Melissa N. McCracken

PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin’s lymphoma. Although it is well established that PD-1–PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1–PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1–PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

An Effective Immuno-PET Imaging Method to Monitor CD8-Dependent Responses to Immunotherapy

The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8(+) T cells and other immune cell subsets. In this study, we describe the generation of (89)Zr-desferrioxamine-labeled anti-CD8 cys-diabody ((89)Zr-malDFO-169 cDb) for noninvasive immuno-PET tracking of endogenous CD8(+) T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T-cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T-cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic.

Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo

Significance Anti-CD8 immuno-PET imaging agents provide the potential to monitor the localization, migration, and expansion of CD8-expressing cells noninvasively in vivo. Shown here is the successful generation of functional anti-CD8 imaging agents based on engineered antibodies for use in a variety of preclinical disease and immunotherapeutic models. The noninvasive detection and quantification of CD8+ T cells in vivo are important for both the detection and staging of CD8+ lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8+ T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8+ T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for 64Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both 64Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.

Immuno-PET of Murine T Cell Reconstitution Postadoptive Stem Cell Transplantation Using Anti-CD4 and Anti-CD8 Cys-Diabodies

The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole-body T lymphocyte dynamics noninvasively in vivo, we generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for 89Zr-immuno-PET detection of helper and cytotoxic T cell populations. Methods: Anti-CD4 and -CD8 cDbs were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cDbs were site-specifically conjugated to maleimide-desferrioxamine for 89Zr radiolabeling and subsequent small-animal PET/CT acquisition and ex vivo biodistribution in both wild-type mice and a model of hematopoietic stem cell (HSC) transplantation. Results: Immuno-PET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild-type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 wk after HSC transplantation using the 89Zr-radiolabeled anti-CD4 and -CD8 cDbs. Conclusion: These newly developed anti-CD4 and -CD8 immuno-PET reagents represent a powerful resource to monitor T cell expansion, localization, and novel engraftment protocols. Future potential applications of T cell–targeted immuno-PET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.

Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene

Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [18F]-L-FMAU (1-(2-deoxy-2-18fluoro-β-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.

Anti-CD47 Treatment Stimulates Phagocytosis of Glioblastoma by M1 and M2 Polarized Macrophages and Promotes M1 Polarized Macrophages In Vivo

Tumor-associated macrophages (TAMs) represent an important cellular subset within the glioblastoma (WHO grade IV) microenvironment and are a potential therapeutic target. TAMs display a continuum of different polarization states between antitumorigenic M1 and protumorigenic M2 phenotypes, with a lower M1/M2 ratio correlating with worse prognosis. Here, we investigated the effect of macrophage polarization on anti-CD47 antibody-mediated phagocytosis of human glioblastoma cells in vitro, as well as the effect of anti-CD47 on the distribution of M1 versus M2 macrophages within human glioblastoma cells grown in mouse xenografts. Bone marrow-derived mouse macrophages and peripheral blood-derived human macrophages were polarized in vitro toward M1 or M2 phenotypes and verified by flow cytometry. Primary human glioblastoma cell lines were offered as targets to mouse and human M1 or M2 polarized macrophages in vitro. The addition of an anti-CD47 monoclonal antibody led to enhanced tumor-cell phagocytosis by mouse and human M1 and M2 macrophages. In both cases, the anti-CD47-induced phagocytosis by M1 was more prominent than that for M2. Dissected tumors from human glioblastoma xenografted within NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and treated with anti-CD47 showed a significant increase of M1 macrophages within the tumor. These data show that anti-CD47 treatment leads to enhanced tumor cell phagocytosis by both M1 and M2 macrophage subtypes with a higher phagocytosis rate by M1 macrophages. Furthermore, these data demonstrate that anti-CD47 treatment alone can shift the phenotype of macrophages toward the M1 subtype in vivo.

