Development of a Stable Peptide-Major Histocompatibility Complex (MHC) via Sortase and Click Chemistry.

T cells play a crucial role in antitumor immune responses and the clearance of infected cells. They identify their targets through the binding of T-cell receptors (TCRs) to peptide-major histocompatibility complex (pMHC) molecules present in cancer cells, infected cells, and antigen-presenting cells. This interaction is often weak, requiring multimeric pMHC molecules to enhance the avidity for identifying antigen-specific T cells. Current exchangeable pMHC-I tetramerization methods may overlook TCRs recognizing less stable yet immunogenic peptides. In vivo applications targeting antigen-specific T cells demand the genetic synthesis of a pMHC fusion for each unique peptide antigen, which poses a significant challenge. To address these challenges, we developed a sortase and click chemistry-mediated approach for generating stable pMHC molecules. Leveraging sortase technology, we introduced an azide click-handle near the N-terminus of ß2m, proximal to the MHC-peptide-binding groove. Simultaneously, the peptide was engineered with a multi glycine linker and a C-terminal alkyne click-handle. Azide-alkyne click reactions efficiently immobilized the peptide onto the MHC molecule, providing a versatile and efficient method for pMHC generation. The resulting peptide-clicked-MHC specifically binds to its cognate TCR and remains stable for over 3 months at 4 °C in the absence of any additional free peptide. The stability of the pMHC and its affinity to cognate TCRs are influenced by the linker's nature and length. Multi glycine linkers outperform poly(ethylene glycol) (PEG) linkers in this regard. This technology expands the toolkit for identifying and targeting antigen-specific T cells, enhancing our understanding of cancer-specific immune responses, and has the potential to streamline the development of personalized immunotherapies.

Multiparametric Immunoimaging Maps Inflammatory Signatures in Murine Myocardial Infarction Models.

In the past 2 decades, research on atherosclerotic cardiovascular disease has uncovered inflammation to be a key driver of the pathophysiological process. A pressing need therefore exists to quantitatively and longitudinally probe inflammation, in preclinical models and in cardiovascular disease patients, ideally using non-invasive methods and at multiple levels. Here, we developed and employed in vivo multiparametric imaging approaches to investigate the immune response following myocardial infarction. The myocardial infarction models encompassed either transient or permanent left anterior descending coronary artery occlusion in C57BL/6 and Apoe-/-mice. We performed nanotracer-based fluorine magnetic resonance imaging and positron emission tomography (PET) imaging using a CD11b-specific nanobody and a C-C motif chemokine receptor 2-binding probe. We found that immune cell influx in the infarct was more pronounced in the permanent occlusion model. Further, using 18F-fluorothymidine and 18F-fluorodeoxyglucose PET, we detected increased hematopoietic activity after myocardial infarction, with no difference between the models. Finally, we observed persistent systemic inflammation and exacerbated atherosclerosis in Apoe-/- mice, regardless of which infarction model was used. Taken together, we showed the strengths and capabilities of multiparametric imaging in detecting inflammatory activity in cardiovascular disease, which augments the development of clinical readouts.

Enhanced Tumor Accumulation of Multimodal Magneto-Plasmonic Nanoparticles via an Implanted Micromagnet-Assisted Delivery Strategy.

One of the major shortcomings of nano carriers-assisted cancer therapeutic strategies continues to be the inadequate tumor penetration and retention of systemically administered nanoformulations and its off-target toxicity. Stromal parameters-related heterogeneity in enhanced permeability and retention effect and physicochemical properties of the nanoformulations immensely contributes to their poor tumor extravasation. Herein, a novel tumor targeting strategy, where an intratumorally implanted micromagnet can significantly enhance accumulation of magneto-plasmonic nanoparticles (NPs) at the micromagnet-implanted tumor in bilateral colorectal tumor models while limiting their off-target accumulation, is demonstrated. To this end, novel multimodal gold/iron oxide NPs comprised of an array of multifunctional moieties with high therapeutic, sensing, and imaging potential are developed. It is also discovered that cancer cell targeted NPs in combination with static magnetic field can selectively induce cancer cell death. A multimodal caspase-3 nanosensor is also developed for real-time visualization of selective induction of apoptosis in cancer cells. In addition, the photothermal killing capability of these NPs in vitro is evaluated, and their potential for enhanced photothermal ablation in tissue samples is demonstrated. Building on current uses of implantable devices for therapeutic purposes, this study envisions the proposed micromagnet-assisted NPs delivery approach may be used to accelerate the clinical translation of various nanoformulations.

