NIH and CDC SBIR Annual Contract Solicitation

Background

Neglected tropical diseases (NTDs) are bacterial, parasitic, and viral infections that disproportionately affect poor and underserved populations around the world, and are primarily associated with high levels of morbidity due to the chronic nature of the infections. Adults affected by NTDs often have decreased productivity. School aged children are also affected by NTDs, resulting in decreased physical and scholastic performance.  A subset of NTDs, including lymphatic filariasis (1 billion people at risk in 73 countries), onchocerciasis (120 M people at risk in 37 countries), schistosomiasis (700 M at risk in 74 countries), trachoma (540 M at risk in 55 countries) and soil transmitted helminth (STH) infections (4 billion at risk, 1 billion infected, worldwide), can be targeted effectively through mass drug administration (MDA). In recent years, there have been significant increases in the number of countries implementing public health programs to combat NTDs, and in the number of persons being treated for NTDs. This progress is the direct result of generous donations of drugs from pharmaceutical manufacturers as well as funding support from the U.S. Agency for International Development (USAID) and the UK Department for International Development (DFID), among others. Reducing the morbidity caused by NTDs is an objective of the U.S. Government Global Health Initiative (GHI) with specific targets for the global elimination of lymphatic filariasis and trachoma.

Currently available laboratory and epidemiological tools to achieve the new elimination goals set for several NTDs require improvement. For example, there is significant geographic overlap in the distribution of most NTDs, but programs continue to use disease-specific and labor intensive clinical or parasitologic exams for mapping and surveillance.  This approach does not maximize the limited resources available to these programs.  Treatment drugs for some NTDs can cause serious adverse events if other infections are also present. For example, serious neurologic adverse events can result when MDA takes place with the use of ivermectin in regions where onchocerciasis and Loa loa overlap (i.e., Central and West Africa).  An integrated diagnostic platform for NTDs could produce significant cost-savings for mapping and surveillance and improved safety for MDA programs.

 Project Goal 

Field-compatible antibody and antigen (as appropriate for the particular disease, as delineated in the table below) tests can be used to define treatment areas, guide MDA program implementation and to conduct post-treatment surveillance. Development of multiplexed strip tests would facilitate integrated, cost-effective surveillance and mapping activities.

(+) = test is needed

The specific project goal is to have prototype field-compatible tests that can address the following issues currently faced by national NTD programs

·       the need  for rapid  determination of infection prevalence in support of micro mapping;

·       the detection of co-infections that hamper MDA activities (for example lymphatic filariasis endemicity in areas where ivermectin has been previously used for onchocerciasis MDA,  Loa loa infections in areas endemic for onchocerciasis);

·       epidemiological surveillance, evaluation of program impact through serological monitoring, and surveillance for infection or exposure following apparent interruption of transmission.

The proposed assays should be developed towards use of a standard platform, therefore opening opportunities for integrated surveillance for NTDs.

Phase I Activities and Expected Deliverables 

1)     Prototype device or methodology for point of care application (field compatible) for simultaneous or consecutive detection of one or more NTDs. A rapid diagnostic, field compatible serological assay is highly desirable.  Such a device will help identify infected persons in specific areas, therefore facilitating fast mapping could be the basis of program monitoring and evaluation activities. A desirable prototype should include either two or more of the NTDs listed above, including but not limited to:

§  Schistosomiasis and lymphatic filariasis

§  Loa loa and onchocerciasis

§  Loa loa and LF

§  Schistosomiasis and intestinal helminth infections (e.g. Strongyloides stercoralis)

2)     Determination of basic assay performance characteristics: preliminary sensitivity and specificity desired but not required.

3)     Field compatibility characteristics: performance outside a fully equipped laboratory and the stability, shelf life, and storage requirements of the tests.

Projected Phase II activities

Phase II activities for a successful Phase I prototype will include expanded testing for sensitivity and specificity, and small-scale production of beta prototypes for field testing. Following this, modifications of the beta prototypes towards a final field-compatible test will be done. Finally, data will be generated to further characterize the test performance characteristics and assay compatibility with NTD program needs for mapping and program monitoring and evaluation, including post-treatment surveillance.

Impact

Development of improved diagnostic tools supports CDC’s efforts to address lymphatic filariasis in the Americas and the NTD GHI targets. These tools would also encourage the commitment of donors and policy makers to NTD control and elimination programs by allowing program integration across diseases and enhanced efficiency. New devices or assays may provide more reliable detection of infection rates, which would lead to increased confidence towards meeting public health goals. Significant savings in human and financial resources could be obtained through the development of improved diagnostic tools.

Commercialization Potential

NTDs are by definition neglected and diagnostic tests for NTDS are not necessarily compatible with standard commercialization strategies.  However, there is a need for new diagnostic methods. Small businesses are the frontrunners on developing novel technologies and approaches for addressing unmet needs. Market opportunities arise from the presence of donors and policy makers already committed in NTD elimination and control efforts, who could encourage manufacturers to promote production and commercial availability of these devices.

National Center for Emerging Zoonotic and Infectious Diseases (NCEZID)

The mission of the National Center for Emerging and Zoonotic Infectious Diseases aims to prevent disease, disability, and death caused by a wide range of infectious diseases. NCEZID focuses on diseases that have been around for many years, emerging diseases (those that are new or just recently identified), and zoonotic diseases (those spread from animals to people). NCEZID’s work is guided in part by a holistic “One Health” strategy, which recognizes the vital interconnectedness of microbes and the environment. Through a comprehensive approach involving many scientific disciplines, NCEZID can attain better health for humans and animals and improve our environment.

NCEZID’s Web site:  http://www.cdc.gov/ncezid

For this solicitation NCEZID invites Phase I proposals in the following areas:

Background

Long range de novo genome assembly from short sequence reads is still one of the greatest challenges in genomics despite vast and rapid improvements in obtaining those short reads.  Numerous viral, bacterial, and parasitic agents causing human and veterinary diseases are carried and transmitted by arthropods including but not limited to ticks, mosquitos, triatomids, sandflies, mites, lice, and fleas. The prevalence, diversity, and range of these arthropod disease vectors render them an important subject of study for improving our understanding of their roles in human disease transmission, and consequently for the prevention of those diseases. The impact of assembly of the human genome sequence on medicine has been tremendous.  Similarly, high-quality genome assemblies of arthropod vectors are a critical precondition to new approaches to studying vector-pathogen interactions and for controlling vector populations. Such genome assemblies will be used to underpin demographic, phylogenetic, host/parasite, and population genetic analyses. Unfortunately, very few reference or even draft-quality assemblies exist for arthropod vectors of public health importance, with the exception of species with small genomes like mosquitoes.  This is true despite the huge economic and public health impacts of many other vector species. Assemblies of larger, more complex arthropod genomes, such as ticks, were first proposed in 2006 (Lyme disease vector) but they are still very poor despite their importance to public health. A consequence of the lack of cost-effective assembly technologies to complete even single sequences is that large-scale comparative assembly efforts are virtually nonexistent. This is partly a consequence of the costs required to generate high-quality assemblies for this massively diverse phylum, even as the cost per read has declined.  However, the read length of efficient sequencers with contemporary libraries is too short for effective construction of chromosome length assemblies.  Higher order assembly requires very expensive and cumbersome approaches that are still being applied to correction of the human genome sequence.  New approaches  may substantially reduce the time and effort required for higher order genome assemblies and thus make fuller and more cost effective use of the short read sequence libraries which are the norm for NextGeneration Sequencers but quite inadequate for achieving the goal of accurate full genome assemblies. Currently genomes are often released as assemblies containing tens of thousands of contigs (e. g., Rhodnius prolixus, the triatomine vector of Chagas Disease, even at 8 X coverage has 58,559 contigs and 27,872 supercontigs).

Two recent Institute of Medicine of the National Academies Workshops by expert panels have focused on the costs and public health threats associated with vector-borne disease.  The more general 2008 workshop was entitled “Vector-Borne Diseases, Understanding the Environmental, Human Health, and Ecological Connections, Workshop Summary (Forum on Microbial Threats).” And published by the National Academy Press.  This was followed by another more focused National Academy Press publication in 2011 o f a workshop proceeding which was entitled “Critical Needs and Gaps in Understanding Prevention, Amelioration, and Resolution of Lyme and Other Tick-Borne Diseases: The Short-Term and Long-Term Outcomes - Workshop Report.”  These lengthy documents fully explore the public health problem, costs, and research directions needing effort in order to reduce the health burden posed by vector-borne diseases.  Suffice it to say, greater genetic understanding of the target species, the focus of this solicitation, was fully addressed as a pressing need in these publications.  Those same considerations apply to a wide range of arthropods of veterinary and agricultural importance.

Project Goals

The goals of the proposed research are to rapidly and cost-effectively assemble high-quality arthropod genomes de novo. The innovation should ultimately enable large numbers of genomes to be assembled in multi-megabase scaffolds rapidly and affordably.  A scalable, parallelizable approach will enable much broader surveys and targeted studies of arthropod genomes to better understand their role in disease transmission and myriad costs to society. Technologies designed to meet these needs will need to employ computational and assay-based innovations. Projects must start with input DNA and yield assembled genomes, not just data from which assembly may be done eventually.  This will be the first such effort attempted with large arthropod genomes for higher order and more complete assemblies.  Technologies previously found to be effective for human and alligator genome assemblies (e.g., Chromosome-scale shotgun assembly using an in vitro method for long-range linkage ArXiv: 1502.05331v1 [q-bio.GN] 18 Feb 2015) using emerging sequencing and bioinformatic technologies may be used to achieve this goal.

Phase I Activities and Expected Deliverables:

Phase I must demonstrate the feasibility of an advanced methodology pipeline for rapid and high-quality de novo genome assembly of several arthropod genomes. Specifically, at least three tick vector genomes of public health importance (e. g., Ixodes scapularis, Dermacentor variabilis, Amblyomma americanum) with different genomic characteristics (total estimated genome sizes of >1 Gbp and different amounts of repetitive DNA families) must be assembled to reasonable contiguity (N50 > 200 Kbp) and quality by the responder to the solicitation. For Phase I, only the final data demonstrating successful de novo assembly of these targets will need be provided. Additional high quality conventional annotation and chromosome mapping confirmation that these assemblies are indeed correct will be required in Phase II for each of these three targets as well as the additional genomes to be analyzed in Phase II.

Projected Phase II activities

Phase II projects must demonstrate the scalability and cost-effectiveness of the technology approach demonstrated in Phase I, as well as quality annotations for the assemblies. For each respective year of the phase II project period, a total of 8 (including the three Phase I targets) (Phase II-yr1) and 10 (Phase II-yr2) additional arthropod vector genome assemblies and annotations, including other arthropod species of public health interest in at least four arthropod orders (e. g., fleas, ticks, lice, mites, triatomines) with genomes > 500 Mbp, must be produced that meet the same annotation quality, N50 and contiguity criteria established for the three Phase I assemblies. Furthermore, each assembly in the second phase must be completed, or a credible roadmap demonstrated to reach, a total reagent cost reduced to less than $10,000 per assembly for those performed in the last year of Phase II. Generated assemblies must be released to the public domain, and applicant must perform and demonstrate gene annotation and synteny comparisons of qualities comparable to the state of the art generally achieved for well-assembled reference vertebrate genomes.

Impact

Higher-quality arthropod genome assemblies will support public health research and interventions on a number of fronts. Better assemblies and broader sampling are highly conducive to comparative analyses for understanding phylogenetic relationships between closely and distantly related disease vectors. Population genetic analyses will become more powerful by allowing for more accurate characterization of population growth or decline, changes in geographic distribution through time, and evolutionary forces such as selection and drift. Higher-quality assemblies also allow better genome annotation via both syntenic and computational analyses, which in turn offer insight into host/symbiont relationships and the genetic basis of disease transmission. All of these benefits positively impact the ability to understand arthropod-borne disease transmission and, consequently, capabilities for effective prevention and intervention.  Arthropods are increasingly resistant to pesticides used in their control.  A fundamental understanding of the genetics of vectors is essential to developing new strategies for reducing the huge economic and public health burdens they cause.  The first step in this process is to obtain high quality reference genome sequences which can provide the basis for development of other novel methodologies applicable to related species and diverging populations.   The methods demonstrated should be applicable to a much wider selection of species of interest, both vertebrate and for other invertebrate groups, and provide a commercially viable future for a successful responder to this solicitation.

Commercialization Potential

More than 5000 arthropod genomes have been proposed for sequencing (i5K project) and numerous pests of agricultural importance as well medically important vectors are on this list.  Insects alone comprise the largest number of species of any life form except bacteria and arthropods are the most successful animals on the planet. Given the importance of arthropods in disease transmission, the vast diversity of the phylum, and the importance of high quality genome assemblies for effective scientific investigations, the proposed technologies embody substantial market potential. Whereas the i5K project is estimated to take 5 or 10 years to complete, our goal is to use advanced technologies to obtain results sooner than this and of better quality. The ultimate service or product provided will find extensive use with all eukaryotic subjects of genomics research. Thorough characterization of the many important arthropod genomes will be a long term effort by the scientific community providing an ongoing need for the proposed product from the solicitation responder for many years to come.

Background

Antibiotic resistance causes over 2 million infections and 23,000 deaths annually in the United States alone and is a global public health challenge that has reached critical levels in healthcare settings, and the evolution of multidrug-resistant organisms (MDROs) threaten to move the care of hospitalized patients into a pre-antibiotic era. These MDROs include organisms such as vancomycin-resistant enterococcus (VRE), carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum beta-lactam resistant Enterobacteriaceae (ESBLs), and Clostridium difficile, all of which primarily colonize patients in the lower intestine where they undergo clonal expansion and often dominate the microbiome.  Prerequisite for colonization by these intestinal MDROs are disruption and shift in diversity of the lower intestinal microbiome that usually result from exposure to antibiotics, but may be contributed to by other medications, dietary changes, and diarrhea from viral and non-infectious causes.  Following colonization, dominance of the lower intestinal microbiome by a particular MDRO (as defined by constituting >30% of the microbiome) is a risk factor for infection. Moreover, intestinal MDRO dominance, over and above low-level colonization, is associated with increased skin and environmental contamination, and risk of transmission.

Investigations are underway to identify critical taxonomic and functional components of the intestinal microbiome that, when absent, confer risk for colonization or infection with MDROs. Meanwhile, current infection control and public health recommendations often include active surveillance testing to detect and contain transmission from patients who are asymptomatically colonized with the aforementioned MDROs.

Project Goals

Develop a proof of concept assay that could be used as the basis of a diagnostic method for stool that quantitatively detects not only the presence and relative amount of one or more of the previously described MDROs (i.e., CRE, VRE, ESBL, and/or C. difficile), but also the taxonomic components and diversity of the gut microbiome.  The approach to both MDRO detection and microbiome description may utilize a number of different existing technologic platforms and combinations thereof including, but not limited to, single or multiplex PCR platforms, 16S ribosomal RNA-encoding DNA amplification and sequencing, deep DNA sequencing, or other advanced metagenomic or metabolomic methods.

The overall objectives are to: 1) detect colonization  by one or more MDRO(s) using a molecular approach expected to yield a clinically meaningful sensitivity and specificity; 2) determine the abundance of the MDRO(s) relative to important taxonomic components of the lower intestinal microbiome (e.g., degree of dominance); 3) determine relative abundance and diversity of the important taxa of the lower intestinal microbiome to describe disruptions that may portend future near-term risk of MDRO colonization or, if already colonized, the future risk for transmission and infection and; 4) generate results with a clinically useful turnaround time.

Phase I Activities and Expected Deliverables

1.      Determine a workable strategy to achieve the above outlined goals.  

2.      Develop pertinent wet-lab protocols to identify and modify, if necessary, any existing software or bioinformatics tools necessary for interpretation, and 

3.      Demonstrate, using spiked human stool or waste clinical specimens from which MDRO have been cultured, the detection of MDROs and, using stool from antibiotic-naïve and antibiotic-experienced patients, the ability to discern major microbiome disruptions.

Projected Phase II Activities

1.      Build-out of modular components for commercialization of a clinical assay including adaptation of assay and results interpretation for use with rectal swabs

2.      Perform a clinical demonstration study divided into two phases:

a.      Testing at-risk patients periodically throughout their hospitalization, correlating results of the combined microbiome and MDRO assay performed on a rectal swab with antibiotic and other drug exposures as well as microbiologic evidence (i.e., perform sampling) of patient skin, patient care area environment (i.e., high-touch surfaces), and healthcare worker hand contamination caused by the target MDRO. Select patient population (based on underlying clinical risk) and power the sample size to examine the capability of assay to predict ongoing risk for colonization (in the previously non-colonized) and transmission or infection events among those already colonized. No clinical or infection control intervention will be based upon assay results, observational only. The goal will be to demonstrate the predictive capability of the combined assay results of microbiome disruption and MDRO detection (and the degree of MDRO dominance in the microbiome), over and above qualitative MDRO detection alone, for the likelihood of colonization or, if already colonized, the likelihood to serve as a source for transmission.

b.      A proof of concept infection control intervention focused on enhanced environmental cleaning and glove use triggered by assay results, examining its impact on transmission and compared to either a historic (i.e., quasi-experimental) or concurrent ward, unit, or facility control.

3.      Engage either developers of an advanced probiotic or academic investigators studying fecal microbiota transplantation (under an FDA IND) to design study for future implementation in which one of these interventions is offered to patients on the basis of assay results as a means to reduce their risk for colonization and infection as well as transmission to other patients.

Impact

Having a means to monitor the level of microbiome disruption in a patient, while simultaneously detecting colonization with selected MDROs, will allow proactive identification of the infection control risk of patients, both in terms of their vulnerability to colonization with an MDRO (i.e., if they are disrupted) and their risk of transmission if they are already MDRO-colonized or (especially) if they are MDRO-dominated. Moreover, because MDROs are pathobionts, it is likely the identification of MDRO domination will become regarded as an important independent risk factor (along with others) for infection in many, if not all, patient populations. Meanwhile, microbiome restorative therapies are currently under clinical development. The data generated from assays such as this, once integrated into clinical care, will provide not only direction to antibiotic stewardship and infection control but also form the basis for an entirely new frontier of patient management. It is not an over statement that the development and use of ‘microbiome disruption indexes’ in patient management will revolutionize current infection control and MDRO prevention in all of healthcare.

Commercialization Potential

With the appropriate level of intellectual and fiscal capital invested into a relatively easy to use, straightforward platform with good bioinformatics support, the commercialization potential is tremendous. At least in the case of C. difficile there are already national third-party payer incentives for hospitals to reduce publically reported rates, such that it is plausible that hospitals will utilize available advanced diagnostics that stratify patient risks for colonization, transmission, and infection. In addition, it is likely that advanced probiotics and other microbiome restorative therapies will become available in the next 5-10 years and, coupled with the appropriate risk-stratifying diagnostics, these may become administered routinely to patients with microbiome disruption following antibiotic or other drug therapies.

National Center For HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP)

The mission of the National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention (NCHHSTP) is to maximize public health and safety nationally and internationally through the elimination, prevention, and control of disease, disability, and death caused by HIV/AIDS, Viral Hepatitis, other Sexually Transmitted Diseases, and Tuberculosis.

NCHHSTP Web site:  http://www.cdc.gov/nchhstp/

For this solicitation NCHHSTP invites Phase I proposals in the following areas:

Background

Hepatitis B virus (HBV) infection is a global public health concern. Worldwide more than 350 million people are chronically infected with hepatitis B virus. HBV infection causes acute and chronic hepatitis leading to liver cirrhosis and hepatocellular carcinoma. After HBV infection, viral DNA is transferred to nuclei of the infected hepatocytes and the double-stranded, open circular DNA is converted to covalently closed circular DNA (cccDNA). Persistence of cccDNA remains an obstacle to clearing HBV in chronically infected people, who remain at risk of developing advanced liver disease. This is because cccDNA acts as a template for continued virion production in the hepatocyte nucleoplasm. As long as the infected hepatocyte survives, cccDNA remains in the nucleus, maintaining a viral ′pool′. Further, in patients undergoing antiviral therapy who discontinue treatment, HBV can reactivate from cccDNA.  To monitor the persistence of cccDNA in the liver, repeated liver biopsies are required, which are hazardous and uncomfortable to the patient, and costly.

Previous studies have shown the presence of cccDNA in serum of chronically infected patients. Serum cccDNA levels correlate well with intrahepatic cccDNA content. Serum cccDNA may thus be used for sequential monitoring of intrahepatic cccDNA levels without the requirement for repeated liver biopsies. Further, the appearance of cccDNA in the serum can be a marker of liver damage.

Quantitative detection of cccDNA in serum thus has potential to evaluate the severity of liver damage and the efficacy of antiviral therapy. Methodologies for the detection of HBV cccDNA in serum have been reported but assays for its detection and quantification are not commercially available. Development of a facile quantitative assay is required for quantitative detection of cccDNA in peripheral blood, whose manufacture can be scaled up and marketed to diagnostic laboratories.

Project Goal

The purpose of this project is to identify a panel of sera from treated and untreated HBV-infected patients, validate and develop an assay for quantitative detection of cccDNA in serum or plasma, establish the performance characteristics of assay, and establish and validate the cccDNA detection kit.

Phase I Activities and Deliverables

1.      Design assay for quantitative detection of HBV cccDNA in serum or plasma from HBV-infected patients.

2.      Validate assay and determine sensitivity and specificity using seroconversion panels.

Projected Phase II Activities:

1.      Validation of the assay using specimens from HBV-infected patients and controls; optimize and validate the assay using clinical samples from patients and controls and establish and improve performance characteristics of assay.

2.      Validation of the assay using specimen from HBV infected patients and Report Writing; produce prototype HBV cccDNA assay and explore feasibility of transferring technology to commercial and state and public health laboratories.

Impact

After infection or antiviral therapy, HBV remains dormant in the infected person by adopting the cccDNA form in the liver. As cccDNA can also be found in the blood, especially after liver damage, its detection in serum or plasma allows the efficacy of antiviral therapy and the extent of liver damage to be evaluated without resorting to liver biopsies. Worldwide more than 350 million people are chronically infected with HBV. Improved antivirals are in the pipeline that potentially cure instead of suppress HBV. In the next 5-10 years, a substantial proportion of HBV infected persons who undergo antiviral therapy will benefit from access to a facile diagnostic method for the detection of cccDNA.

Commercialization Potential

Assays for detection and quantification of cccDNA in serum are not available. A simple, cost-effective and sensitive test kit, whose manufacture is then scaled up, should become marketable for use in diagnostic laboratories. 

Background

Hepatitis B is a major public health problem in United States, where 1.4 million persons are estimated to be infected with the virus. HBV surface antigen (HBsAg) is the mainstay serological marker used for identifying HBV infection and evaluating the efficacy of antiviral therapy. However, it is not a reliable marker of HBV found in blood, as it is shed from the liver in much greater abundance than HBV virions. Further, HBsAg can be shed from HBV even when it is residing in the liver in the latent (non-replicating) state.

HBV core antigen (HBcAg) is a constituent of the HBV nucleocapsid, which after encapsidation is released into the circulation.  HBcAg in serum or plasma is a better indicator than HBsAg of the extent of shedding of HBV virions from the liver to peripheral blood, i.e., ‘productive’ HBV infection.  Critically, as transcription and translation of HBcAg production are not inhibited by nucleotide analogues currently used for antiviral therapy, patients treated with these drugs continue to produce HBcAg for considerable periods of time. Immunoassays to detect HBcAg can therefore help in identifying HBV infections without resorting to HBsAg or HBV DNA testing.

Previous studies have shown that HBc antigenimia correlates positively with HBcAg production in the liver.  Several methodologies for the detection of HBcAg core antigen in serum have been published but assays for its detection and quantification are not commercially available for use in diagnostic procedures. Development of a reliable immunoassay that can also be an alternative to HBsAg and HBV DNA testing, is needed.

Project Goals

·          Identify a panel antibodies that have the potential to detect HBV core antigen in clinical samples.

·          Validate and develop a serological assay for quantitative detection of HBV core antigen

·          Validate the performance characteristics of the assay using commercial panels of serum samples from HBV-infected persons.

·          Validate the performance characteristics of the assay using with prospectively obtained serum samples from HBV-infected persons.

·          Establish protocols to scale up production of validated assay. 

Phase I Activities and Deliverables

Deliverable: Design and develop a simple immunoassay for detection and quantification of HBcAg in human serum or plasma from persons with acute and chronic HBV infection

Activity: Identify a panel of antibodies with the potential to be capture agents for HBcAg in serum or plasma.

Projected Phase II Activities

Deliverable: Optimize and validate the assay using clinical samples from HBV infected patients and controls

Activity: Establish sensitivity and specificity of the assay, continue to refine the assay and improve performance characteristics of the assay

Deliverable: Establish and validate prototype assay.

Deliverable: Produce final report and explore feasibility of transferring technology to commercial and state and public health laboratories.

Impact

Serologic testing for hepatitis C virus (HCV) core antigen is increasingly being used to identify persistent HCV infection and evaluating the efficacy of anti-HCV therapy. Similarly, testing for HBcAg has potential for use to identify productive HBV infection and evaluating the efficacy of anti-HBV therapy. Worldwide more than 350 million people are chronically infected with HBV. Improved antivirals are in the pipeline that potentially cure instead of suppress HBV. In the next 5-10 years, a substantial proportion of HBV-infected persons can then benefit from access to testing for HBVcAg. This start- up initiative, when funded and successfully developed, should lead to a marketable product for use in commercial and publically funded diagnostic laboratories.

Commercialization Potential

A simple assay for serologic detection and quantification of Hepatitis B virus core antigen with high sensitivity and specificity and low cost can lead to a marketable product for use in commercial and publically-funded diagnostic laboratories. The market potentially comprises the 350 million people who are chronically living with HBV worldwide.

National Center for Immunization and Respiratory Diseases (NCIRD)

The mission of the National Center for Immunization and Respiratory Diseases (NCIRD) is the prevention of disease, disability, and death through immunization and by control of respiratory and related diseases. Our challenge is to effectively balance our efforts in the domestic and global arenas as well as accommodate the specific needs of all populations at risk of vaccine preventable diseases from children to older adults.

