Aaron Mayer

All Publications

• Vaccine nanocarriers: Coupling intracellular pathways and cellular biodistribution to control CD4 vs CD8 T cell responses BIOMATERIALS Rincon-Restrepo, M., Mayer, A., Hauert, S., Bonner, D. K., Phelps, E. A., Hubbell, J. A., Swartz, M. A., Hirosue, S. 2017; 132: 48-58

  Abstract

  Nanoparticle delivery systems are known to enhance the immune response to soluble antigens (Ags) and are thus a promising tool for the development of new vaccines. Our laboratory has engineered two different nanoparticulate systems in which Ag is either encapsulated within the core of polymersomes (PSs) or decorated onto the surface of nanoparticles (NPs). Previous studies showed that PSs are better at enhancing CD4 T cells and antibody titers, while NPs preferentially augment cytotoxic CD8 T cells. Herein, we demonstrate that the differential activation of T cell immunity reflects differences in the modes of intracellular trafficking and distinct biodistribution of the Ag in lymphoid organs, which are both driven by the properties of each nanocarrier. Furthermore, we found that Ags within PSs promoted better CD4 T cell activation and induced a higher frequency of CD4 T follicular helper (Tfh) cells. These differences correlated with changes in the frequency of germinal center B cells and plasma cell formation, which reflects the previously observed antibody titers. Our results show that PSs are a promising vector for the delivery of Ags for B cell vaccine development. This study demonstrates that nanocarrier design has a large impact on the quality of the induced adaptive immune response.

  View details for DOI 10.1016/j.biomaterials.2017.03.047

  View details for Web of Science ID 000401210000005

  View details for PubMedID 28407494

• Practical Immuno-PET Radiotracer Design Considerations for Human Immune Checkpoint Imaging JOURNAL OF NUCLEAR MEDICINE Mayer, A. T., Natarajan, A., Gordon, S. R., Maute, R. L., McCracken, M. N., Ring, A. M., Weissman, I. L., Gambhir, S. S. 2017; 58 (4): 538-546

  Abstract

  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 (64)Cu-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 (68)Ga. At 1 h after injection, (68)Ga-NOTA-HACA-PD1 and (68)Ga-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.

  View details for DOI 10.2967/jnumed.116.177659

  View details for Web of Science ID 000398249600012

  View details for PubMedCentralID PMC5373501

• Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model. Molecular imaging and biology Natarajan, A., Mayer, A. T., Reeves, R. E., Nagamine, C. M., Gambhir, S. S. 2017

  Abstract

  It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade.We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγ(null) (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo.[(89)Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3-5, 3.2 ± 0.4 MBq/15-16 μg/200 μl) and [(64)Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 μg/200 μl) were administered in mice. PET-CT scans were performed over 1-144 h ([(89)Zr] Keytruda) and 1-48 h ([(64)Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([(89)Zr] Keytruda), and 6.4 ± 0.7 ([(64)Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([(89)Zr] Keytruda) and 48 h ([(64)Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively.Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.

  View details for DOI 10.1007/s11307-017-1060-3

  View details for PubMedID 28247187

• Imaging B cells in a mouse model of multiple sclerosis using (64)Cu-Rituximab-PET. Journal of nuclear medicine : official publication, Society of Nuclear Medicine James, M. L., Hoehne, A., Mayer, A. T., Lechtenberg, K., Moreno, M., Gowrishankar, G., Ilovich, O., Natarajan, A., Johnson, E. M., Nguyen, J., Quach, L., Han, M., Buckwalter, M., Chandra, S., Gambhir, S. S. 2017

