@article{vrabel_schulman_gillam_mantooth_nguyen_zaharoff_2023, title={Focal Cryo-Immunotherapy with Intratumoral IL-12 Prevents Recurrence of Large Murine Tumors}, volume={15}, ISSN={["2072-6694"]}, DOI={10.3390/cancers15082210}, abstractNote={Focal ablation technologies are routinely used in the clinical management of inoperable solid tumors but they often result in incomplete ablations leading to high recurrence rates. Adjuvant therapies, capable of safely eliminating residual tumor cells, are therefore of great clinical interest. Interleukin-12 (IL-12) is a potent antitumor cytokine that can be localized intratumorally through coformulation with viscous biopolymers, including chitosan (CS) solutions. The objective of this research was to determine if localized immunotherapy with a CS/IL-12 formulation could prevent tumor recurrence after cryoablation (CA). Tumor recurrence and overall survival rates were assessed. Systemic immunity was evaluated in spontaneously metastatic and bilateral tumor models. Temporal bulk RNA sequencing was performed on tumor and draining lymph node (dLN) samples. In multiple murine tumor models, the addition of CS/IL-12 to CA reduced recurrence rates by 30–55%. Altogether, this cryo-immunotherapy induced complete durable regression of large tumors in 80–100% of treated animals. Additionally, CS/IL-12 prevented lung metastases when delivered as a neoadjuvant to CA. However, CA plus CS/IL-12 had minimal antitumor activity against established, untreated abscopal tumors. Adjuvant anti-PD-1 therapy delayed the growth of abscopal tumors. Transcriptome analyses revealed early immunological changes in the dLN, followed by a significant increase in gene expression associated with immune suppression and regulation. Cryo-immunotherapy with localized CS/IL-12 reduces recurrences and enhances the elimination of large primary tumors. This focal combination therapy also induces significant but limited systemic antitumor immunity.}, number={8}, journal={CANCERS}, author={Vrabel, Maura R. and Schulman, Jacob A. and Gillam, Francis B. and Mantooth, Siena M. and Nguyen, Khue G. and Zaharoff, David A.}, year={2023}, month={Apr} }
 @article{zaharoff_nguyen_2022, title={IDENTIFICATION OF VASOACTIVE INTESTINAL PEPTIDE (VIP)-SPECIFIC SINGLE-CHAIN ANTIBODY FRAGMENTS (SCFVS) VIA YEAST SURFACE DISPLAY}, volume={10}, ISSN={["2051-1426"]}, DOI={10.1136/jitc-2022-SITC2022.1371}, abstractNote={<h3>Background</h3> Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide expressed in various tissues including the pancreas, intestines and central nervous system.<sup>1, 2</sup> The overexpression of VIP and its receptors is associated with increased growth and metastasis of breast, prostate, and lung malignancies.<sup>3</sup> In addition, the interaction of VIP with its receptors on activated T cells results in immune suppression which further supports tumor growth.<sup>4, 5</sup> Furthermore, tumor-supporting regulatory T cells have been found to be promoted by VIP-dependent mechanisms.<sup>6</sup> Altogether, prior literature implies that blockade of VIP signaling may inhibit tumor-mediated immune suppression and augment antitumor immune responses. Recent preclinical studies in acute myeloid leukemia and T lymphoblastic leukemia demonstrated that VIP receptor antagonists increase T cell-dependent anti-tumor responses.<sup>2</sup>. Unfortunately, the short-half lives of peptide antagonists limit their clinical utility. A more translatable approach is the development of long circulating antibodies that bind VIP and inhibit its immunosuppressive activities. <h3>Methods</h3> In this study, we utilized a yeast display of a non-immune human single-chain variable fragment (scFv) library to identify VIP-binding scFvs.<sup>7-9</sup> VIP binders were screened by several rounds of selection using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). The enriched binder population was cloned into single colonies of yeast cells by limited dilution. The binding affinities of VIP-binding clones were evaluated via flow cytometry by titrating fluorescence-labeled VIP. Clones with high binding affinity (Kd < 500 nM) were selected for sequencing (figures 1-5). <h3>Results</h3> Sequences of the isolated scFv revealed that a unique section of complementarity-determining region 3 (CDR3) of the heavy chain played an important role in VIP binding. Multiple clones with similar but distinct CDR3 sequences produced a useful range of binding affinities for further development (figure 6). <h3>Conclusions</h3> Yeast display is an effective technology for identifying human scFvs that bind to the immunosuppressive neuropeptide, VIP. CDR3 of scFv heavy chains were influential in VIP recognition. Ongoing studies are focused on the production, purification, and validation of novel anti-VIP human antibodies. <h3>Acknowledgements</h3> We thank Dr. K. Dane Wittrup (Massachusetts Institute of Technology) for kindly providing yeast libraries, Drs. Edmund K. Waller and Jens Wrammert (Emory University) for helpful discussions and comments, and Dr. Ryan W. Paerl (North Carolina State University) for technical advice on cell sorting. <h3>References</h3> Petersen CT, Li JM, Waller EK. Administration of a vasoactive intestinal peptide antagonist enhances the autologous anti-leukemia T cell response in murine models of acute leukemia. <i>Oncoimmunology</i>. 2017;<b>6</b>(5):e1304336. Epub 2017/06/24. Li JM, Darlak KA, Southerland L, Hossain MS, Jaye DL, Josephson CD, <i>et al</i>. VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice. <i>PLoS One</i>. 2013;<b>8</b>(5):e63381. Epub 2013/06/01. Moody TW, Nuche-Berenguer B, Jensen RT. Vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide, and their receptors and cancer. <i>Curr Opin Endocrinol Diabetes Obes</i>. 2016;<b>23</b>(1):38–47. Epub 2015/12/26. Forghani P, Petersen CT, Waller EK. Activation of VIP signaling enhances immunosuppressive effect of MDSCs on CMV-induced adaptive immunity. <i>Oncotarget</i>. 2017;<b>8</b>(47):81873–9. Epub 2017/11/16. Liu L, Yen JH, Ganea D. A novel VIP signaling pathway in T cells cAMP-->protein tyrosine phosphatase (SHP-2?)-->JAK2/STAT4-->Th1 differentiation. <i>Peptides</i> 2007;<b>28</b>(9):1814–24. Epub 2007/04/28. Delgado M, Chorny A, Gonzalez-Rey E, Ganea D. Vasoactive intestinal peptide generates CD4+CD25+ regulatory T cells in vivo. <i>J Leukoc Biol</i> 2005;<b>78</b>(6):1327-38. Epub 2005/10/06. Angelini A, Chen TF, de Picciotto S, Yang NJ, Tzeng A, Santos MS, <i>et al</i>. Protein engineering and selection using yeast surface display. <i>Methods Mol Biol</i> 2015;<b>1319</b>:3–36. Epub 2015/06/11. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Yeung YA, <i>et al</i>. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. <i>Nat Biotechnol</i>. 2003;<b>21</b>(2):163–70. Epub 2003/01/22. Kelly RL, Le D, Zhao J, Wittrup KD. Reduction of Nonspecificity Motifs in Synthetic Antibody Libraries. <i>J Mol Biol</i>. 2018;<b>430</b>(1):119–30. Epub 2017/12/01.}, journal={JOURNAL FOR IMMUNOTHERAPY OF CANCER}, author={Zaharoff, David and Nguyen, Khue}, year={2022}, month={Nov}, pages={A1424–A1425} }
 @article{nguyen_mantooth_vrabel_zaharoff_2022, title={Intranasal Delivery of Thermostable Subunit Vaccine for Cross-Reactive Mucosal and Systemic Antibody Responses Against SARS-CoV-2}, volume={13}, ISSN={["1664-3224"]}, DOI={10.3389/fimmu.2022.858904}, abstractNote={Despite the remarkable efficacy of currently approved COVID-19 vaccines, there are several opportunities for continued vaccine development against SARS-CoV-2 and future lethal respiratory viruses. In particular, restricted vaccine access and hesitancy have limited immunization rates. In addition, current vaccines are unable to prevent breakthrough infections, leading to prolonged virus circulation. To improve access, a subunit vaccine with enhanced thermostability was designed to eliminate the need for an ultra-cold chain. The exclusion of infectious and genetic materials from this vaccine may also help reduce vaccine hesitancy. In an effort to prevent breakthrough infections, intranasal immunization to induce mucosal immunity was explored. A prototype vaccine comprised of receptor-binding domain (RBD) polypeptides formulated with additional immunoadjuvants in a chitosan (CS) solution induced high levels of RBD-specific antibodies in laboratory mice after 1 or 2 immunizations. Antibody responses were durable with high titers persisting for at least five months following subcutaneous vaccination. Serum anti-RBD antibodies contained both IgG1 and IgG2a isotypes suggesting that the vaccine induced a mixed Th1/Th2 response. RBD vaccination without CS formulation resulted in minimal anti-RBD responses. The addition of CpG oligonucleotides to the CS plus RBD vaccine formulation increased antibody titers more effectively than interleukin-12 (IL-12). Importantly, generated antibodies were cross-reactive against RBD mutants associated with SARS-CoV-2 variants of concern, including alpha, beta and delta variants, and inhibited binding of RBD to its cognate receptor angiotensin converting enzyme 2 (ACE2). With respect to stability, vaccines did not lose activity when stored at either room temperature (21-22°C) or 4°C for at least one month. When delivered intranasally, vaccines induced RBD-specific mucosal IgA antibodies, which may protect against breakthrough infections in the upper respiratory tract. Altogether, data indicate that the designed vaccine platform is versatile, adaptable and capable of overcoming key constraints of current COVID-19 vaccines.}, journal={FRONTIERS IN IMMUNOLOGY}, author={Nguyen, Khue G. and Mantooth, Siena M. and Vrabel, Maura R. and Zaharoff, David A.}, year={2022}, month={May} }