NCIRD Web site:  http://www.cdc.gov/ncird/

For this solicitation NCIRD invites Phase I proposals in the following areas:

Background

Currently licensed live oral rotavirus vaccines, RotaTeq and Rotarix, are effective in preventing severe diarrhea among children in developed and middle income countries, but are significantly less effective in the developing world. While the causal mechanisms for this lower efficacy have not been clearly defined, we hypothesize that multiple factors, such as high titers of pre-existing maternal antibody, breast feeding and interference by other flora and viruses in the gut, might play a role in reduced vaccine efficiency among children.  Consequently, rotavirus remains a major killer among children in low-income countries of Africa and Asia. In addition, both vaccines are associated with a low risk of diarrhea and intussusception (i.e., blockage of the small intestine) among infants who receive the first dose of vaccine. To improve the safety and efficacy of oral rotavirus vaccines, CDC scientists have developed a proprietary inactivated rotavirus vaccine (IRV) technology (new human strains and a novel method for rotavirus inactivation) and demonstrated the immunogenicity and protective efficacy in piglets of this IRV by intramuscular (IM) injection and transcutaneous administration using microneedles. With the establishment of proof of concept for parenteral (i.e., non-oral) immunization, CDC has licensed this technology to a number of vaccine manufacturers for further R&D and clinical development of an IM IRV.

We now propose a SBIR topic for the formulation and fabrication of a dissolving microneedle patch to deliver an IRV for transcutaneous immunization against rotavirus in collaboration with a contract manufacturing organization. We have recently demonstrated enhanced immunogenicity of our IRV using an innovative metal microneedle patch technology, achieving comparable antibody titers with a 1/10th of the antigen dose compared to those induced by a full IM dose of vaccine. Microneedles provide a simple and painless method to administer vaccines without using hypodermic needles. They are inexpensive to manufacture and may not need the cold chain with large volume of cold storage and high cost, a major advantage for immunization campaigns in the developing world.

Transcutaneous immunization using a dissolvable microneedle patch is a novel and innovative approach to the prevention against infectious diseases, but no such vaccines have been licensed for use in humans yet. Currently this technology to deliver influenza vaccine is being tested and evaluated in phase I clinical trials. Similar clinical trials for inactivated polio vaccine (IPV) using a dissolvable microneedle patch are being planned for the next few years. However, no development and proof of concept work have been done for IRV.

Project Goal

The goal of this project is to conduct formulation and process development and a feasibility study to manufacture a dissolving microneedle patch for skin immunization against rotavirus. This program area will provide small business companies with opportunities to apply for necessary funds and work with CDC scientists to further optimize the fabrication process and prepare a dissolving microneedle patch for clinical trials of a patch IRV.

Phase I Activities and Expected Deliverables 

1.      Develop an outline for the project goals described above.

2.      Develop a draft scalable manufacturing process for a dissolving microneedle patch, including formulation and fabrication of IRV and necessary assays.

Expected Phase II Activities

1.      Develop and validate a scalable manufacturing process for a dissolving microneedle patch.

2.      Develop and implement manufacturing methods to make microneedle patches for IRV vaccination under good manufacturing practice (GMP) conditions.

3.      Support regulatory approval to conduct a phase I clinical trial of IRV vaccination using a microneedle patch.

4.      Support for a phase I clinical trial to assess the safety, immunogenicity, reactogenicity, and acceptability of IRV vaccination using a microneedle patch.

Impact

The findings from this SBIR research may allow us to enhance public health through the development of a low cost vaccine with an improved safety and efficacy profile and thus help advance CDC’s Global Immunization Winnable Battle that includes increasing Global Health Impact of rotavirus vaccination.

Commercialization Potential

Demonstration of the feasibility for the manufacture of a dissolving microneedle patch for IRV will bode well for a serious investment and more expeditious and effective development of this new and innovative IRV for commercialization. This IRV would be more efficacious in resource-poor settings because of its parenteral administration. As the world is transitioning to IPV from oral polio vaccine (OPV), a combined IPV and IRV in the expanded program on immunization (EPI) would ultimately increase global health impact through large immunization campaigns and help save more lives.

Background

Ongoing disease and death associated with seasonal influenza and the threat of an influenza pandemic are two of the highest priority issues for global public health. Vaccination is a powerful tool for preventing influenza, however, current vaccines have several limitations. Inactivated influenza vaccines (IIV) given by needle injection require skilled health care workers and can lead to needle-associated injuries and infections. Liquid nasal live attenuated influenza vaccine (LAIV) is needle-free but has limitations which can result in restricted distribution of vaccine.  The limitations of IIV and LAIV are critical because unlike many other vaccines, almost everyone needs flu vaccine every year. Also, efficient rapid distribution is essential especially in a pandemic situation. An ideally distributable vaccine could be shipped without restriction, by mail for example, and self-administered.  Every step away from this ideal decreases the accessibility of the vaccine. Both IIV and liquid LAIV require strict adherence to cold chain requirements (shipping and storage at 2-8⁰C) which restricts distribution to facilities with monitored refrigeration.  This is expensive in the developed world and can significantly restrict distribution in the developed world, where cold chain capacity is stretched by routine EPI vaccination requirements and has little or no surge capacity for influenza vaccines or pandemic situations.

While the needle-free nasal administration LAIV is an advantage over vaccination by injection the liquid delivery format has several limitations. The FluMist™ LAIV available in the US is shipped and delivered in a prefilled liquid in a glass syringe which imposes shipping requirements and increases shipping costs. More importantly vaccine by liquid nasal spray results in suboptimal vaccine deposition. The large droplets administered by liquid nasal spray devices accumulate in the nares and much the dose is wasted by dripping out of the nose does not reach the target nasopharyngeal tissues.  Also, liquid nasal sprays require an experienced vaccinator as differences in the force and speed of plunger depression results in variable droplet size and the placement of the spray tip can significantly affect vaccine deposition.    LAIV based on the Leningrad donor virus is also delivered as a liquid nasal spray. This vaccine is shipped as a lyophilized cake and requires reconstitution with a separate liquid diluent, which adds another layer of complexity to vaccine delivery. These two limitations in the distributability of liquid LAIV, cold chain requirements and inefficient liquid delivery are the gaps this project seeks to address.

Recent studies in anatomic models of nasal airways have shown dry powder nasal delivery provides markedly improved distribution and retention compared to liquid nasal spray delivery. Dry powder nasal delivery was also less sensitive to vaccinator variability and can potentially be self-administered. (CDC unpublished data) A nasal thermostable dry powder LAIV would retain the advantages of needle-free nasal delivery and improve upon them by removing cold chain restrictions and improving the consistency and efficiency of delivery and reducing the skill level needed to deliver nasal LAIV. 

Project Goal

The goal of the proposed research is to develop a thermostable dry powder LAIV for nasal delivery as a platform technology and assess immunogenicity following nasal powder vaccination in a ferret model. It is expected that this platform technology of thermostable dry powder nasal vaccine will be expanded to use for other vaccines.

Phase I Activities and Expected Deliverables

1.      Create dry powder LAIV

a.      Acquire high titer single strain LAIV bulk lot vaccine

b.      Dry LAIV into a thermostable format

c.      Process dry LAIV into a powder  with a size suitable for nasal delivery ( approximately 20 micron average particle size)

2.      Assess potency and 1 month thermostability of the dry powder LAIV

a.      Freeze aliquots of bulk lot vaccine at -70⁰C for potency test controls

b.      Store samples of powder vaccine at 4-8⁰C, 24⁰ and 37⁰C for testing

c.      Compare potency of powder vaccine at various temperatures to frozen and lyophilized LAIV potency at 1 week, 2 weeks, 1 month

d.      Test powder vaccine potency by EID₅₀ and TCID₅₀ compared to frozen LAIV

e.     Assess formulation and process parameters for optimum thermostability

Projected Phase II Activities

1.      Optimize formulation and process parameters

2.      Package powder into a nasal delivery device

3.      Assess potency and 1year thermostability of the optimum dry powder LAIV

a.      Freeze aliquots of bulk lot vaccine at -70⁰C for potency test controls

b.      Store samples of powder vaccine at 4-8⁰C, 24⁰ and 37⁰C for testing

c.      Store samples of powder vaccine in nasal delivery packages at 4-8⁰C, 24⁰ and 37⁰C for testing

d.      Test powder vaccine by EID₅₀ and TCID₅₀ compared to frozen LAIV

e.      Test packaged powder vaccine by EID50 and TCID₅₀ compared to frozen LAIV

f.      Compare potency of powder vaccine at various temperatures to frozen and lyophilized LAIV potency at 1 week, 2 weeks, 1 month and then every other month for one year total storage time.

4.      Assess immune responses to and efficacy of  dry powder LAIV in the ferret model, which is the gold standard animal model for assessing the influenza vaccines

a.      Package powder into nasal powder delivery system

b.      Adapt delivery system to ferret nasal delivery if needed

c.      Vaccinate ferrets with dry powder LAIV and Liquid LAIV

d.      Assess serologic immune response

e.      Challenge with homologous influenza virus and measure viral load

Impact

The increased distributability of thermostable dry powder nasal LAIV could significantly improve coverage in the developed and developing world. In addition to the reduction in influenza morbidity and mortality, it is intended that this platform technology be expanded to other vaccines, increasing their accessibility and decreasing the morbidity and mortality of the respective diseases.  New thermostable vaccines could be stored and delivered at ambient temperatures, without refrigeration, deceasing energy usage and equipment costs for refrigeration and lightening loads for vaccine transport and delivery. This would be especially helpful in the developing world where hauling icepacks and coolers can make an already rough journey to a village more difficult. Thermostable vaccines can also eliminate the potentially catastrophic loss of vaccine potency that results when cold-chain methods fail, and facilitate distribution in pandemic responses, mass-vaccination campaigns, and agricultural applications. The disadvantages of liquid vaccine delivery format- increased shipping requirements and costs, suboptimal vaccine deposition, need for experienced vaccinators and onsite reconstitution are described above. A thermostable powder vaccine could be potentially be shipped by mail or hand carried by anyone and self-administered or administered by people with minimal skill level.   This is the ideal in making vaccines accessible to everyone who needs them.

Commercialization Potential

The Advisory Committee on Immunization Practices (ACIP) recommends every person over age 6 months receive annual flu vaccination, with few exceptions, which would require over 300 million doses a year. In recent seasons over 130 million doses of flu vaccine were distributed annually in the US, almost equaling the total of all other vaccines combined. Over the recent years, the global influenza vaccine market has witnessed double digit growth rate due to fear of an impending pandemic and is expected to cross USD 4 billion in 2015.  The high volume demand for influenza vaccine fosters competition which is leading to increased innovation in vaccine delivery. Vaccine manufacturers are willing to invest in improved delivery systems to increase market share by making their vaccines more acceptable and accessible to the population.  Recent licensed innovations influenza vaccine include needle-free nasal spray LAIV (MedImmune FluMist™ AccuSpray syringe), intradermal delivery using minineedles (Sanofi Fluzone ID™ with Soluvia™ minineedles), and vaccination by jet injection (bio CSL’s Afluria™ with PharmaJet Stratis™ jet injector. 

The most likely business model for the small business developer of a formulation and process for thermostable dry powder LAIV would be to license the technology to vaccine manufacturers for fees and or royalties.

Adversehumanhealthoutcomes – a.k.a., “toxicity” – caused by pharmaceutical or environmental compounds are a major cause of drug development failure and public health concern. Methods to evaluate the potential of chemical compounds to induce toxicity are based largely on animal testing, are low-throughput and expensive while giving little insight into mechanisms of compound toxicity, and have not changed appreciably in the last 50 years despite enormous advances in science. Multiple efforts, including Tox21 program in the U.S., REACH Program in the E.U., and multiple industrial collaborations, are attempting to develop in vitro methods using induced pluripotent stem cell (iPSC)-derived cells to assess chemical toxicity. Stem cells or iPSC-derived cell types have great potential to provide more physiological relevance than immortalized/transformed cell lines and to provide larger quantities and higher assay reproducibility than primary cells. These programs must assess toxicity potential in every organ system and identify pathways and/or targets affected.

Given the protean nature of these effects, it is likely that hundreds of in vitro assays will need to be developed and evaluated for their ability to profile chemical effects on particular cell types and pathways. Progress in the field is currently limited by the relatively small number of pathways and cell types that have been developed into high- throughput screening (HTS)-ready assays, and the artificial nature of many of the assays that have been developed (e.g., immortalized/transformed cell lines, heterologous expression with lack of physiologically accurate regulation).

The development of HTS-ready assays using stem cells or iPSC-derived cells, which can report on particular pathways and cellular phenotypes across the full spectrum of pathway space and toxicological outcomes, is needed. Such assays would need to meet strict performance criteria of robustness, reproducibility, and physiological relevance. The assays developed would need to be capable of being run in 384-well or (ideally) 1536-well format and must allow the testing of >100,000 samples per week.

Mainrequirements

Theoutcomeofthiscontractisexpectedtobeoneormorenovelassays that can be performed in stem cells or iPSC-derived cells fortargets,pathways,andcellularphenotypesrelatedtoanytypeofxenobiotictoxicity.Theseassayswouldutilizehumancells,includingprimarycellsandstemcellderivedcells,andmustbefunctionalinmulti-wellformatwithcharacteristicssuitableforautomatedhigh-throughputscreening.Suchassaysshouldbenovel, having metabolic capability, reflecting new pathways or cellular endpoints than are currently available, and be clearlyconnectedtosometypeofhumantoxicologicalresponse.Suchassayscouldfindutilityasinchemicalassessmentandriskmanagementafter validation.

DeliverablesPhase1

Anassaythatmeetstherequirementslistedaboveandalsomeetsthefollowing:

·          Developaworkingassayin96-wellordenser(384,1536)micro-wellformat

·          Characterizethesensitivity,specificity,variability,reproducibility,signal:background,dynamicrange,and accuracyoftheassay,utilizingstandardpositiveandnegativecontrols,Z’values>0.5

·          Demonstratetheutilityoftheassaybycharacterizingitsabilitytodetecttheeffectsofcompoundsknowntoaffectthepathway/cellularphenotype,withathroughputofatleast10,000samples/daywithworkstationautomation

·          Arenotduplicativeofassaysalreadyavailablecommercially

·          Delivertheassay/SOPtoNCATSforevaluation

DeliverablesPhase2

·          Demonstrateminiaturizationofassaytoworkinatleast384-well(preferably1536-well)formatwithsametechnicalspecificationsaslistedabove

·          DemonstrateamenabilityforHTSbysuccessfultestingof>100,000samples/dayinfullyautomatedroboticformatwithmaintenanceofassayperformance

·          Deliverfinalassay/SOPtoNCATSforevaluation.

Microtitre plates are nearly ubiquitous in many life sciences laboratory settings, used as a standard tool to act as the vessel in which a wide variety of experiment types are performed.  There are a variety of key parameters that dictate the application a particular plate type will be used for, such as the number of wells present on a plate, the color of the plate depending upon the detection method to be used, the material the plate is made from to ideally be as chemically inert as possible, the surface treatment or coating of the wells within a plate geared towards a specific experiment type and other parameters for more specialty plate types.

One nearly universal parameter regardless of plate type is that these plates are typically assumed to be a consumable product, used once and then discarded, making them in effect a disposable item.  Depending on the laboratory in question, it is not uncommon for thousands if not tens of thousands of these plates to be consumed and discarded within a single year.  Given the disposable nature of these parts, they have been manufactured with the idea that each plate will be a consumable product, and are typically made out of a variety of polymers using injection molding techniques, most commonly polystyrene and polypropylene.  Given the consumable nature of the product and the materials used to manufacturer them on a large scale at low cost, the typical features that distinguish one plate from another typically come down to the physical properties of the materials the plates are made of and any additional additives to them, potentially limiting the plate from being anything more than a vessel for an experiment.

Over the last ten years, High-Throughput Experimental Sciences (High-Throughput Screening) have evolved at a very rapid pace- mostly enabled by ever more sensitive assay constructs, and the evolution of detection and miniaturization schemes. Ten years ago a typical high-throughput assay would have 2-3 reagent constituents and a single absorbance or fluorescence-based readout. Today, modern screening facilities are commonly running very high-content, very data rich assays with optical microscopy- based readouts.  These assays produce terabytes of data leading to information that is uncovering cellular behavior characteristics never before measured. These progressive steps have far out-paced the evolution of the microplate—as a “dumb” article made of plastic. The microplate component of the “system” needs to be significantly updated in its’ ability to harvest data, monitor environmental and atmospheric conditions, and report in real time any micro-climate variances-which are critical systemic contributors to cell performance.

Main Requirements:

The purpose of this project is to treat a microtitre plate not as a disposable product meant to act as a vessel for one experiment, but instead to potentially be a resource that can be used multiple times against a variety of different experiments.  Instead of limiting these plates to only being a variety of plastics with a lifespan of one use, if different materials and manufacturing techniques were utilized it could greatly impact what a plate could be used for.  Imagining the plate as being used for a variety of monitor and control applications be built directly into the plate, such as temperature, relative humidity, CO2 and O2 levels etc., instead of relying on external pieces of instrumentation to perform these measurements.

The title of this solicitation is 'Smart Plate' in reference to idea of the Smart Phone; once a platform was built to create a phone that could perform a variety of functions as opposed to simply one a huge amount of innovative ideas sprang forth.  The goal of this SBIR solicitation is to do the same, to fundamentally transform the idea of a microtitre plate from being a single use vessel for an experiment to nearly becoming an instrument itself which could provide more data about the samples under test to actually providing measurements in the plate itself.  A key goal tied to this is to break the idea of a plate being completely disposable and instead treating each plate as a resource that can be used many times.

One important point of this solicitation is that we are NOT looking for the creation of a plate that only works with a specific piece of instrumentation; the goal is a feature rich plate that can be used in a variety of existing instrumentation.

Phase I Activities and Expected Deliverables:

·       Develop a prototype that has the following features:

·       Adheres as closely as possible to current ANSI/SLAS Microplate Standards

§  ANSI/SLAS 1-2004 (R2012) Microplates – Footprint Dimensions (formerly ANSI/SBS 1-2004)

§  ANSI/SLAS 2-2004 (R2012) Microplates – Height Dimensions (formerly ANSI/SBS 2-2004)

§  ANSI/SLAS 3-2004 (R2012) Microplates – Bottom Outside Flange Dimensions (formerly ANSI/SBS 3-2004)

§  ANSI/SLAS 4-2004 (R2012) Microplates – Well Positions (formerly ANSI/SBS 4-2004)

·       The plate should provide the ability to work with different well types, shapes and materials.

§  The wells do not necessarily have to store sample; they could in fact be components that perform various functions within a plate.

·       Incorporates automated sensing of variables such as temperature, relative humidity, volume, CO2, O2, pH and others of the samples within a well.  Not all variables are represented and the prototype is not required to monitor all of these, these are just given as examples.

§  The values should ideally be accessible in real time; so in the best case scenario the ‘Smart Plate’ would have some sort of integrated messaging capabilities over common network protocols such as SMS, MMS, RFID or TCP/IP.

§  If messaging is not an option in real time, the ability to store these values to memory for retrieval at a later time is required.

·       The device will ideally have some capacity to allow flow between wells through microfluidic channels.  The pumps or devices used to generate the flow are not required as part of the plate itself but access ports should be available.

·       Provide a detailed requirements and design document for the device, including mechanical and electrical drawings, in addition to hardware specifications and communications protocols used.

·       Cost estimates to manufacture a device capable of meeting the specifications listed above.

·       Provide NCATS with all data resulting from Phase I Activities and Deliverables.

Phase II Activities and Expected Deliverables:

·       Build a prototype plate that meets the Phase I specifications.

·       Provide a test plan to evaluate every feature of the device

§  Provide NCATS with all data from each executed test to properly evaluate each test condition

·       Develop a robust manufacturing plan for the device, using off the shelf OEM components where possible to minimize expense.

·       Provide NCATS with all data resulting from Phase II Activities and Deliverables.

National Heart, Lung, and Blood Institute (NHLBI)

The NHLBI plans, conducts and supports research, clinical trials and demonstration and education projects related to the causes, prevention, diagnosis, and treatment of heart, lung, and blood (including blood vessel), and sleep disorders. It also supports research on the clinical use of blood and all aspects of the management and safety of blood resources. The NHLBI SBIR/STTR program fosters basic, applied, and clinical research on all product and service development related to the mission of the NHLBI.

For more information on the NHLBI SBIR/STTR programs, visit our website at: http://www.nhlbi.nih.gov/sbir

NHLBI Phase IIB Programs

The NHLBI would like to provide notice of two SBIR Phase IIB funding opportunities. This notice is for informational purposes only and is not a call for Phase IIB proposals. This informational notice does not commit the government to making such awards to contract awardees.

The NHLBI offers Phase IIB opportunities through the NHLBI Bridge Award and the NHLBI Small Market Award using separate funding opportunity announcements (Bridge Award: RFA-HL-16-009; Small Market Award: RFA-HL-14-012). The purpose of the NHLBI Bridge and Small Market Awards is to accelerate the transition of SBIR Phase II projects to the commercialization stage by promoting partnerships between SBIR or STTR Phase II awardees and third-party investors and/or strategic partners. The Small Market Award is designed to support technologies addressing rare diseases or pediatric populations. The Bridge and Small Market Awards encourage business relationships between applicant small business concerns and third-party investors/strategic partners who can provide substantial financing to help accelerate the commercialization of promising new products and technologies that were initiated with SBIR/STTR funding. In particular, applicants are expected to leverage their previous SBIR/STTR support, as well as the opportunity to compete for additional funding through the NHLBI Bridge Award or Small Market Award programs, to attract and negotiate third-party financing needed to advance a product or technology toward commercialization.

Budgets up to $1 million in total costs per year and project periods up to three years (a total of $3 million over three years) may be requested. Development efforts may include preclinical R&D, which is needed for regulatory filings (e.g., IND or IDE) and/or clinical trials.

An SBIR Phase IIB Bridge or Small Market Award application must represent a continuation of the research and development efforts performed under a previously funded SBIR or STTR Phase II award. The NHLBI welcomes applicants previously funded by any NIH Institute or Center or any other Federal agency, as long as the proposed work applies to the NHLBI mission. Applications may be predicated on a previously funded SBIR or STTR Phase II grant or contract award. Applicants with Phase II contracts or awards from another Federal agency must contact the NHLBI to ensure their application can be received.

Applicants are strongly encouraged to contact Jennifer Shieh, Ph.D. at 301-443-8785 or jennifer.shieh@nih.gov for additional information.

Limited Amount of Award

For budgetary, administrative, or programmatic reasons, the NHLBI may not fund a proposal and does not intend to fund proposals for more than the budget listed for each topic.

This solicitation invites proposals in the following areas.

In the short term, this topic aims to 1) develop bioinformatic methods to integrate metabolite data across various laboratory platforms and analytical technologies, including liquid-chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and NMR; and 2) develop scalable software tool(s) to automate these methods for use by the cancer and overall public health research communities. Valid and reliable data harmonization of metabolomics data also builds a critical foundation for the longer term goal of integration of metabolomics data with other ‘omics data (e.g., genomics, proteomics, transcriptomics, epigenomics, etc.). The development of methods to integrate a wide range of -omics data will position the research community to better leverage existing data for the discovery of novel cancer biomarkers of etiology, diagnosis, and prognosis.

Responses to this topic are expected to address the development of efficient bioinformatic methods to:

1. Demonstrate bioinformatic methods for the integration of metabolite data across different laboratory platforms and analytical technologies with high accuracy;

2. Store metabolite data from the different data sources in databases that can be easily used for data integration and quality control protocols;

3. Implement valid quality control (QC) checks; and

4. Appropriately secure data at each stage of transfer and storage.

An essential task for each proposal is the development of bioinformatic tools in the form of scalable software that can be used by the research community at-large to automate complex data integration tasks for metabolomics data sources.

Phase I activities should provide evidence that metabolite data integration bioinformatic methods, using identified metabolite data, have been effectively developed, can be implemented across data inputs from diverse laboratory platforms and at least two analytical technologies, and demonstrate readiness to proceed to Phase II. Additionally, Phase I will be used to demonstrate the framework for scalable software tool(s) that apply the bioinformatic methods to automate the integration of metabolite data.

Phase I Activities and Deliverables

• Establish a project team including proven expertise in metabolomics analytical technologies, epidemiology, biostatistics/bioinformatics, and computer technology. Additionally, a team including expertise in biochemistry/clinical chemistry is preferred.

• Develop bioinformatic methods for metabolite data integration for identified metabolites across data inputs from diverse laboratory platforms and at least two analytical technologies (preferably liquid-chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and/or NMR).

• Participate in the development of a collaboration agreement between the offeror, NCI, and NCI-identified third party sources to access relevant input data types for the proposed project. NCI staff will work with established cohort studies and consortia to provide metabolomics data (identified metabolite data) to successful offerors.

• Develop database formats that support the import and export of individual datasets and “combined” datasets, store structured data from different sources of metabolite data, and are readily used for data integration and QC protocols.

o Finalize database formats and structure, data collection, transport and importation methods for targeted Phase I data inputs.

• Provide wireframes and user workflows for the proposed Graphical User Interface (GUI) and software functions that:

o Support the import and export of individual datasets and “combined” datasets;

o Implement, script or automate all features and functions of the data integration tool(s);

o Conduct QC of “combined” datasets.

• Provide a report including a detailed description and/or technical documentation of the following:

Page 63

o Specific approach to metabolite data integration;

o Specific approach to QC;

o Data standards for transfer and importation of individual metabolite data and storage of individual and “combined” metabolite data;

o Data visualization, feedback, and reporting systems for individual and “combined” metabolite data;

o Technology compatibility matrix for Phase I and Phase II metabolomics data sources by laboratory platform, analytical technology, and identified metabolites (Phase I) / unidentified metabolite peaks (Phase II).

o Software tool(s);

o Transparent, documented, and non-proprietary bioinformatic methods; and

o Description of additional software and hardware required for use of the tool.

o Finalized database formats and structure, data collection, transport, and importation methods for targeted data inputs; and

o Funds in budget to present Phase I findings and demonstrate the wireframes and user workflows for the GUI and software functions to an NCI evaluation panel.

• Develop functional prototype software that integrates data from planned Phase I technology compatibility matrix data sources using automated algorithms and methods.

• Include funds in the Phase I budget to present project deliverable and the prototype software tools to an NCI panel for evaluation.

Phase II Activities and Deliverables

• Expand the bioinformatic methods to include unidentified metabolite peaks, in addition to identified metabolite data, and demonstrate metabolite data integration across data inputs from diverse laboratory platforms and at least two analytical technologies (preferably liquid-chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and/or NMR).

