RECOMBINANT SYNTHESIS OF MICELLE-FORMING DIBLOCK POLYPEPTIDES FOR CANCER IMMUNOTHERAPY

by

Stephanie Zelenetz

Duke University, Department of Biomedical Engineering

Grand Challenge Scholars Thesis

Focus: Engineer Better Medicines

Supervisor: Ashutosh Chilkoti

April 21, 2020

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CONTENTS

Research...........................................................................................................................................3

Introduction............................................................................................................................

Results................................................................................................................................. 4

Materials and methods.......................................................................................................6

Future Directions.................................................................................................................7

Conclusion...........................................................................................................................9

Figures and data.................................................................................................................10

Innovation and Entrepreneurship...................................................................................................17

Global.............................................................................................................................................17

Service Learning........................................................................................................................... 18

Interdisciplinary Curriculum..........................................................................................................19

Acknowledgements........................................................................................................................20

Citations.........................................................................................................................................21

Appendix........................................................................................................................................22

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RESEARCH

Checkpoint blockades and adoptive T cell therapy have highlighted recent breakthroughs in cancer immunotherapy, but clinically effective cancer vaccines have yet to be achieved. By leveraging biomaterials for the delivery of antigen and adjuvant it is possible to fine-tune an anticancer immune response. Nanoparticles have demonstrated tremendous potential for the enhancement of immunotherapies by promoting increased antigen uptake, programming accumulation in the lymph nodes, and decreasing clearance. Here we describe the use of a diblock consisting of reslin-like polypetides (RLPs), hydrophobic repetitive proteins, and elastin-like polypeptides (ELPs), hydrophilic repetitive protein biopolymers for a cancer vaccine consisting of antigen and negatively charged adjuvants, such as CpG. RLP-ELP diblocks, a novel class of amphiphilic diblock polypeptides, are a strong choice for such as they provide a robust platform for creating micelles of a given size and morphology. Here, we created a library of Ova-RLP-ELP-K12 constructs and successfully expressed and purified Ova-RLP140-ELP180-K12 and Ova-RLP180-ELP1160-K12. We have shown the binding of Ova-RLP140-ELP180-K12 to CpG and the protein’s self-assembling behavior. Further characterization of different RLP-ELP variants will be used to determine the fusion that is the ideal candidate for a nanoparticle-based vaccine platform.

Introduction

Checkpoint blockade inhibitors and CAR T cells have demonstrated tremendous breakthroughs in cancer treatment in the past decades. Cancer immunotherapy has already shown significant clinical impact, and vaccines have demonstrated some clinical benefit. In a trial conducted entailing the use of GM-CSF secreting tumor vaccines to treat stage 2-3 pancreatic cancer, vaccinated patients experienced as survival rate of 26 months, whereas unvaccinated patients have a median survival time of 21 months4. One approach to cancer vaccines entails dendritic cell recruitment, antigen loading, activation and proliferation, due to their ability to promote cytotoxic T lymphocyte responses1. Despite the success of such treatments, the results of cancer vaccines have been disappointing in the clinic. Only 5 percent of individuals treated with these vaccines demonstrate a significant response6.

Biomaterials have the potential to augment the response elicited by traditional immunotherapies by providing additional control over immunological signaling and the context of antigen presentation. Elastin-like-polypeptides (ELPs), hydrophilic repetitive biopolymers that exhibit temperature-dependent phase behavior, and resilin-like polypeptides (RLPs), a class of hydrophobic repetitive protein polymers, are ideal biomaterials for enhancing immunotherapies. The development amphiphilic RLP-ELP polypeptides diblocks provides a novel and customizable platform for nanoscale micelle formation. The size and morphology of these micelles can be engineered by altering the hydrophilic weight fraction of the diblock polypeptide and the molecular weight of its components5.

Recent work has demonstrated that micelle size and morphology impact cellular uptake, accumulation and clearance. The accumulation of antigen and adjuvant in the lymph is particularly important to the development of immunotherapies as the lymph nodes are the site of lymphocyte priming, which initiates cell-mediated immunity. The size of a nanomaterial is crucial to lymph node targeting. For a subcutaneously administered particle molecules smaller than 9 nm in diameter diffuse into the blood, while those with a diameter greater than 100 nm can be entrapped

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by the extracellular matrix, and can only be transported into the lymph via cell-mediated processes. Additionally, particles with a diameter greater than 60 nm may accumulate in the lungs due to the small size of the vasculature. Other features of nanoparticles that can impact immunotherapies include the morphology of the nanoparticle (i.e. spherical or wormlike) and linked antigen and adjuvant. The simultaneous delivery of antigen and adjuvant to antigen presenting cells can lead to antigen presentation with the proper costimulatory molecules, leading to a robust immune response, with minimal tolerogenic responses5.

RLP-ELP diblocks provide a strong platform for the delivery of cancer antigen, adjuvants and cytokines for cancer immunotherapies, allowing for the customization of micelle morphology and size. Here, we describe the incorporation of antigen, Ova, and adjuvant, CpG (bound electrostatically to a polylysine tail) into the RLP-ELP diblock system, with the eventual goal of developing a nanoparticle-based cancer vaccine. An Ova-RLP ELP-K12:CpG library was created with the intention of discovering the ideal formulation that results in optimal micelle size and morphology for accumulation in the lymph. Additionally, the design of the RLP-ELP polypeptide will ensure for the simultaneous delivery of the antigen and adjuvant, allowing for an optimal immune response with minimal tolerogenic effects. We will investigate the optimal route of administration (intraperitoneal, intramuscular, intravenous or subcantaneous injection) to develop micelle-based cancer vaccine with accumulation in the lymph nodes, preferable uptake and thus an enhanced immune response.

