Nanotechnology Characterization Laboratory Frederick National Laboratory for Cancer Research Advanced Technology Research Facility 8560 Progress Drive Frederick, MD 21701

Phone: 301-846-6939Fax: 301-846-6399Email: ncl@mail.nih.gov

Web: https://ncl.cancer.gov

Highlights and Accomplishments

2020 Annual Report

https://ncl.cancer.gov

A national resource and knowledge base for the advancement of nanotechnologies for cancer.

Our research is dedicated to advancing applied sciences which enable translation of promising nanotechnology concepts for cancer diagnosis and therapy. Our team comprises experts in various disciplines, including chemistry, pharmacology, toxicology, immunology, and biomedical engineering. We are here to support you by conducting nanomedicine characterization, providing scientific consultation and training in protocols developed by our team, and helping you uncover the nuances of nanomedicine structure-activity relationships. We are immensely grateful to all NCL collaborators and are excited for your achievements and being able to contribute to your science.

— Marina A. Dobrovolskaia, PhD, MBA, PMP and Stephan T. Stern, PhD, DABT Laboratory Directors

https://ncl.cancer.gov

CONTENTS

1. NCL’s Assay Cascade Numbers

2. Nanomedicine Characterization

3. New Protocols

4. Structure-Activity Relationships

5. Regulatory Advancements

6. Support of Regulatory-Directed Research

7. Publication Highlights

8. Workshops and Training

Characterize nanomedicines using standardized methods.

Conduct structure activity relationship (SAR) studies.

Facilitate regulatory review of nanotech constructs.

Engage in educational and knowledge sharing efforts.

NCL’s research activities are focused on meeting four objectives:

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New for 2020 and 2021—SARS-CoV-2

In an effort to help combat the global COVID-19 pandemic, the NCL began accepting applications for novel nanomaterials for prevention and treatment of COVID-19. The NCL is continuing to accept COVID-19 related applications until further notice. Application deadlines follow those for oncology indications.

Assay Cascade Deadlines

The NCL accepts applications year-round, with quarterly review of submissions. The remaining cut-off dates for 2021 Assay Cascade applications are June 1, September 1, and December 1, 2021.

Average Acceptance Rate

Declined62%

Accepted38%

NCL’S ASSAY CASCADE CHARACTERIZATION PROGRAMThe NCL’s Assay Cascade program provides free preclinical characterization of nanomaterials with an oncology indication. The service is awarded through a competitive application process wherein successful applicants have demonstrated proof-of-concept efficacy for their formulation in comparison to standard of care.

Despite the challenges of 2020, the NCL received more than two dozen applications and accepted 10 projects into the program. These formulations will be subjected to thorough analyses, including elucidation of the physicochemical, immunological, pharmacological and toxicological properties.

Access more information about the NCL’s Assay Cascade characterization program on our website:https://ncl.cancer.gov/working-ncl/ncl-assay-cascade-application-process

• James Adair, Penn State University• Young Kwon, University of California Irvine• Gregory Lanza, Washington University Medical

School• BW Therapeutics• Haima Therapeutics

• MonTa Biosciences• Salvacion USA, LLC• SignaBlok• Sitka Biopharma• Tyndall Formulations Services

26White Paper

Applications in 2020

15Proposals Advancing

to Phase II

10Projects Accepted

2020 ASSAY CASCADE AWARDEES

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Unique Concepts and Applications

The NCL is interested in improving our experience and understanding of imaging-related nanomaterials, as well as exploring other novel concepts and unique applications. If you have something outside the box, reach out to us. We can help assess whether your technology may be right for NCL’s Assay Cascade.

Email us at ncl@mail.nih.gov

Collaborators

Academic42% Industry

48%

Government10%

NANOPARTICLE CHARACTERIZATIONThe NCL receives a broad range of nanomedicine technologies through its Assay Cascade program, and has experience with virtually all platforms, therapeutic agents and applications employed in the pursuit of better treatments and diagnostics for cancer.

In 2020, the NCL expanded efforts towards the evaluation of nanomedicine bioequivalence and assessment of personalized vaccine strategies, immunotherapies, and imaging constructs, as well as nanomedicines produced using continuous manufacturing strategies.