  Abstract

  B lymphocytes are a key pathological feature of multiple sclerosis (MS), and are becoming an important therapeutic target for this condition. Currently, there is no approved technique to non-invasively visualize B cells in the central nervous system (CNS) to monitor MS disease progression and response to therapies. Here we evaluated (64)Cu-Rituximab, a radiolabeled antibody specifically targeting the human B cell marker CD20, for its ability to image B cells in a mouse model of MS using positron emission tomography (PET). Methods: To model CNS infiltration by B cells, experimental autoimmune encephalomyelitis (EAE) was induced in transgenic mice that express human CD20 on B cells. EAE mice were given subcutaneous injections of Myelin Oligodendrocyte Glycoprotein fragment1-125 (MOG1-125) emulsified in complete Freund's adjuvant. Control mice received complete Freund's adjuvant alone. PET imaging of EAE and control mice was performed 1, 4, and 19h following (64)Cu-Rituximab administration. Mice were perfused and sacrificed after final PET scan, and radioactivity in dissected tissues was measured with a gamma-counter. CNS tissues from these mice were immunostained to quantify B cells or further analyzed via digital autoradiography. Results: Lumbar spinal cord PET signal was significantly higher in EAE mice compared to controls at all evaluated time points (e.g., 1h post-injection: 5.44 ± 0.37 vs. 3.33 ± 0.20 %ID/g, p<0.05). (64)Cu-Rituximab-PET signal in brain regions ranged between 1.74 ± 0.11 and 2.93 ± 0.15 %ID/g for EAE mice compared to 1.25±0.08 and 2.24±0.11%ID/g for controls, p<0.05 for all regions except striatum and thalamus at 1h post-injection. Similarly, ex vivo biodistribution results revealed notably higher (64)Cu-Rituximab uptake in brain and spinal cord of huCD20tg EAE, and B220 immunostaining verified that increased (64)Cu-Rituximab uptake in CNS tissues corresponded with elevated B cells. Conclusion: B cells can be detected in the CNS of EAE mice using (64)Cu-Rituximab-PET. Results from these studies warrant further investigation of (64)Cu-Rituximab in EAE models and consideration of use in MS patients to evaluate its potential for detecting and monitoring B cells in the progression and treatment of this disease. These results represent an initial step toward generating a platform to evaluate B cell-targeted therapeutics en route to the clinic.

  View details for DOI 10.2967/jnumed.117.189597

  View details for PubMedID 28687602

• Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells. Breast cancer research : BCR Amanatullah, D. F., Tamaresis, J. S., Chu, P., Bachmann, M. H., Hoang, N. M., Collyar, D., Mayer, A. T., West, R. B., Maloney, W. J., Contag, C. H., King, B. L. 2017; 19 (1): 121

  Abstract

  Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear.Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry.ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation.These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.

  View details for DOI 10.1186/s13058-017-0910-x

  View details for PubMedID 29141657

  View details for PubMedCentralID PMC5688761

• Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging. Proceedings of the National Academy of Sciences of the United States of America Maute, R. L., Gordon, S. R., Mayer, A. T., McCracken, M. N., Natarajan, A., Ring, N. G., Kimura, R., Tsai, J. M., Manglik, A., Kruse, A. C., Gambhir, S. S., Weissman, I. L., Ring, A. M. 2015; 112 (47): E6506-14

  Abstract

  Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1-directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti-PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm(3)) and large tumors (150 mm(3)), whereas the activity of anti-PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1-positive and PD-L1-negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

  View details for DOI 10.1073/pnas.1519623112

  View details for PubMedID 26604307

  View details for PubMedCentralID PMC4664306

• Novel Radiotracer for ImmunoPET Imaging of PD-1 Checkpoint Expression on Tumor Infiltrating Lymphocytes. Bioconjugate chemistry Natarajan, A., Mayer, A. T., Xu, L., Reeves, R. E., Gano, J., Gambhir, S. S. 2015; 26 (10): 2062-2069