Results

The MHC I restricted antigenic peptide from ovalbumin (Ova) amino acid sequence SIINFEKL was codon optimized for E. Escherichia coli (E. Coli) using IDT. A library of several different Ova-RLP-ELP-K12 constructs was successfully cloned as follows and the status of these constructs is presented in Table 1. Several new RLP-ELP diblocks, RLP140-ELPS160, RLP240-ELPS160 and RLP240-ELP2160, were successfully cloned. Ova and K12 were fused to the N and C terminals, respectively, of RLP140-ELP180, RLP1100-ELP180, RLP180-ELP1160, RLP140-ELP2160, RLP140-ELPS160, and RLP240-ELPS160. K12 was successfully ligated to the C terminus of RLP240-ELP2160. The expression and purification of Ova-RLP140-ELP180-K12 is depicted in Figure 2. The construct was expressed solubly in BL21. It was successfully purified via inverse transition cycling (ITC), as noted by the presence of a single band in the third cold spin supernatant at the molecular weight of the fusion (67.1 kDa). The expression and purification of Ova-RLP1100-ELP180-K12 and Ova-RLP180-ELP1160-K12 is shown in Figure 3. The expression of Ova-RLP180-ELP1160-K12 and Ova-RLP1100-ELP180-K12 were moderately soluble. Ova-RLP180-ELP1160-K12 was successfully purified with ITC, as noted by the presence of a single band in the third cold spin supernatant at the molecular weight of the fusion (131.3 kDa). Ova-RLP1100-ELP180-K12 was unable to be purified with ITC, evident by the absence of a band corresponding to the molecular weight of the protein (118.6 kDa) in the third cold spin supernatant.

The ability of Ova-RLP180-ELP1160-K12 to bind the CpG was verified with a gel shift assay, as seen in Figure 4. CpG alone is able to migrate down the lane. In the presence of protein, the adjuvant is entrapped in the well, indicating that it is bound to the protein, which cannot migrate down the lane due to its large size. E4-60-K12, the non-micelle forming version of this polypeptide had previously been shown to bind the adjuvant. Thus, the formation of micelles does not hinder the protein’s ability to bind the CpG electrostatically. A Cary Scan was run to assess the extent of

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temperature-dependent phase behavior of the protein. In the absence of CpG, as seen in Figure 5, the Ova-RLP140-ELP180-K12 exhibits a slight phase transition. Though the magnitude of this increase in absorbance is much smaller than that of E4-60-K12. In the presence of CpG, the phase behavior of the protein is notably different. At 50uM, the absorbance constantly decreases with temperature. At 100uM, the absorbance generally decreases with temperature. There is notable peak around 30°C, before the absorbance declines significantly. It is possible that the additional negative charge, which serves to neutralize the polylysine tail, alters micelle formation. In addition, the unexpected temperature-dependent phase behavior of the protein may be due to micelle inversion, driven by the hydrophobic effect. The RLP and ELP components of the diblock each have their own phase behavior. The RLP component exhibits UCST phase behavior and has its own transition temperature (Tt), while the ELP component exhibits LCST phase behavior and has a different Tt. It is likely that when the temperature is below the ELP’s Tt, the soluble ELP is towards the corona of the diblock, minimizing the need for water cages around the hydrophobic RLP. This increases the entropy and decreases the free energy of the solvent. At intermediate temperatures (between the Tt of the ELP and the Tt of the RLP), neither component is soluble. The fusion is insoluble, as indicated by an increase in absorbance. When the temperature is above the RLP’s Tt, the RLP is soluble. If presented on the corona of the micelle, the free energy of the system can be minimized (as the ELP had done previously). Additional imaging studies are needed to verify this theory.

Figure 6 shows the results of DLS at 25°C for Ova-RLP140-ELP180-K12 in the absence of the adjuvant, and at 25°C. 37°C and 55°C in the presence of CpG. At 25°C in the absence of CpG, the Ova-RLP140-ELP180-K12 had an average radius of hydration of 27.9 nm. At 25°C. 37°C and 55°C Ova-RLP140-ELP180-K12:CpG had an average radius of hydration of 15.4 nm, 22.4 nm and 31.1 nm, respectively. The average radius increases with temperature, which was also noted by Dzuricky et al2. The size of the radius suggests micelle formation, rather than unimers or aggregates. Additionally, the average radius indicated by the DLS is an ideal size for targeting of the lymph nodes as it is not small enough to be transported in the blood (<9 nm), or large enough that it becomes entrapped in the lungs or extracellular matrix (>60nm, >100nm, respectively). The results of cryoTEM, as seen in Figure 7, confirm the self-assembly of the protein. In the absence of CpG, Ova-RLP140-ELP180-K12 forms both spherical and wormlike micelles. In the presence of CpG, Ova-RLP140-ELP180-K12 forms spherical and wormlike micelles in addition to larger, zipper-like structures. These zipper-like structures range in size, and are on the scale of 100s of nm. These self-assembling behavior of this construct may be dependent on factors such as N:P, the size and hydrophilic weight fraction of the RLP-ELP diblock, and time.