Enzymatic Construction of DARPin-Based Targeted Delivery Systems Using Protein Farnesyltransferase and a Capture and Release Strategy.

Protein-based conjugates have been extensively utilized in various biotechnological and therapeutic applications. In order to prepare homogeneous conjugates, site-specific modification methods and efficient purification strategies are both critical factors to be considered. The development of general and facile conjugation and purification strategies is therefore highly desirable. Here, we apply a capture and release strategy to create protein conjugates based on Designed Ankyrin Repeat Proteins (DARPins), which are engineered antigen-binding proteins with prominent affinity and selectivity. In this case, DARPins that target the epithelial cell adhesion molecule (EpCAM), a diagnostic cell surface marker for many types of cancer, were employed. The DARPins were first genetically modified with a C-terminal CVIA sequence to install an enzyme recognition site and then labeled with an aldehyde functional group employing protein farnesyltransferase. Using a capture and release strategy, conjugation of the labeled DARPins to a TAMRA fluorophore was achieved with either purified proteins or directly from crude E. coli lysate and used in subsequent flow cytometry and confocal imaging analysis. DARPin-MMAE conjugates were also prepared yielding a construct manifesting an IC50 of 1.3 nM for cell killing of EpCAM positive MCF-7 cells. The method described here is broadly applicable to enable the streamlined one-step preparation of protein-based conjugates.

Probing immune infiltration dynamics in cancer by in vivo imaging.

Cancer immunotherapies typically aim to stimulate the accumulation and activity of cytotoxic T-cells or pro-inflammatory antigen-presenting cells, reduce immunosuppressive myeloid cells or regulatory T-cells, or elicit some combination of effects thereof. Notwithstanding the encouraging results, immunotherapies such as PD-1/PD-L1-targeted immune checkpoint blockade act heterogeneously across individual patients. It remains challenging to predict and monitor individual responses, especially across multiple sites of metastasis or sites of potential toxicity. To address this need, in vivo imaging of both adaptive and innate immune cell populations has emerged as a tool to quantify spatial leukocyte accumulation in tumors non-invasively. Here we review recent progress in the translational development of probes for in vivo leukocyte imaging, focusing on complementary perspectives provided by imaging of T-cells, phagocytic macrophages, and their responses to therapy.

Molecular Immune Targeted Imaging of Tumor Microenvironment.

Novel targeted therapies are rapidly emerging for the treatment of cancer. With the advent of new immune targeting agents, understanding the changes in the tumor microenvironment (TME) is critical. Given the complexity and several cellular mechanisms and factors that play a role in the TME, novel imaging methods to assess and evaluate the dynamic changes in the TME during treatment are needed. Several techniques are being developed for imaging TME including optical, fluorescence and photoacoustic methods. Positron emission tomography (PET) imaging can be used to track the dynamics of different molecular targets in the TME in live animals and in humans. Several novel PET imaging probes including radiolabeled antibodies, antibody fragments, and small molecules have been developed with many more that are under development preclinically and in early human studies. This review is a brief overview of some of the PET agents that are either in the preclinical developmental phase or undergoing early clinical studies.

Design and synthesis of gold nanostars-based SERS nanotags for bioimaging applications.