  Abstract

  Immune checkpoint signaling through the programmed death 1 (PD-1) axis to its ligand (PD-L1) significantly dampens anti-tumor immune responses. Cancer patients treated with checkpoint inhibitors that block this suppressive signaling have exhibited objective response rates of 20-40% for advanced solid tumors, lymphomas, and malignant melanomas. This represents a tremendous advance in cancer treatment. Unfortunately, all patients do not respond to immune checkpoint blockade. Recent findings suggest that patients with tumor infiltrating lymphocytes (TILs) expressing PD-1 may be most likely to respond to αPD-1/PD-L1 checkpoint inhibitors. There is a compelling need for diagnostic and prognostic imaging tools to assess the PD-1 status of TILs in vivo. Here we have developed a novel immunoPET tracer to image PD-1 expressing TILs in a transgenic mouse model bearing melanoma. A (64)Cu labeled anti-mouse antibody (IgG) PD-1 immuno positron emission tomography (PET) tracer was developed to detect PD-1 expressing murine TILs. Quality control of the tracer showed >95% purity by HPLC and >70% immunoreactivity in an in vitro cell binding assay. ImmunoPET scans were performed over 1-48 h on Foxp3+.LuciDTR4 mice bearing B16-F10 melanoma tumors. Mice receiving anti-PD-1 tracer (200 ± 10 μCi/10-12 μg/200 μL) revealed high tracer uptake in lymphoid organs and tumors. BLI images of FoxP3(+) CD4(+) Tregs known to express PD-1 confirmed lymphocyte infiltration of tumors at the time of PET imaging. Biodistribution measurements performed at 48 h revealed a high (11×) tumor to muscle uptake ratio of the PET tracer (p < 0.05). PD-1 tumors exhibited 7.4 ± 0.7%ID/g tracer uptake and showed a 2× fold signal decrease when binding was blocked by unlabeled antibody. To the best of our knowledge this data is the first report to image PD-1 expression in living subjects with PET. This radiotracer has the potential to assess the prognostic value of PD-1 in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.

  View details for DOI 10.1021/acs.bioconjchem.5b00318

  View details for PubMedID 26307602

• On-Command Drug Release from Nanochains Inhibits Growth of Breast Tumors PHARMACEUTICAL RESEARCH Peiris, P. M., Tam, M., Vicente, P., Abramowski, A., Toy, R., Bauer, L., Mayer, A., Pansky, J., Doolittle, E., Tucci, S., Schmidt, E., Shoup, C., Rao, S., Murray, K., Gopalakrishnan, R., Keri, R. A., Basilion, J. P., Griswold, M. A., Karathanasis, E. 2014; 31 (6): 1460-1468

  Abstract

  To evaluate the ability of radiofrequency (RF)-triggered drug release from a multicomponent chain-shaped nanoparticle to inhibit the growth of an aggressive breast tumor.A two-step solid phase chemistry was employed to synthesize doxorubicin-loaded nanochains, which were composed of three iron oxide nanospheres and one doxorubicin-loaded liposome assembled in a 100-nm-long linear nanochain. The nanochains were tested in the 4T1-LUC-GFP orthotopic mouse model, which is a highly aggressive breast cancer model. The 4T1-LUC-GFP cell line stably expresses firefly luciferase, which allowed the non-invasive in vivo imaging of tumor response to the treatment using bioluminescence imaging (BLI).Longitudinal BLI imaging showed that a single nanochain treatment followed by application of RF resulted in an at least 100-fold lower BLI signal compared to the groups treated with nanochains (without RF) or free doxorubicin followed by RF. A statistically significant increase in survival time of the nanochain-treated animals followed by RF (64.3 days) was observed when compared to the nanochain-treated group without RF (35.7 days), free doxorubicin-treated group followed by RF (38.5 days), and the untreated group (30.5 days; n=5 animals per group).These studies showed that the combination of RF and nanochains has the potential to effectively treat highly aggressive cancers and prolong survival.

  View details for DOI 10.1007/s11095-013-1102-8

  View details for Web of Science ID 000336745800010

  View details for PubMedID 23934254

  View details for PubMedCentralID PMC3875625

• Treatment of cancer micrometastasis using a multicomponent chain-like nanoparticle JOURNAL OF CONTROLLED RELEASE Peiris, P. M., Toy, R., Abramowski, A., Vicente, P., Tucci, S., Bauer, L., Mayer, A., Tam, M., Doolittle, E., Pansky, J., Tran, E., Lin, D., Schiemann, W. P., Ghaghada, K. B., Griswold, M. A., Karathanasis, E. 2014; 173: 51-58