The activity of the Ova-RLP140-ELP180-K12 with CpG was found to be superior to that of E4-60-K12 with CpG as seen in Figure 8. Each protein showed negligible activity in the absence of CpG, with nitrite concentrations of 1.08uM and 0.52uM, respectively. In the presence of CpG, Ova-RLP140-ELP180-K12 had a nitrite concentration of 44.09uM, while E4-60-K12 had a nitrite concentration of 26.19uM. This difference is statistically significant. It is likely that this increase in activity is due to enhanced adjuvant uptake mediated by the self-assembling behavior of the biomaterial.

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Materials and methods

Vector Creation and Extraction The Ova peptide amino acid sequence was obtained and was codon optimized for E. Escherichia coli (E. Coli). Forward and reverse primers were ordered from IDT. The Ova peptide was ligated into the JMD-5 vector via PCR. The K12 sequence was B-cut digested with the BgII and BseRI restriction enzymes. VA5-RLP140-ELP180, VA4-RLP180-ELP180, RLP140-ELP180, RLP1100-ELP180, RLP180-ELP1160, RLP140-ELP2160, RLP140-ELPS80, RLP240, ELPS160 ELPS80 and ELP280 DNA sequences were obtained (ELP1 =((A/G)GVPG)n, ELP2 =(AGVPG)n, ELPS =(SGVPG)n RLP1=(QYPSDGRG)n, RLP2=(GRGDSPYS)n).

RLP140-ELPS80, and RLP240 were A cut digested using the AcuI and BgII restriction enzymes. ELPS160 and ELPS80, and ELP280 were B cut digested using the BseRI and BgII restriction enzymes. The RLP140-ELPS80 A cut was ligated to the ELPS80 B cut to obtain RLP140-ELPS160. The RLP240 A cut was ligated to the ELPS160 and ELP280 B cuts to obtain RLP240-ELPS160 and RLP240-ELP280, respectively. The DNA vectors were transformed into EB5α Escherichia coli (E. Coli). The cells were incubated in SOC media for 1 hour at 37°C. The cells were plated on agar plates with 2XYT containing kanamycin (45 µg/mL) and allowed to grow overnight. Large, isolated colonies were grown in 3 mL 2XYT inoculated with kanamycin (45 µg/mL) overnight. The vectors were isolated using a standard miniprep procedure and were sequenced with T7- reverse primers. The positive RLP240-ELP280 sequence was A cut digested using the AcuI and BgII restriction enzymes and ligated to the ELP280 B cut sequence to get RLP240-ELP2160. The DNA vectors were transformed into EB5α as described above, allowed to grow overnight, miniprepped, and sequenced. All RLP-ELP constructs except VA5-RLP140-ELP180, VA4-RLP180-ELP180 were A cut digested using the AcuI and BgII restriction enzymes and ligated to the K12 B cuts. DNA vectors were transformed into EB5α as described above, allowed to grow overnight, miniprepped, and sequenced. Positive RLP-ELP-K12 sequences were B cut digested using BgII and BseRI restriction enzymes. The RLP-ELP B cuts were ligated to the Ova A cuts and transformed into EB5 α E. Coli, as described, above. Colonies were grown in 3 mL 2XYT inoculated with kanamycin overnight. The vectors were isolated using a standard miniprep procedure and were sequenced with T7 primers. Positive sequences were transformed into BL21 E. Coli, using the standard NEB protocol. The recovered cells were plated on agar plates containing kanamycin and grown overnight.

Protein Expression Ova-RLP140-ELP180-K12, Ova-RLP1100-ELP180-K12, Ova-RLP180-ELP1160-K12, Ova-RLP140-ELPS160-K12 and Ova-RLP240-ELPS160-K12 were expressed and purified. The transformed competent bacteria were grown overnight in 2XYT media, composed of 0.25 g NaCl, 0.8 g tryptone, 0.5g yeast extract and 50 mL water and kanamycin (45 µg/mL). The starter cultures were added to 1-L flasks of 2XYT media (25 mL/ flasks) containing kanamycin (45 µg/mL). The cultures were allowed to shake in at 25°C to promote cell growth. After reaching an OD of ~0.6 (approximately 3.5 hours), the temperature of the incubator was reduced to 16°C and cells were inducted Isopropyl-β-D-thiogalactopyranoside (500uM). Expression was promoted overnight. Cells were harvested via centrifugation at 3500 rpm for a total of 15 min. The cells were resuspended in PBS. The cell suspensions were sonicated on ice for 3 min (10 seconds on, 40 seconds off; 75% amplitude, 60-80 watts).

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Purification The Ova-RLP140-ELP180-K12, Ova-RLP1100-ELP180-K12, Ova-RLP180-ELP1160-K12, Ova-RLP140-ELPS160-K12 and Ova-RLP240-ELPS160-K12 were purified via inverse transition cycling (ITC). The soluble PEI supernatant was warmed to room temperature. Ammonium sulfate was added until the solution transitioned, which was characterized by the formation of an opaque solution. The solution was transferred into Nalgene tubes, and centrifuged at 35°C and 14,000 RPM for 10 min. The hot spin supernatant was removed. Each pellet was resuspended in 10 mL of cold tris-buffer, and placed on the rotator to suspend for approximately one hour. The solution was spun 4°C and 14,000 RMP for 10 min. The supernatant was warmed to room temperature. Sodium sulfate was added until the protein transitioned. A second hot spin (35°C, 14,000 RPM, 10 min) was completed. The pellet was resuspended in 8mL tris buffer and allowed to resolubilize. The solution was centrifuged at 4°C and 14,000 RMP for 10 min.