Surface-enhanced Raman spectroscopy (SERS) nanotags hold a unique place among bioimaging contrast agents due to their fingerprint-like spectra, which provide one of the highest degrees of detection specificity. However, in order to achieve a sufficiently high signal intensity, targeting capabilities, and biocompatibility, all components of nanotags must be rationally designed and tailored to a specific application. Design parameters include fine-tuning the properties of the plasmonic core as well as optimizing the choice of Raman reporter molecule, surface coating, and targeting moieties for the intended application. This review introduces readers to the principles of SERS nanotag design and discusses both established and emerging protocols of their synthesis, with a specific focus on the construction of SERS nanotags in the context of bioimaging and theranostics.

Direct N- or C-Terminal Protein Labeling Via a Sortase-Mediated Swapping Approach.

Site-specific protein labeling is important in biomedical research and biotechnology. While many methods allow site-specific protein modification, a straightforward approach for efficient N-terminal protein labeling is not available. We introduce a novel sortase-mediated swapping approach for a one-step site-specific N-terminal labeling with a near-quantitative yield. We show that this method allows rapid and efficient cleavage and simultaneous labeling of the N or C termini of fusion proteins. The method does not require any prior modification beyond the genetic incorporation of the sortase recognition motif. This new approach provides flexibility for protein engineering and site-specific protein modifications.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

Employing nanobodies for immune landscape profiling by PET imaging in mice.

Noninvasive immunoimaging holds great potential for studying and stratifying disease as well as therapeutic efficacy. Radiolabeled single-domain antibody fragments (i.e., nanobodies) are appealing probes for immune landscape profiling, as they display high stability, rapid targeting, and excellent specificity, while allowing extremely sensitive nuclear readouts. Here, we present a protocol for radiolabeling an anti-CD11b nanobody and studying its uptake in mice by a combination of positron emission tomography imaging, ex vivo gamma counting, and autoradiography. Our protocol is applicable to nanobodies against other antigens. For complete details on the use and execution of this protocol, please see Priem et al. (2020), Senders et al. (2019), or Rashidian et al. (2017).

Nanobodies for Medical Imaging: About Ready for Prime Time?

Recent advances in medical treatments have been revolutionary in shaping the management and treatment landscape of patients, notably cancer patients. Over the last decade, patients with diverse forms of locally advanced or metastatic cancer, such as melanoma, lung cancers, and many blood-borne malignancies, have seen their life expectancies increasing significantly. Notwithstanding these encouraging results, the present-day struggle with these treatments concerns patients who remain largely unresponsive, as well as those who experience severely toxic side effects. Gaining deeper insight into the cellular and molecular mechanisms underlying these variable responses will bring us closer to developing more effective therapeutics. To assess these mechanisms, non-invasive imaging techniques provide valuable whole-body information with precise targeting. An example of such is immuno-PET (Positron Emission Tomography), which employs radiolabeled antibodies to detect specific molecules of interest. Nanobodies, as the smallest derived antibody fragments, boast ideal characteristics for this purpose and have thus been used extensively in preclinical models and, more recently, in clinical early-stage studies as well. Their merit stems from their high affinity and specificity towards a target, among other factors. Furthermore, their small size (~14 kDa) allows them to easily disperse through the bloodstream and reach tissues in a reliable and uniform manner. In this review, we will discuss the powerful imaging potential of nanobodies, primarily through the lens of imaging malignant tumors but also touching upon their capability to image a broader variety of nonmalignant diseases.

HIV-infected macrophages resist efficient NK cell-mediated killing while preserving inflammatory cytokine responses.