NCL Offers:• Comprehensive preclinical characterization• Customized research plans• Regular interim updates• Inclusive written reports• Defense of NCL-generated data

156Collaborative

Projects

947Total Nanomaterials

Characterized

429Unique Nanomaterials

Characterized

Common Nanoparticle Platforms• liposomes• polymeric-based• metallic/metalloid• lipid-based• nucleic acids• dendrimers

Common Active Pharmaceutical Ingredients• cytotoxics• therapeutic nucleic acids• neoantigens• adjuvants/immunomodulators

Common Application Types• chemotherapy• immunotherapy• vaccine• imaging

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Protocol Categories• Sterility, Endotoxin and β-Glucans (STE)• Physicochemical Characterization (PCC)• Immunotoxicity (ITA)• General Toxicity (GTA)• In Vitro Efficacy (IEA)• Pharmacology (PHA)

Popular Downloads• PCC-1, Dynamic Light Scattering• STE-1.1, End-point Chromogenic LAL Assay• GTA-2, In Vitro Cytotoxicity in Hep G2 Cells• GTA-1, In Vitro Cytotoxicity in LLC-PK1 Cells• PCC-7, Transmission Electron Microscopy• ITA-1, In Vitro Hemolysis• PCC-2, Zeta Potential• STE-1.2, Kinetic Turbidity LAL• STE-2.1, Microbial Contamination• ITA-10, Cytokines, Chemokines and Interferons

NEW PROTOCOLSThe NCL has a series of standardized protocols on our website, all free to download, intended to provide researchers with the knowledge and resources to thoroughly characterize their nanomaterials. In an expansion of these efforts, the NCL posted several new protocols to the website over the last year, devoted to topics such as β-glucan contamination and in vitro detection of cytokines, as well as several physicochemical analyses including asymmetric-flow field-flow fractionation, particle concentration and quantitation of polymeric prodrug formulations.

• STE-4: Detection of β-Glucan Contamination• PCC-18: Quantitation of APIs in Polymeric Prodrug Formulations• PCC-19: Asymmetric-Flow Field-Flow Fractionation• PCC-20: Particle Concentration and Size using the Spectradyne nCS1• PCC-21: Measuring Size and Number Concentration of Metallic Nanoparticle

using single particle-ICP-MS• ITA-27: Multiplex Enzyme-Linked Immunosorbent Assay (ELISA) for Detection

of Human Cytokines in Culture Supernatants

Download Protocols

You can find links to all NCL protocols on our website: https://ncl.cancer.gov/resources/assay-cascade-protocols

Questions?

NCL staff are happy to answer questions pertaining to protocols.

Email us at ncl@mail.nih.gov

74Protocols available for download

2,243Downloads over the last year

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Other Areas of SAR Study• Silica-based nanoparticles• Lipid-based nanoparticles• Dendrimers• Surface functionalities (e.g., PEG length,

end-group modifications)• Complement activation• Infusion reactions

4Collaborations on nucleic

acid nanoparticles

13Publications on nucleic

acid nanoparticles

Recent Publications on Nucleic Acid Nanoparticles• Afonin KA, Dobrovolskaia MA, Church G, and Bathe M. Opportunities, Barriers, and

a Strategy for Overcoming Translational Challenges to Therapeutic Nucleic Acid Nanotechnology. ACS Nano. 2020, 14(8), 9221-9227. PMID: 32706238

• Dobrovolskaia MA, Afonin KA. Use of human peripheral blood mononuclear cells to define immunological properties of nucleic acid nanoparticles. Nat Protoc. 2020, 15(11), 3678-3698. PMID: 33097923

• Dobrovolskaia MA and Bathe M. Opportunities and challenges for the clinical translation of structured DNA assemblies as gene therapeutic delivery and vaccine vectors. WIREs Nanomedicine and Nanobiotechnology. 2021, 13(1), e1657. PMID: 32672007

Have a Particular Area of Interest?

We want your feedback. Where do you think the nanomedicine field is headed? What areas do you think would benefit from more in-depth SAR studies?

Email us at ncl@mail.nih.gov

STRUCTURE-ACTIVITY RELATIONSHIPSInvestigation of structure-activity relationships (SAR) is an important part of nanoparticle research and aids in a more complete understanding of how a particles’ physical and chemical properties influence its biological performance.

Therapeutic nucleic acid nanoparticles have been at the forefront of nanomedicine research for many years. NCL has partnered with several well-respected researchers in the field to explore these materials in greater detail, including Prof. Peixuan Guo, The Ohio State University, Prof. Kirill Afonin, University of North Carolina-Charlotte, Prof. Mark Bathe, Massachusetts Institute of Technology, and Dr. Bruce Shapiro, NCI among others.

Study of these materials has highlighted the challenges associated with their translation, including complex design and architecture, along with challenging manufacturing, scale-up and purification strategies. In collaboration with the researchers noted above, in vitro and in vivo studies to better assess efficacy, potential toxicity, and immunogenicity of therapeutic nucleic acid formulations are being explored to facilitate the advancement of this novel and promising class of nanomaterials.

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REGULATORY ADVANCEMENTSNCL’s charter is to advance the applied science of nanoparticle characterization. Towards this, NCL characterization is often used to support the regulatory advancement of nanomaterials submitted through NCL’s Assay Cascade characterization program.

To date, NCL has had 17 collaborators advance their formulations into clinical trials, supporting a combined 67 clinical trails for various cancer indications.

NCL data has not only been used to directly support regulatory review and clinical evaluation of a candidate nanomedicine, but has also been used to evaluate next-generation improvements of a candidate formulation, explore alternate cancer indications, and to support bioanalytical analysis of follow-on formulations.

Collaborators are free to use any or all of their NCL-generated data in support of their Investigational New Drug (IND), Investigational Device Exemption (IDE), New Drug Application (NDA), and/or Abbreviated New Drug Application (ANDA).

NCL will supply comprehensive written reports on all studies conducted to support your regulatory pursuit. In addition, NCL will attend any regulatory meetings to defend and support NCL-generated data.

As an example of NCL regulatory activities, in 2020, NCL staff attended a type B CMC meeting with the FDA in support of a collaborator’s NDA. The NCL continues to work with this company to address remaining regulatory questions of the formulation.

AREAS OF SUPPORT

• Preclinical characterization to support CMC• Explorative toxicity studies to facilitate

IND-enabling GLP safety studies• Method development and transfer to CRO• Exploration of alternate indications• Analysis of generic and next-generation

nanomedicines

• Address regulatory questions and concerns• Attend regulatory meetings to defend NCL data• Comprehensive written reports to supplement

IND/IDE/NDA/ANDA• Non-GLP characterization of clinical samples

17Collaborators in

Clinical Trials

67Clinical

Trials

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SUPPORT OF REGULATORY-DIRECTED RESEARCHPredicting Innate Immune Responses

In 2019, the NCL and FDA entered into a 2-year interagency agreement (IAA) to identify non-clinical assays for assessing and predicting potential innate immune responses to generic versus reference listed drugs (RLD) for peptide drug products. The findings from these studies will be used to identify an assay or a framework of assays that can assess potential immunogenicity from the proposed generics and the RLD arising from a products’ potential contamination with innate immunity modulating impurities (IIMIs).

The first phase of the IAA was completed in 2020, with the study of Teriparatide (TP) as the model RLD and ten model IIMIs and formulation buffer (FB) for comparison. The assays used to assess the compounds included endotoxin and beta-glucan detection, complement activation, leukocyte proliferation, and cytokine secretion, with the latter assay having the highest sensitivity to IIMIs. The key findings from these studies are currently being prepared for publication.

The second phase of the IAA is underway now and will include the study of two additional RLD, Liraglutide and Semaglutide. This work will again evaluate the RLD for induction of cytokines as well as validate a multiplex panel of cytokines for detecting the unique profiles.

The successful validation of in vitro tests to evaluate innate immune responses is critical to support the review and approval process of generic synthetic peptide drug products. This battery of in vitro assays may also be used for assessing innate immune responses of other complex drug products as well.

Bioequivalence of Nanomedicines

The NCL also recently completed a 2-year IAA with the FDA evaluating the bioequivalence of several nanomedicine products. The results of this study are detailed in a recent manuscript.