Protein samples were analyzed via SDS-Page. Lammeli buffer was added to all samples. Protein gels were run at 300 V for 20 min. A stain free procedure was used in addition to staining with Simply Blue®dye.

Gel shift assay A 1% agarose gel was prepared by adding 0.7g of agarose to 70 mL of 1X TAE buffer. The solution was heated in the microwave until dissolved. Sybr safe (7uL) was added to the solution before the gel was poured and left to set. Once solidified, a sample of CpG alone, and CpG mixed with Ova-RLP140-ELP180-K12 was loaded. The gel was run at 100V for 25 minutes. The results were then visualized user the standard sybr safe protocol.

Cryo-transmission electron microscopy The self-assembly of Ova-RLP140-ELP180-K12 was characterized with cryoTEM. Samples of Ova-RLP140-ELP180-K12 were prepped to a final protein concentration of 10uM, and samples with Ova-RLP140-ELP180-K12 and CpG were prepped using a N:P=1. The cryoTEM protocol was performed in the SMIF facility thanks to the help of Dr. Micahel Dzuricky. RAW 264.7 CpG ODN Assay RAW 264.7 cells were cultured according to the protocol recommended by ATCC. Once the cells became confluent they were concentrated to a final value of 2 million cells/mL. 500uL of cells were plated into each well of a 24 well plate, and allowed to adhere for 30 minutes. Once adherent, Ova-RLP140-ELP180-K12, E4-60-K12 and PBS, either in the presence of absence of CpG, was added to the corresponding well. The cells were incubated at 37°C for 24 hours. At this point, nitric oxide production was measured with a Griess Assay, following the standard protocol. N:P=1 was used for this experiment. Future directions

Verification of antigen presentation The proper antigen presentation of Ova-RLP140-ELP180-K12 must be verified. To assess this, flow cytometry will be performed on DC2.4 cells stimulated with the protein. Specifically, DC2.4 cells will be stimulated with Ova-RLP140-ELP180-K12 or the Ova peptide, or unstimulated, and then prepared for flow cytometry. Flow cytometry will be run on all the samples and the MHC-I presentation of Ova will be confirmed.

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The cross-presentation of Ova will be verified by performing flow cytometry as described above, and the additional incubation with brefeldin A. Brefeldin A is a molecule that has been shown to inhibit cross-presentation by limiting transport out of the ER-Golgi system2. If an antigen is processed in the cross-presentation pathway, no MHC-I presentation of Ova will be observed in the presence of brefeldin A. Thus, to determine if the Ova peptide is properly cross-presented, DC2.4s will be incubated with Ova-RLP140-ELP180-K12 in the presence or absence of brefeldin A. Flow cytometry will be run on all samples, including unstimulated cells. If Ova-RLP140-ELP180-K12 is cross-presented, the MHC I presentation of Ova will not be observed for the samples treated with brefeldin A. Characterization of Ova-RLP140-ELP180-K12 with cryoTEM Further work must be done to characterize the self-assembling behavior of Ova-RLP140-ELP180-K12 with and without CpG. Figure 7. shows that in the absence of CpG, Ova-RLP140-ELP180-K12 forms both spherical and wormlike micelles. In the presence of CpG (N:P=1), an additional zipper-like structure is formed. The self-assembling nature of Ova-RLP140-ELP180-K12:CpG may be dependent on factors such as the N:P and time. To investigate the former, cryoTEM will be run on samples of Ova-RLP140-ELP180-K12:CpG with N:P=0.5, 3,7. Additionally, to assess the time-dependent nature of self-assembly, samples of Ova-RLP140-ELP180-K12 will be incubated at CpG, and imaged with cryoTEM at 0, 1, 2, 4, and 6 hours after the addition of the adjuvant. This information will provide useful information about the ideal route of administration for the vaccine, and whether Ova-RLP140-ELP180-K12 is an ideal candidate for a nanoparticle-based vaccine platform. Confocal Imaging Confocal imaging will be used to confirm the uptake of CpG. Specifically, HEK-BlueTM TLR9 cells will be incubated with FITC labeled CpG and washed. The cells will then be imaged using confocal microscopy to locate CpG within the cell and verify cellular uptake. HEK-BlueTM TLR9 Assay The HEK-BlueTM TLR9 Assay will be used to determine the activity of CpG, a TLR agonist, in complexed with Ova-RLP140-ELP180-K12. Previously, the results of the RAW 264.7 CpG ODN Assay, as seen in Figure 8, indicated that the activity of the adjuvant was significantly higher for Ova-RLP140-ELP180-K12:CpG than for E4-60-K12:CpG. However, the activity of CpG alone was much higher than that of both Ova-RLP140-ELP180-K12:CpG and E4-60-K12:CpG. This suggests that the assay requires further optimization. Moving forward, the HEK-BlueTM TLR9 Assay will be used in place of the RAW 264.7 CpG ODN Assay. HEK-BlueTM TLR cells, unlike the RAW 264.7 cells, are not sensitive to TLR4 agonists, such as endotoxin, which may have influenced the results of the RAW 264.7 CpG ODN Assay. We suspect that the HEK-BlueTM TLR9 Assay will produce more reliable results, and will be preferable for analyzing CpG activity. In addition, Ova-E4-60-K12 will be used as the soluble unimer control, in place of E4-60-K12. Ideally, the CpG only control, will have minimal activity compare to the both Ova-RLP140-ELP180-K12:CpG and Ova-E4-60-K12:CpG samples. If the activity of Ova-RLP140-ELP180-K12:CpG is significantly higher than that of Ova-E4-60-K12:CpG, it is possible that the self-assembly of this biomaterial enhances CpG uptake.