Natural killer (NK) cells are innate cytolytic effectors that target HIV-infected CD4+ T cells. In conjunction with antibodies recognizing the HIV envelope, NK cells also eliminate HIV-infected targets through antibody-dependent cellular cytotoxicity (ADCC). However, how these NK cell functions impact infected macrophages is less understood. We show that HIV-infected macrophages resist NK cell-mediated killing. Compared with HIV-infected CD4+ T cells, initial innate NK cell interactions with HIV-infected macrophages skew the response toward cytokine production, rather than release of cytolytic contents, causing inefficient elimination of infected macrophages. Studies with chimeric antigen receptor (CAR) T cells demonstrate that the viral envelope is equally accessible on CD4+ T cells and macrophages. Nonetheless, ADCC against macrophages is muted compared with ADCC against CD4+ T cells. Thus, HIV-infected macrophages employ mechanisms to evade immediate cytolytic NK cell function while preserving inflammatory cytokine responses. These findings emphasize the importance of eliminating infected macrophages for HIV cure efforts.

Noninvasive Imaging of Cancer Immunotherapy.

Immunotherapy has revolutionized the treatment of several malignancies. Notwithstanding the encouraging results, many patients do not respond to treatments. Evaluation of the efficacy of treatments is challenging and robust methods to predict the response to treatment are not yet available. The outcome of immunotherapy results from changes that treatment evokes in the tumor immune landscape. Therefore, a better understanding of the dynamics of immune cells that infiltrate into the tumor microenvironment may fundamentally help in addressing this challenge and provide tools to assess or even predict the response. Noninvasive imaging approaches, such as PET and SPECT that provide whole-body images are currently seen as the most promising tools that can shed light on the events happening in tumors in response to treatment. Such tools can provide critical information that can be used to make informed clinical decisions. Here, we review recent developments in the field of noninvasive cancer imaging with a focus on immunotherapeutics and nuclear imaging technologies and will discuss how the field can move forward to address the challenges that remain unresolved.

Direct and Indirect Regulators of Epithelial-Mesenchymal Transition-Mediated Immunosuppression in Breast Carcinomas.

The epithelial-to-mesenchymal transition, which conveys epithelial (E) carcinoma cells to quasi-mesenchymal (qM) states, enables them to metastasize and acquire resistance to certain treatments. Murine tumors composed of qM mammary carcinoma cells assemble an immunosuppressive tumor microenvironment (TME) and develop resistance to anti-CTLA4 immune-checkpoint blockade (ICB) therapy, unlike their E counterparts. Importantly, minority populations of qM cells within a tumor can cross-protect their more E neighbors from immune attack. The underlying mechanisms of immunosuppression and cross-protection have been unclear. We demonstrate that abrogation of qM carcinoma cell-derived factors (CD73, CSF1, or SPP1) prevents the assembly of an immunosuppressive TME and sensitizes otherwise refractory qM tumors partially or completely to anti-CTLA4 ICB. Most strikingly, mixed tumors in which minority populations of carcinoma cells no longer express CD73 are now sensitized to anti-CTLA4 ICB. Finally, loss of CD73 also enhances the efficacy of anti-CTLA4 ICB during the process of metastatic colonization. SIGNIFICANCE: Minority populations of qM carcinoma cells, which likely reside in human breast carcinomas, can cross-protect their E neighbors from immune attack. Understanding the mechanisms by which qM carcinoma cells resist antitumor immune attack can help identify signaling channels that can be interrupted to potentiate the efficacy of checkpoint blockade immunotherapies.This article is highlighted in the In This Issue feature, p. 995.

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Immuno-PET identifies the myeloid compartment as a key contributor to the outcome of the antitumor response under PD-1 blockade.

Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used noninvasive immuno-positron emission tomography (PET), using 89Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8+ T cells and CD11b+ myeloid cells in response to anti-PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8+ and CD11b+ cells. Anti-PD-1 treatment mobilized CD8+ T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8+ T cells went on to complete resolution. All tumors contained CD11b+ myeloid cells from the outset of treatment, with later recruitment of additional CD11b+ cells. As tumors grew, the distribution of intratumoral CD11b+ cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b+ population in the center of the tumors. The changes in distribution of CD8+ and CD11b+ cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti-PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45+ cells showed that CD11b+ cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45+ population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti-PD-1 treatment not only affects interactions of CD8+ T cells with the tumor but also impacts the intratumoral myeloid compartment.