• Participate in the development of a collaboration agreement between the offeror, NCI, and NCI-identified third party sources to access relevant input data types for the proposed project. NCI staff will work with established cohort studies and consortia to provide metabolomics data (identified metabolites and unidentified peak data) to successful offerors that would serve to: 1) train and validate the expanded bioinformatic methods; and 2) demonstrate the application of these methods through scalable software to automate complex data integration tasks for metabolomics data sources.

• Demonstrate usability of scalable software through the following:

o Beta-test and finalize automated file transfer, database importation protocols, metabolite data integration applications and reporting tools developed in Phase I

o Develop beta-test, finalize, and demonstrate the GUI

o Demonstrate the software systems ability to integrate data from planned Phase II technology compatibility matrix data sources using automated algorithms and analytic methods

• Conduct usability testing of the GUI elements of the metabolite data integration tool(s).

• Develop systems documentation where applicable to support the software and bioinformatic methods.

• In the first year of the contract, provide the program and contract officers with a letter(s) of commercial interest.

• In the second year of the contract, provide the program and contract officers with a letter(s) of commercial commitment.

Mobile health technologies have grown exponentially in the past few years. The ubiquity of mobile phone use provides a platform for health assessment, monitoring and interventions previously unavailable to health research and clinical practice. The penetration of mobile phone use, even in remote areas, provides a vehicle for health care delivery to individuals with limited access to care. Wireless sensor technologies have also rapidly expanded in availability and function in the past few years. When paired with mobile devices, these sensor technologies provide real-time data on a variety of image-based, physiological, behavioral, or environmental variables.

The range of health research and clinical practice affected by this technology revolution is quite broad. Preventive health assessment and intervention applications for cancer associated behavioral risk factors have increased dramatically. Mobile technologies have been developed for medical screening and diagnostic purposes, providing low cost and portable diagnostic tools for use in rural and underserved settings. Mobile technologies have also been used to support cancer survivorship care and improve chronic disease management for cancer risk factors such as obesity and diabetes, allowing healthcare providers to more intensively monitor patient status and intervene as needed while providing patients a resource to more effectively self-manage their disease.

The NCI Division of Cancer Control and Population Sciences aims to reduce risk, incidence, and deaths from cancer, as well as enhance the quality of life for cancer survivors. Emerging mobile technologies provide an opportunity to support innovation and progress towards NCI’s mission of cancer prevention & control by 1) improving quality or access & reducing cost or burden of screening, diagnostic, treatment and follow-up care for cancer and associated chronic diseases; and 2) improving lifestyle intervention efficacy and scalability for cancer related behavioral risk factors. The number of mobile and wireless health tools grows each year, but the majority of these tools have been inadequately validated in clinical research and practice. Adoption of these technologies in support of cancer treatment and survivorship requires more evaluation in clinical and behavioral settings. This topic is not intended to support new technology development, but instead to clinically validate promising but insufficiently tested tools for cancer prevention & control.

Project Goals

The purpose of this topic is to support validation of mobile technologies for clinical assessment, screening, diagnostics, monitoring or intervention delivery focused on cancer prevention, and control objectives. Examples of technologies may include monitoring or diagnostic sensors & paired smartphone applications, cancer treatment or survivor care planning & remote monitoring systems, behavioral analytics and decision support systems, or intervention delivery systems. In the short term, the topic aims to develop research evidence to support adoption of innovative mobile technologies which support cancer prevention, treatment, disease management, or survivorship. Longer term goals are the integration of these technologies in clinical assessment, care & intervention delivery within health systems, accountable care organizations (ACO), and health research.

Within the context of this topic, "mobile" health technologies are defined broadly to include anyhealth technologies that wirelessly transmit data and that are intended for portable use. The early focus of these technologies has primarily been devices worn on or carried by the individual throughout the day. However, devices that provide a level of portability not previously available (e.g. smaller and more portable version of a diagnostic scanner that transmits data wirelessly to the healthcare provider) is consistent with the scope of this initiative.

As noted previously, this topic in not intended to support the development of new technologies. Some additional programming may be required to customize or integrate the technology into the target clinical, health system, or related software environments, but these efforts should be sufficiently limited to retain a focus on validation and expanded evidence of commercial potential and value for health assessment or outcomes.

Responses to this topic are expected to address one or more of the following areas of mobile/wireless health research;

1) Evaluation of the reliability of mobile screening, diagnostic, assessment or monitoring technologies & methods

2) Evaluation of the validity of mobile screening, diagnostic, assessment or monitoring technologies & methods

3) Evaluation of the efficacy and effectiveness of mobile technology and systems for behavioral analytics, clinical decision support, or intervention delivery.

Although extension of existing usability, acceptability, and feasibility of the mobile/wireless health tool may be considered as secondary research questions, they should not be the primary objectives of applications in response to this topic.

This topic will prioritize research that will rapidly validate existing mobile technologies in clinical care & monitoring, clinical decision support or intervention applications. It is anticipated that the clinical screening, diagnostic, assessment, and monitoring technologies will provide the "gold standard" comparator for the new mobile or wireless tool being evaluated, but additional clinical measures may be needed to validate the new tool. However, in some instances, novel measures may not directly translate to existing clinical “gold standard” measures/technologies, and alternative validation approaches may be required. Validation analyses could include but are not limited to agreement rates, sensitivity/specificity, and receiver operating curves (ROC). Research evaluating the reliability of the technology is consistent with this topic. For outcome monitoring purposes, assessment of sensitivity to change are also consistent with this topic.

○ Validation of mobile technologies and systems for intervention delivery or decision support are particularly encouraged. Dependent on the research question and technology under evaluation, research designs may include randomized controlled trials (RCTs), series of single case designs, optimization designs (e.g. factorial, sequential) or quasi-experimental approaches such as interrupted time series and stepped-wedge designs. Projects that integrate and automate ongoing validation and/or outcomes evaluation (e.g. automated RCTs) in the commercial product are particularly encouraged. For additional information on evaluation of mHealth technologies please see (http://www.ajpmonline.org/article/S0749-3797(13)00277-8/abstract). Primary clinical or behavioral outcomes may be supplemented with cost-effectiveness analyses where appropriate.

Milestones for Direct-to-Phase II Technologies

All proposals submitted under this topic must provide evidence that specific mobile technology or systems development milestones have been achieved to demonstrate readiness for a Direct-to-Phase II contract. These milestones will be evaluated in addition to standard review criteria for all submissions.

1. Provide evidence that a working prototype, including all major functional components of the technology, is ready for formal validation in Phase II with minimal further development other than that required to perform the validation or outcomes research.

a. Products in beta version are particularly appropriate for this effort although recently released commercial products that do not have adequate validity or efficacy support are also encouraged.

2. Provide documentation that the product to be evaluated has been developed based on theory and/or empirical evidence.

3. Present evidence that appropriate focus groups, interviews, cognitive or user testing with potential end-users of the device/software tool, etc. have been conducted to demonstrate that the feasibility, acceptability, and usability of the product have been established.

4. Provide evidence that an established project team with appropriate expertise for the scope of work is in place to advise and support the small business on Phase II activities and outcomes. This team should include, but will not be limited to, personnel with training and research experience in clinical or intervention design, implementation, and statistical methods for validation/evaluation as appropriate for the proposed project.

Phase II Activities and Deliverables

• Provide documentation that the established project team with appropriate expertise for the scope of work is in place to advise and support the small business on Phase II activities and outcomes. This team should include, but will not be limited to, personnel with training & research experience in clinical trial or intervention design, implementation, and statistical methods for validation/evaluation as appropriate for the proposed project. Provide a report outlining team member credentials, specific project roles, and timelines for performance.

• Evaluate specific IT customization requirements to support hardware, software, or communications system integration of the technology into the target clinical, health system or service, or other relevant software environment in preparation for validation. Provide a report documenting the specific IT customization requirements and timelines for implementation.

• Evaluate, enhance as necessary and provide documentation that the technology and communications systems maintain compliance with HIPAA, data security, privacy, and consent management protocols as required for the proposed solution.

• Evaluate, enhance as necessary, and provide documentation that data systems, APIs & wireless transmissions for all clinical, laboratory, or behavioral measures sent or received adhere to common data elements standards (e.g. HL7, SNOMED, LOINC, etc.) where available to facilitate data sharing and system integration.

• Evaluate, enhance as necessary, and provide a report detailing communication systems architecture and capability for data reporting to patients/subjects, care providers, clinicians/researchers, electronic medical records, and health surveillance systems as appropriate for the proposed technology solution.

• Test the integration of the technology into the target clinical, health system or service, or other relevant software environment in preparation for validation. Provide a report documenting the results of system testing and timelines for problem mitigation.

• Develop user support documentation to support all applicable potential users of the technology, including but not limited to patients/consumers, family/caregivers, and providers. Provide a report documenting user support resources, including but not limited to, links to online resources and copies of electronic or paper user support resources as appropriate.

• Prior to evaluation, provide a final report of the research plan including at a minimum

o Appropriate human subjects protection / IRB submission packages and documentation of approval for your research plan.

o Final study design including aims, participant characteristics, recruiting plans, inclusion and exclusion criteria, measures, primary and secondary endpoints, design and comparison conditions (if appropriate), power analyses and sample size, and data analysis plan.

o Publication plan outlining potential research and whitepaper publications resulting from the research, including anticipated lead and co-author lists.

• Provide study progress reports quarterly, documenting recruitment and enrollment, retention, data QA/QC measure, and relevant study specific milestones.

• Prepare a tutorial session for presentation at NCI and/or via webinars describing and illustrating the technology and intended use.

• Include funds in budget to present Phase II findings and demonstrate the technology to an NCI evaluation panel.

• In the first year of the contract, provide the program and contract officers with a letter(s) of commercial interest.

In the second year of the contract, provide the program and contract officers with a letter(s) of commercial commitment.

Persistent cognitive deficits are a frequent complaint of the increasing population of cancer survivors, particularly those who have undergone chemotherapy. Cancer patients experience both acute and chronic cognitive effects during both the treatment and survivorship phases of the cancer control continuum. One significant barrier to assessing cognitive symptoms in cancer populations is the inability to administer a brief, scientifically valid cognitive battery, either remotely or within a clinical visit.

The current gold standard in the field is standardized neuropsychological tests. These tests were devised for the purpose of diagnosing severe and focal cognitive impairments, such as stroke, and present consistent research challenges. First, they lack sensitivity to less severe, but still debilitating cognitive impairments such as those observed in chemotherapy patients. Second, they lack specificity; it is difficult to tell which cognitive function is responsible for poor performance. Third, they lack repeatability; tests were designed for a single diagnostic administration, and thus it is difficult or impossible to administer the same test multiple times over the course of a treatment protocol. Fourth, most such tests were originally devised decades ago and thus make no contact with the considerable advances in cognitive and neuro-scientific theory over the last thirty years. Fifth, a trained neuropsychologist must be on site to administer the test, pushing up the costs of research and limiting use in the clinical oncology setting. Therefore, there is a substantial unmet need for a suite of computerized cognitive tests, based on contemporary cognitive psychology and neuroscience research, designed for repeated testing, that could be easily administered remotely.

Project Goals

The goal of this project is to develop a scalable, secure, and privacy-compliant software system, and tools to support computerized administration of brief cognitive assessments specifically focused on measuring the subtle cognitive changes associated with cancer and cancer treatment.

The software system must support assessments of basic cognitive processes that are repeatable and can be remotely administered across diverse settings (i.e., clinic or home) using multiple technology platforms (i.e., PC, tablet, smartphone). In addition, the system must support provider/researcher portal functionality for patient management functions including (but not limited to) adding patients, ordering and scheduling assessments, automated scoring, visualization and triage of results, and standards based data reporting to third party systems. Where appropriate and relevant for project goals, the software system should integrate measures available via third party systems such as PROMIS, NIH Toolbox, and NeuroQOL rather than validate duplicative resources.

In Phase I, offerors will establish a multidisciplinary team for validation/evaluation of the proposed platform, provide documentation that appropriate software-based assessments have been developed, as well scoring protocols in the minimum specified cognitive domains. A report providing a detailed description, visual design, and technical documentation will be required, as well as a functional prototype. Phase I will also include client side user testing. Lastly, documentation detailing output reporting systems feasibility will be included in the demonstration of the final prototype to an NCI evaluation panel via webinar. In Phase II, offerors will evaluate specific IT customization requirements, test and finalize client, server, and data systems, including technical documentation for the software

systems application programming interface. Usability testing and support documentation will be provided, and submission of a research plan and presentation to NCI will be delivered via webinars.

The short term goal of this topic is to

1) Develop innovative software systems which support brief, remotely administered patient assessments and scoring of cognitive processes affected by cancer and cancer treatment

2) Develop paired provider portal tools for remote administration and management of patient assessments and results

3) Conduct user testing of the client side assessment tools and modes of administration

4) Conduct clinical validation of the cancer cognitive assessment instruments delivered via the software system

Longer term goals include the integration of these software tools in clinical assessment and monitoring in both oncology research and care settings, with the eventual goal of embedding assessment results into electronic medical records used in health systems and accountable care organizations (ACO).

This topic aims to support customized development and/or integration of information technology into the cognitive assessment process. The primary focus will be brief administration and scoring of assessments of cognitive processes affected by cancer and cancer treatments. Minimum cognitive domains to be assessed include: Attention, executive function, working memory, verbal abilities, visuospatial ability, motor function, and processing speed.

In addition to technical development, this topic is intended to support validation (e.g., efficacy and/or effectiveness) of the assessment battery, specifically with respect to reliably detecting cognitive changes in cancer populations.

Phase I Activities and Deliverables

• Establish a project team including personnel with training and research experience in cognitive psychology, neuroscience and/or neuropsychology, clinical oncology, implementation, and statistical methods for validation/evaluation as appropriate for the proposed technology platform.

• In addition, technical personnel should have experience in Health IT software standards (i.e., privacy, security, health data exchange protocols, etc.), electronic health records, cross platform client side software development, scalable sever side software development, data visualization, and systems architecture that will effectively address all objectives of the current topic.

• Provide documentation that software-based assessments have been developed based on current cognitive and neuroscience findings and evidence.

• Provide a report including detailed description of proposed assessments (including relevant modification for electronic administration) and scoring protocols planned for Phase I and Phase II development. Specifically address the minimum cognitive domains required including: attention, executive function, working memory, verbal abilities, visuospatial ability, motor function, and processing speed. In addition, patient reported cognitive complaints will need to be included.

• Provide documentation that planned software-based assessments have been developed based on current cognitive and neuroscience findings and evidence.

• Provide a report including detailed description, visual design, and/or technical documentation of the proposed:

o Database structure and data models for the proposed cognitive assessments, as well as system metadata requirements

o Client side graphical user interface and user experience

o Provider side graphical user interface and user experience

o Data standards for capture, transport, importation, and storage of data between client, server, and third party application as applicable

o Data visualization, feedback, or reporting systems, as required for target clinical monitoring and/or research applications

o Systems architecture for implementation of scalable software, as required based on development and commercialization targets.

• Develop a functional prototype system that includes

o Client software and wireframe user interface to facilitate and control the administration and transport of cognitive assessment and any associated metadata used within the system

o Server software and wireframe provider portal that supports automated schedule, administration, data scoring, and management of patient assessments and associated metadata

o Development release of end-to-end software system that connects client and server software & patient or provider portals for administration of planned Phase I assessments.

• Conduct user testing of client side software visual designs (or functional software) and proposed user experience for planned Phase I assessments.

• Provide a report detailing output reporting systems feasibility, proposed timelines, data standards, and communication architecture for reporting summary outputs to patients/subjects, clinicians/researchers, electronic medical records, and health surveillance systems.

• Finalize database formats and structure, data collection, transport, and importation methods for targeted cognitive assessments.

• Present Phase I findings and demonstrate the final prototype to an NCI evaluation panel via webinar

Phase II Activities and Expected Deliverables

• Evaluate specific IT customization requirements to support hardware, software, or communications system integration of the technology (e.g., HL7 compatibility); provide a report documenting the specific IT customization requirements and timeline for implementation.

• Enhance, user test and finalize client side software, patient portals and functionality listed in Phase I

• Enhance, user test and finalize server side software, provider portals and functionality listed in Phase I

• Enhance, beta test and finalize data visualization, feedback and reported systems listed in Phase I.

• Provide a report including technical documentation for the software systems application programing interface (API) for interaction with third party data systems.

• Conduct usability testing of

o Consumer/patient facing portals or mobile applications

o Care team/researcher facing portals or mobile applications

• Develop user support documentation for all applicable potential users of the technology, including but not limited to patients/consumers and providers. Provide a report documenting user support resources, including links to online resources and/or copies of electronic or paper user support resources as appropriate.

• Prior to evaluation/validation of software-based cognitive assessments, provide a final report of the research plan including at a minimum

o Appropriate human subjects protection/IRB submission packages, and documentation of approval for your research plan.

o Final study design including aims, participant characteristics, recruiting plans, inclusion and exclusion criteria, measures, design and comparison conditions (if appropriate), power analyses and sample size, and data analysis plan.

o Publication plan outlining potential research manuscripts and whitepaper publications resulting from the research.

• Prepare a tutorial session for presentation to NCI via webinars describing and illustrating the technology and intended use.

• In the first year of the contract, provide the program and contract officers with a letter(s) of commercial interest.

• In the second year of the contract, provide the program and contract officers with a letter(s) of commercial commitment.

Both normal and cancer tissues shed exosomes and other vesicles into body fluids. Tissue-shed exosomes are found in several body fluids including amniotic fluid, breast milk, bronchoalveolar fluid, cerebrospinal fluid, malignant ascites, plasma, saliva and urine. Exosomes collected from the blood and other body fluids of patients diagnosed with various cancers were shown to contain tumor suppressors, phosphoproteins, proteases, growth factors, bioactive lipids, mutant oncoproteins, oncogenic transcripts, microRNA and genomic DNA fragments. Exosomal trafficking and reciprocal exchange of molecular information among different organs and cell types were reported to contribute to cell-to-cell communication, horizontal cellular transformation, cellular reprogramming, functional alterations, regulation of immune response, and metastasis. In functional studies, exosomes shed by tumors, referred to as oncosomes, were reported to activate normal epithelial cells to form tumors, while exosomes from healthy individuals appear to have anti-tumor characteristics. Comparative molecular profiling of normal tissue-derived exosomes and tumor-derived oncosomes in blood and other body fluids may therefore offer a non-invasive or minimally invasive way to assess carcinogenesis; cancer risk; tumor initiation, promotion, development and progression, metastasis in tissues; survival and treatment response, and the knowledge gained may lead to better cancer prevention/care/control.The major bottleneck for using oncosomes in cancer research or clinical care is in obtaining enriched preparations of oncosomes from body fluids. Existing technologies are based on centrifugation, precipitation/centrifugation or affinity purification, which are labor intensive, time consuming, or biased because they are based on known exosomal markers. Furthermore, existing approaches impose significant stresses on these vesicles and potentially compromise their biological integrity and viability for various downstream uses.  Therefore, the goal of this proposal is to accelerate the development of technologies for differential isolation and enrichment of tissue-derived exosomes and tumor-derived oncosomes which will be useful for comparative molecular profiling or therapeutic purposes. Given the potential of exosomes and oncosomes for basic research and clinical applications, proposed technology platforms should be capable of processing a large number of samples each with significant volumes and be useful for profiling multiple body fluids from multiple cancer types. Of further interest are technology proposals amenable to low-cost production, appropriate for handling large number of samples, and useful for profiling multiple body fluids from multiple cancer types to conduct molecular analysis studies in population science.

The biospecimen sources for exosomes or oncosomes isolation and enrichment can be blood, plasma, serum, urine, saliva, amniotic fluid, breast milk, bronchoalveolar lavage, cerebrospinal fluid, peritoneal fluid, malignant ascites or other types of body fluids or effusions. In Phase I, the technology development should focus on isolation and enrichment and obtaining distinct preparations of exosomes and oncosomes. In Phase II, the focus should be adopting the technology developed in phase I to isolating and enriching exosomes and oncosomes from multiple body fluids in multiple cancer types.

Project Goals

The goal of this contract proposal is 1) to support the development of large scale (capable of handling a large volume of a body fluid) or high-throughput (capable of isolating and exosomes or oncosomes from large number of samples in a finite time) technologies for differential isolation of tissue-specific exosomes and tumor-derived oncosomes from any body fluid(s), and 2) to obtain enriched, distinct preparations useful for downstream comparative molecular profiling or therapeutic use. Applicants must propose to develop an efficient and cost effective platform for complete isolation and segregation of extracellular vesicle populations, with particular emphasis on yielding pure exosome or oncosome populations that are morphologically and functionally intact. The technology should preferably establish automated workflows and reduce human intervention to obtain enriched distinct preparations of exosomes and oncosomes.

To apply for this topic, offerors should have a proof-of-concept prototype platform with demonstrated capability for isolating exosomes from complex solutions. Preference will be given for proposals with demonstrated capability for further isolating oncosomes from the general exosome population. They should demonstrate sufficient expertise and necessary resources for robustly characterizing captured oncosomes, and verifying persistence of their biological integrity.

Applicants are required to obtain distinct preparations of exosomes and oncosomes, which originated in a specific tissue/tumor, from routinely collected fresh or archived body fluids. They should demonstrate integrity, quantity and reproducibility of isolation and separation using physicochemical and functional studies. This solicitation is not intended for developing technologies for molecular profiling exosomal or oncosomal cargo.

Phase I Activities and Deliverables

·       Develop a technology for differential isolation of exosomes with highly selective isolation of oncosomes from the exosome population, which originated in a specific tissue, from body fluid(s) collected from cancer patients (e.g., breast, prostate, colon, lung or brain). High-throughput capacity or large scale abilities must be sufficient for adoption in clinical workflows (therefore demonstrate capability for processing at least 50 sample in 8 h or 10 mL of clinical fluid specimen in <1 hour)

·       Demonstrate that the technology can obtain distinct preparations of exosomes and oncosomes from the routinely collected fresh/archived body fluids, and yields sufficient quantity for downstream analysis. Specifically, demonstrate sufficient yield of nucleic acids for NGS/qPCR and proteins for LC-MS/MS

·       Preferably establish automated workflows sufficient to allow for minimal training for new users

·       Demonstrate that the reproducibility is >90% and yield is >70%

·       Demonstrate the integrity of exosomes/oncosomes is >80% using physicochemical methods (Transmission electron microscopy, AFM, dynamic light scattering, immunostaining/immunofluorescence)

·       Benchmark the developed technology against at least 2 current techniques (e.g. centrifugation, density gradient, immunocapture, size-based filtration, etc.) and demonstrate comparable purity and yield from clinically appropriate sample sizes for the specific bodily fluid.

·       Deliver to NCI the SOPs for exosome/oncosome isolation and the data from physicochemical characterization that demonstrates the quality of the isolated exosomes/oncosomes.

Phase II Activities and Deliverables

·       Adapt the technology to multiple body fluids (i.e., stored or freeze thawed) with varying complexity

·       Demonstrate that the isolated exosomes/oncosomes are morphologically intact by physicochemical methods (Transmission electron microscopy, AFM, dynamic light scattering, immunostaining/immunofluorescence), and functionally active in in vitro systems (transmission of information from exosomes/oncosomes to cells in culture and/or co-culture). 

·       Develop a production prototype kit/tool/device for the deferential isolation of exosomes/oncosomes, and/or established a marketing partnership/alliance with an established strategic partner (e.g. diagnostic or device company) 

Radiotherapy is an important definitive and palliative treatment modality for millions of patients with cancer and is used alone or in combination with drug therapy. However, a variety of patient, tumor, and treatment-related factors will influence its outcome. Significant advances in delivery and distribution of dose for radiotherapy have been made over the years. Currently, treatment decisions in radiotherapy/radiochemotherapy are primarily defined by disease stage, tumor location, treatment volume, and patient co-morbidities, together with general guidelines concerning normal tissue tolerance for surrounding organs. However, treatment planning does not take into account an individual patient’s, or a cohort of patients’ sensitivities to this important modality of treatment. This is an important limitation in personalized care, as there are known variations in individual patient normal tissue sensitivities to radiation, but treatments are based on population normal tissue complication probabilities. As molecularly targeted therapy is being integrated into radiotherapy and chemotherapy, selecting the “right type of treatment” is critical to improve outcomes.

A substantial number of patients treated with radiotherapy suffer from severe to life-threatening adverse acute effects as well as debilitating late reactions. Acute side effects (e.g. skin reactions, mucositis, etc.) are often dose limiting, but may be reversible in contrast to the late effects such as fibrosis in the lung, telangiectasia, and atrophy, which are irreversible and progressive. A biomarker-based test that can predict the risk of developing severe radiotherapy-related complications will allow delivery of suitable alternative treatments. Such stratification may also allow dose escalation to the tumor in less sensitive patients. However, discovery, development, and validation of predictive biomarkers of radiation hypersensitivity are challenging, particularly due to a low incidence of normal tissue complications in the clinic, the need for long-term studies for predicting late effects, and the combination of chemotherapy with radiation as standard of care for most tumors.

Project Goals

The goal of this contract topic is to identify, develop, and validate a simple, cost-effective biomarker(s) to rapidly assess inter-individual differences in radiation sensitivity and predict early and late complications among patients with cancer prior to radiation therapy.

A predictive biomarker of individual radiation sensitivity can measure any biological changes in response to absorbed ionizing radiation, which is able to predict imminent normal tissue injury prior to radiotherapy and help determine radiotherapy suitability and outcomes. Radiation biomarkers are an emerging and rapidly developing area of research, with potential applications in predicting individual radiosensitivity, predicting severity of normal tissue injury among patients, assessing and monitoring of tumor response to radiation therapy as well as in estimating dose to accidentally radiation-exposed individuals. The purpose of this contract topic is to develop a radiation biomarker(s) to specifically identify and exclude likely “over responders” prior to radiotherapy in order to avoid severe complications and to refer them for alternative treatment modalities.

A variety of radiation biomarkers have already been explored or are currently under development at different technology readiness levels (TRLs) at different government agencies and programs. This contract topic intends to leverage on these advances. These assays include but are not limited to (i) fibroblast clonogenic assay, (ii) measurement of DNA damage foci, (iii) damaged base metabolites, (iv) various types of chromosome aberrations studied in different phases of cell cycles, serum biomarkers, gene expression changes, (v) protein and microRNA expression changes, (vi) and genetic tests.

