell plasma membrane proteins. Therefore, a therapeutic nanobody-based strategy seems a valid approach to target and modulate the activity of cell plasma membrane transport proteins. This review paper focuses on methodologies to generate cell plasma membrane transport protein-targeting nanobodies, and the advantages and pitfalls while generating these small antibody-derivatives, and discusses several therapeutic nanobodies directed towards transmembrane proteins, including channels and pores, adenosine triphosphate-powered pumps and porters.

Antibodies Targeting Chemokine Receptors CXCR4 and ACKR3.

Dysregulation of the chemokine system is implicated in a number of autoimmune and inflammatory diseases, as well as cancer. Modulation of chemokine receptor function is a very promising approach for therapeutic intervention. Despite interest from academic groups and pharmaceutical companies, there are currently few approved medicines targeting chemokine receptors. Monoclonal antibodies (mAbs) and antibody-based molecules have been successfully applied in the clinical therapy of cancer and represent a potential new class of therapeutics targeting chemokine receptors belonging to the class of G protein-coupled receptors (GPCRs). Besides conventional mAbs, single-domain antibodies and antibody scaffolds are also gaining attention as promising therapeutics. In this review, we provide an extensive overview of mAbs, single-domain antibodies, and other antibody fragments targeting CXCR4 and ACKR3, formerly referred to as CXCR7. We discuss their unique properties and advantages over small-molecule compounds, and also refer to the molecules in preclinical and clinical development. We focus on single-domain antibodies and scaffolds and their utilization in GPCR research. Additionally, structural analysis of antibody binding to CXCR4 is discussed. SIGNIFICANCE STATEMENT: Modulating the function of GPCRs, and particularly chemokine receptors, draws high interest. A comprehensive review is provided for monoclonal antibodies, antibody fragments, and variants directed at CXCR4 and ACKR3. Their advantageous functional properties, versatile applications as research tools, and use in the clinic are discussed.

Nanobody-Targeted Photodynamic Therapy Selectively Kills Viral GPCR-Expressing Glioblastoma Cells.

Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with near-infrared light. One of the aspects hampering the clinical use of PDT is the poor selectivity of the photosensitizer. To improve this, we have recently introduced a new approach for targeted PDT by conjugating photosensitizers to nanobodies. Diverse G protein-coupled receptors (GPCRs) show aberrant overexpression in tumors and are therefore interesting targets in cancer therapy. Here we show that GPCR-targeting nanobodies can be used in targeted PDT. We have developed a nanobody binding the extracellular side of the viral GPCR US28, which is detected in tumors like glioblastoma. The nanobody was site-directionally conjugated to the water-soluble photosensitizer IRDye700DX. This nanobody-photosensitizer conjugate selectively killed US28-expressing glioblastoma cells both in 2D and 3D cultures upon illumination with near-infrared light. This is the first example employing a GPCR as target for nanobody-directed PDT. With the emerging role of GPCRs in cancer, this data provides a new angle for exploiting this large family of receptors for targeted therapies.

Nanobodies detecting and modulating GPCRs outside in and inside out.

G protein-coupled receptors (GPCRs), belonging to the largest class of membrane proteins, play a prominent role in many (patho)physiological processes and are, therefore, important drug targets. Although most often targeted by small molecules, these receptors have become interesting targets for antibodies and antibody fragments, especially camelid-derived heavy chain-only antibodies and fragments thereof (nanobodies). The small size and molecular structure of nanobodies allow GPCR-binding and modulation, from both the intracellular and extracellular sides. These molecular features make nanobodies attractive tools to study, modulate, and exploit GPCRs. Besides modulating GPCR activity as monovalent or multivalent constructs, nanobodies can also be functionalized for imaging and therapy. Moreover, GPCR-binding nanobodies have been instrumental in obtaining crystal structures of GPCRs, facilitating structure-based drug discovery. Here, we describe the current status and future perspectives of nanobodies targeting GPCRs intra and extracellularly.

Nanobodies: New avenues for imaging, stabilizing and modulating GPCRs.

The family of G protein-coupled receptors (GPCRs) is the largest class of membrane proteins and an important drug target due to their role in many (patho)physiological processes. Besides small molecules, GPCRs can be targeted by biologicals including antibodies and antibody fragments. This review describes the use of antibodies and in particular antibody fragments from camelid-derived heavy chain-only antibodies (nanobodies/VHHs/sdAbs) for detecting, stabilizing, modulating and therapeutically targeting GPCRs. Altogether, it becomes increasingly clear that the small size, structure and protruding antigen-binding loops of nanobodies are favorable features for the development of selective and potent GPCRs-binding molecules. This makes them attractive tools to modulate GPCR activity but also as targeting modalities for GPCR-directed therapeutics. In addition, these antibody-fragments are important tools in the stabilization of particular conformations of these receptors. Lastly, nanobodies, in contrast to conventional antibodies, can also easily be expressed intracellularly which render nanobodies important tools for studying GPCR function. Hence, GPCR-targeting nanobodies are ideal modalities to image, stabilize and modulate GPCR function.

The constitutive activity of the virally encoded chemokine receptor US28 accelerates glioblastoma growth.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251- and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.