Practical ImmunoPET radiotracer design considerations for human immune checkpoint imaging

Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. Here, we sought to optimize noninvasive immuno-PET imaging of human programmed death-ligand 1 (PD-L1) expression, in a preclinical model, using a small high-affinity engineered protein scaffold (HAC-PD1). Six HAC-PD1 radiotracer variants were developed and used in preclinical imaging and biodistribution studies to assess their ability to detect human PD-L1 expression in vivo. Radiotracer design modifications included chelate, glycosylation, and radiometal. HACA-PD1 was adopted as the naming convention for aglycosylated tracer variants. NOD scid γ-(NSG) mice were inoculated with subcutaneous tumors engineered to either be constitutively positive (CT26 hPD-L1) or be negative (ΔmPD-L1 CT26) for human PD-L1 expression. When the tumors had grown to an average size of 1 cm in diameter, mice were injected with 0.75–2.25 MBq (∼10 μg) of an engineered radiotracer variant and imaged. At 1 h after injection, organs were harvested for biodistribution. Of the practical immuno-PET tracer modifications considered, glycosylation was the most prominent design factor affecting tracer uptake, specificity, and clearance. In imaging studies, aglycosylated 64Cu-NOTA-HACA-PD1 most accurately visualized human PD-L1 expression in vivo. We reasoned that because of the scaffold’s small size (14 kDa), its pharmacokinetics may be suitable for labeling with the short-lived and widely clinically available radiometal 68Ga. At 1 h after injection, 68Ga-NOTA-HACA-PD1 and 68Ga-DOTA-HACA-PD1 exhibited promising target-to-background ratios in ex vivo biodistribution studies (12.3 and 15.2 tumor-to-muscle ratios, respectively). Notably, all HAC-PD1 radiotracer variants enabled much earlier detection of human PD-L1 expression (1 h after injection) than previously reported radiolabeled antibodies (>24 h after injection). This work provides a template for assessing immuno-PET tracer design parameters and supports the translation of small engineered protein radiotracers for imaging human immune checkpoints.

HSV-sr39TK positron emission tomography and suicide gene elimination of human hematopoietic stem cells and their progeny in humanized mice.

Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 5173-83. ©2014 AACR.

Noninvasive detection of tumor-infiltrating T cells by PET reporter imaging

Adoptive transfer of tumor-reactive T cells can successfully reduce tumor burden; however, in rare cases, lethal on-target/off-tumor effects have been reported. A noninvasive method to track engineered cells with high sensitivity and resolution would allow observation of correct cell homing and/or identification of dangerous off-target locations in preclinical and clinical applications. Human deoxycytidine kinase triple mutant (hdCK3mut) is a nonimmunogenic PET reporter that was previously shown to be an effective tool to monitor whole-body hematopoiesis. Here, we engineered a construct in which hdCK3mut is coexpressed with the anti-melanoma T cell receptor F5, introduced this construct into human CD34 cells or PBMCs, and evaluated this approach in multiple immunotherapy models. Expression of hdCK3mut allowed engrafted cells to be visualized within recipient bone marrow, while accumulation of [18F]-L-FMAU in hdCK3mut-expressing T cells permitted detection of intratumoral homing. Animals that received T cells coexpressing hdCK3mut and the anti-melanoma T cell receptor had demonstrably higher signals in HLA-matched tumors compared with those in animals that received cells solely expressing hdCK3mut. Engineered T cells caused cytotoxicity in HLA/antigen-matched tumors and induced IFN-γ production and activation. Moreover, hdCK3mut permitted simultaneous monitoring of engraftment and tumor infiltration, without affecting T cell function. Our findings suggest that hdCK3mut reporter imaging can be applied in clinical immunotherapies for whole-body detection of engineered cell locations.

Deoxycytidine Kinase Augments ATM-Mediated DNA Repair and Contributes to Radiation Resistance

Efficient and adequate generation of deoxyribonucleotides is critical to successful DNA repair. We show that ataxia telangiectasia mutated (ATM) integrates the DNA damage response with DNA metabolism by regulating the salvage of deoxyribonucleosides. Specifically, ATM phosphorylates and activates deoxycytidine kinase (dCK) at serine 74 in response to ionizing radiation (IR). Activation of dCK shifts its substrate specificity toward deoxycytidine, increases intracellular dCTP pools post IR, and enhances the rate of DNA repair. Mutation of a single serine 74 residue has profound effects on murine T and B lymphocyte development, suggesting that post-translational regulation of dCK may be important in maintaining genomic stability during hematopoiesis. Using [18F]-FAC, a dCK-specific positron emission tomography (PET) probe, we visualized and quantified dCK activation in tumor xenografts after IR, indicating that dCK activation could serve as a biomarker for ATM function and DNA damage response in vivo. In addition, dCK-deficient leukemia cell lines and murine embryonic fibroblasts exhibited increased sensitivity to IR, indicating that pharmacologic inhibition of dCK may be an effective radiosensitization strategy.