To be of practical value in the clinic, where radiation exposures are well-defined in terms of dose, distribution and timing, and thus quite different from radiation accidents, a predictive radiation biomarker of individual radiation sensitivity should be (i) able to predict heterogeneity of radiation responses among patients in clinic, (ii) specific to radiation, (iii) sensitive, (iv) able to show signal persistence as applicable to radiation therapy or have known time-course kinetics of signal, (v) amenable for non-invasive or minimally-invasive sampling, (vi) amenable to automation to improve quality control and assurance, (vii) have a quick turn-around time between sampling and results (though speed is not as critical as in the countermeasures scenarios), (viii) and be cost effective.

This contract topic aims to encourage the development and validation of predictive radiation biomarkers for clinical applications as described above. Both the FDA and the Centers for Medicare and Medicaid Services (CMS) through Clinical Laboratory Improvement Amendment (CLIA) regulate diagnostic tests. A reasonable predictive radiation biomarker development process for identifying likely “over-responders” to radiation treatment may involve biomarker discovery, assay design and validation, determination of assay feasibility, assay optimization and harmonization, assessing the assay performance characteristics (reproducibility, sensitivity, specificity etc.), determining the effect of confounders, if any, determination of suitable assay platforms and platform migration as may often be needed, and clinical validation with a locked-down assay before regulatory submission and commercialization. Early pre-IDE interaction with FDA is therefore critical. The following activities and deliverables are applicable to both biomarkers for acute early effects and surrogate endpoints for late effects.

Phase I Activities and Deliverables

Phase I contract proposals must describe (i) a quantitative estimate of the patient population that will benefit from the availability of such predictive radiation biomarkers for the applicable cancer type/organ site, (ii) a plan for generating evidence that the proposed biomarker or biomarkers are relevant in the prediction of radiation hyper-sensitivity among patients with cancer and logical approach in the developmental pathway to clinic from discovery, (iii) a description of assay characteristics including sensitivity and specificity and the effects of known confounders, if any, (iv) level of technological maturity, describing critical technology elements allowing technology readiness assessment by the reviewers, (v) and a description of the proposed regulatory pathway for approval and pre-IDE consultation with FDA. In such meetings with FDA it is expected that the applicant will invite NCI’s participation, where applicable.

Activities and deliverables include the following:

·       Discovery and early development

o   Demonstrate biomarker prevalence and utility

o   Develop a working qualitative test correlating the presence or absence of the biomarker(s) with potential outcome or a quantitative assay to assess radiation sensitivity

o   Demonstrate feasibility

·       Preclinical development and technical validity

o   Provide assay characteristics, including but not limited to performance, reproducibility, specificity, and sensitivity data using frozen (or other) samples from past clinical trials, or retrospective clinical studies providing adequate power calculations

o   Illustrate the performance of the biomarker(s) with receiver operating characteristic (ROC) data

o   Demonstrate suitability of the test for use in the clinic, including kinetics of biomarker, if transient.

o   Determine the effect of confounders, such as any induction or concurrent chemotherapy regimens.

o   Provide defined metrics for measurements of success

o   Deliver the SOP of the working test or assay to NCI.

o   Benchmark the technology against quantitative milestones proposed by offers to measure success

o   Provide description of proposed regulatory pathway for approval and pre-IDE consultation with FDA

Phase II Activities and Deliverables

Phase II contract proposals must describe (i) the setting and intended use of the predictive biomarker(s) in retrospective or prospective studies using human tissue samples (frozen or fresh), (ii) a logical approach to regulatory approval, (iii) a description of assay platform and platform migration, if necessary, (iv) a demonstration of clinical utility and clinical validation, (v) a proposed schedule for meeting with FDA regulators regarding approval. In such meetings with FDA it is expected that the applicant will invite NCI’s participation, where applicable.

Activities and deliverables include the following:

·       Provide a schedule of proposed meetings with FDA regarding approval

·       Early-trial development

o   Retrospective tests using archived, frozen samples from past clinical trials, or prospective trials using fresh human samples.

·       Full development

o   Demonstrate clinical utility

o   Demonstrate clinical validity in a large prospective randomized clinical trial 

Targeted radionuclide therapy (TRT) enables personalized cancer treatment by combining the therapeutic effect of radiation therapy with the targeting capability of molecular therapies.  In TRT, a cytotoxic dose of a radioactive isotope is attached to monoclonal antibodies, receptor ligands, or synthetic molecules that target malignant tumor cells selectively. The ability of these molecules to bind specifically to a tumor-associated structure ensures that the tumor gets a lethal dose of radiation, while normal tissue gets only a minimal dose. This minimizes toxicity to normal tissues and can increase therapeutic efficacy (therapeutic index) leading to a reduction of overall treatment costs.

Currently available TRT compounds such as Zevalin and Bexxar have been developed and approved in the United States for use in the treatment of non Hodgkins Lymphoma (NHL).  Although these drugs have shown a response rate of approximately 80%, they have failed to show a survival advantage in patients. Large multicenter trials to study long- term survival are currently underway.  Because these drugs have had modest commercial success to date, private investment in molecularly-targeted radiation pharmaceuticals remains at low levels.  As this class of treatments shows tremendous clinical potential, there is a need to encourage the development of next-generation technologies (see below) for cancers other than NHL, including solid tumors, where the clinical need is most acute.

Project Goals

This contract solicitation seeks to stimulate research, development, and commercialization of innovative TRT techniques that could potentially shorten treatment cycles and reduce toxicity to normal tissues. Proposals addressing the following technology areas are encouraged: new treatment strategies; design, synthesis and evaluation of innovative ligands and radiotracers for TRT; novel radioisotope generators and radioisotope production techniques; dosimetry techniques; and new conjugation chemistries that can link the radioisotopes to targeting agents other than antibodies (e.g. existing small molecule chemotherapeutic drugs) are also encouraged.

The short-term goal of the project is to perform feasibility studies for development and use of possible radioimmunotherapeutics for the treatment of cancer.  The long-term goal of the project is to enable a small business to bring a fully developed TRT compound to the clinic and eventually to the market.

Phase I Activities and Deliverables

Phase I activities should support the technical feasibility of the innovative approach. Specific activities and deliverables during Phase I should include:

·       Proof-of-concept of the conjugation or attachment of the radioisotope to the antibody or other targeting moiety.

·       Radiation dosimetry studies in an appropriate small animal model

·       Proof-of-concept small animal studies demonstrating an improved therapeutic efficacy and improved therapeutic index, assessment of toxicity to normal tissues, and pharmacokinetic/pharmacodynamic studies utilizing an appropriate animal model.

Phase II Activities and Deliverables

Where cooperation of other vendors or collaborators is critical for implementation of proposed technology, the offeror should provide evidence of such cooperation (through written partnering agreements, or letters of intent to enter into such agreements) as part of the Phase II proposal.

Specific activities and deliverables during Phase II should include:

·       Demonstration of the TRT manufacturing and scale-up scheme

·       IND-enabling studies, preferably in consultation with FDA, carried out in a suitable pre-clinical environment.

·       When appropriate, demonstration of similar or higher specificity and sensitivity of the technology when compared to other technologies.

·       Offerors are encouraged to demonstrate knowledge of appropriate FDA regulations and strategies for securing insurance reimbursement.

Accurate detection of specific markers is crucial for the diagnosis of malignant disease, monitoring drug therapy and patient screening. The development of sensitive and reliable strategies for the detection of biomarkers at ultra-low concentration is of particular importance to cancer medicine.  Efforts have been made to develop techniques for the amplification of signals, but there is still a paucity of novel approaches used specifically to improve the simplicity, selectivity and sensitivity of cancer biomarker assays, especially for the interrogation of tumor tissue.

The current work focus of this SBIR topic is for incorporation of an existing or novel signal amplification system  into  high value cancer biomarker antibody based  assays  (ELISA type or slide-based  IFA/IHC) to enable an increase in accuracy and sensitivity (at least 100-fold) for  detection of the specific analyte at the lowest possible  concentration level. Detection at attomolar/sub-femtomolar concentrations is desired. These assays must be optimized for performance on tumor tissues and tissue extracts, which requires a fundamentally different approach than those commonly used for high-sensitivity serum assays already in use in the field.

In vitro immunoassays are probably the most common, simple and relatively inexpensive methods used in clinical laboratories for the diagnosis and management of disease. Despite continued efforts to improve the performance of immunoassays, there is an urgent need for assays with increased sensitivity (i.e., attomolar sensitivity) and accuracy for detection of low-level disease markers specifically in tumor tissue.

There are many amplification systems that have been published in the past 10-15 year but most have limited effect,  demonstrating improved sensitivity < 10 fold,  and have not been widely adopted in the clinical lab.  One issue is low signal-to-noise ratio; noise often increases with signal amplification.  There is a big technological gap as we learn more and more about signaling molecules in cancer; many are tightly regulated with low levels of protein expression. Simply put, the existing assays are not of sufficient sensitivity to detect low abundance biomarkers.  Finally, the most commonly requested utility for these existing assays is on serum or cell line lysates, not solid tumor tissues. There is a great need for the development of highly sensitive/specific antibody based assays that can detect analytes in tumor lysates (ELISA) and in tumor tissue sections (IFA/IHC).

The current techniques used in molecular tag detection assays use radiolabels, electrical, light scattering, fluorescent, and chemiluminescent molecules—see table below. Most of the commercially available labels have inherent limitations in signal strength. These assay limitations lead to numerous drawbacks in our ability to measure low abundant biomarkers to improve the clinical care of the patient – to include:

• The assay is limited (i.e., the sensitivity of an assay is not sufficient to detect biomarkers in majority of clinical specimens) to the measurements of biomolecules in clinical situations where they are present in high abundance (e.g., protein overexpression or high gene copy number)
• The assays can only be applied to biological samples where biomolecules are more abundant such as tumor tissue, but not to more easily obtained non-invasive specimens, such as blood, where the biomolecules are often present but in extremely low- abundance.
• The current lower sensitivity of antibody based assays limits detection of many signaling molecules, and where they can be detected, often it will not be possible to detect a drug-mediated decrease in the signal (e.g., for pharmacodynamics [PD] applications).
• If the biomarker is of low abundance, then the reliable measurement via an ELISA assay requires the consumption of large amounts of biological samples (e.g., tumor needle biopsies)  to bring up the protein level  high enough to quantitate biomarkers at the  limits of detection (i.e., in the process,  large amount of precious tumor biopsy is lost to further molecular analyses) .

*David A. Giljohann & Chad A. Mirkin.  Nature 462, 461-464, 2009

Recently, a few new technologies have been described in the literature that is reported to achieve 10²-10⁶ fold improvement in signal strength.  Many of the technologies bring together large aggregates of immune complexes to produce amplified detection signals several magnitudes greater than reagents in which unitary labels are coupled directly to the secondary antigen or antibody without using multi-label scaffolds.   A short description of some of the more promising technologies is listed below with greater details found in Appendix A. These technologies described below are representative, but not exclusive; offerors are welcome to propose solution technologies not listed below, provided they address the aims of this solicitation.

Plasmonic absorbers, especially in the infrared (IR) range, have potential applications for biochemical sensing, imaging, energy conversion, and other medical diagnostics. One technology referred to as “Nanobar shaped disk-coupled dots-onpillar antenna-array” (Bar-D2PA) claims up to 1 million fold improvement of immune sensitivity. – see DocLINK. The D2PA is composed of an array of dense three-dimensional nanoantennas that can be layered with immunological reagents to create a specific assay. The benefits of the bar-D2PA technology are:

(a) low manufacturing costs;

(b) multiple identifying resonance peaks;

(c) tunable transmission with high absorption;  and

(d) high-field regional cross-section for analyte detection.  The bar-D2PA structure shows unique mid-IR light response with polarization-dependent plasmonic resonances.

Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins have attomolar sensitivity. This ultrasensitive method for detecting protein analytes is referred to as the bio-barcode method and has been shown to amplify the signal from extremely low levels of protein or oligonucleotide in solution as published by Mirkin and colleagues (Nam et al., 2003). The principle is to get low-abundant proteins to bind to particles containing a target specific antibody – this particle is also tagged with thousands of identical single strands of DNA to the target analyte gene which is used to amplify the signal. A magnetic particle tagged with another analyte specific antibody (different epitope) pulls the analyte-particle complex out of solution, and the DNA is separated from the captured particle and quantified.

Another approach of amplification technology is 3DNA® developed by Genisphere which is a 3-dimensional structure made entirely out of DNA. For many applications, a four-layer arrangement is used, which has an average of 280 +/- 20 arms per molecule. The arms are modified with labels and targeting moieties.  As examples, the labels can be fluorescent, enzymatic (HRP, AP), nanogold, or a hapten (biotin, FITC, DIG); and the targeting moiety may be an antibody, peptide, specified RNA/DNA sequence, aptamer, PNA, or a hapten (biotin, FITC, DIG). Mixing and matching a variety of labels and targets on the same 3DNA core creates a highly customized reagent. Genisphere’s 3DNA technology has been used to improve the limit of detection by up to 100-fold in a variety of assay platforms, including microarray, ELISA, bead-based flow cytometry, and lateral flow.

Another novel signal amplification strategy in lateral flow immunoassay (LFIA) utilizes three amplification steps: (a) biotin-streptavidin amplification; (b) polylysine amplification; and (c) fluorescence dye signal amplification.  The resulting conjugates achieved a detection limit 100-fold lower than that of the magnetic beads-based ELISA and gold-based LFIA.

The Enzyme-cascade-amplification strategy (ECAS-CIA) allows detection of low-abundance proteins by coupling with enzyme cascade amplification strategy (DocLINK).  In the presence of target analyte, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification.

It is believe that these technologies and other new technologies could have broad applicability to improving the sensitivity of ELISA and slide-based immunoassays for target proteins as well as for nucleic acid detection assay platforms. However, most of these reagents and associated instrumentations are not commercially available to laboratory researcher or adapted to clinical quality assays.

References:

David A. Giljohann & Chad A. MirkinNature 462, 461-464, 2009

Liangcheng Zhou; Fei Ding; Hao Chen; Wei Ding; Weihua Zhang; Stephen Y. Chou; Anal. Chem.  2012, 84, 4489-4495.

Chao Wang, Qi Zhang, Yu Song, and Stephen Y. Chou*.  Plasmonic Bar-Coupled Dots-on-Pillar

Cavity Antenna with Dual Resonances for Infrared Absorption and Sensing: Performance and Nanoimprint Fabrication.  ACS Nano. VOL. 8 ’ NO. 3 ’ 2618–262, 2014 DocLINK

Nam JM, Thaxton CS, Mirkin CA. Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science. 2003 Sep 26;301(5641):1884-6. PubMed.

Chem. Commun., 2012, 48, 12207–12209 Nanogold-based bio-bar codes for label-free immunosensing of proteins coupling with an in situ DNA-based hybridization chain reaction.

Jun Zhou, Mingdi Xu, Dianping Tang,* Zhuangqiang Gao, Juan Tang and Guonan Chen*

Expert Rev Mol Diagn. 2006 Sep;6(5):749-60.Signal amplification systems in immunoassays: implications for clinical diagnostics. Dhawan S

Sensors (Basel). 2012; 12(9): 11684–11696. A Fast and Sensitive Quantitative Lateral Flow Immunoassay for Cry1Ab Based on a Novel Signal Amplification Conjugate Chunxiang Chen1 and Jian Wu2

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein Zhuangqiang Gao,1, Li Hou,1, Mingdi Xu1, & Dianping Tang1, Scientific Reports4, Article number:3966Volume

Project Goals

With rapid advances in biomedical research and the growing biotechnology industry, development of highly sensitive and economical assays will meet an unmet need and serve to promote the use of biomarkers in the personalized care of patients.  These enhanced detection technologies can also be directed towards designing fully automated, highly sensitive assays to identify multiple disease markers in a single clinical specimen using multiple assay platforms and to improve the amount of molecular information that a clinician obtains for more precise care of the cancer patients.

The increasing demand for screening assays at the early stage of disease development and from minimally-sized specimens calls for ultrasensitive detection of biologically relevant biomarkers at an extremely low level of expression. To keep pace with expectations in clinical assays, there is still the quest for more flexible, yet highly sensitive, quantitative, and easy-to-use methods.  This SBIR topic is to support assay development that pushes the level of analyte detection to the absolute maximum.

The goal of this SBIR topic is to incorporate recent advances in signal amplification methods into the development of quantitative ELISA and/or slide-based antibody assays (IFA/IHC) to low abundance but high value cancer biomarkers. The amplification system could be chemical, bio-chemical, nano-particle, or any other component based.  The desired aim is to improve the sensitivity of an assay by at least 100-fold and preferably thousand-fold.  Demonstrating the diagnostic potential of existing or novel amplification systems (i.e., platform, tagging, enrichment)  in antibody based assays to protein analytes will provide the foundation for their use in other molecular assays (FISH, RNA in situ, etc) – i.e. this SBIR will support a significant technological advance in molecular test development in general by promoting the integration of emerging technology into the diagnostic paradigm.

There are two objectives:

Objective 1. Select an appropriate signal enhancing system to improve the signal strength /sensitivity of an ELISA or slide-based antibody assays (IFA/IHC)  using tumor tissue by 10²-10⁶ fold (preferably at attomolar levels) for two high value cancer biomarkers, preferably a NCI designated target listed below .

NCI may advise on the appropriate assay reagents and non-clinical models.   In special cases, NCI may provide reagents to selected PD biomarkers and/or the associated xenograft tumors or cell line models to awardees. 

Objective 2. The enhanced sensitivity system must maintain the integrity of ELISA and/or slide-based antibody assay designs and be easily adaptable to widely used formats/platforms in clinical laboratories (i.e.,  96 well microtiter plate assay, detection instrumentation, bead based detection, automated slide strainers, flurosecent microscopes, etc.).

Phase I Activities and Deliverables

·       The amplification technology must provide a significant improvement in assay sensitivity (10²-10⁶ fold) to high value low abundant cancer biomarkers using tumor tissue. Two assays are to be developed with the new amplification system. 

·       The amplification technology must be consistently manufacturable, and if new instrumentation is required, size and cost of prototype instrumentation should be within reach of a clinical lab.

·       Any alteration in the assay design or assay protocol as an attempt to increase the sensitivity of the assay constitutes a critical issue and introduces bias resulting from the changes made. The assay design must be adjusted to optimize the analytic performance of the assay for clinical utility while ensuring analytic validity using proper controls to minimize false results. Suggested activities to test for optimal assay performance are:

o   Optimize the assay to increase signal over background noise and maintain the optimal kinetics of the assay (in fact faster assays with improved kinetics should be possible with a strong amplification system). Appropriate controls and calibrators are to be used.

o   Evaluate the specificity of the higher sensitivity assay to make sure that it is unchanged by challenging the system with interfering substance es and related proteins.

o   The reproducibility and precision of the enhanced antibody based assay are to be evaluated by calculating the intra- and inter-batch variation coefficients (CVs of the assays using the same batch of signal enhancing reagent should be <20%).

o   The batch-to-batch reproducibility using at least 2 different batches of the signal enhancing reagent, performed on different days with different operators should have CV <20%.

·       Assay performance must be tested in the appropriate non-clinical models for the chosen immunoassay analytes.  NCI will recommend at least two models and expects that at least 6 separate specimen preparations from the models will be tested for statistical determinations.

·       The assay, associated methodology, and if applicable, instrumentation will be independently verified by an external laboratory (NCI may be available for this activity). 

Phase II Activities and Deliverables

·       Expand and ‘optimize’ enhanced detection for the selected ELISA or slide-based antibody assays (at least 100 fold to bring analysis in attomolar range) to at least 4 high value oncology biomarkers, preferably from NCI’s list of desired analytes.  Test in appropriate models using target specific calibrators and controls. The assays/associated equipment must be affordable and easily adaptable to standard clinical laboratories.

·       Reproducibly manufacture signal amplification reagents for the 4 high value oncology biomarkers (3 lots) and do at least 6 months stability testing under different storage conditions. If new instrumentation or equipment is required, then the design and manufacture must be optimized.

·       Show reproducibly/robustness of the antibody-based assay quantitation of the designated biomarkers in selected models/tumor specimens performed by 2 users on 3 different days.

·       Statistical quantification of the signal should be provided to demonstrate the enhanced sensitivity and the reliability of the technique.

·       The assay, associated methodology, and if applicable, instrumentation should be independently verified by an external laboratory (NCI may be available for this activity).

·       Show adaptability of the signaling reagents for use in other assay formats (e.g., FISH). 

·       Provide the program and contract officers with a letter of commercial interest. 

Personalized medicine approaches allow the treatment of patient tumors with drugs tailored to their tumors, which increase the probability of a beneficial response.  In the last decade, a number of pharmacodynamic (PD) markers have been identified that help the physician know that the drug is hitting the target or target pathway in the patient’s tumor.  Recently approved cancer treatments target either the cell surface receptors at the head of these signaling pathways or the intermediate phosphoproteins and kinases in the pathway signaling cascades. Understanding the phosphoprotein activation state of key signaling molecules in tumor cells can yield critical information on the type, stage and status of those cells, aiding in the diagnosis, prognosis and treatment of an individual’s disease.  Unfortunately, target analyte lability, especially with phosphoproteins, requires adoption of rapid and highly controlled tissue collection and handling before widespread clinical use of the assays in predicting drug response.  Also, in some cases, the magnitude of drug modulation of biomarker may be small but still significant in correlating to overall tumor response, which mandates the use of quantitative assays in enriched samples of tumor.  NCI/DCTD has developed analytic multiplexed ELISA type immunoassays to cancer drug targets/pathways that quantitate actual analyte levels in tumor lysates.

However, there is an inherent problem in the use of tissue lysates in that a tissue is comprised not only of tumor cells but also of normal parenchyma, stromal cells, inflammatory cells, vessels, and often significant necrosis, so when a specimen sample is homogenized the macromolecules extracted is a sum of  all cellular/matrix components, proportional to each representative element. Thus, the presence of non-target cells and necrosis can significantly affect the quantification of protein levels in ‘viable’ tumor cells.  Even the most sophisticated testing methods are of limited value when the input DNA, RNA, or protein is contaminated or diluted by non-target cells and necrotic/acellular matrix. The requirement for relatively pure cell populations has led to various technical solutions, most notable Laser Capture Microdissection (LCM). Microdissection techniques permit analysis of various molecular signatures within a specific cell population of a tissue and reduce the interference from non-target cell populations and acellular matrix such as fibrosis/necrosis. Although there are numerous reports of genetic and gene expression analyses of microdissected tumor populations, and of proteomic assessment using Western blot, 2D-PAGE, mass spectrometry, and peptide sequencing, studies using ELISA/immunoassay quantitation of key drug targets are limited. Tumor enrichment techniques that allow the rapid capture of tumor rich zones from core needle biopsies (not sections) have the potential to provide sufficient tumor amounts for analyte quantitation in tumor cells residing within a tissue with the sensitivity and specificity of ELISA. This technology will have significant clinical value. 

The purpose/goal of this SBIR topic is the development and commercialization of a visualization/microdissection system that is capable of identifying and capture of ‘viable’ tumor rich zones in frozen solid tumor biopsies under conditions that preserve labile pharmacodynamics (PD) biomarkers for antibody mediated quantitation. 

Microdissection technologies are powerful tools for the isolating enriched populations of tumor cells from cellular heterogeneous tissues. It has been shown that the harvested cells can be used for many molecular investigations including DNA, RNA, protein, microRNA, and protein analyses (See Figure 1 below). The goal of this SBIR topic is to adapt / improve existing visualization strategies for frozen solid tumor needle biopsies allowing microdissection of large tumor rich zones with 50% tumor region enrichment while preserving labile biomarkers. The desire is to include quantitative ELISA/immunoassay in the list of molecular analyses (see chart below) that can be performed reliably on microdissected cell populations by developing a system for the rapid identification and dissection of enriched tumor cell zones from frozen biopsies. These tumor rich zones should provide sufficient material for multiplex ELISA quantitation of biomarkers. This system will be much more time-effective and productive than the labor intensive microdissection of multiple tissue sections from a biopsy. The ability to quantify biomarkers via immunoassay of isolated biopsy zones enriched for ‘viable’ tumor cells is especially important when a biomarker is only modulated to a small extent requiring very sensitive and specific analytic assays. Of note, the development of this technology has the potential to improve the ‘specificity’ of any molecular test that involves homogenization of the tissue and macromolecular isolation.

Applied Immunohistochemistry & Molecular Morphology. 21(1):31-47, January 2013

References

1.      Wikipedia: http://en.wikipedia.org/wiki/Laser_capture_microdissection

2.      Applied Immunohistochemistry & Molecular Morphology: January 2013 - Volume 21 - Issue 1 - p 31–47, Cheng, Liang et al.

3.      Veterinary Pathology Onlinevet.sagepub.com

4.      Veterinary PathologyJanuary 2014vol. 51 no. 1 257-269.

5.      Laser Capture Microdissection for the Investigative Pathologist, H. Liu et al

6.      Published online before print November 13, 2013, doi: 10.1177/0300985813510533

7.      Espina V, Heiby M, Pierobon M, Liotta LA (2007). "Laser capture micro-dissection technology". Expert Rev. Mol. Diagn. 7 (5): 647–57. doi:10.1586/14737159.7.5.647. PMID 17892370.

8.      Orba Y, Tanaka S, Nishihara H, Kawamura N, Itoh T, Shimizu M, Sawa H, Nagashima K (2003). "Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma". Cancer 99 (4): 198–204. doi:10.1002/cncr.11331. PMID 12925980.

9.      Laser Microdissection & Pressure Catapulting". University of Gothenburg.

10.   LCM User Community: http://abcommunity.lifetechnologies.com/community/laser_capture_microdissection_%28lcm%29

11.   Am J Pathol. 1999 Jan;154(1):61-6.Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis.Fend F1, Emmert-Buck MR, Chuaqui R, Cole K, Lee J, Liotta LA, Raffeld M.

12.   Molecular & Cellular Proteomics 9:2529–2544, 2010.In Situ Proteomic Analysis of Human Breast Cancer Epithelial Cells Using Laser Capture Microdissection: Annotation by Protein Set Enrichment Analysis and Gene Ontology*□SSangwon Cha§, Marcin B. Imielinski¶, Tomas Rejtar§, Elizabeth A. Richardson¶,Dipak Thakur§, Dennis C. Sgroi¶‡**, and Barry L. Karger‡**

Project Goals

ELISA/Immunoassays have the capacity to quantify the levels of a specific analytes in a specimen.  There are two important considerations in the reliability of immunoassays to accurately measure the levels of PD biomarkers and the use of the information in clinical care:

1.      Many of the drug biomarkers are very labile and require immediate processing after collection which usually involves rapid freezing or lysis in buffers with specific inhibitors (e.g., phosphatase inhibitors).