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Cloning, expression, purification and characterization of other Ova-RLP-ELP-K12 constructs Table 1 shows the status of the remaining Ova-RLP-ELP-K12 constructs that must be expressed and purified. Ova and K12 must be ligated onto the N and C terminals, respectively of VA5-RLP140-ELP180 and VA4-RLP180-ELP180. Ova must be ligated to RLP280-ELP2160-K12 to obtain Ova-RLP280-ELP2160-K12. Ova-VA5-RLP140-ELP180-K12, Ova-VA4-RLP180-ELP180-K12, Ova-RLP280-ELP2160-K12, Ova-RLP140-ELP2160-K12 and Ova-RLP240-ELP2160-K12 must be expressed and purified with ITC. All Ova-RLP-ELP-K12 constructs successfully expressed and purified must be characterized. Specifically, Cary scans, DLS and cryoTEM must be performed for all constructs in the presence (N:P=1) and absence of CpG. The temperature-dependent phase behavior, and self-assembling nature of these proteins will help to determine which construct is the most viable as a vaccine candidate. Proteins that self-assemble (in the presence of CpG) into structures with radius of hydration of up to 30 nm will be suitable for subcutaneous, intravenous or intramuscular injection. Once the ideal Ova-RLP-ELP-K12 construct is selected, further experiments must be done, as outlined above, to verify proper antigen cross-presentation, CpG uptake and CpG activity.

Conclusion

This project has ultimately resulted in the creation of a library of novel Ova-RLP-ELP-K12 fusions. Ova-RLP140-ELP180-K12 and Ova-RLP180-ELP1160-K12 were successfully expressed and purified via ITC purification, as verified by SDS-page analysis. A gel shift assay confirms the ability of the Ova-RLP140-ELP180-K12 to electrostatically bind the adjuvant. DLS results confirmed self-assembly formation. CryoTEM shows the formation of both spherical and wormlike micelles, in addition to larger, zipper-like structures in the presence of CpG. Cary Scans indicate that this fusion did not exhibit the steep, reversible thermoresponsive behavior that is characteristic of ELPs. Instead the samples without the adjuvant only experienced a slight increase in absorbance as the temperature was temperature. Additionally, the samples with the CpG showed an overall decrease in absorbance as temperature increased. It is possible that the additional negative charge (or the neutralization of the polylysine tail) altered micelle formation, or that the micelles are inverting. Finally, the activity of Ova-RLP140-ELP180-K12 with CpG was found to be superior to that of the E4-60-K12 with CpG. This suggests that the self-assembly of the protein enhances the activity of the adjuvant, perhaps by increasing cellular uptake. Recommendations for future research include additional characterization with cryo-TEM to explore factors such as N:P and time on the self-assembling behavior of Ova-RLP140-ELP180-K12:CpG. Additionally, the cross presentation of Ova must be verified with flow cytometry, and the uptake of CpG uptake must be confirmed with confocal imaging. The activity of CpG should be verified with the HEK-BlueTM TLR9 Assay using the proper protein control. Finally, the expression, purification and characterization of other Ova-RLP-ELP-K12 fusions is recommended to determine the construct (micelle morphology, size) that is optimal for vaccine development.

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Figures and data

Figure 1. Ova-RLP-ELP-K12 forms spherical or wormlike micelles with Ova at the core and K12, electrostatically bound to CpG at the corona. Micelle size and morphology can be altered based on the hydrophilic weight fraction and molecular weight of the diblock polypeptide.

Table 1. Cloning, expression and purification of constructs in the Ova-RLP-ELP-K12 library

RLP-ELP Diblock K12 cloned?

Ova cloned?

Expressed? Solublility Successfully Purified?

RLP140-ELP180 Yes Yes Yes Soluble Yes RLP1100-ELP180 Yes Yes Yes Moderately soluble No RLP180-ELP1160 Yes Yes Yes Moderately soluble Yes VA5-RLP140-ELP180

No No No N/A N/A

VA4-RLP180-ELP180

No No No N/A N/A

RLP180-ELP2160 Yes Yes No N/A N/A RLP180-ELPS160 Yes Yes Yes Moderately soluble No RLP280-ELP2160 Yes No No N/A N/A RLP280-ELPS160 Yes Yes Yes Moderately soluble No

Table 1. shows the progress of the cloning, expression and purification for the constructs in the Ova-RLP-ELP-K12 library. Ova and K12 were successfully ligated to the N and C terminals, respectively, of RLP140-ELP180, RLP1100-ELP180, RLP180-ELP1160, RLP180-ELP2160, RLP180-ELPS160, and RLP180-ELPS160. RLP140-ELP180 was expressed solubly in BL21 and was successfully purified. RLP1100-ELP180, RLP180-ELP1160, RLP180-ELPS160, and RLP280-ELPS160 all expressed somewhat solubly in BL21. RLP180-ELP11600 was successfully purified.

.

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Figure 2. Ova-RLP140-ELP180-K12 expresses solubly in BL21 as shown by the presence of a 67.1 kD band in the soluble fraction (SS). The presence of a single band at ~67.1 kDa in the cold spin 3 supernatant indicates that the protein was successfully purified via ITC.

Figure 3. The expression of Ova-RLP180-ELP1160-K12 and Ova-RLP1100-ELP180-K12 were moderately soluble in BL21, as indicated by the presence of bands in the soluble fraction (SS) at molecular weights of 131.3 and 118.6 kDa, respectively. Inverse transition cycling (ITC) resulted in the successful purification of Ova-RLP180-ELP1160-K12 as indicated by the presence of pure protein of the correct molecular weight in the cold spin 3 supernatant (CS3S). Ova-RLP1100-ELP180-K12 was not successfully purified with ITC as indicated by the presence of the 118.6 kDa band in the cold spin 1 pellet (CS1P) and cold spin 2 pellet (CS2P), and lack of the band in the cold spin 2 supernatant (CS2S).

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Figure 4. The results of a gel shift assay indicate that Ova-RLP140-ELP180-K12 binds CpG. CpG is unable to migrate down the gel in the presence of Ova-RLP140-ELP180-K12 (lane 1), and able to migrate in the absence of protein, as indicated by the 19 base pair band in lane 2.

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Figure 5. Characterization of LCST and UCST phase transition. Ova-RLP140-ELP180-K12does not exhibit the sharp and reversible phase behavior characteristic of ELPs. The phase behavior of the protein changes upon the addition of CpG. There is a mild concentration dependence.

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Figure 6. Dynamic light scattering (DLS) was used to determine the size of Ova-RLP140 -ELP180- K12 in PBS. The DLS data indicates micelle formation, and temperature dependence, A) At 25°C and in the absence of CpG, there is a narrow peak with an average radius of 27.9 nm (12.5% Pd, 8125 kD, 100% Int, 100 % mass) nm. B) At 25°C, and in the presence of CpG, there is a single, broad peak with an average radius of 15.4 nm (65.2% Pd, 2037 kD, 100% Int, 100 % mass). C) At 37°C, and in the presence of CpG, the average radius is 22.4 nm (64.8% Pd, 4862 kD, 100% Int, 100 % mass). D) In the presence of CpG at 55°C there is broad peak with an average radius of 31.1 nm (62.4% Pd, 10464 kD, 100% Int, 100 % mass).

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Figure 7. Characterization of Ova-RLP140-ELP180-K12 with cryoTEM. A) In the absence of CpG, Ova-RLP140-ELP180-K12 forms both spherical and wormlike micelles. B) In the presence of CpG (N:P=1), Ova-RLP140-ELP180-K12:CpG forms zipper-like structures, on the scale of 100s of nm, in addition to spherical and wormlike micelles.

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Figure 8. A Griess Assay was conducted on samples containing RAW 264.7 cells that were incubated with Ova-RLP140-ELP180-K12 and E4-60-k12, in both the presence and absence of CpG. The nitrite concentration of the samples incubated with Ova-RLP140-ELP180-K12 and E4-60-K12 were low 1.08uM and 0.52uM, respectively. In the presence of adjuvant, these values were 44.09uM and 26.19uM. The differences in nitrite production between the two samples incubated with CpG is statistically significant (p<0.00001).

0

5

10

15

20

25

30

35

40

45

50

Nitrite

Con

centratio

n(uM)

Ova-RLP1-40-ELP1-80-K12 Ova-RLP1-40-ELP1-80-K12+CpG E4-60-K12 E4-60-K12+CpG

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INNOVATION AND ENTREPRENEURSHIP My involvement in biotechnology design, a two-semester biomedical engineering course taught by Dr. Mike Lynch, has allowed me to gain invaluable innovation and entrepreneurship (I&E) skills related to my GC focus of engineering better medicines. As a student in biotechnology design I learned about both the business and research and development (R&D) techniques commonly practiced in the industry. We covered topics such as patents, copyrights, and FDA regulation-all of which I will need to understand to achieve my goal of developing a novel medicine that can be translated into the clinic. During the class, I had the opportunity to practice marketing skills such as cumulative and discounted cash flow analysis and business elevator pitches. Additionally, I learned about R&D processes such as large-scale fermentation. I studied the process that would be necessary to take a project, like the biomaterial-based cancer vaccine I am developing for the GC program, from paper into the clinic. Biotechnology design fortified my GCS experience by allowing me to apply the I&E and engineering techniques I learned in the classroom to develop another therapeutic. Each student in the class proposed a project to complete, and had to justify its intellectual merit. I pitched the synthesis of a broadly neutralizing antibody for a Hepatitis C (HCV) therapy. Broadly neutralizing antibodies target all viral strains, and have already demonstrated potential in the context of HIV treatment. Broadly neutralizing antibodies can provide a robust response against all genotypes, and therefore have the potential to mitigate the issue of drug resistance. My project was selected to complete for the course, and I worked in a group to complete a more comprehensive needs-finding and market analysis. We also worked to design and synthesize the antibody. Unfortunately, I did not get to complete the remaining expression, purification, and characterization protocols and in vitro assays we had outlined due to the transition to remote learning. However, I did have the opportunity to apply some of the I&E skills I learned during the course. My experience in biotechnology design provided with an additional opportunity to address my GC focus of engineering better medicines. Moreover, in addition to practicing the scientific and engineering skills that are required to create a novel therapeutic, I applied the I&E techniques (such as a comprehensive cash flow analysis) that are essential to translate a product from the laboratory to the clinic (and market). These newfound skills will unquestionably be of benefit to me during and after my PhD, as I work both in the laboratory and the pharmaceutical industry, to engineer better medicines.

GLOBAL For the global component of my GCS experience I attended the Cancer Research UK-AACR Joint Conference on Engineering and Physical Sciences in Oncology in London in October of 2019. The conference covered topics such as cancer research in the era of big data, cancer biophysics and clinical challenges, emerging detection strategies, engineered solutions to enhance therapy, and novel therapeutic approaches. I learned about many different aspects of cancer research to which I had not previously been exposed. This enhanced background in oncology has allowed me to better understand the motivation behind my GC focus to engineering better (cancer) medicines, to appreciate the merit of my cancer vaccine, and to acknowledge some of its short comings.

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The conference provided me with the opportunity to interact with several different leaders in the field of cancer research, including professors, physicians, and members of industry. I spoke briefly with a professor at Harvard, who presented on a cancer immunotherapy that has largely impacted my project. In fact, I am using the same components in my vaccine, with the added dimension of biomaterials to increase efficacy (see Research for more details). One aspect of the conference that was especially informative was the ability to speak with individuals about the different healthcare systems in the U.S. and U.K. I had a conversation with a professor at Queen’s College, England, who believed that personalized medicine would only ever succeed in the U.S. Personalized medicine was a “hot topic” in medicine and engineering for a while, and many believed that it would change the future of medicine. However, it is too expensive (and lacks the required validation) to be covered by the U.K.’s current healthcare system. Both practical (i.e. economic, political) and scientific (i.e. safety, efficacy) measures both can inform a therapeutics’ success, and it was interesting to consider the former in a global context. This conversation made me appreciate my GC project, a cancer vaccine which is not personalized, but is customized in the sense that immunotherapies recruit the patient’s own immune system to initiate a response against disease. This conference expanded my horizons by increasing my familiarity with the field of oncology, specifically why and how we can engineer better cancer therapeutics. I appreciated the exposure to new ideas, and the ability to discuss both the scientific and practical advances that need to be made to institute a global response against cancer.

SERVICE LEARNING During my junior year, I participated in the Duke Organization Females Excelling More in Math, Engineering and Science (FEMMES), an outreach program which engages girls and young women in STEM. I participated in an after-school program that entails interactive science experiments with elementary school girls. We completed activities such as constructing electric circuits with lemons and potatoes, and reflecting upon which materials were most successful. It was gratifying to see the girls’ excitement about the activities we completed and their pride when they comprehend the experiments’ underlying scientific principles. I also participated in the FEMMES mentorship program. As a volunteer, I was a friend, a role model and a constant source of advice. I engaged in activities such as visits to the Duke Lemur Center, and holiday fashion shows in which we constructed clothes out of wrapping paper, that sought to develop meaningful relationships between college women and underprivileged girls. By participating in the program, these girls are exposed to women who epitomize that passion and hard work can result in success. I hope to inspire young girls as others have done for me. This experience relates to my GC focus of engineering better medicines and to the GC focus of advancing personalized learning. By participating in the FEMMES program, I exposed young girls to basic scientific techniques, and supplemented the hands-on education they may have lacked in a large school with less resources. I worked hard to encourage them to become engaged in STEM, and perhaps even inspired them to one day engineer better medicines.

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In addition, my involvement with the Durham Literacy Center during the Fall of my sophomore year enabled me to tutor young adults seeking to receive their general education diploma (GED). I worked with students individually (once a week in three hour sessions) to review practice exams and concepts they found difficult. Passing the GED test is the first step for many of these students to achieve the numerous and diverse aspirations they have shared with me. I am humbled to have played a small part in their journeys. This experience relates to the GC focus of advancing personalized learning. Many of the individuals receiving tutoring had dropped out of high school because they felt as though their school was not able to provide the support they needed. Classes were not taught at the appropriate speed, and teachers had too many students to slow down for one. In other words, classes were tough because schools did not have the resources to customize the curriculum (and teaching style) to each student’s needs. By working as a tutor in this program, I had the opportunity to address this issue by providing students with a personalized education. I varied my teaching style from student to student, and worked with each student on a curriculum customized to his strengths and weaknesses. I saw the direct impact that I made on these individuals, and some even achieved their goal of obtaining a GED.

INTERDISCIPLINARY CIRRICULUM Throughout my time at Duke, I have taken several courses outside of the biomedical engineering curriculum. In addition to the courses (organic chemistry and biochemistry) to satisfy my chemistry minor, I have enrolled in several classes in the humanities. My freshman year I took a gender studies class and writing 101. My sophomore year I took educational psychology. My junior year I took a class on migration and human trafficking. My senior year I took an art history class on Impressionism. While these classes do not directly relate to my GC focus these courses have certainly enhanced my critical thinking and communication. These skills have undoubtedly ameliorated my GC experience, but allowing me to think more deeply about my experiences, and to communicate my ideas more concisely. See the Appendix for justification for the interdisciplinary curriculum (had previously been discussed with Dr. Schaad).

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ACKNOWLEDGEMENTS

Thank you to my supervisor Dr. Ashutosh Chilkoti for the opportunity to pursue research in your laboratory. Thank you to my mentor, Garrett Kelly, for the support and direction. Thank you to Dr. Michael Dzuricky for the RLP sequences, and for conducting cyroTEM. Thank you to the Grand Challenge Scholars Program Committee for the opportunity to complete such a fulfilling project, and the resources that have enriched this experience. Finally, thank you to Dr. Schaad for the guidance, and for the providing me with flexibility needed to pursue a project so meaningful to me.

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CITATIONS

1. Ali, Omar, et al. “Inflammatory Cytokines Presented from Polymer Matrices Differentially Generate and Activate DCs In Situ.” Advanced Functional Materials vol. 23, 36 (2013):4621-4628. doi: 10.1002/adfm.201203859.

2. Chardin, P., McCormik, F. “Brefeldin A: The Advantage of Being Uncompetitive.” Cell. vol. 97, (1999): 153-155.

3. Dzuricky, M., Xiong, S. Weber, s., & Chilkoti, A. “Avidity and Cell Uptake of Integrin-Targeting Polypeptide Micelles is Strongly Shape-Dependent.” Nano Letters vol. 19, 9 (2019): 6124-6132. doi: 10.1021/acs.nanolett.9b02095

4. Gupta, Richa, and Leisha A Emens. “GM-CSF-secreting vaccines for solid tumors: moving forward.” Discovery medicine vol. 10,50 (2010): 52-60. 5. Irvine, Darrell J et al. “Synthetic Nanoparticles for Vaccines and Immunotherapy.” Chemical reviews vol. 115,19 (2015): 11109-46. doi:10.1021/acs.chemrev.5b00109

6. Klebanoff, C. A., Acquavella, N., Yu, Z. & Restifo, N. P. “Therapeutic cancer vaccines: are we there yet?: Therapeutic cancer vaccines: moving forward.” Immunol. Rev. vol. 239, (2011):27–44.

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APPENDIX A. Supplemental information regarding in-depth research experience

Table A1. Summary of in-depth research experience

Table A1. Shows information about in-depth research experiences including experience, research project, supervisor, date, time commitment and grade. B. Requirements for entrepreneurship, global and service learning components One medium depth and two minimum depth (or higher): Yes At least one related to GC focus: Yes Others related to other GCs: Yes Entrepreneurship: Depth: In-depth (2 semesters) Related to GC focus: Yes Enrolled in a two-semester design class called biotechnology design, which covered topics such as patents and copyrights, the FDA, industrial research and development and discounted cash flow analysis. Learned business and laboratory skills such as fermentation, and protein purification techniques, used in the biotechnology industry. Had first-hand experience developing an I&E project by working on the synthesis of a novel Hepatitis C (HCV) therapeutic compatible with all genotypes. Such a drug has the potential to initiate a robust response against HCV, and eliminate the issue of drug resistance. Therefore, it is related to my GC focus of engineering better medicines. I the project, justified its merit with a needs-finding and financial analysis, designed the construct, began the production process, and planned downstream characterization and validation experiments. Unfortunately, due to the transition to online learning, I was unable to complete production, characterization and validation, as planned. Global: Depth: Minimum Related to GC focus: Yes Attended the CRUK-AACR Joint Conference on Engineering and Physical Sciences in Oncology in London in October 2019. Topics of the conference included breakthrough research in cancer treatment and diagnostics, many of which used engineering principles, and some even biomaterials, like my own project. Other topics, such as cancer research in the era of big data, expanded my understanding of the field as a whole, why the need to engineer better cancer therapeutics is dire, and promising strategies for doing so. Therefore, this conference was directly related to my GC focus of engineering better (cancer) medicines. Service Learning:

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Depth: Medium Related to GC focus: No Other GC focus: Advance personalized learning Participated in FEMMES by conductive science experiments with young girls, and mentoring underprivileged adolescent girls. This STEM outreach is related to the GC program broadly, by encouraging young girls to become involved in STEM. Additionally, by supplementing the girls’ education in the classroom with hands-on activities in small groups (or on-on-one), this relates to the GC focus of advancing personalized learning. Volunteered at Durham Literacy center tutoring young adults seeking to receive their GED.

Table B1. Service learning experiences

Table B1. Shows information about service learning experiences including date and time commitment. C. Justification for interdisciplinary curriculum I have taken several courses outside of STEM during my time at Duke (see Interdisciplinary Curriculum section). When I originally was accepted to GCS, I was told that any non-STEM (and non-global health) classes satisfied this requirement. I was not aware that these classes were supposed to relate to my GC focus, and was told that global health classes did not satisfy the interdisciplinary curriculum requirement. This was discussed with Dr. Schaad in Spring 2019, and from my knowledge, this requirement was made more flexible for my GC experience. I do believe that they have enhanced my critical thinking and communication skills, though I acknowledge that they are not related to my GC focus.

Table B2. Interdisciplinary curriculum

Table B2. Shows information about interdisciplinary curriculum including course, date and grade.