  Abstract

  While potent cytotoxic agents are available to oncologists, the clinical utility of these agents is limited due to their non-specific distribution in the body and toxicity to normal tissues leading to use of suboptimal doses for eradication of metastatic disease. Furthermore, treatment of micrometastases is impeded by several biobarriers, including their small size and high dispersion to organs, making them nearly inaccessible to drugs. To circumvent these limitations in treating metastatic disease, we developed a multicomponent, flexible chain-like nanoparticle (termed nanochain) that possesses a unique ability to gain access to and be deposited at micrometastatic sites. Moreover, coupling nanochain particles to radiofrequency (RF)-triggered cargo delivery facilitated widespread delivery of drug into hard-to-reach cancer cells. Collectively, these features synergistically facilitate effective treatment and ultimately eradication of micrometastatic disease using a low dose of a cytotoxic drug.

  View details for DOI 10.1016/j.jconrel.2013.10.031

  View details for Web of Science ID 000329157100007

  View details for PubMedID 24188960

  View details for PubMedCentralID PMC3873646

• Imaging Metastasis Using an Integrin-Targeting Chain-Shaped Nanoparticle ACS NANO Peiris, P. M., Toy, R., Doolittle, E., Pansky, J., Abramowski, A., Tam, M., Vicente, P., Tran, E., Hayden, E., Camann, A., Mayer, A., Erokwu, B. O., Berman, Z., Wilson, D., Baskaran, H., Flask, C. A., Keri, R. A., Karathanasis, E. 2012; 6 (10): 8783-8795

  Abstract

  While the enhanced permeability and retention effect may promote the preferential accumulation of nanoparticles into well-vascularized primary tumors, it is ineffective in the case of metastases hidden within a large population of normal cells. Due to their small size, high dispersion to organs, and low vascularization, metastatic tumors are less accessible to targeted nanoparticles. To tackle these challenges, we designed a nanoparticle for vascular targeting based on an α(v)β(3) integrin-targeted nanochain particle composed of four iron oxide nanospheres chemically linked in a linear assembly. The chain-shaped nanoparticles enabled enhanced "sensing" of the tumor-associated remodeling of the vascular bed, offering increased likelihood of specific recognition of metastatic tumors. Compared to spherical nanoparticles, the chain-shaped nanoparticles resulted in superior targeting of α(v)β(3) integrin due to geometrically enhanced multivalent docking. We performed multimodal in vivo imaging (fluorescence molecular tomography and magnetic resonance imaging) in a non-invasive and quantitative manner, which showed that the nanoparticles targeted metastases in the liver and lungs with high specificity in a highly aggressive breast tumor model in mice.

  View details for DOI 10.1021/nn303833p

  View details for Web of Science ID 000310096100037

  View details for PubMedID 23005348

  View details for PubMedCentralID PMC3487383

• Enhanced Delivery of Chemotherapy to Tumors Using a Multicomponent Nanochain with Radio-Frequency-Tunable Drug Release ACS NANO Peiris, P. M., Bauer, L., Toy, R., Tran, E., Pansky, J., Doolittle, E., Schmidt, E., Hayden, E., Mayer, A., Keri, R. A., Griswold, M. A., Karathanasis, E. 2012; 6 (5): 4157-4168

  Abstract

  While nanoparticles maximize the amount of chemotherapeutic drug in tumors relative to normal tissues, nanoparticle-based drugs are not accessible to the majority of cancer cells because nanoparticles display patchy, near-perivascular accumulation in tumors. To overcome the limitations of current drugs in their molecular or nanoparticle form, we developed a nanoparticle based on multicomponent nanochains to deliver drug to the majority of cancer cells throughout a tumor while reducing off-target delivery. The nanoparticle is composed of three magnetic nanospheres and one doxorubicin-loaded liposome assembled in a 100 nm long chain. These nanoparticles display prolonged blood circulation and significant intratumoral deposition in tumor models in rodents. Furthermore, the magnetic particles of the chains serve as a mechanical transducer to transfer radio frequency energy to the drug-loaded liposome. The defects on the liposomal walls trigger the release of free drug capable of spreading throughout the entire tumor, which results in a widespread anticancer effect.

  View details for DOI 10.1021/nn300652p

  View details for Web of Science ID 000304231700056

  View details for PubMedID 22486623

  View details for PubMedCentralID PMC3358486