2.      Tumors are not of a homogeneous cell type and often contain significant amounts of necrotic and normal cell zones which will impinge on the accurate measurement of an analyte in the tumor population if the entire tissue sample is homogenized. 

It is believed that the development of microdissection techniques for identification and collection of viable tumor zones with at least 50% tumor enrichment from frozen solid tumor biopsies under conditions that preserve the stability of labile biomarkers can lead to detailed quantitative assessment of key protein targets modulated by drugs in tumor cells in their natural microenvironment and increase the PD utility of selected biomarkers.

The use of an appropriate visualization technologies and microdissection method such laser capture microdissection (LCM) is proposed.  The LCM process has the advantage of working with tissue in various physical states, particular frozen (a state that would preserve labile phosphoprotein biomarkers), and does not alter the morphology or biochemistry of the sample collected. LCM has demonstrated success in collecting selected cells for molecular analyses, particular when frozen sections are used. Limitations of LCM include difficulties of microdissection due to decreased optical resolution of tissue sections.  Possible options to improve cell resolution include the use of specialized optics to identify tumor zones via architectural/morphometric/light refraction/density parameters or the use of special stains or immunohistochemistry/immunofluorescence. This topic goal is to develop ‘identification’ or visualization and capture methods for frozen solid tumor needle biopsies or thick sections.  These techniques must not impinge on the stability of the labile biomarkers (i.e., frozen conditions must be maintained). 

The goalof this SBIR topic is to develop a reliable visualization approach to identify and capture zones of ‘viable’ tumor cells from frozen solid tumor biopsies that lead to at least 50% enrichment of tumor zones.  The technology should focus on distinguishing tumor regions from non-tumor zones and acellular matrix, such as necrosis, upon scanning frozen solid tumor biopsies or thick sections. This technology will result in the collection of relatively large areas of biopsy material that is enriched in tumor cells with preserved labile biomarkers and of sufficient amounts to be amenable to quantitation by ELISA/Immunoassay. 

The first objective should be to develop reliable methods to distinguish enriched ‘viable’ tumor zones from necrotic zones or viable tumor zones from normal cell types in frozen solid tumor biopsies or thick sections. Malignant transformation is associated with structural, genotypic/phenotypic cellular modifications, and biochemical changes, which as a consequence, alter the spectroscopic, metabolic and microscopic properties. These and other alterations may be exploited to develop a means to scan frozen solid tumor biopsies /thick sections and identify zones that correlate to cellular/acellular areas in the biopsy. The optimal scanning system should be able to extract information about the morphological/ architecture/ spectroscopic properties in the frozen biopsy that may be used to distinguish normal and malignant areas; such as, the measurement of color, overall density or light reflectance which may correlate to cellular density and/or vascularity which in turn may reflect tumor zones.

The second objective is to microdissect and capture the enriched tumor zones from frozen solid tumor biopsies/thick sections in a manner that preserves the label PD biomarkers, while maintaining the tumor zone as frozen throughout the process. Such a product will enable the reliable identification and capture of relatively large amounts of pure populations of tumor cells from frozen solid tumor biopsies which are amenable for quantification of label biomarkers via immunoassays, thus increasing the sensitivity and specificity of the assays for the target in tumor cells and their PD utility.   

Phase I Activities and Deliverables

The essential characteristics of a tumor enrichment microdissection system of frozen solid tumor biopsies should include all or some of the following features:

1)     a biopsy/thick section scanning/microdissection method that is adaptable for use with most common microscopic/microdissection systems with the capability to maintain the specimens under freezing temperatures;

2)     able to generate an easily interpretable signal indicative of parameters that can distinguish between necrotic and viable tumor, and if possible, also between tumor and non-tumor tissue;

3)     be capable of microdissection of enriched tumor zones without inducing significant cellular damage or change in labile PD biomarkers (i.e. maintain frozen state); and

4)     able to perform as designed and intended in fit-for-purpose studies in relevant clinical veterinary models (i.e. the method  has to produce tumor tissue of sufficient quantity and quality for ELISA based  immunoassay).

To accomplish the goal of this SBIR topic to develop microdissection techniques that reliably identifies and captures tumor-rich ‘viable’ zones in frozen, unfixed tumor biopsy cores or thick sections in a manner that preserves labile drug target/pathway PD biomarkers for quantitative ELISA/Immunoassay analyses, the Phase I deliverables are:

·       Develop a microscopic visualization/microdissection method to identify and capture ‘viable/cellular’ tumor zones from frozen tumor biopsies/thick sections from at least two solid tumor types while maintaining the frozen state of the specimen.  The use of the entire needle tumor biopsy is preferred. Any gauge needle biopsy can be used for Phase I development, but 18 gauge is desired for Phase II. The readout from the visualization/scan of the frozen biopsy should be easily interpretable (e.g. cellular rich or acellular rich) and is associated with the level of necrosis and ‘viable’ cell-rich tumor zones, and if possible, between tumor and non-tumor ‘normal’ zones.  Xenograft tumors may be used for developing this technology.  NCI can recommend specific xenografts that have sufficient levels of specific labile PD biomarkers and are known for developing necrosis. (See Table 1 below).

·       The purity and cellularity of the captured zones can be assessed by H&E staining and imaging.  Initially the histology of the entire biopsy will need to be analyzed to associate the visualization measurement with the histology (i.e. the cellular quantity and cellular types present throughout the biopsy).  One option is that during the development phase, the biopsy can be cut longitudinally into 2 halves with one side being subjected to frozen sectioning and H&E staining for histological evaluation and the 2^nd half to being scanned via the visualization method. This will allow the visualization measures to be related to the histology of the biopsy. As to the quantitative needs, it will depend on the assay; for example, for the DCTD apoptosis multiplex immunoassay, 20 ug of protein is required per panel, preferably with 50% tumor cell content.

·       Demonstrate that the visualization/capture technology preserves in the enriched tumor cells at least one of labile protein biomarkers of interest to NCI (see Table 1 below).  Maintaining the biopsy specimen under freezing conditions is mandatory and it is preferred that the enriched tumor zones be kept frozen. If specimen loss after capture is an issue, then the use of special lysis buffer may be approved by NCI. NCI may provide assistance in the analysis of the biomarker levels in isolated tumor zones. 

·       The device and methodology need to be independently tested  at a different laboratory.  NCI may be willing to perform the independent validation/field testing of the breadboard prototype device and associated methodology.

Table 1: Suggested Phospho-protein PD biomarkers and associated xenograft models

Phase II Activities and Deliverables

The Phase II activities should be focused on the design criteria/specification and fabrication of the alpha prototype biopsy visualization/microdissection system. The size of the needle biopsy is recommended to be 18 gauge for Phase II activities.  The activities/deliverables are:

·       Processes/instrumentation should be optimized to reproducibly identify and allow microdissection of ‘viable’ cell rich tumor zones from frozen tumor biopsies/thick sections of at least 4 different tumor types, evaluating a minimum of 6 specimens from each type (e.g., xenograft models).  Reproducibility should be demonstrated by 2 users on 3 different days. Images of H&E stained slides of the isolated tumor zones should be provided to demonstrate reliability of the technique.

·       Demonstrate preservation of 2 labile PD biomarkers via ELISA/immunoassay. NCI may provide assistance in this task.

·       The alpha prototype device and associated methodology should undergo independent validation/field testing at a separate laboratory. NCI may be willing to do this.  

·       Provide the program and contract officers with a letter of commercial interest. 

The cancer community has developed a series of single-plex and multiplex immunofluorescent assays (IFA) to evaluate oncology biomarkers in tumor sections on slides.  Many of the assay targets are key DNA damage response and signaling molecules (γH2AX, Nbs1, ERCC1, RAD51, RAD50, pATR, pchk2, cdk/PY15, pKAP, MET – total-pY1234/pY1235-pY1236, AKT- pT308pS473, ERK1-pY202pT204, and ERK2-pY185pT187).  Our understanding of DNA damage response and signaling in tumors is critically dependent on our ability to visualize and quantify specific signaling molecules with high spatial resolution in the cellular context.  However, these slide-based assays are at most semi-quantitative. ELISA/Immunoassays have the capacity to precisely quantify the levels of a specific analytes in a specimen. ELISA analysis of whole tissue homogenates would be more informative if the analysis could be done on a pure population of tumor cells; however, tumors are not of a homogeneous cell type and often contain significant amounts of necrotic and normal cell zones which will impinge on the accurate measurement of an analyte in the tumor population if the entire tissue sample is homogenized. So, there is an un-met need to improve the specificity and sensitivity of slide-based immunoassays that visualize analytes on single cell types to approach or exceed that of quantitative ELISAs. It is clear that the current proximity technology has the potential to provide a robust foundation for significant improvements in the design and construction of cell type specific quantitative slide-based, cancer biomarker, IFA for the interrogation of tumor sections. 

The applications of proximity technology such as Fluorescence Resonance Energy Transfer (FRET) or Radio Frequency have expanded tremendously in the last 25 years. Proximity technology has enabled the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes.

Proximity technology shows great promise for further development in the utility and scope of biological applications due to dramatic improvements in instrumentation, particularly with respect to time-resolved techniques. Advances in signaling tag such as fluorescent probe development have produced smaller and more stable molecules with new mechanisms of attachment to biological targets. For example, fluorophores have also been developed with a wide range of intrinsic excited state lifetimes, and a significant effort is being placed on development of a greater diversity in genetic variations of fluorescent proteins. Entirely new classes of tag materials, many of which are smaller than previous fluorophores, allow for the evaluation of molecular interactions at lower separation distances, promise to improve the versatility of labeling and lead to new applications of the proximity techniques.

This topic is focused on developing slide-based proximity technologies using two specific antibodies at different epitopes of the target to enable more accurate quantitation of cancer biomarkers in tumor cells in tissue sections.   Initial focus should be on epitopes within the same target molecule. This new technological approach has the potential to improve slide-based IFA sensitivity and specificity to approach or exceed that of sandwich ELISA testing of tissue homogenates and have the advantage of visualization of the cell types that express the biomarker and enable the quantitation of the state biomarker (e.g., activation) in specific cell types.

It is believed that the development of the proximity reagents for dual antibody staining of tissue sections to high value cancer biomarkers will have great research value and have a significant clinical impact; i.e., direct visualization and quantitation of informative biomarkers in the tumor population that the drug targets.

References:

1.      Wikipedia: http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer

2.      D. Geißler, S. Stufler, H. G. Löhmannsröben, and N. Hildebrand, J. Am. Chem. Soc. 135 (2013) 1102.

3.       K. D. Wegner, Z. Jin, S. Linde´n, T. L. Jennings, and N. Hildebrandt, Acs Nano 7 (2013) 7411.

4.      M. Schifferer and O. Griesbeck, J. Am. Chem. Soc. 134 (2012) 15185.

5.      S. A., Hussain et al. (2015). "Fluorescence Resonance Energy Transfer (FRET) sensor". J. Spectrosc. Dyn. 5 (7): 1–16.

6.      Herman,Brian et al. Microscopy resource Center: http://www.olympusmicro.com/primer/techniques/fluorescence/fret/fretintro.html, 2012

7.      Peter König, et al. FRET–CLSM and double-labeling indirect immunofluorescence to detect close association of proteins in tissue sections. Investigation (2006) 86, 853–864.

8.      http://www.perkinelmer.com/Resources/TechnicalResources/ApplicationSupportKnowledgebase/AlphaLISA-AlphaScreen-no-washassays/AlphaLISA-AlphaScreen-no-wash-assays.xhtml#AlphaLISAAlphaScreenno-washassays-Assayprinciple

9.      Ewa Heyduk,et al.  Molecular pincers – new antibody-based homogenous protein sensors. Anal Chem. 2008 Jul 1; 80(13): 5152–5159.

10.   Kattke, MD, et al. FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami. AntibodyChain 11:6396, 2011.  http://www.antibodychain.com/print/32258

11.   Sharma N, Hewett J, Ozelius LJ, et al. A close association of torsinA and alpha-synuclein in Lewy bodies: a fluorescence resonance energy transfer study. Am J Pathol 2001;159:339–344.

12.   Mills JD, Stone JR, Rubin DG, et al. Illuminating protein interactions in tissue using confocal and two photon excitation fluorescent resonance energy transfer microscopy. J Biomed Opt 2003;8:347–356.

Project Goals

Over the past decade, biosensors based on fluorescent proteins, FRET, and recently radio-frequency tags have emerged as major classes of probes that are capable of tracking a variety of cellular signaling events, such as second messenger dynamics and enzyme activation/activity, in time and space. For example, a donor chromo/fluorophore, initially in its electronic excited state, may transfer energy to an acceptor chromo/flurophore through nonradiative dipole–dipole coupling.  The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.  Depending on the types of detection probes used, the distance /proximity between donor and acceptor can be between 1 nm and 10 nm to generate a signal.

There is increased use of proximity biosensors, particularly tagged-antibodies in combination with signaling factors (electric, optical, etc.) to provide a specific signal directly related to the concentration of an analyte. These proximity reagents have the potential to allow rapid detection of the target with the sensitivity and specificity of a sandwich assay.

The goal of this topic is to develop reagents/methods for use of dual primary antibodies to different epitopes of the same analyte or to different subunits of a target that, upon binding of the molecule(s) in cells within a tissue section, will generate a proximity signal due to close spatial association of the antibody reagents containing donor/acceptor tags, respectively (e.g., flurochromes).  This signal can be captured and visualized to cell types as well as quantitated via conventional microscopy. 

The benefit is that antibody based proximity assays to high value cancer biomarkers will represent a significant advancement in detection capabilities for slide-based immunoassays of tumor sections.  These assays will provide specific, sensitive and reliable detection of targets in a cell specific manner and approach or exceed ELISA level quantitation which has significant clinical applications.

Phase I Activities and Deliverables

The goal is to replicate or exceed the sensitivity and specificity of an ELISA in slide-based immunoassays of tumor specimens. The objective is to develop and test the applicability of double antibody labeling using proximity technology for the detection of two epitopes on TWO high value oncology biomarkers, preferably choosing NCI designated biomarkers.  This may involve the development of new proximity tags adapted for use in dual antibody labeling of histological sections.  The readout will be the successful localization of protein based analytes in the appropriate cell type when expressed at varying levels (i.e., specificity/sensitivity) and quantitation of the cell specific signal that agrees with known protein levels measured by other biochemical methods – initially using non-clinical models to evaluate.  

Biomarkers important to NCI are oncology-relevant proteins that have two epitopes of interest; for example, one could select epitope specific antibodies to allow for cell specific visualization and quantitation of the amount of a receptor in a cell that is also phosphorylated at a specific epitope.

The NCI biomarkers and possible epitopes of interest for developing these assays are listed below:

NCI is available to advise on biomarker reagents and xenograft models. In special cases, NCI may provide antibody reagents to selected PD biomarkers and the associated xenograft tumors to awardees. 

Phase I Activities and Deliverables

·       Reagent parameters [proximity tags], assay parameters, imaging platform parameters, and image capture and analysis strategy for the proximity measurements (e.g. FRET) are to be developed.

·       Select appropriate donor and acceptor probes for the 2 analyte specific antibodies chosen for each target and determine the manner in which they are employed as molecular labels to obtain optimal energy transfer/signal.

·       Detailed SOPs written foreach of TWO high value oncology biomarkers, preferably choosing from NCI designated biomarkers. 

·       Optimize reaction and stabilization conditions.

·       Develop measurement strategy for capturing the intensity of signal.

·       Relate the proximity signal to the quantitation provided by other biochemical measurements in appropriate non-clinical models. 

·       Prove that proximity signals are emanating from the same protein molecules, rather than adjacent or nearby protein molecules.

·       Carry out independent verification of prototype assay reagents/instrument (NCI may be available to do this). 

Phase II Activities and Deliverables

·       Develop ‘optimized’ dual antibody proximity assays to two epitopes on each of THREE high value oncology biomarkers, preferably choosing from NCI designated biomarkers.

·       Reproducibly manufacture proximity-reagents and do at least 6 months stability testing.

·       Show reproducibly/robustness of the proximity -dual antibody staining and quantitation in sections of a human tumor xenograft(s).

·       Provide images of the dual antibody stained xenograft tumor sections and quantification of the signal to demonstrate reliability of the technique.

·       Carryout independent verification of performance of reagents/ instrumentation (NCI may be available to do this). 

·       Provide the program and contract officers with a letter of commercial interest. 

The generation and dynamic interplay of redox effector molecules (e.g., oxygen, free radicals, peroxides, nitrogen oxides, and hydrogen sulfide) are fundamental features underlying the genomic, structural, metabolic and functional alterations observed in cancers.  Alterations in redox balance impact all phases of disease including carcinogenesis, disease progression, response to treatment and prevention.  For example, the DNA damaging effects of free radicals can mutagenize key oncogenic sites.  Redox imbalances occur by abnormalities commonly associated with cancers including mutations in p53, myc and ras pathways.  Redox effectors operate to modify protein function at the post-translational level, which plays a significant mechanistic role in the phenotypic plasticity cancer cells demonstrate in the face of oxidative and reductive (hypoxia) stresses.  Redox tone is a key regulator of the self-renewal properties of stem-like cancer cells, which has been shown to contribute to tumor resistance to current therapies.

Progress in the cancer biology and pre-clinical space has been limited by the lack of tools that can accurately measure redox parameters in animal models with sufficient spatio-temporal resolution and minimal perturbation of the system.  NCI seeks input from the small business community to develop and optimize a new generation of quantitative and specific technologies that will enable and accelerate basic research aimed at understanding basic redox effector mechanisms and the roles they play in the cellular adaptations and complex biology of tumors.

Supporting the development of these technologies will allow researchers to validate and benchmark data obtained across different 3D cell culture platforms and pre-clinical animal model systems with the goal of accurately mimicking tumor environments experienced by patients with cancer.  Moreover, an enhanced ability to screen, manipulate, or analyze redox dynamics is an invaluable index in the evaluation of cancer cell-tumor responses to therapeutic interventions in the critical pre-clinical testing phase.  These redox data have potential to significantly improve our understanding of tumor biology and ability to better predict treatment responses and long-term efficacy when translated into patients.

Project Goals

There is an unmet need in basic cancer research for probes or technologies that can better measure, characterize, profile, or resolve the spatiotemporal dynamics of redox effectors at the subcellular to cellular levels.  Genomic profiles, for instance, cannot capture post-translational redox regulation that occurs with changes in the tumor microenvironment.   Redox probes have been traditionally reliant on organic dyes that experience spectral shifts with redox.  The current state of the art is genetically encoded redox indicators that couple redox responsive enzyme motifs with indicator proteins.  These genetically engineered redox probes have improved response kinetics, but may have limited optical qualities.  Given the critical role played by redox effectors, developing a range of new tools will help us better understand how redox effectors regulate cell phenotypes in functional tumor populations.

The goal of this FOA is to develop quantitative tools to measure redox dynamics in biological systems.  Ideally, probes or biosensor tools should be minimally invasive as to not significantly perturb the system.  The technical approach should: (1) allow for in vivo measurements of redox effector spatiotemporal dynamics; and/or (2) be useable in high throughput systems, for example to allow the screening of cellular response to experimental perturbations, such as exposure to cytotoxic agents.  The long term goal is that the technologies developed through this contract can help validate whether data gathered in model experimental systems faithfully represents the redox dynamics of human tumors.

To successfully meet this goal, offerors shall develop a technology for the minimally to non-invasive measurement of one or more redox effectors, including but not limited to oxygen, free radicals, reactive oxygen species, peroxides, nitrogen oxides, and hydrogen sulfide.  Phase I studies should focus on developing the technology and demonstrating proof of concept in an in vitro system. Phase II studies further refine the technology and demonstrate the use of the technology to measure redox effectors.  Offerors shall justify their choice of approach with respect to the scientific utility and commercial potential, and specify quantitative milestones that can be used to evaluate the success of the technology being developed.

It is anticipated that offerors shall develop a probe or similar agent that facilitates the measurement of redox effectors by one or more imaging modalities; however, offerors are not restricted to any particular technical approach and label or probe free approaches that can meet the requirements of this contract are welcome.

Offerors are not restricted to any particular technical approach and can propose resource and tool development that incorporates high-risk/high-impact technologies.  Examples can include, but are not limited to:

·       Redox probes that provide significant advances in sensitivity, selectivity, ratiometric capability, or resolution in reporting the spatial concentration gradients and temporal dynamics of redox effectors at the subcellular, cellular and/or tissue compartment levels.

·       Genetically encoded redox biosensors that are expressed in a cell or tissue selective manner in small animal models of cancer for interrogation by non-invasive to minimally invasive imaging modalities.

·       Biology-inspired redox sensors (e.g., based on bacterial chemosensors) that through synthetic biology techniques are genetically encoded for expression in a cell or tissue selective manner.

·       Nanotechnology scaffolds multiplexed with sensors that permit functional parallel profile analyses of a combination of redox effectors (i.e., oxygen, nitric oxide, hydrogen peroxide, superoxide) and/or related species (e.g., proton, glutathione, ascorbate) across both time and space at the subcellular, cellular and/or tissue compartment levels.

Technologies that have the potential for in vivo use, especially those with potential clinical applications in the long term will be of particular interest, but methods that will be restricted to pre-clinical research applications are also of interest.

Phase I Activities and Deliverables

·       Identify and justify development of a sensing tool or probe for specific redox effector species from both a cancer biology and commercial perspective.

·       Offerors shall describe the current state of the art technologies for sensing and measuring the redox effector being addressed by their proposal, and outline the advantages that their approach will offer.

·       Develop and characterize a redox probe or biosensor. Offerors shall specify quantitative milestones that can be used to evaluate the success of the technology being developed, and justify these milestones from the viewpoint of both scientific utility and commercial value.

·       Develop an assay or system that demonstrates proof-of-concept testing and benchmarking of specificity and sensitivity parameters of the agent or system for a range of redox effector species (e.g., oxygen, free radicals, hydrogen peroxide, nitric oxide, hydrogen sulfide).

·       For each redox effector or parameter, a technical description of methodology for each assessment shall be provided that includes how each measurement is calibrated.  If measurements are collected serially, the rationale for the order of measurements shall be specified.

·       Demonstrate feasibility to sense, interrogate, detect or resolve the spatiotemporal dynamics of redox effector species in live cells or animal model, ideally with a minimally invasive perturbation of the system.

·       Provide NCI with proof-of-concept assay SOP. 

Phase II Activities and Deliverables

The goal of the Phase II product is an optimized commercial resource, product, reagent, kit or device that can allow researchers to measure the relevant redox effector molecules in their laboratory.  Decisions for continued project development into Phase II will be based on probes, biosensors, assays or systems that:

·       Can demonstrate reliability and robustness. Offerors shall provide a technical evaluation and quality assurance plan with specific detail on shelf life, best practices for use, and equipment required for use.

·       Can be scaled up at a price point that is compatible with market success and widespread adoption by the basic research community.

·       Have potential to benchmark data obtained across different cancer model systems (e.g. 2D and 3D tissue culture systems, and in vivo animal models of cancer).

Deliverables for the Phase II projects are:

·       Scaled up synthesis or manufacture of necessary agents, chemicals, device, or products.

·       Design and implementation of quality assurance controls and assays to validate production.

·       Validate scaled up device, chemical or product. Offeror shall demonstrate the utility, reliability and sensitivity of their device, chemical or product across in vitro and/or in vivo models relevant to cancer research.

·       Refine SOPs to allow for user friendly implementation of technology by the target market for the agents, chemicals, device, or products.

Metagenomic studies in humans and animal models have established that there are alterations of the GI microbiota community during development of neoplastic and pre-neoplastic disease, and in tumor-bearing vs. healthy individuals. Understanding the impact of human host/microbiota interactions on the initiation, progression and treatment of cancer, and the molecular mechanisms that govern the outcomes of these interactions, will provide new therapeutic strategies and new targets for the treatment of many human tumors.

One promising approach emerging from recent research is alteration of microbiome function designed to enhance the efficacy of cancer therapies.  Recent work demonstrated that individual variability in patient drug response to chemo (and other) therapies can be attributed to actions of the gastrointestinal (GI) microbiota, either through direct metabolic activity on the agent itself, or by effects on host barrier function and immunomodulation that affect drug bioavailability. For example, microbial β-glucuronidase activity results in re-activation of toxic metabolites that affect the dose-limiting range of CPT-11, a prodrug form of the topoisomerase inhibitor Irinotecan that is widely used to treat a variety of solid tumors. Antibiotic co-therapy and specific inhibition of bacterial β-glucuronidase activity reduced chemotherapy-induced GI toxicity in several animal models. Other studies have shown that depletion of NOX-inducing Lactobacillus species by antibiotics, results in reduced tumoricidal activity of platinum based drugs, which rely on induction of reactive oxygen species (ROS) to mediate tumor cell killing. Similarly, the antitumor effects of radiotherapy and several cytotoxic chemotherapeutic drugs such as cyclophosomide (CTX), oxaliplatin, and CpG-ODN, are achieved in part by an immune-mediated bystander effect that requires the recruitment and activation of an intense inflammatory infiltrate to regress tumors. 

As we learn more about how the microbiome affects disease progression and response to treatments, the opportunity to exploit the microbiome for therapeutic benefit is an exciting new approach that should be explored. 

Project Goals

The purpose of this SBIR contract solicitation is to develop innovative technologies and methods designed to modulate the GI microbiota in order to enhance the therapeutic efficacy of existing or novel cancer therapies, or ameliorate side effects of these therapies. The goal is to develop effective adjuvant strategies that specifically target critical microbial activities or populations that affect drug efficacy and/or tolerability. Ultimately, this activity will accelerate the development of novel strategies based on the rational targeting and manipulation of human GI microbiome functions for the treatment of human tumors.