Skoczen, S.L., Snapp, K.S., Crist, R.M., Kozak, D., Jiang, X., Liu, H., and Stern, S.T., Distinguishing Pharmacokinetics of Marketed Nanomedicine Formulations Using a Stable Isotope Tracer Assay. ACS Pharmacol Transl Sci. 2020, 3(3), 547-558. PMID: 32566919

AREAS OF RESEARCH

4Interagency Agreements

with the FDA

• Dermal penetration of nanoparticles used in cosmetics

• Effects of sterilization techniques on nanomaterials

• Bioequivalence studies of generic nanomedicines

• Immunogenicity studies of generic vs RLD peptide drug products

• Physicochemical characterization of environmental nanomaterials NIEHS

• Physicochemical characterization of nanomaterials for DoD

2Interagency Agreements with other Government Agencies

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A full list of NCL publications is found on the website, https://ncl.cancer.gov/resources/ncl-scientific-bibliography.

A collection of more than 200 publications has been organized into several categories to aid researchers in quickly extracting publications within their topics of interest.

• General Nanomedicine/Preclinical Characterization• Autophagy• Immunology• Pharmacology/Toxicology

• Physicochemical Characterization• Reviews• Safety, Efficacy and Imaging• Sterility, Endotoxin and Beta-Glucans

PUBLICATION HIGHLIGHTSPhysicochemical Characterization

Growing interest in asymmetric-flow field-flow fractionation (AF4) characterization of nanoparticles prompted this research article describing the broad utility of AF4 when coupled with additional analytical techniques, such as DLS, MALS, TEM and RP-HPLC. To supplement this article, a detailed protocol describing AF4 was also published on the NCL website. Download PCC-19 here.

The utility of asymmetric-flow field-flow fractionation for preclinical characterization of nanomedicines.Yingwen Hu, Rachael M. Crist, Jeffrey D. ClogstonAnalytical and Bioanalytical Chemistry, 2020, 412(2), 425-438. PMID: 31776639

Immunology

Beta-glucan levels are of interest for biotechnology therapeutics and for complex formulations containing components with potential immunogenicity concerns and could be an important attribute in explaining immuno-stimulation. This manuscript highlights NCL’s approach to assessing beta-glucan contamination and highlights trends in beta-glucan levels within nanoparticle formulations submitted to the NCL. Our approach to quantifying beta-glucan contamination was also summarized in a new protocol on the NCL website. Download STE-4 here.

Detection of beta-glucan contamination in nanotechnology-based formulations.Barry W. Neun, Edward Cedrone, Timothy M. Potter, Rachael M. Crist, Marina A. DobrovolskaiaMolecules, 2020, 25(15), 3367. PMID: 32722261

Pharmacology

Pharmacokinetics of nanomedicine products and drug delivery to tumors using nanoparticles has been the subject of several high-profile papers in the field over the last few years. Here, in collaboration with the University of North Carolina Chapel Hill, we provide a reanalysis of the drug delivery data using classical pharmacokinetic approaches.

A reanalysis of nanoparticle tumor delivery using classical pharmacokinetic metrics.Lauren S. L. Price, Stephan T. Stern, Allison M. Deal, Alexander V. Kabanov, Willian C. ZamboniScience Advances, 2020, 6(29), eaay9249. PMID: 32832614

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WORKSHOPS AND TRAININGMolecular and Cellular Immunology: From Basics to Applications in Preclinical Development of Nanotechnology-Formulated Drugs

In 2019 and 2020, NCL offered a 9-day training course in immunology. The workshop included lectures covering fundamental aspects of the immune system structure and function at both molecular and cellular levels, principles of immunotoxicology, regulatory requirements for assessing the immunotoxicity of new pharmaceutical products, methodology relevant to the immunotoxicity assessment of nanomaterials and case-studies focusing on the critical structure-activity relationship between nanoparticles and the immune system.

Sign-up for NCL News Alerts to learn about upcoming workshops and training opportunities.

New Workshops

The NCL is interested in hearing your input. If there is a topic you would be interested in learning more about, let us know so that we can consider putting together a workshop or webinar.

Email us at ncl@mail.nih.gov

Something for everyone. Workshops are tailored to meet the needs and interests of a broad range of researchers.

Breakdown of Participating Institutions

Academic64%

Industry31%

Government5%

2Immunology Workshops

169Workshop Attendees

57%Principal/Senior

Investigators

43%Post-Doctoral Fellows and

Graduate Students

16Countries Reached

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The NCL is part of the Frederick National Laboratory for Cancer Research (FNLCR). FNLCR is operated by Leidos Biomedical Research, Inc., for the National Cancer Institute, under contract 75N91019D00024.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.