 @article{paidi_troncoso_harper_liu_nguyen_ravindranathan_rebello_lee_ivers_zaharoff_et al._2022, title={Raman spectroscopy reveals phenotype switches in breast cancer metastasis}, volume={12}, ISSN={["1838-7640"]}, DOI={10.7150/thno.74002}, abstractNote={The accurate analytical characterization of metastatic phenotype at primary tumor diagnosis and its evolution with time are critical for controlling metastatic progression of cancer. Here, we report a label-free optical strategy using Raman spectroscopy and machine learning to identify distinct metastatic phenotypes observed in tumors formed by isogenic murine breast cancer cell lines of progressively increasing metastatic propensities. Methods: We employed the 4T1 isogenic panel of murine breast cancer cells to grow tumors of varying metastatic potential and acquired label-free spectra using a fiber probe-based portable Raman spectroscopy system. We used MCR-ALS and random forests classifiers to identify putative spectral markers and predict metastatic phenotype of tumors based on their optical spectra. We also used tumors derived from 4T1 cells silenced for the expression of TWIST, FOXC2 and CXCR3 genes to assess their metastatic phenotype based on their Raman spectra. Results: The MCR-ALS spectral decomposition showed consistent differences in the contribution of components that resembled collagen and lipids between the non-metastatic 67NR tumors and the metastatic tumors formed by FARN, 4T07, and 4T1 cells. Our Raman spectra-based random forest analysis provided evidence that machine learning models built on spectral data can allow the accurate identification of metastatic phenotype of independent test tumors. By silencing genes critical for metastasis in highly metastatic cell lines, we showed that the random forest classifiers provided predictions consistent with the observed phenotypic switch of the resultant tumors towards lower metastatic potential. Furthermore, the spectral assessment of lipid and collagen content of these tumors was consistent with the observed phenotypic switch. Conclusion: Overall, our findings indicate that Raman spectroscopy may offer a novel strategy to evaluate metastatic risk during primary tumor biopsies in clinical patients.}, number={12}, journal={THERANOSTICS}, author={Paidi, Santosh Kumar and Troncoso, Joel Rodriguez and Harper, Mason G. and Liu, Zhenhui and Nguyen, Khue G. and Ravindranathan, Sruthi and Rebello, Lisa and Lee, David E. and Ivers, Jesse D. and Zaharoff, David A. and et al.}, year={2022}, pages={5351–5363} }
 @article{nguyen_wagner_vrabel_mantooth_meritet_zaharoff_2021, title={Safety and Pharmacokinetics of Intravesical Chitosan/Interleukin-12 Immunotherapy in Murine Bladders}, volume={7}, ISSN={["2352-3735"]}, DOI={10.3233/BLC-211542}, abstractNote={BACKGROUND: Intravesical administration of interleukin 12 (IL-12) co-formulated with the biopolymer, chitosan (CS/IL-12), has demonstrated remarkable antitumor activity against preclinical models of bladder cancer. However, given historical concerns regarding severe toxicities associated with systemic IL-12 administration in clinical trials, it is important to evaluate the safety of intravesical CS/IL-12 prior to clinical translation. OBJECTIVE: To evaluate the pharmacokinetics as well as the local and systemic toxicities of intravesical CS/IL-12 immunotherapy in laboratory mice. METHODS: Local inflammatory responses in mouse bladders treated with intravesical IL-12 or CS/IL-12 were assessed via histopathology. Serum cytokine levels following intravesical and subcutaneous (s.c.) administrations of IL-12 or CS/IL-12 in laboratory mice were compared. Systemic toxicities were evaluated via body weight and liver enzyme levels. RESULTS: Intravesical IL-12 and CS/IL-12 treatments did not induce significant local or systemic toxicity. IL-12 dissemination and exposure from intravesical administration was significantly lower compared to s.c. injections. Weekly intravesical CS/IL-12 treatments were well-tolerated and did not result in blunted immune responses. CONCLUSIONS: Intravesical CS/IL-12 is safe and well-tolerated in mice. In particular, the lack of cystitis and acute inflammation justifies continued investigation of intravesical CS/IL-12 immunotherapy in larger animals and patients with bladder cancer.}, number={4}, journal={BLADDER CANCER}, author={Nguyen, Khue G. and Wagner, Ethan S. and Vrabel, Maura R. and Mantooth, Siena M. and Meritet, Danielle M. and Zaharoff, David A.}, year={2021}, pages={427–437} }