To successfully meet this goal, applicants will need to demonstrate that their approach accomplishes the specific perturbation or modulation of the microbiome that is desired, and that these approaches have demonstrable benefits in addressing a significant unmet medical need relevant to cancer (e.g. reduction of off-target toxicity). Phase I studies should focus on developing and refining the approach that will be used to modulate GI microbiota or functions performed by the microbiota (such as metabolic activity). Applicants should establish appropriate criteria to benchmark or evaluate the success of their approach, and these should be related to the expected level of perturbation or modulation that is required to have therapeutic benefits. Phase II studies should focus on demonstrating that the approaches developed in Phase I studies are effective in an appropriate in vivo model system. Lead candidates should be developed and tested for efficacy in appropriate animal models, and Phase II studies should also measure drug delivery (e.g., probiotics,  engineered phage, lipids, nano-particles) and pharmacokinetic  targeting (e.g., reduction/increase of specific microbial enzyme activity, signaling ligand, or host interaction) in addition to measured endpoints of tumor regression and/or ablation in vivo.

Applicants are not limited to specific cancer types, but are required to identify and justify a cancer type and unmet medical need that can be addressed by their approach. They should also provide a scientifically justified rationale for exploring particular approach(es) for perturbing or modulating the microbiome, and justify the choice of model system to evaluate their approach(es).

It is anticipated that applicants will test perturbations of the GI microbiome, such as antibiotic treatments, bacteriophage therapies, probiotic supplements, dietary metabolites, drug metabolizing enzymes, modulators of bacterial metabolism, and immunomodulators. However, applicants are free to employ any approach.

The focus of this contract topic is not to search for new mechanisms or effects by which the microbiome affects cancer therapy or progression, but rather to explore microbiome directed intervention strategies that have a rational basis. The contract topic is not intended to develop screening approaches, though applicants may propose to refine or optimize lead compounds or other agents designed to modulate or perturb GI microbiota.

Phase I Activities and Deliverables

·       Define and characterize a microbial activity/interaction that affects therapeutic efficacy, demonstrated through appropriate in vitro and in vivo experiments.

·       Develop targeted microbiota regulated/directed intervention strategies designed to improve, either alone or in combination, patient outcomes for new or current therapeutic agents. Approaches may involve, but are not limited to:

o   Narrow spectrum antibiotics

o   Bacteriophage therapies

o   Probiotics/Prebiotics

o   Dietary metabolites

o   Expression or delivery of novel drug metabolizing enzymes

o   Inhibitors of bacterial enzymes

o   Immunomodulators/vaccines

·       Test and refine therapeutic approaches in order to identify lead candidates or agent (e.g. bacteriophage, bacterial strain, enzyme, dietary metabolite, vaccine, etc.)  to develop further in Phase II studies

·       The lead candidate or agent should be able to successfully accomplish the desired perturbation or modulation of the microbiome to a level that can reasonably be expected to be have an impact on the efficacy of the therapeutic interventions and demonstrate proof of concept for the efficacy of their approach

o   Offeror should demonstrate proof of concept in an appropriate in vivo model

·       Offeror should determine and justify the assays and endpoints that will be used to evaluate the success of their approach (e.g., biomarkers, enzymatic activity, presence or absence of specific microbial populations)

o   If needed, offeror should develop alternative tools/methods to evaluate candidate effects on microbiome function.

·       Submit a statement to NCI that specifies the metrics and criteria used to evaluate the success of the approach being developed, and justification for these metrics and criteria from a commercial and scientific perspective.

Phase II Activities and Deliverables

·       Demonstrate the efficacy of lead candidate(s) or agent(s) from Phase I studies in an appropriately characterized  in vivo model

o   Identify and measure appropriate pharmacokinetic, pharmacodynamics,  and therapeutic endpoints

o   Evaluate toxicity and efficacy of therapeutic candidate(s) or agent(s)

o   Evaluate immune response to therapeutic approach where appropriate

·       Determine the toxicology and safety profile of the lead candidate(s) or agent(s) using appropriate animal models and assays relevant to the specific therapeutic approach being pursued

·       Optimize or scale up lead candidate(s) or agent(s) (e.g. bacteriophage, bacterial strain, enzyme, dietary metabolite, vaccine, etc.) from Phase I studies. Activities may include, but are not restricted to:

o   Medicinal chemistry to optimize small molecules for in vivo studies

o   Scale up production of lead therapeutic candidate(s) or agent(s)

o   Optimize delivery method for therapeutic candidate(s) or agent(s)

·       Develop a plan for obtaining regulatory approval to conduct human studies. Offerors should provide plans and a detailed time table for obtaining this regulatory approval

Cancer health disparities (CHDs) are defined as differences in the incidence, prevalence, morbidity, and mortality that contribute to an unequal burden of cancer and represent a major public health concern both nationally and globally. In the United States, several racial/ethnic populations demonstrate increased incidence and/or more aggressive disease for numerous cancer types. The causes of these CHDs are multifactorial, including differences in access to health care, diet and lifestyle, cultural barriers, environmental exposures, and ancestry-related factors. Recent data suggest that biological factors may contribute to CHDs. The NCI specifically encourages and funds investigations of such biological factors to better understand mechanisms that contribute to CHDs. One limitation in conducting basic, translational, and clinical  research investigating the causes of CHDs is a substantial lack of relevant in vitro and in vivo-based models. The development and validation of appropriate models to study underrepresented population groups would greatly advance this field of research.

Program Goals

The primary goal of this topic is to develop new, commercially available models relevant to diverse racial/ethnic populations.  These models may be used to enhance research capabilities of basic scientists and/or provide novel tools to pharmaceutical companies for preclinical oncology studies. Establishing these novel models may influence CHD research in multiple ways including 1) attracting additional researchers to this largely underexplored area of research, 2) improving the quality and acceptance of CHD research data, and 3) improving validation and commercialization of cancer therapeutics relevant to diverse patient populations. Lastly, achieving these goals will contribute to the overarching goal of facilitating the reduction of CHDs. 

Small businesses are invited to submit proposals to develop a panel of cell lines, primary cells, or patient-derived xenograft (PDX) mouse models established from racially/ethnically diverse patient populations. Additionally, competitive applications may propose novel genetically engineered mouse (GEM) models to investigate cancers or co-morbid conditions that are more frequent and aggressive amongst diverse racial/ethnic populations.

·       Cancer cell lines and primary cancer cells: The scientific integrity of cancer cell lines and primary cells is critical for maintaining high standards in research.  Any cells established via this solicitation must be fully confirmed through a rigorous and validated authentication and be contamination-free. Furthermore, offerors must have access to fully annotated tumor tissues from diverse racial/ethnic populations with appropriate approval(s) in place (i.e., IRB).

·       PDX Mouse Models: PDX models have recently gained huge recognition in clinical and preclinical oncology research. Molecular profiling studies have shown the similarity between patient tumor and PDX models is greater than between patient tumor and traditional cell lines. Therefore, new initiatives have been proposed to use PDX models in a number of clinically relevant research areas including characterization of tumor heterogeneity, in vivo therapeutic target validation studies, clinically relevant mechanism of action studies, and sensitivity and resistance to therapy studies.  Furthermore, PDX models have even been suggested to be a useful tool to mimic human clinical trials using animals. Similarly, offerors must have access to fully annotated tumor tissues from diverse racial/ethnic populations with appropriate approval(s) in place (i.e., IRB).

·       GEM Models: Numerous cancer types are more prevalent in specific racial/ethnic populations.  An example of one such disease is triple negative breast cancer (TNBC). Although diagnosed less often, breast cancer in African American women display different characteristics compared to breast cancer in Caucasian women, including earlier onset, less favorable clinical outcome, and an aggressive tumor phenotype. The reason for this aggressive phenotype is currently widely studied however progress is hampered by the lack of suitable TNBC model systems. Development of GEMs (including knock-in mice, knock-out mice, and mice with chemically induced mutations) to study cancers disproportionately effecting racial/ethnic populations would advance the field.  Offerors must provide data or cite literature justifying the GEMs proposed and have relevant technical expertise.

Phase I Activities and Deliverables

·         Establish an experimental model relevant to CHD research.  This may include one of the following:

o  Cancer cell line or primary cells established from racial/ethnic minorities

o  PDX animal model established from racial/ethnic minorities

o  GEM model

·         Cancer cell line and primary cells deliverables: Establish a stable cell line from tumor cells and passage the cells in culture to determine viability. 

o   Detailed documentation must be provided including patient clinical characteristics, passage history, mycoplasma testing results, and appropriate growth/preparation conditions.

o   Develop a standardized, working protocol for establishment of additional cell culture models.

·         PDX animal model deliverables: Establish a serially transplantable, phenotypically stable, human cancer xenograft model in immunocompromised mice.

o   Transplant fresh surgical tissue or biopsy (either subcutaneous or intraperitoneal) into recipient immunodeficient mice (Transplant generation 1)

o   Subsequent serial transplantations must be conducted following establishment of initial xenograft outgrowths, typically >10mm in diameter (A minimum of three generations of transplantation is required to establish a stable line)

o   After three generation of transplantations, confirm genetic and phenotypic heterogeneity of the tumors.

o   Freeze and bank tumors.

o   Develop a standardized, working protocol for establishment of additional models. 

o   Perform comprehensive molecular characterization of patient samples and earliest PDXs, including whole exome sequencing and mutational status analysis using a CLIA-approved panel.

·       GEM model deliverables: Develop a GEM model to support investigations on cancers disproportionately effecting racial/ethnic populations.

o   Develop transgenic constructs and strategy to create GEM models

o   Transfer fertilized mouse embryos with transgenic constructs to foster mouse mothers

o   Identify potential transgenic founders and mate  to generate F1 progeny

o   Analyze to identify and confirm successful transgenic mice

o   Determine validation and development plan for transgenic mice

·         Validate the genetic ancestry of patients (if applicable) from which a model was established using a panel of ancestry informative makers (AIMs).  The AIM panel(s) selected should be relevant to the patient populations being investigated and capable of specifying admixture proportions.

Phase II Activities and Deliverables

·         Cancer cell lines and primary cells: Generate a panel of no less than 50 cell lines from different patient sources.

·         PDX animal models: Generate a panel of no less than 20-50 models (depending on tumor type being used) from unique patient sources using established protocols.

·         GEM Models: Demonstrate preclinical utility and merit of the generated transgenic mouse model(s) by conducting sufficient experiments.

The evidence that cell-free circulating DNA is present in cancer patient’s blood was first reported over half century ago.  Since then studies that addressed the clinical significance of the cell free DNA quantification in plasma/serum for cancer diagnosis have grown steadily.  Research findings indicated that most patients with solid tumors in lung, breast, prostate, colon, cervix, ovary, testes, and bladder have increased DNA levels that allow for discriminating patients with malignancies from those with non-malignant disease.  The first application of cell free circulating nucleic acids (cfNA) in the diagnosis and prognosis of cancer was demonstrated in 1977 when higher level of circulating DNA was detected in the serum of cancer patients; these levels decreased in response to radiation therapy. 

In recent years, it has been recognized that circulating DNA may be altered in fragmentation pattern, microsatellite stability, and DNA methylation. In addition, the cfNA sequences may be mutated and tumor-specific allowing for increased sensitivity and specificity in evaluation and detection of cancer compared to mere quantification of cfNA levels.  Besides circulating cell free DNA, evidence has indicated that tumor-derived RNA, (especially the quantification of the tumor-derived microRNA in plasma/serum) may be an excellent biomarkers for the diagnosis and prognosis of cancer.  Furthermore, alterations of cfNA are also found in other sources of body fluids or effusions such as urine or sputum.  Clearly, cfNA as a biomarker, which is easily accessible, reliable, and reproducible, can offer many advantages in their implementation into clinical use.

To date, however, there are no currently effective cfNA-based assays that are approved for clinical use in the diagnosis or prognosis of cancer. The low abundance of cfNA from all body fluids and effusions remains a major challenge in the assay development.  Many early developments need to be further verified and validated before they can be translated to clinical use. With the latest technology advancement in sample collection, processing, and analysis for nucleic acids, the likelihood of clinical utilization of cfNA becomes more reachable. 

The purpose of this initiative is to provide much needed support for the development of a cfNA-based assay for cancer diagnosis and/or prognosis.  The selected applicants will develop an assay for detection of cancer or its subtype, so that cancer or subtypes can be identified specifically.  Since a single alteration in cfNA may not be sufficient to detect a specific cancer, offerors are encouraged to use a panel of cfNA alterations that could be more robust for their assay development.  The cfNA alterations may include, but not limited to, cfNA concentration, fragmentation pattern, microsatellite stability, and DNA methylation, tumor-specific sequences, DNA mutations or tumor-derived RNA.  The sources for cfNAs can be from plasma, serum, urine, sputum or other types of body fluids or effusions.  In Phase I, the development of molecular diagnostic assay should focus on proof of concept.  In Phase II, the assay developed in Phase I will be validated in the clinic setting under a plan developed with the NCI project officer.

Program Goals

The goal of the project is to develop a cfNA-based assay for clinical use in the evaluation of cancer diagnostics, prognostics, and response to therapy. The levels of sensitivity and specificity required will depend on the clinical question and unmet need the assay is attempting to answer.  The assay may also be used to provide a better mechanistic understanding of tumor development and progress with the idea that this knowledge may lead to better therapeutic targets and improve patient outcome.  Preference will be given to the assays that are platform driven, meaning that the technology platform should be portable and easily used for diagnosis of multiple cancer types.

To apply for this topic, offerors need to outline and indicate the clinical question and unmet clinical need that their assay will address. Offerors are also required to use validated cfNA markers.  This solicitation is not intended for biomarker discovery. 

Phase I Activities and Expected Deliverables

·       Select one or a set of validated cfNA markers with samples of a choice (e.g., plasma, serum or/and urine) for detection of a cancer or subtype (e.g., breast cancer or triple negative breast cancer).  If novel or proprietary markers are used, offerors must show that the markers have been validated.

·       Develop an assay to identify these markers effectively to distinguish the cancer samples from healthy samples.  The offerors should also demonstrate that the assay is able to differentiate the cancer from other cancer types.

• Demonstrate high reproducibility and accuracy with the assay.
• Demonstrate high specificity and sensitivity of the assay.  Specificity and sensitivity will depend on the application (e.g., high specificity will be required if the assay is used to provide specific molecular information for the lesion that was detected by CT imaging).
• Deliver to NCI the SOPs of the cfNA-based assay for cancer diagnosis.

·       Demonstration of a plan that is necessary to file a regulatory application.

Phase II Activities and Expected Deliverables

• Demonstrate the assay that enables a test to be finished within one day.
• Validate the assay in the clinical setting.
• Submit a regulatory application to obtain approval for clinical application.

The field of cancer immunotherapy has expanded rapidly over the last few years with the development of several new immunomodulatory agents that have shown promising clinical results.  As one example, in 2011, the FDA approved a therapeutic antibody for the treatment of melanoma, ipilimumab (YERVOY®), which blocks cytotoxic T-lymphocyte antigen 4 (CTLA-4), a receptor found on T-cells that downregulates the immune system.  In 2014, the FDA approved another agent for the treatment of melanoma, pembrolizumab (KEYTRYDA®), which is the first approved therapeutic antibody targeting the programmed cell death protein 1 (PD-1), another T-cell receptor that plays a role in immune inhibition.  These two therapeutic antibodies are representative of a major class of cancer immunotherapies known as immune checkpoint inhibitors, and several other therapeutic agents are currently in development targeting CTLA-4, PD-1, and other immune checkpoint proteins.  Other major classes of cancer immunotherapies currently being developed include therapeutic cancer vaccines, as well as therapeutic approaches that involve ex vivo manipulation and engineering of immune cells, including chimeric antigen receptor (CAR) therapy.

Cancer immunotherapies offer several advantages over current standard-of-care cancer treatments, including the potential to eradicate cancer cells not visible to the surgeon, as well as disseminated metastases that remain undetectable using current imaging modalities.  Immunotherapy approaches may also prove effective at targeting slowly dividing or quiescent tumor cells that do not respond well to chemotherapy and/or radiation, and certain immunotherapy approaches are expected to suppress re-emergence of the cancer (following initial treatment) by exploiting the immune system’s memory.  In fact, early results have shown positive and dramatic clinical outcomes for some of the more recent cancer immunotherapies, even in patients with advanced disease; however, it is often the case that only a subset of patients respond to such therapies for reasons that are often poorly understood. 

As the field of cancer immunotherapy continues to evolve, and as more cancer immunotherapies advance through clinical development, there will be an increasing need for companion diagnostic assays capable of predicting responders (and non-responders) to cancer immunotherapies.  Moreover, such assays will become critically important as these therapies are eventually utilized as part of routine clinical practice.

Program Goals

The goal of this contract topic is to develop companion diagnostic assays and technologies capable of identifying individual patients for whom a particular cancer immunotherapy regimen will be safe and effective.  This includes cancer immunotherapies that have already received marketing approval from the FDA, as well as cancer immunotherapies currently in clinical development.  This topic is specifically intended to address cancer immunotherapies that depend upon eliciting an immune response.  Projects that do not meet this requirement will not be considered responsive.  For example, a monoclonal therapeutic antibody that exerts a direct antitumoral effect either by neutralizing the antigen or by activating signaling pathways within the target tumor cell, but does not elicit an immune response for its clinical activity, is not considered an immunotherapy and would not be considered responsive.

The goal of this contract topic is NOT to solicit any particular technology or approach, i.e., this contract topic is technology agnostic.  Technologies employed may include, but are not limited to, genetic analysis, other molecular diagnostic approaches, cell culture and cell expansion technologies, imaging modalities, radio-labeling approaches, and data science/analytics.  This contract topic is specifically intended to support the development of assays that provide predictive and/or prognostic information for a specific cancer immunotherapy.  Projects that do not meet this requirement will not be considered responsive.  For example, development of an assay for the sole purpose of measuring whether an agent modulates its intended molecular target (e.g., pharmacodynamic assay) would not be considered responsive.  Likewise, development of an assay for providing information that is useful in cancer diagnostics or prognostics but not in determining the safe and effective use of a therapeutic product/regimen would also not be considered responsive.  Noninvasive and minimally invasive sampling methods (e.g., body fluids and mouth swab) are preferred.  Other sampling methods are acceptable if they provide significantly improved predictive value, accuracy, and clinical applicability.

Phase I Activities and Expected Deliverables

• Develop a working companion diagnostic test for a specific cancer immunotherapy regimen, which meets the criteria described above
• Characterize the variation, reproducibility, and accuracy of the test, and implement a QA/QC plan
• Demonstrate suitability of the test for use in the clinic, and conduct benchmarking studies against current tests (if available); algorithms must be tested with datasets other than those used for their development
• In cases where a companion diagnostic test is proposed for a specific immunotherapeutic that is not yet commercially available (i.e., approved for marketing), the applicant must provide proof of collaboration or partnership with the entity that is developing the therapeutic agent or with an established diagnostic company
• All offerors must establish a collaboration or partnership with a diagnostic and/or pharmaceutical company and/or clinical/research institution that can provide relevant clinical trial specimens; offerors must provide a letter of support from the partnering organization in the Phase II application
• Deliver the SOP of the working test to NCI for evaluation

Phase II Activities and Expected Deliverables

• Incorporate the assay into a standard kit for clinical testing and eventual distribution and sale
• Demonstrate clinical utility and value by testing sufficient numbers of patients from multiple sites to unequivocally demonstrate statistical significance with regard to patient selection for the therapy
• If the primary conclusions reached during the Phase I studies were based on animal experiments or ex vivo modeling, then a correlation study between these models and treatment in human subjects is expected
• Establish marketing partnership or alliance with the company developing the therapy, unless the therapy is already approved for marketing
• It is preferred that the test be performed in at least one independent CLIA-certified laboratory
• Deliver the final SOP to NCI for evaluation

National Center for Advancing Translational Sciences (NCATS)

The mission of the National Center for Advancing Translational Sciences is to catalyze the generation of innovative methods and technologies that will enhance the development, testing, and implementation of diagnostics and therapeutics across a wide range of human diseases and conditions.  For additional information, please visit our home page at http://www.ncats.nih.gov.

It is strongly suggested that potential offerors not exceed the total costs (direct costs, facilities and administrative (F&A)/indirect costs, and fee) listed under each topic area.

Extra-anatomic bypass or vascular shunts divert blood flow.  In congenital heart disease, these surgical procedures are critical for management.  Children born with one functional ventricle or cardiac pumping chamber require two to three major cardiac surgery procedures for palliation.  The management goal is to divert systemic venous return (deoxygenated blood) from the heart directly to pulmonary artery circulation such that the single ventricle can pump oxygenated blood returning to the heart from the lungs to the body.  These multiple surgical procedures carry significant morbidity and mortality, as well as incur substantial hospital costs secondary to lengthy hospital stays.  A minimally invasive transcatheter approach would revolutionize the management of these children with congenital heart disease.  No commercial alternatives exist for off-label medical use.  Children born with “single ventricle physiology” represent 7.7% of congenital heart disease diagnosed in childhood and have a birth incidence of approximately 4–8 per 10,000.  In the United States, this represents approximately 2,000 children born each year.  The commercial market is small enough to discourage the early development costs of a transcatheter cavopulmonary bypass endograft.  There is a considerable unmet need for a purpose-built cavopulmonary anastomosis device.

Project Goals

The goals of this project are to develop and test a transcatheter cavopulmonary bypass endograft prototype in vivo in Phase I, and to develop a clinical device and obtain an FDA Investigational Device Exemption (IDE) for first-in-human testing in the United States in Phase II.

Phase I Activities and Expected Deliverables

Expected deliverables are transcatheter endografts to be delivered using conventional interventional cardiovascular techniques including guiding catheters or sheaths, trans-lesional guidewires, and balloon-expandable or self-expanding delivery systems. Conventional and novel approaches are welcomed.

Specific requirements of the endografts include:

·       Delivery systems (10-12 French or smaller);

·       Sufficient radial force to resist elastic recoil (with specific focus at anastomosis site);

·       Nominal calibers suitable for the most common lesions;

·       Freedom from “pull-through” of the anastomosis once deployed; and

·       Accommodation for growing children (ultimately dilatable to adult size vessels).

Proposed endograft nominal geometry should be diameter 10-14mm, length range 25-50mm, and delivery system 10-12 French or smaller. The radial hoop strength of the deployed device should approach that of commercial endovascular stent grafts such as Gore Viabahn or Atrium iCast. Percutaneous vascular access routes would be trans-venous.  The implant and the delivery system should be conspicuous under the intended image-guidance modality; MRI compatibility is considered important.  Offerings should specifically provide the high radial force required to overcome immediate recoil of the intended applications, and should allow “direct stent” treatment technique.

Considerable detail should be supplied about the intended mechanical and biological performance of the graft-pulmonary anastomosis, including resistance to inadvertent separation and pull-through, hemorrhage, thrombosis, neointimal overgrowth, angulation, distortion or failure by patient and cardiovascular motion, and anticipated flow characteristics.

Phase I should focus on mechanical and biological performance of the proposed endograft, taking into account the mechanical strength required for the application; geometry of the access vessels and geometry and morphology of target vessels; “growth” accommodation to achieve larger size and delivery, implantation, and visualization strategies.

At the conclusion of Phase I, a candidate device design should be selected for clinical development based on in vivo performance of a mature prototype resembling a final design. Consideration for transition to Phase II funding will include progress toward regulatory clearance. Consideration may include the status of the contractor’s interactions with the Food and Drug Administration (FDA); therefore, contractors are encouraged to provide a detailed report of pre-IDE interactions with the FDA identifying requirements for IDE development under Phase II, including the summary of mutual understanding, if available. NHLBI encourages contractors to consider requesting designation to the FDA’s Expedited Access for PMA Devices (EAP) program (http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM393978.pdf) during the Phase I award period. 

The sponsoring NHLBI laboratory is willing to perform a limited number in vivo proof-of-principal experiments in swine (by mutual agreement) to confirm mechanical performance.

Phase II Activities and Expected Deliverables

The activities in Phase II should align with the required testing and development milestones agreed upon with the FDA in Phase I. The device should fit the specifications as described in the Phase I Activities and Expected Deliverables. The offeror should provide clear project milestones.

At the conclusion of Phase II, the offeror should submit an IDE for a US-based first-in-human research protocol, involving at least 10 subjects.

If the IDE is not granted during Phase II, the offeror must provide an FDA response that indicates that the specific deficiencies are limited to Current Good Manufacturing Design Verification and Validation, and that the offeror-proposed plan to address these deficiencies would be considered acceptable.

Offerors are encouraged to consider the NHLBI Phase IIB Small Market Award program (http://www.nhlbi.nih.gov/research/funding/sbir/small-market-awards.htm) to support additional development beyond Phase II. The NHLBI Phase IIB Small Market Awards provide up to an additional $3M over 3 years, with an expectation that applicants secure independent third-party investor funds.

The sponsoring NHLBI laboratory is willing to perform a limited number of in vivo proof-of-principal experiments in swine (by mutual agreement). 

NHLBI offers, but does not require, to perform the clinical trial at no expense to the offeror, to participate in the development of the clinical protocol, and to provide clinical research services.   The vendor is expected to perform or obtain safety-related in vivo experiments and data to support the IDE application.  

Catheter access to the left atrium is a fundamental step to numerous transcatheter therapies including catheter ablation of rhythm disorders, diagnostic catheterization in pediatric and structural heart disease, and future treatments for mitral valve and left atrial appendage disease.   MRI operation would enable radiation free catheterization and superior image guidance that are expected to enhance clinical outcomes.  No MRI safe and conspicuous atrial transseptal needle is commercially available.  “Active” MRI catheter devices contain electronic elements to produce MRI visibility.  This topic aims to support the development of an active MRI transseptal needle catheter and accessories.

Project Goals

The goals of this project are to develop and test an active MRI transseptal needle catheter prototype and accessories in vivo in Phase I, and in Phase II to develop a clinical device and obtain an FDA Investigational Device Exemption (IDE) for first human testing in the United States.

Phase I Activities and Expected Deliverables

A Phase I award would support the development and testing of actively visualized atrial transseptal needle system prototypes.

The deliverable is a complete clinically-relevant system including:

1.      A transseptal needle; 

2.      Accompanying electronics, if needed to enable safe active visualization and interface to the host MRI hardware;

3.      A transseptal introducer sheath, which should be visualized passively or actively using real-time balanced steady state free precession MRI;

4.      A matched dilator to allow safe delivery of the introducer sheath over the needle, which must also be clearly visualized, preferably using active visualization. “Active” refers to visualization by virtue of serving as a resonant antenna connected or coupled to the MRI hardware system.

The system should be free from clinically-important heating during continuous MRI at 1.5T.   Proposals for novel alternative visualization and heat-mitigation strategies are welcomed.

The sheath should be 8.5Fr or smaller and approximately 71cm in length.   Shapes should be available to accomplish transseptal puncture in a range of clinical applications; a deflectable sheath would be attractive. 

The transseptal “needle” functionality can be conferred using any combination of mechanical, electrical, acoustic, or photonic energy.

A solution must be provided for visualization of the “active” electronic components using a real-time MRI system.

At the conclusion of Phase I, a candidate device design should be selected for clinical development based on in vivo performance of a mature prototype resembling a final design. Consideration for transition to Phase II funding will include progress toward regulatory clearance. Consideration may include the status of the contractor’s interactions with the Food and Drug Administration (FDA); therefore, contractors are encouraged to provide a detailed report of pre-IDE interactions with the FDA identifying requirements for IDE development under Phase II, including the summary of mutual understanding, if available. NHLBI encourages contractors to consider requesting designation to the FDA’s Expedited Access for PMA Devices (EAP) program (http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM393978.pdf) during the Phase I award period. 

The contracting DIR lab is willing to provide feedback about design at all stages of development.  The contracting DIR lab will test the final deliverable device for success in vivo in swine. This requires specific hardware compatibility with the NIH Siemens Aera 1.5T MRI system.

Phase II Activities and Expected Deliverables

Phase II activities should include testing and regulatory development for the device with specifications as described in the Phase I Activities and Expected Deliverables section to be used in first-in-human investigation in the United States, whether under IDE or 510(k) marketing clearance.  IDE license or 510(k) clearance would constitute the deliverable.  

Aortic coarctation is a common congenital heart condition that is usually recognized during the neonatal period early after birth. The usual treatment is open surgery. Non-surgical catheter-based stent therapy is not available to treat neonatal aortic coarctation because children outgrow commercially available metallic stents. Absorbable stents might revolutionize the treatment of aortic coarctation in children, especially in neonates.  Neonates require small delivery systems for relatively large nominal diameter implants, which is technically challenging.  No commercial alternatives are available for off-label medical use.  There is a considerable unmet need for a purpose-built, absorbable scaffold stent for neonatal aortic coarctation.

Project Goals

The Phase I award is intended to support the development of a mature prototype with the requisite geometry, strength, deliverability, and absorption characteristics required for the clinical product.

The Phase II award is intended to result in an Investigational Device Exemption (IDE) for a first human clinical test in the United States. 

Phase I Activities and Expected Deliverables

Expected deliverables are transcatheter stents to be delivered using conventional interventional cardiovascular techniques including guiding catheters or sheaths, trans-lesional guidewires, and balloon-expandable or self-expanding delivery systems. Conventional and novel approaches are welcomed.

Specific requirements of the stents include:

·       small delivery systems (5 French or smaller);

·       sufficient radial force to resist elastic recoil for the coarctation;

·       sustained radial strength suited to the application for at least 6 months;

·       controlled degradation within 6-12 months;

·       inflammatory response that does not cause significant stenosis, restenosis, or aneurysm;

·       resistance to downstream embolization or toxicity;

·       geometry that does not threaten patency of the subclavian artery;

·       nominal calibers suitable for aortic coarctation.

Proposed stent nominal geometry should be diameter 6-10mm, length range 10-25mm, delivery system 5-6 French or smaller. The radial hoop strength of the deployed device should approach that of commercial balloon-expandable stent such as the Cordis Palmaz Genesis. Percutaneous vascular access routes for aortic coarctation application include transvenous-transeptal antegrade and retrograde transfemoral artery.  The implant and the delivery system should be conspicuous under the intended image-guidance modality to allow precise positioning.  Offerings should specifically provide the high radial force required to overcome immediate recoil of the target tissue, and should allow “direct stent” treatment technique for native and iatrogenic lesions.

Phase I should focus on mechanical and biological performance of the proposed biodegradable stents in the intended use for aortic coarctation, taking into account mechanical strength required for the application; geometry of the access vessels and geometry and morphology of target vessels; strategies to avoid inflammatory restenosis or constriction; and delivery, implantation, and visualization strategies.

At the conclusion of Phase I, a candidate device design should be selected for clinical development based on in vivo performance of a mature prototype resembling a final design. Consideration for transition to Phase II funding will include progress toward regulatory clearance. Consideration may include the status of the contractor’s interactions with the Food and Drug Administration (FDA); therefore, contractors are encouraged to provide a detailed report of pre-IDE interactions with the FDA identifying requirements for IDE development under Phase II, including the summary of mutual understanding, if available. NHLBI encourages contractors to consider requesting designation to the FDA’s Expedited Access for PMA Devices (EAP) program (http://www.fda.gov/downloads/MedicalDevices/DeviceRegulationandGuidance/GuidanceDocuments/UCM393978.pdf) during the Phase I award period. 

The sponsoring NHLBI laboratory is willing to perform a limited number in vivo proof-of-principal experiments in swine (by mutual agreement) to confirm mechanical performance. 

Phase II Activities and Expected Deliverables

The activities in Phase II should align with the testing and development requirements agreed upon with the FDA in Phase I. The device should fit the specifications as described in the Phase I Activities and Expected Deliverables. The offeror should provide clear project milestones.

The sponsoring NHLBI laboratory is willing to perform a limited number of in vivo proof-of-principal experiments in swine (by mutual agreement). 

At the conclusion of Phase II, the offeror should obtain an IDE for a US-based first-in-human research protocol, involving at least 10 subjects.

NHLBI offers, but does not require, to perform the clinical trial at no expense to the offeror, to participate in the development of the clinical protocol, and to provide clinical research services.   The vendor is expected to perform or obtain safety-related in vivo experiments and data to support the IDE application. 

Offerors are encouraged to consider the NHLBI Phase IIB Small Market Award program (http://www.nhlbi.nih.gov/research/funding/sbir/small-market-awards.htm) to support additional development beyond Phase II. The NHLBI Phase IIB Small Market Awards provide up to an additional $3M over 3 years, with an expectation that applicants secure independent third-party investor funds.

Cardiotoxicity is increasingly recognized as a significant challenge to many existing therapies and as a potential barrier to the development of new therapies.  For example, despite improved survival from cancer, chemotherapy-induced cardiotoxicity has emerged as a significant problem.  Cardiovascular complication, particularly heart failure, is an important cause of morbidity and mortality among cancer survivors.  In small studies, cardioprotective strategies against cancer therapy-induced cardiac dysfunction are effective if implemented early at the subclinical phase.  However, detection of the frequency of subclinical disease and subsequent ability to protect against further functional decline are limited by inadequacy of current technologies to accurately assess and monitor changes in cardiac structure and function.  Novel non-invasive strategies that detect early subclinical changes in cardiac structure, function, and/or tissue are needed to improve detection and monitoring of cardiac injury in order to improve cardioprotection and effectiveness of cancer therapeutics or other toxic exposure.  Studies that demonstrate increased sensitivity and precision of existing or enhanced imaging technologies with respect to normal and altered cardiac structure, function, energetics, and metabolism are sought.  Pre-clinical or patient studies using molecular changes or biomarkers to enhance early detection of cardiac derangements are also responsive.

Project Goals

The goal of this initiative is to encourage the development of innovative methods to detect and monitor cancer therapy-induced cardiac injury as early as possible through minimally invasive means.  Early monitoring of cardiac injury will enhance both cardiac safety and treatment efficacy of cancer therapies.

Phase I Activities and Expected Deliverables

Phase I activities include proof-of-concept studies to demonstrate the feasibility of the method that will be fully developed in the Phase II.  Examples of Phase I research and expected deliverables may include, but are not limited to:

·       Design, testing, and initial in vivo validation of imaging methods or probes capable of assessing subclinical myocardial injury by cardiac MR, PET, or x-ray based imaging

·       Identification, selection, and initial testing of biomarker-based monitoring methods of cardiac injury, specifically aimed for cardiac injury due to chemotherapy

·       Studies to demonstrate innovative advances in ultrasound-based methods, including echocardiography, to improve sensitivity and resolution in order to assess early cardiac structural or functional changes

Phase II Activities and Expected Deliverables

Phase II activities are expected to include full development of the method whose feasibility was successfully demonstrated in Phase I, including additional validation in order to apply for regulatory approval and attract funding from industry.  A detailed report of interactions with the Food and Drug Administration (FDA) identifying the requirements for regulatory clearance or approval of the method is needed.  Examples of Phase II activities may include, but are not limited to:

·       Development and validation of imaging methods to assess subclinical myocardial injury by cardiac MR, PET, or x-ray based imaging

·       Validation of biomarker-based monitoring methods of cardiac injury, specifically aimed for cardiac injury due to chemotherapy

·       Development of novel ultrasound-based methods, including echocardiography, to assess early cardiac structural or functional changes

National Institute on Alcohol Abuse and Alcoholism (NIAAA)

The mission of the National Institute on Alcohol Abuse and Alcoholism (NIAAA) is to conduct and support biomedical and behavioral research, health services research, research training, and health information dissemination with respect to the prevention of alcohol abuse and the treatment of alcoholism, and to conduct a study of alternative approaches for alcoholism and alcohol abuse treatment and rehabilitation.

This solicitation invites proposals in the following area.

Background

Efforts to develop medications for the treatment of alcohol use disorder have expanded rapidly in recent years. Developing novel compounds for alcohol treatment is high priority for NIAAA Medications Development Program. Three agents directed at the addictive behavior in the use of alcohol —disulfiram, naltrexone, and acamprosate—are now approved for use in the United States and many other countries. Still, these medications do not work for everyone. Because of this, further research is needed to develop additional medications to treat Alcohol Use Disorder and organ damage caused by alcohol consumption.

During the past decade, many new targets in the brain and liver have evolved that alter alcohol-seeking and drinking behavior. Brain effects and behavior may be influenced by agents directed at CRF, adrenergic, opioid kappa, vasopressin V1b, NK1, orexin, NPY, NOP, glutamate mGluR2/3, mGluR5, GABAa α-1 and α-5 receptors. Several intracellular targets in additional (peripheral) organs have also been identified that alter outcomes of chronic alcohol use, including ALDH-2; PKC; PPARg; epigenetic modulators, (HDAC inhibitors, methylases, demethylases, and microRNAs); rapamycin complex 1; and GDNF. Tissue damage induced by the influence of alcohol or acetaldehyde on any of the above have serious negative consequences including development of steatohepatitis, cirrhosis or hepatocellular carcinoma.  Agents affecting these, and any other validated targets, may be synthetically derived or developed from plant materials.

Summary

This solicitation seeks to support the preliminary work required for the development of novel compounds to interact with recently identified targets to alter alcohol-seeking, drinking behavior and/or organ tissue damage caused by excessive alcohol consumption. 

Project Goals

The goal of this solicitation is to provide support for the development of novel therapeutic agents to treat alcohol use disorder and tissue damage caused by excessive alcohol consumption. 

Phase 1 Activities

If compounds have not yet been identified, activities include conducting high-throughput screening of libraries for novel compounds for further development. For identified candidate compounds, conducting preclinical animal studies to demonstrate proof of concept and safety; drug formulation and pharmacokinetic testing; drug optimizations and GMP manufacturing; IND-directed animal toxicology.

Phase 1 Expected Deliverables

·       Conduct animal toxicology and pharmacology studies as appropriate for the agent(s) selected.

·       Develop a detailed plan for future regulatory activities.

Phase II Activities and Expected Deliverables

·       Complete IND-enabling experiments and assessments according to the plan developed in Phase I (e.g., demonstration of desired function and favorable biochemical and biophysical properties, PK/PD studies, safety assessment, preclinical efficacy, GMP manufacturing, and commercial assessment). The plan should be re-evaluated and refined as appropriate.

·       Develop and execute an appropriate regulatory strategy. If warranted, provide sufficient data to file an IND or an exploratory IND for the candidate therapeutic agents (i.e., oncologic indications for CSCs).

·       Demonstrate the ability to produce a sufficient amount of clinical grade material suitable for an early clinical trial.

National Institute of Allergy and Infectious Diseases (NIAID)

The National Institute of Allergy and Infectious Diseases (NIAID) conducts and supports basic and applied research to better understand, treat, and ultimately prevent infectious, immunologic, and allergic diseases. For more than 60 years, NIAID research has led to new therapies, vaccines, diagnostic tests, and other technologies that have improved the health of millions of people in the United States and around the world. To learn more about the NIAID, please visit our web page at http://www.niaid.nih.gov/about/whoWeAre/Pages/moreWhoWeAre.aspx. 

Background

Highly active antiretroviral therapy is now optimized to control HIV-1 replication long-term, but the virus remains integrated into the host genome in a latent form and poses a threat for re-emergence. In search for more potent therapeutic agents geared towards HIV cure, newly developed chimeric nucleases, which allow the precise modification of viral and human genomes, have recently been explored for HIV reservoir elimination. These designer enzymes have the ability to disrupt the integrated HIV genome by double-stranded DNA break, so integrated proviruses become permanently defective, and to modify host genes essential for HIV replication, so cells become resistant to HIV infection.  The gene-editing enzymes currently available to the scientific community are zinc finger nucleases (ZFNs), transcription activator like-effector nucleases (TALENs), homing endonucleases, and clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9.  Each of these restriction enzymes is associated with unique strengths, but also with off-target effects.

Project Goals

The primary goal is to design improved nucleases for disruption of integrated HIV provirus and/or modification of host genes, so HIV replication is no longer supported.  There is also a need for alternative delivery strategies for these nucleases to substitute for lentiviral gene delivery or plasmid transfections. An additional goal is to evaluate off-target effects and immune responses induced by the delivered nucleases and their vectors. 

Phase 1 activities may include

·       Design and test chimeric nucleases that irreversibly disrupt or excise HIV provirus in infected cell lines and peripheral blood mononuclear cells (PBMC)

·       Design modified host genes and test their ability to impede HIV infection

·       Evaluate off-target effects in cell lines and primary PBMC

·       Develop strategies for eliminating off-target effects, including software tools for designing nucleases with reduced off-target sites

Phase 2 activities may include

·       Develop and test improved delivery strategies

·       Evaluate efficacy and adverse reactions of delivered nucleases in humanized mouse and nonhuman primate models

·       Evaluate immune responses against nucleases and vectors in vivo and develop strategies to reduce the immunogenicity of the delivered constructs

Background

One of the most significant hurdles to overcome in evaluating strategies to cure HIV infection is the lack of a simple method for quantifying changes in the size of the latent reservoir of replication-competent HIV in resting CD4+ memory T cells in individuals on highly effective antiretroviral therapy. Most of the HIV DNA in these cells represents defective virus; less than 0.01% of highly purified resting CD4 cells harbor replication-competent provirus.  As a result, PCR-based methods tend to over-estimate the size of the reservoir and do not correlate with the number of cells producing functional virus in a viral outgrowth assay. However, viral outgrowth assays are labor-intensive and require large volumes of blood.

Project Goal

The goal of this project is to design a high-throughput assay platform that can be used to reproducibly quantify changes in the size of the replication-competent latent HIV reservoir in resting CD4+ memory T cells isolated from individuals on highly effective antiretroviral therapy. Applicants must provide a plan for validating the assay by demonstrating correlation with quantitative viral outgrowth assays (QVOA) and/or functional non-induced HIV proviruses using cells isolated from virally suppressed HIV+ individuals on optimized antiretroviral therapy.

Phase 1 activities

·       Development of technologies for detecting replication-competent latent proviruses

·       Validation of detection methods using standardized controls

·       Optimization of sensitivity to detect low-frequency latently infected cells

·       Demonstration of correlation with replication-competent provirus vs. defective provirus

Phase 2 activities

·       Further optimization of the assay platform technology and validation of assay reproducibility

·       Increased throughput

·       Comparison of assay to other methods published in the literature

·       Testing of clinical samples from diverse cohorts of HIV+ individuals with varying levels of residual viral reservoirs

·       Comparison of blood vs. tissue samples from virally suppressed individuals

·       Modification of assay to detect latent HIV in humanized mouse models and latent SIV in nonhuman primate models in the context of optimized antiretroviral therapy

·       Use of assay to demonstrate changes in the size of the latent HIV/SIV reservoir in response to an intervention

Background

Antiretroviral therapy (ART) reduces mortality and morbidity in HIV-infected individuals. With successful therapy HIV RNA becomes undetectable, but drug resistance may occur.  Specific HIV mutations are associated with resistance and these mutations can be detected through standard genotypic resistance tests, which have the ability to detect mutations only when they are present in approximately 20% of the virus population within an individual. Importantly, it is now known that the presence of certain resistance mutations, even at very low concentrations within a patient’s virus population (1% or more), can contribute to virological failure.  These drug resistant minor variants can reflect the early emergence of acquired resistance during therapy, and can also be transmitted to newly infected individuals. These minor variants are not detected by standard drug resistance assays and methods to detect minor variants that contribute to HIV virological failure are needed.  These assays would need to detect mutations causing resistance to each of the antiretroviral drug classes (NRTI, NNRTI, PI and INI) in all HIV subtypes, and must be inexpensive, since large numbers of patients would need to be screened.   

Project Goal

The goal of this solicitation is to develop an assay to detect minor populations of resistant variants in blood specimens from HIV-infected individuals with HIV RNA viral loads above 1000 copies/ml.  The test must detect resistant variants that comprise 1% or more of the virus population or quasispecies, and must detect mutations causing resistance to NNRTIs, NRTIs, PIs and INIs in all subtypes of HIV. Sensitivity for qualitative detection of the minor variant at 1% or more must be at least 95% and specificity at least 98%, but the method must also yield quantitative results, showing the percentage of each resistant variant in the overall quasispecies. Methods that detect a set of relevant point mutations and methods that collect full sequences are both acceptable. For methods that detect point mutations, a set of relevant mutations should be proposed in the application, but will be finalized in cooperation with DAIDS program staff.  The method must be appropriate for use in centralized clinical laboratories with a target turn-around-time of less than 1 week and an initial target cost of $100 or less.      

Phase I activities

·       Development of a method for the quantitative detection of minor populations of HIV resistance mutations comprising 1% or more of the viral quasispecies

a.      Must detect major mutations causing resistance to all drug classes (NNRTI, NRTI, PI and INI)

b.      Sensitivity must be at least 95%, specificity at least 98%

c.      Must be suitable for clinical laboratory use, with turn-around time of less than 1 week

d.      Cost must be less than $100 per test

·       Initial testing of the product with combinations of drug-susceptible and –resistant laboratory strains of HIV spiked into HIV-negative blood

·       Additional testing on clinical isolates

Phase II activities

·       Validation testing with controls as well as clinical isolates, including assessment of sensitivity, specificity, precision, accuracy, and linearity

·       Production of the test under good manufacturing practices (GMP)

·       Development of a quality control program to ensure lot-to-lot consistency

Multi-site evaluation

Background

Tuberculosis (TB) continues to cause significant mortality and morbidity throughout the world, especially in HIV-infected individuals.  Increases in drug resistant TB cases have been occurring in many high endemic countries that have limited resources to identify and diagnose patients. Standard diagnostic techniques for TB include sputum smear microscopy to detect acid fast bacilli and microbiological culture confirmation. As a result, diagnosis of TB is both difficult and time consuming, especially in smear-negative, HIV-infected and pediatric patients.  Molecular technologies are under development for TB case detection and identification of drug resistance mutations in lower levels of the health care system. These technologies will make use of sputum samples, the most important specimen type for TB diagnosis. Processing of sputum prior to PCR amplification will be necessary. A simple, inexpensive device to purify DNA from sputum and re-suspend the DNA in a buffer along with a compatible transfer system to molecular diagnostic assays is being sought. The purified DNA sample from the device should be compatible with many different technologies, thus removing the very difficult step of sample processing from development of the molecular test for Mtb detection and drug resistance testing. This would also allow the sputum processing and molecular testing to be delinked, with processing done immediately at the point-of-care.  

Project goal

The goal of this solicitation is to develop an inexpensive (less than $10), easy to use device for processing sputum samples to obtain purified DNA for TB testing to be used in a rural clinic setting with minimal infrastructure.  Processing of the sputum must be performed in less than 30 minutes, without the need for external electricity (battery power can be proposed).  The resulting material must: 1) be stable over a wide range of ambient temperatures in TB endemic areas (approximately 5 to 50⁰C), 2) must provide DNA comparable in quality and quantity to DNA prepared using standard laboratory methods , and 3) must provide results comparable to those obtained using DNA from standard laboratory methods in molecular assays for Mtb. 

NOTE: The use of TB positive sputum samples may be proposed, but due to the difficulty in obtaining these samples, the use of spiked TB negative sputum samples from donors or artificial sputum samples may also be proposed for initial studies.    

Phase I activities

·       Development of a method for processing sputum to purify DNA for TB testing. 

a.      Sample processing time must be no more than 30 minutes with no more than 2-3 steps performed by the operator.

b.      DNA recovery must be at least 50% compared to a gold standard laboratory method and must allow detection of Mtb in sputum containing at least 5-20 CFU/ml using a standard molecular detection method.

c.      The CV for inter-operator variability must be no more than 20%.

d.      Processed specimens must be stable for at least 7 days at temperatures ranging from 5 to 50⁰C.

·       Initial evaluation of the product compared to DNA obtained using standard laboratory methods with at least one molecular TB test technology

Phase II activities

·         Production of a small lot to be used for validation testing with at least one molecular Mtb test.  Pre-defined validation targets should be specified.

·         Validation testing with at least one Mtb test, to include precision, accuracy, sensitivity, and specificity with a standard laboratory method of DNA preparation as the comparator

·         Manufacture of the product under good manufacturing practices (GMP) and compliant with the requirements of ISO 13485

·         Development of a quality control program to ensure lot-to-lot consistency

Background

Traditional methods for the diagnosis and clinical management of infectious diseases require the direct assessment of a patient’s symptoms, vital signs, and often the collection and analysis of clinical specimens by health care practitioners, usually in a health care setting.  Recent scientific advances in mobile and remote monitoring technologies have enabled home-based telemonitoring of multiple physical parameters in patients with chronic medical conditions including cardiovascular disease, diabetes, hypertension, and asthma. Additionally, advances in rapid diagnostic platforms to detect pathogens have also been recently realized.  There is now a unique opportunity to leverage and integrate the advances made in these two areas of research and their associated technologies and to apply them to the development of portable and continuous monitoring systems to revolutionize the early detection, progression, and/or effects of treatment of patients with infectious diseases.

The emerging development of portable monitoring devices and technologies have yet to be applied to infectious diseases and may have a significant impact on clinical management. Systems that are able to remotely monitor and report physiological status with minimally-invasive specimen collection would be useful ways to inform and support the clinical management of disease, e.g., in premature infants at risk for (or already diagnosed with) RSV, in elderly individuals with chronic illness.  Remote monitoring of patients for the occurrence or worsening of an infectious disease may result in early clinical intervention and optimize the use of available therapies.  Such “real-time” monitoring of symptoms and/or disease progression would also add value by avoiding unnecessary trips to the ER/physician, and reducing healthcare costs.  While this type of monitoring system is envisioned to be broadly applicable, some patient populations (infants, elderly adults, and immunocompromised individuals) may have greater benefit as they may not show symptoms on examination, yet they may have an increased chance of developing infections due to recent medical procedures or to rapid worsening of a previously diagnosed infectious disease.

Project goal

The overall goal of this solicitation is to develop a device that can, in a non-clinical setting, monitor and report data that reflects the emergence and/or progression of an infectious disease.  The complexity of such a device, and its operation, should allow for its use in patient environments such as a home or a nursing facility and be capable of communicating “actionable” data to professional healthcare workers.  Ideally, such a device would have an integrated architecture consisting of a physiological monitoring component, and/or a specimen collection apparatus with equipment to analyze the sample, and a communications functionality for data transmission; however, the capability for specimen collection is not required for applications that involve physiological measurements only (i.e. that do not require evaluation of analytes).  A highly desirable feature for a device that requires sample collection might be a visual menu that provides simple instructions for outpatient specimen collection when needed, so that family members or care providers could support the analytical device with minimal training.  The types of information to be collected may include, but are not limited to: physiological measurements (vital signs), analytes (biomarkers) that are present in bodily fluids or breath and measures of behavioral responsiveness to environmental stimuli.  Evidence must be presented that the data collected by the device is directly relevant to the occurrence and/or progression of an infectious disease and that its ultimate use will be to support clinical decision-making.  

Phase I activities include but are not limited to:

·       Development of appropriate tests / physiological measurements to be used for monitoring of disease

·       Proof of concept that the information can be used to infer a clinically-relevant change in disease state 

·       Initial design of integrated device for outpatient monitoring including, if proposed, specimen collection / analysis  

Phase II activities include but are not limited to:

·       Development of prototype device for monitoring and reporting of infectious disease status

·       Development of analytic software (beta version) to evaluate and report results

·       Optimization of any reagents that are required for analytic tests

·       Validation of test procedures to address issues of reproducibility, sensitivity and specificity

·       Proof of concept that device can integrate measurement / analysis steps with data transmission 

Background

There is a persistent need to develop alternative, simple to administer formulations of FDA approved anti-infective agents for use by children and by adults who have difficulty taking traditional tableted drugs.  These formulations will simplify administration for caretakers and patients and ensure compliance.

There are few child-friendly formulations of pediatric anti-infective medications available to practitioners in the U.S. and especially globally. It is standard practice to cut or crush un-scored adult tablets and administer them to children in juice or other palatable substances, such as food. This practice has significant potential to deliver incorrect and highly variable doses to children, contributing to ineffective treatment. For some adult patients, especially those with difficulty swallowing or those with dementia or other mental impairments, taking standard pills or syrups may be problematic and also affect compliance.

For infectious diseases, completion of drug therapy is critical to assure cure and reduce development of resistance. Resistance often develops when therapy is terminated early or drugs taken intermittently, rather than the prescribed daily doses. Furthermore, for infectious diseases that require long term dosing such as tuberculosis or HIV/AIDS, there is evidence of decrease of patient compliance as time goes on, particularly if drugs are not very palatable. Customized oral formulations are needed that facilitate long term compliance and are of sufficient stability to be suitable for use in resource limited countries.

Examples of the types of oral formulations that may address these issues include, but are not limited to:

·       Oral thin-films

·       Porous, chewable matrix systems (scorable “Taffy” based on patient weight)

·       Candy-like formulations,  including gummies and jellybeans

It is recognized that these formulations may only be suitable for highly active anti-infective drugs.

While consideration of pediatric applications is a recent regulatory requirement for novel drugs in general, this requirement does not apply to a majority of anti-infective drugs that are already off patent and therefore, development of new formulations should be an attractive commercial goal for small businesses. In addition, offering these easier to take formulations to adults who have difficulty taking oral tablets would further expand the utility of these innovative dosing forms, as well as facilitate overall compliance with taking medicines.

Project Goal

The goal of this project is to develop innovative oral formulations for FDA approved anti-infective agents (antibacterials, antifungals, antiparasitics, and antivirals, including anti-retrovirals for HIV/AIDS) other than nanoparticles or pills to facilitate administration to patients who are either too young or have difficulty taking oral medications.  The final product should be simple to manufacture, stable under ambient conditions and ready for testing in Phase I bioequivalence and PK studies.

Phase I activities include but are not limited to

·       Develop prototype formulations that address the goals of this solicitation

·       Develop analytical assays to characterize the chemical composition, purity and stability of prototype formulations

·       Assess the pharmacokinetic profile and safety of the formulations when delivered in the intended way, in appropriate systems

·       Conduct or develop drug potency assays for bioequivalence studies

PhaseII activities include but are not limited to             

·       Scale-up the formulations (activity need not be compliant with cGMP) for further preclinical studies

·       Conduct additional pharmacology and toxicology evaluations of the formulations in appropriate systems

·       Conduct other pre-clinical studies necessary for subsequent human bioequivalence studies

This SBIR will not support:

·       The design and conduct of clinical trials (see http://www.niaid.nih.gov/researchfunding/glossary/pages/c.aspx#clintrial) for the NIH definition of a clinical trial). For SBIR phase II clinical trial support, see the NIAID SBIR Phase II Clinical Trial Implementation Cooperative Agreement program announcement.

Background

There is an urgent need to develop vaccines against pathogens affecting a relatively small segment of the US population.  While the market or segment of the overall population affected may be statistically small, the morbidity and mortality in some cases can be quite substantial. NIAID is interested in receiving proposals to develop vaccines against small or limited market-type pathogens.  Specific examples of unmet vaccine needs that would fit this request would be Coccidioidomycosis/San Joaquin Valley Fever (VF), Lyme disease, as well as vaccines for selected high risk populations.

Project Goal

·       To promote identification, characterization, validation, and ultimately product development of potential vaccines against pathogens with limited market potential. 

·       To encourage collaboration between academic researchers and small business entities to discover, validate and produce vaccines for pathogens with small market potential.

Phase I activities include but are not limited to

·       Identification, characterization, and validation of vaccine candidates

·       Development of in vitro assays to qualify vaccine candidates for future product development

·       Selection of adjuvant, as appropriate, for further development and antigenicity testing

Phase II activities include but are not limited to

·       Additional testing of the lead vaccine candidate(s) in the vaccine product development pathway leading to IND-enabling studies, including but not limited to testing to improve safety, efficacy, and QA/QC

·       Pilot lot of cGMP manufacturing for further refinement of the vaccine candidate(s)

·       Formulation, stability, and toxicology studies, as appropriate, for later stages of the vaccine product development pathway

This SBIR will not support:

·       The design and conduct of clinical trials (see http://www.niaid.nih.gov/researchfunding/glossary/pages/c.aspx#clintrial) for the NIH definition of a clinical trial). For SBIR phase II clinical trial support, see the NIAID SBIR Phase II Clinical Trial Implementation Cooperative Agreement program announcement.

·       Platform development such as vehicle or delivery systems.

National Institute on Drug Abuse (NIDA)

NIDA’s mission is to lead the nation in bringing the power of science to bear on drug abuse and addiction, through support and conduct of research across a broad range of disciplines and by ensuring rapid and effective dissemination and use of research results to improve prevention, treatment, and policy.

This solicitation invites proposals in the following areas:

Objective

This topic addresses the demand to promote awareness and knowledge of the best practice in management of Neonatal Abstinence Syndrome (NAS). The need is caused by clinical rigor and raised concerns among neonatal and pediatric practitioners regarding a constellation of various withdrawal symptoms and treatment approaches. The ultimate goal of this solicitation is to develop a skill-building Primer and Reference Tool to assist clinicians in identifying, interpretation, scoring and responding to NAS symptoms toward improving neonatal outcomes.

Background

NAS is a group of withdrawal problems that occur in a newborn who was exposed to addictive drugs while in the mother’s womb. NAS characterized by gastrointestinal, respiratory, autonomic, and central nervous system disturbances from drug withdrawal that affect critical regulatory areas for postnatal life adaptation.  Withdrawal signs may develop in 37% to 94% of neonates exposed by various addictive substances including illegal drugs or prescribed analgesics and antidepressants.   

In the past decade, the use of prescription opioids and the incidence of opioid addiction among women of childbearing age increased substantially. According the American Academy of Pediatrics, the number of opioid-dependent NAS diagnoses increased threefold reaching more than 13,000 babies across the United States annually. Currently, the consistent use of pain relievers with other drugs become more common that causes the NAS symptoms to be more severe and poorly manageable. At the consequence, newborns with NAS present new challenges for the neonatal hospitals and may require prolonged hospitalization in order to alleviate the symptoms. The national health cost to care for such infants jumped from $190 million in 2000 to $720 million per year in 2009. In addition, there is the growing concern, that hospitals may discharge some newborns before the symptoms appearance, if newborns were exposed by long-acting opioids with five or more days to show signs of withdrawal.  These newborns present the significant challenge for diagnostics at hospital emergency rooms or out-patient pediatric units outside of the nation’s epicenters of drug abuse.

To date, there is neither a standard diagnostic tool, preventive treatment strategy, nor a comprehensive educational program to manage NAS. Among educational resources, few existing online applications (Neonatal Drug Withdrawal Protocol App, Kaiser Permanente; Neonatal Abstinence Syndrome CE581, Nurse.com) are limited in the context and provide general text-based information with no interactivity rather than helping practitioners to adapt the clinical symptoms to the established diagnostic and treatment guidelines.

Responding to the demand for NAS awareness and medical care standardization, the National Institute on Drug Abuse (NIDA) supports development of a medical Primer and Reference Tool to specifically address the information gap in this critical pediatric health problem. With the long-term goal of improving neonatal health care, NIDA is soliciting proposals for a SBIR contract to develop and evaluate a bedside assisting App for NAS management. 

Currently, the modified Neonatal Abstinence Scoring System (or Modified Finnegan Scoring system) is the predominant scoring tool used in the United States. Despite of its complexity and bulkiness as the 21-item related observation checklist, the Modified Finnegan Scoring remains more comprehensive system.  The value of NAS severity, calculated by this tool, is a critical feature for any NAS assessment which serves as the basis for treatment selection and start. Recently, the Modified Finnegan Scoring was incorporated into the electronic medical record (EMR) platforms in the national centers of pediatric care excellence and the nation’s epicenters of drug abuse. However, outside the recognized medical centers, most nurses and pediatricians have little experience in NAS evaluation and Finnegan Scoring. These hospital codes are unfamiliar with the criteria of NAS and may give the neonatal an alternative diagnosis that shares signs with NAS such as infection, hypoglycemia, hypocalcemia, hyperthyroidism, intracranial hemorrhage, hypoxic-ischemic encephalopathy, and hyperviscosity. At presentation, signs of NAS vary and usually include excessive cry, irritability, short sleep, tremors, stiffened muscles, gastro-intestinal and respiratory problems. NAS unawareness or different practice standards cases the number of challenges in diagnostics and therapeutic strategies.  

Thus, the lack of the standard management and bed-side references causes bias and subjectivity in NAS scoring and assessment. To address this problem, the new electronic tool should adapt and unify the existing knowledge regarding NAS risks, symptoms, Finnegan Scoring, diagnostic and therapeutic approaches.  New tool is sought to better explain the withdrawal symptoms in newborns and provide bed-side instructions how to assess the Modified Finnegan Score.  The primary target audience for the proposed tool includes neonatal practitioners, pediatricians, and nurses. However, the material are sought to be useful for a wide range of health care professionals  from family or GYN physicians, pain prescription provides to medical and nurse students who wish to continue their medical carrier to pediatrics.

Phase I Activities and Expected Deliverables:

Technical Requirements

1.      Assemble a consultant team and determine availability of data, references, educational and clinical guidelines, and presentation strategies. Offerors are expected to have in house capabilities or the established practice or experience to contact consultative and CME educational services, neonatal centers, hospitals, professional associations and medical practitioners including but not limited to neonatal providers, nurses, pediatricians, and pain prescription physicians.

2.      Develop a curriculum for education modules and interactive resources. An electronic Tool should adapt the skill-building multimodal Primer and serve as a bed-site Reference Tool for neonatal and pediatric providers. 

A Primer and Reference Tool proposed in response to this solicitation should provide the repository of necessary information in following areas:

a.      Epidemiology and pathophysiology of NAS.

b.      The clinical phenomenon of NAS symptoms. Clinical signs, frequency, severity and duration of NAS. Mechanisms of opioids withdraw. Clinical facts of multi-drug exposures and their impact to NAS development and representation.

c.      Interpretation of the Modified Finnegan Score system. Guidelines in assessment and scoring.

d.      Overview of available and appropriate toxicology tests to determine the exposure level. All drug screening procedures should contain corresponding references to the test sensitivity, efficiency, time for analysis, cost and diagnostic limitations.

e.      Description of pharmacologic and non- pharmacologic interventions. Interactive referral and references how to select the appropriate therapy based on the symptoms appearance and NAS scoring. The materials should describe the importance of breastfeeding in stable mothers.

3.      Define and collect all reference materials such as medical publications, scientific references, best-practice guidelines, and other downloadable tools. A Tool may be built with the option for interactive questions.

4.      Identify an electronic platform for the software implementation. A Tool’s App should have full compatibility with both Flash and FULL compatibility with HTML5 standards.

5.      Develop a detailed project plan for Phase II activities which includes, but is not limited to:

a.      Project Gantt

b.      Task linked budget for Phase II Activities

c.      Mock Ups outlining of Phase I Deliverables 2 (a-e)  and Description of the Software

d.      Plan for the case study recording

e.      Examples of surveys to be used in Phase II Activities

f.      Plan for piloting, evaluation, and refinement of draft modules

g.      Plan to address FDA-regulations if a Tool is to be disseminated as a NAS scoring and a clinical/treatment reference device w/wo compatibility with electronic medical records (EMR) software.

Phase I Activities and Expected Deliverables:

Technical Requirements

1. Develop a multimodal awareness building program of a Primer and Reference Tool:

a.      Collect case presentations (video and descriptive) to supplement tool’s modules and address the challenges in NAS symptoms recognition and interpretation; 

b.      Create reliable software as required. Code the device as needed.

2. Conduct a Pilot study of the draft module with professionals representing the target audience to a Primer and Reference Tool. Conduct community’s feedback survey and analyze data.
3. Revise and improve software in response to perceived needs. Complete the web-based lesson context, iterative design and development of the software operation tools.
4. Conduct efficiency study and evaluate the effectiveness of a Primer and Reference Tool. Complete satisfaction and acceptance testing.
5. Prepare strategy for implementation and dissemination.

Objective

To develop a pulmonary delivery device that can administer therapeutic doses of the non-psychoactive cannabinoid CBD. The ultimate goal is to generate a sufficiently characterized clinical tool such that the Food and Drug Administration (FDA) would allow it to be used to evaluate the efficacy of inhaled CBD as a therapeutic agent in clinical trials.

This opportunity is open to all Small Business Innovation Research (SBIR) award-eligible organizations. However, it is anticipated that a Small Business Concern (SBC) best equipped to produce such a characterized device and data package within the time and budget constraints might currently be marketing a similar or related inhalation product.

Aspects of a Market analysis

The NIDA foresees a niche for the SBC with a pulmonary CBD delivery device that has previously successfully undergone Federal IND review. This product would be marketed to clinical researchers wishing to conduct clinical studies into potential therapeutic effects of CBD. The SBC would supply the researchers with devices and allow them to cross-reference the original IND in their own FDA application(s). All proprietary information would be kept between the SBC and the FDA.

·       Diversified Income Stream- Clinical researchers in the US (and beyond) represent limited / no-competition market niche, protected by the costs involved in characterizing the device and obtaining an IND.

·       In a market where designs are rapidly evolving this representsa situation where a stable device design is prized and minimal future R&D investment will be required.

·       Informing Product Development- Characterizing your existing product will aid future design efforts and perhaps “future-proof” against a scenario where greater regulatory oversight might require characterization.

·       “Free” Clinical Data Studies The product would be described as a generic NIH device, butthe SBC would be welcome to reveal their involvement in NIH clinical studies in their marketing materials.

Background

CBD is a compound found in marijuana that has no euphoric properties but appears to have other pharmacological activities. Current understanding of CBD pharmacology is limited; a number of laboratory studies have been conducted and a few have progressed into early clinical phase investigations, the most successful demonstrating CBD as an anxiolytic agent. Potential applications for the anxiolytic properties of CBD include reduction of craving and relapse in Substance Use Disorders, and reduction of anxiety in Post-Traumatic Stress Disorder. In addition, CBD is currently under clinical investigation for the treatment of childhood intractable epilepsies, where it is added to the existing medication regimen (usually, to 2-3 other drugs).  However, CBD is well known as an inactivator of drug metabolizing enzymes and so can significantly disturb the patient’s exposure to their current medications and potentially contribute to serious drug interactions. If CBD is to be administered via a pulmonary (inhaled) mechanism rather than via oral route, the liver exposure would be much lower, resulting in less enzyme inactivation and drug-drug interactions. Furthermore, inhaled CBD provides 2-3x greater bioavailability and shows substantially lower inter-dose and inter-subject variability than with oral administration. when used in a clinical trial a pulmonary delivery device would result in more reproducible CBD dosing, less risk of drug interactions and ultimately less variable and more reproducible clinical data.

Project Overview

• The solicitation is open to all small businesses.  SBCs with similar / related existing technologies are especially encouraged to apply.
• Products based on herbaceous material will not be acceptable, all formulations will need to be liquids or solids manufactured according to current Good Manufacturing Practices (cGMP).
• The delivery device could be in the form of a vaporizer, nebulizer, dry-powder inhaler or any other FDA-approvable pulmonary delivery device.
• Phase 1 of the project characterizes the quantity and reproducibility of CBD delivered in a single 5-second puff, as well as the identities and the amounts of all other agents in the vapor/aerosol.
• All components of the device and liquid are to be manufactured according to Good Manufacturing Practices (cGMP) or when such standards do not exist manufacturing should be performed according to the “spirit of GMP”. 
• Analytical Studies are to generate Certificates of Analyses demonstrating an appropriate and reproducible CBD output as well as quantities of all other emissions. Studies are to be conducted by an ISO 17025 laboratory. 
• The Phase I deliverables also include the minutes of an FDA pre-IND submission meeting outlining FDA expectations for any additional studies / data that would be required for a successful IND application.
• If selected to progress into phase II, the SBC is to conduct the studies required by the FDA to achieve a successful IND to examine the pharmacokinetics of CBD delivery by the device in normal healthy adults.  In addition, in Phase II, a 10 person study would be conducted to evaluate the pharmacokinetics of single dose/session administration of CBD using the device. It is recognized that a pharmacokinetic study may not always require an IND, but a protocol is required for the FDA to consider the suitability of the data package for issuance of IND. Once the data package has been allowed for one study, that IND can then be cross-referenced in future studies.
• It is expected that the data from the pharmacokinetic study would be published in a peer reviewed journal). 

·       Phase II projects will also require elucidation of the strategy that will be used to move the proposed research tool to a marketable product. Evidence of a track record of commercialization and / or commitment of additional investment from private sector or other non-SBIR funding sources will be expected.

• Phase II would consist of studies aimed to satisfy FDA requirements for issuing an IND and the conduct of a 10 person study to evaluate the pharmacokinetics of single dose/session administration of CBD using the device. It is recognized that a pharmacokinetic study may not always require an IND, but a protocol is required for the FDA to consider the suitability of the data package for issuance of IND.

·       The SBC would agree to market this device as characterized (without further change) to all clinical researchers who have been granted NIH funds to conduct the relevant studies.

·       The device would be available for a sufficient period after the completion of the project to allow clinical studies to be conducted (i.e. 5-10 y).

·       The SBC would retain the proprietary data held within the Drug Master File but would allow NIH researchers (and other customers) to cross reference the original Investigational New Drug Application (IND) using that data in order when seeking FDA approval for their study.

• The battery used in the device must be rechargeable using a USB port and not meet the definition of hazardous waste as described in 40 CFR 261, subpart C.
• The device should deliver at least 40 mg CBD to the vapor / aerosol over 10 minutes of use 
• Availability of other formulations such as a placebo formulation would be desirable, but not essential. If several formulations are proposed, the budget may potentially be modified accordingly during the negotiation phase (if the government determines negotiations are necessary). A formulation that contains a defined low amount of THC in combination with the required CBD delivery would also be viable as an additional formulation, although NIDA would need to be the source of the THC during the course of the contract. 
• Phase II would consist of studies aimed to satisfy FDA requirements for issuing an IND and the conduct of a 10 person study to evaluate the pharmacokinetics of single dose/session administration of CBD using the device. It is recognized that a pharmacokinetic study may not always require an IND, but a protocol is required for the FDA to consider the suitability of the data package for issuance of IND.

The Phase I contract proposal must include:           

• A description of the device appearance and characteristics, including (but not limited to)
• The liquid reservoir volume
• The device dimensions and weight
• An image of the device
• Information regarding protections to prevent a user’s exposure to the CBD containing liquid.
• Estimation of the number of puffs per cartridge, tank fill or disposable device (as appropriate) using a defined puffing topography (see Phase I Activities and Expected Deliverables).
• Estimation of the number of 5 sec puffs on a single battery charge (where applicable). The number of times a battery can be discharged to less than 20% of full charge and then recharged to >90% of specified full charge (estimation of battery life) should be described.
• Estimation of device time to failure.
• Concentration of CBD in the liquid formulation(s).
• Indicate the differential characteristics of each formulation (if several formulations are planned).
• Describe and provide examples of the data output, if the device possesses a data recording capability (not a requirement). 
• The number units developed under this contract that are anticipated to be supplied annually and an indication of number of related units sold in a similar market place over last 1-2 years
• Documentation regarding capability to provide the device for a minimum of 5 years after the end of the development contract.
• Demonstration of the knowledge and capability to provide cGMP reagents in the completed device (see Guidance for Industry Q7A Good Manufacturing Practice Guidance for Active Pharmaceutical Ingredients).
• The anticipated cost of the device and any required accessories such as cartridges or battery chargers (where applicable).  Cost should also be described in terms of equivalence to a typical herbal extract / nicotine vaporizer as well as the cost over the lifetime of a single device.
• It is recommended (but not essential) to describe experience of the key investigators or organization in the development of marketing of similar products to that proposed in this project.
• Demonstration of manufacturing and supply chain stability, is strongly advised including letters of support where applicable.

Phase I Activities and Expected Deliverables

This phase focuses on characterizing the chemical and mechanical characteristics of the device, including:

• Number of puffs per cartridge, tank fill (as appropriate).  This is to be determined using a standardized puffing topography: 10 sec per puff, 60 ml puff volume, 20ml/sec flow rate, 30 sec puff interval.  Grantees should also describe and test the optimal puff topography for their device, if different.
• The approximate number of 10 sec puffs, using the standard puffing topography, on a single battery charge (where applicable).
• Analysis of device time to failure. 
• Chemical and Manufacturing Control (CMC) information for filing a Drug Master File (DMF) with the FDA.  Batch reproducibility will also be covered in the CMC.  Data will be generated from an ISO 17025 accredited laboratory.
• Information to be included in the DMF should be in agreement with the following FDA guidelines:
• Guideline for Submitting Supporting Documentation in Drug Applications for the Manufacture of Drug Substances.
• Guideline for the Format and Content of the Chemistry, Manufacturing, and Controls Section of an Application.
• Guideline for Submitting Documentation for the Manufacture of and Controls for Drug Products.
• Guideline for Submitting Samples and Analytical Data for Methods Validation.
• Guidance for Industry INDs for Phase 2 and Phase 3 Studies - Chemistry, Manufacturing, and Controls Information.
• List of ingredients and a Certificate of Analysis (CoA) for each liquid formulation. Constituents analyzed must include: CBD, other cannabinoids present, cannabinoid breakdown products and vehicle components, for example, ethanol, propylene glycol, glycerin, acrolein and formaldehyde. Reproducibility of liquid constituents should be demonstrated for at least 3 consecutive lots
• CoA will be generated from an ISO 17025 accredited laboratory.
• CoA for the vapor /aerosol produced by the device using the standardized puffing topography. Data should show the amount of CBD in the inhalant collected from the first 10 puffs and the amounts of all constituents (above 1 µg) present in the vapor collected from the first 150 puffs. 
• Indicate highest temperature of vapor / aerosol exiting device during a standard puff.
• Indicate aerosol droplet size range where appropriate. 
• Indicate variation in the vapor constituents over the lifetime of the device.
• Long term and accelerated stability that will be initiated in Phase I for the final device with each of the different liquid formulations. To be included in the Phase I report is the 30 day accelerated (40°C, 75% relative humidity) stability testing.  The testing should be conducted in a manner consistent with the following FDA guidance: Guidance for Industry Q1A(R2) Stability Testing of New Drug Substances and Products.

• A  Drug Master File for the device including all liquid formulations, completed to FDA specifications.

Objective

Provide in-home access to coordinated comprehensive pain treatment through a mobile treatment platform.  This platform may range from a fully equipped mobile clinic, to a mobile extension of a traditional pain clinic, or a virtual network of mobile treatment services.  Note that opioids may be part of this comprehensive treatment plan and may be prescribed, if warranted, but will not be delivered through the pain mobile platform.

Background

Pain is a major health crisis in America, where chronic pain afflicts nearly a third of our population.  Opioids can be a powerful tool in fighting pain; however they are too often used as substitute for a comprehensive interprofessional pain treatment program.  Patients are merely sent home with a bottle of pills.  The result is pain is often inadequately treated and opioids are over used.   

In an effort to improve pain treatment and reduce dependence on opioids, NIDA is soliciting applications to create a mobile pain management system.   Core to this solicitation is the delivery of a comprehensive pain treatment system to a pain patient’s home, as it is difficult for many pain patients to make numerous needed visits to clinical settings. Opioids may be prescribed as part of this comprehensive treatment plan, but will not be dispensed.  The patients will have to get opioids from pharmacies (either in person or via a delivery system). 

This “pain mobile” can be an actual portable clinic that is equipped and staffed to treat pain. Alternatively, it can be more of a virtual remote pain treatment system, where health care providers get to patients homes using various means other than a devoted vehicle. It can also be a hybrid of these approaches.  However, it is crucial that visits and treatments are coordinated in a way that delivers comprehensive and appropriate pain treatment.  This coordination can be done from the actual pain mobile or via a remote site.

The health care providers that visit the pain patients need to have appropriate training (e.g. nurse practitioner); but they do not have to be clinicians.   However, it is essential that clinicians are involved in many aspects of the patients care, including assessment, diagnosis, and management, planning, and prescribing of drugs.  This clinician input can take many forms, and can include occasional office visits by the patients and having the clinician remotely see the patients using various telemedicine technologies (e.g., Skype).

In many cases, as part of a complete pain treatment program, we expect that various other health care providers would visit the pain patients in a coordinated fashion.  These may include acupuncturists, physical therapists, massage therapists, cognitive/behavioral psychologists and others depending on patient needs.   Again, these visits need to be coordinated, and progress needs to be monitored.  Further, this treatment systems needs to be flexible and change with the needs of the patients.  

Given that the “pain mobile” approach involves going to the patients’ homes, it offers some unique opportunities not available with visits to clinics.  With home visits, it is possible to assess the home environment and also to educate patient and those living with the patient. As part of the “pain mobile” program, we expect that at least one of the health care providers that go to the patient’s residence be trained to evaluate the living conditions of the patient and when appropriate, suggest improvements that will allow the patient to function better in their home environment despite their condition.   If the patient is using opioid pain medications, how these drugs are stored and secured will be examined.   The health care provider will also be expected to educate the pain patient and co-inhabitants of the home about the safe use of opioids. 

While this is a small scale project, we would like the chosen model to be economically viable and potentially expanded.   To make this more likely, the offeror must describe a plan of how they will make this project not only self-sustaining, but also expandable.  Agreements with existing health care providers, health insurance companies, health services providers or the like is required.   If the pilot project funded under this contract is shown to be economically viable on a small scale, we envision that this will encourage provider partners to expand this system within their existing networks, and thus expand the impact of this effort.

Phase I Activities and Expected Deliverables:

Technical Requirements

1.      Assemble a team of professionals to work together to provide comprehensive pain treatment.

2.      Develop a plan to deliver coordinated comprehensive pain treatment, including determined the frequency of home visits, the need for in clinic consultations and testing, the coordination plan of treatment delivery (who goes where and when), the development of hardware and software needed in the coordination of care and store/access data, and a plan to insure data security.

3.      Built/equip the pain mobile itself with all the needed medical and communication requirements.

4.      Develop a plan for compensation of services that does not sole or largely rely on the patient paying for services out of pocket.  This may take many forms but could include a contractual relationship with a heath care insurer.

5.      Recruit a patient population.

6.      Perform pilot testing of the system and services for feasibility.

Phase II Activities and Expected Deliverables:

Technical Requirements

1.      Provide pain treatment from the pain mobile full-time on a cohort of chronic pain patients.

2.      Test for efficacy of the pain mobile.  This could include surveys of patient satisfaction as well as impact of treatment on the patient’s pain and quality of life.  These measures could include measures of pain, depression, mood, function, level of use of opioids, activity levels and other indicators of successful pain treatment.  These data could be compared across time within this cohort of pain patients, and/or related to a comparison group of pain patients treated in a traditional clinical setting.

3.      Results from the above testing should be disseminated via conference presentations and manuscript publications.

Center for Global Health (CGH)

The Center for Global Health (CGH) leads the execution of the CDC’s global strategy; works in partnership to assist Ministries of Health to plan, manage effectively, and evaluate health programs; achieves U.S. Government program and international organization goals to improve health, including disease eradication and elimination targets; expands CDC’s global health programs that focus on the leading causes of mortality, morbidity and disability, especially chronic disease and injuries; generates and applies new knowledge to achieve health goals; and strengthens health systems and their impact.

CGH Internet site:  http://www.cdc.gov/globalhealth/

For this solicitation CGH invites Phase I proposals in the following area: