Method of Use for Benzofuran Compounds

Abstract

An orally bioavailable benzofuran is provided which possesses in vitro and in vivo capabilities able to overcome issues in loading of antigenic peptide on major histocompatibility class I complexes, including on the surface of professional antigen presenting cells. The design of immunotherapies such as dendritic cell vaccines, optimal binding of the antigenic peptides to MHC class I complexes is a major challenge. Current therapeutic peptide loading is expensive, labor-intensive, or requires in vitro manipulation. Models demonstrate that the benzofuran enhances T-cell activation through increased peptide binding to cell surface MHC class I complexes. Molecular docking studies indicate the benzofuran binds the F pocket of MHC class I in a similar manner to high-affinity peptides and TAPBPR, aiding in the targeted loading of exogenous peptides. The therapeutic potential was demonstrated when using PLGA particles of BzF were injected intramuscularly, and significantly inhibited the development of E.G7-OVA tumors.

Claims

1. A method of modulating an immune response, comprising: altering an antigen-presenting cell by exposing the antigen-presenting cell with a therapeutically effective amount of a first compound and a therapeutically effective amount of a second compound; wherein the first compound is (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid; wherein the second compound is an antigen molecule; and contacting a lymphocyte with the antigen-presenting cell.

2. The method of claim 1, wherein the antigen-presenting cell is altered by: contacting the antigen-presenting cell with the first compound for a first compound incubation period; wherein the first compound incubation period is at least 15 minutes; and subsequently contacting the antigen-presenting cell with the second compound for a second compound incubation period after expiration of the first compound incubation period.

3. The method of claim 2, wherein the first compound incubation period is between 1 hour and 24 hours.

4. The method of claim 2, wherein the second compound incubation period is between 1 hour and 24 hours.

5. The method of claim 1, wherein the antigen-presenting cell is altered by: concurrently contacting the antigen-presenting cell with the first compound and with the second compound for a determined period of time.

6. The method of claim 1, wherein the antigen-presenting cell is altered in vivo by: administering the first compound to a patient orally, intravenously, or intramuscularly; and wherein the first compound contacts the antigen-presenting cell in vivo.

7. The method of claim 5, wherein the first compound and the second compound contact the dendritic cell ex vivo to form a primed dendritic cell, and wherein the method further comprises administering the primed dendritic cell to a patient in need thereof.

8. The method of claim 1, wherein the antigen molecule is a small molecule antigen vaccine, an epitope vaccine, a DNA vaccine, a recombinant DNA vaccine, a messenger RNA vaccine, a subunit vaccine, a recombinant vaccine, a conjugate vaccine, a immunotherapy cancer antigen, or a toxoid vaccine.

9. A method of enhancing a vaccine, comprising: altering the presentation of an antigen-presenting cell by contacting the antigen-presenting cell with a therapeutically effective amount of (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid and a therapeutically effective amount of the vaccine; and stimulating a lymphocyte by contacting the lymphocyte with the antigen-presenting cell.

10. The method of claim 9, wherein the antigen-presenting cell is stimulated by: contacting the antigen-presenting cell with the (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid for between 1 hour and 24 hours; and subsequently contacting the antigen-presenting cell with the vaccine for between 1 hour and 24 hours.

11. The method of claim 9, wherein the (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid contacts the antigen-presenting cell ex vivo or in vitro at between 5 g/mL and 100 g/mL.

12. The method of claim 9, wherein the vaccine contacts the antigen-presenting cell ex vivo or in vitro at between Ing/mL and 100 g/mL; and wherein the vaccine contacts the antigen presenting cell at a range of 1 ng/mL to 10 g/mL for a peptide vaccine, a range of 1 g/mL to 100 g/mL for a DNA vaccine, a range of 1 g/mL to 25 g/mL for a mRNA vaccine, or a range of 1 g/mL to 20 g/mL for a whole subunit protein vaccine.

13. The method of claim 9, wherein the antigen-presenting cell is a dendritic cell and wherein the dendritic cell is stimulated by: contacting the dendritic cell with the 2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid for between 1 hour and 24 hours; and subsequently contacting the dendritic cell with the vaccine for between 1 hour and 24 hours.

14. The method of claim 13, wherein the dendritic cell is contacted by the (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid ex vivo at between 5 g/mL and 100 g/mL.

15. The method of claim 13, wherein the dendritic cell is contacted by the vaccine ex vivo at between 1 ng/mL and 100 g/mL; and wherein the vaccine contacts the antigen presenting cell at a range of 1 ng/mL to 10 g/mL for a peptide vaccine, a range of 1 g/mL to 100 g/mL for a DNA vaccine, a range of 1 g/mL to 25 g/mL for a mRNA vaccine, or a range of 1 g/mL to 20 g/mL for a whole subunit protein vaccine.

16. The method of claim 10, further comprising: administering the stimulated lymphocyte to a patient in need of the vaccine; and administering (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid a second time, wherein the second time of administering (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid is orally, intravenously, intra-arterially, intrathecally, or intraventricularly to the patient.

17. A method of preparing an immunotherapy, comprising: obtaining an antigen-presenting cell from a patient in need thereof; exposing the antigen-presenting cell with a therapeutically effective amount of a first compound and a therapeutically effective amount of a second compound; wherein the first compound is (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid; wherein the second compound is an antigen molecule; incubating the antigen-presenting cell with the first compound and the second compound for a at least one hour to form a primed antigen-presenting cell; and injecting the primed antigen-presenting cell into the patient.

18. The method of claim 17, wherein the exposing step further comprises: contacting the antigen-presenting cell with the first compound for a first compound incubation period; and wherein the first compound incubation period is at least 15 min.

19. The method of claim 18, wherein the first compound incubation period is between 30 min and 2 h.

20. The method of claim 18, wherein the first compound is exposed to the antigen-presenting cell at between 1.62 g/mL and 50 g/mL.

21. The method of claim 18, wherein the second compound incubation period is between 1 hour and 24 hours.

22. A method of preparing an in vivo immunotherapy, comprising: testing dendritic cell populations and CD8+ t-cells of a patient in need of tumor treatment; confirming the dendritic cell populations and CD8+ t-cells of the patient in need of tumor treatment meets or exceeds a minimum threshold value; administering a therapeutically effective amount of an antitumor therapy to the patient in need of tumor treatment; wherein the antitumor therapy is chemotherapy, radiation, immunotherapy, hyperthermia, or photodynamic therapy; and administering a therapeutically effective amount of a first compound to the patient in need of tumor treatment orally, intravenously, intra-arterially, intrathecally, or intraventricularly; wherein the first compound is (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid.

23. The method of claim 22, wherein the first compound is administered concurrently with the antitumor therapy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

[0026] FIG. 1 is a line drawing showing the structure of (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid (BzF). Molecular weight: 188.18 g/mol.

[0027] FIG. 2 is a graph showing the evaluation of BzF toxicity to murine and human immortalized cell lines, and to murine primary splenocytes was examined 24 h after being treated with increasing doses of BzF. Shown is the percent cell viability as compared to untreated cells.

[0028] FIG. 3 is a graph showing BzF significantly enhances the ability of peptide pulsed DC2.4 to activate the CD8+ T cell hybridoma, B3Z. DC2.4 were treated with BzF as indicated for 6 h and then pulsed with 0.5 nM SIINFEKL peptide for 1 h. Co-treatment of B3Z with the treated DC2.4 for 19 h resulted in significant enhancement of B3Z activation at doses above 6.25 g/mL (p<0.001). Data shown is the mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0029] FIG. 4 is a graph showing BzF rapidly enhances the ability of DC2.4 to bind to peptide. Staining of DC2.4 cells with the MHC class I/SIINFEKL specific antibody (25-D1.16) shows that BzF is able to enhance binding of the peptide within 1 h (p<0.001). Data shown is the mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0030] FIG. 5 is a graph showing BzF significantly enhances the ability of peptide pulsed DC2.4 to activate the CD8+ T cell hybridoma, B3Z. BzF was found to significantly enhance peptide pulsed DC2.4 activation of B3Z cells as rapidly as 5 min (p<0.01). Data shown is the mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0031] FIG. 6 is a graph showing BzF significantly enhances the ability of peptide pulsed DC2.4 to activate the CD8+ T cell hybridoma, B3Z.Co-treatment of DC2.4 with 25 g/mL BzF plus 2-fold serial dilutions of SIINFEKL (starting at 1 nM) for 1 h, activated the B3Z cells to a significantly greater extent than did DC2.4 treated with only the peptide (p<0.0001, error bars shown but not visible). Data shown is the mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation. Open circles (untreated), grey squares (BzF treated).

[0032] FIG. 7 is a graph showing BzF significantly enhances the high dose peptide binding to DC2.4 as detected by antibody (25-D1.16). (p<0.0001), error bars shown but not visible). Open circles (untreated), grey squares (BzF treated).

[0033] FIG. 8 is a graph showing the effects of BzF on cross-presentation of an exogenous protein. DC2.4 cells co-treated for 6 h with increasing concentrations of BzF in the presence of 0.5 mg/mL OVA were able to significantly activate B3Z (p<0.001 for all doses tested). Data shown as mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0034] FIG. 9 is a graph showing the effects of BzF on cross-presentation of an exogenous protein. Co-treatment of freshly isolated murine splenocytes with concentrations of BzF >3.13 g/mL in the presence of 0.5 mg/mL OVA were able to significantly enhance activation of B3Z (p<0.0001). Treatment of the splenocytes overnight with BzF and then a 1 h pulse with 0.5 nM SIINFEKL also enhanced B3Z activation. Data shown as mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0035] FIG. 10 is a graph showing the effects of BzF on cross-presentation of an exogenous protein. B16F10 cells demonstrated a significant BzF dose dependent increase in the ability to activate the B3Z response when they were pulsed with 2 nM SIINFEKL (25 g P=0.0190, 50 g P=0.0017), but not when they were co-treated with BzF and 1 mg/mL OVA. A response similar to that generated by BzF was obtained from B16F10 cells treated with 10 mM (1.88 mg/mL) of the dipeptide, GL (p=0.0013). Data shown as mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0036] FIG. 11 is a graph showing the effects of BzF on cross-presentation of an exogenous protein. Treatment of DC2.4 with BFA along with either BzF or GL for 6 hrs affected B3Z activation by OVA, but not by the SIINFEKL peptide. The significance was calculated against the response of the cells in each treatment group that did not receive BFA. OVA associated activation was significantly inhibited by BFA treatment (vehicle p<0.001, BzF p=0.0442, GL p=0.0358). Data shown as mean of n3 experiments analyzed using a two-tailed t-test compared to control, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0037] FIG. 12A is a graph showing BzF time-dependently affects the overall surface expression of SIINFEKL-bound MHC class I. DC2.4 cells were treated for up to 4 h with g/mL BzF and then pulsed for 1 h with 0.5 nM SIINFEKL. These cells were then stained with the PE-label clone 25-D1.16 antibody specific for the MHC class I/SIINFEKL complex. Examination of the DC2.4 and IMG cells by flow cytometry revealed a significant enhancement (p<0.001 and 0.0001, respectively) of MHC class I/SIINFEKL complexes that increased over time. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0038] FIG. 12B is a graph showing BzF does not affect the overall surface expression of MHC class I. When identically treated DC2.4 cells, as outlined in FIG. 12A, were stained with the MHC class I H-2Kb specific antibody (AF6-88.5.5.3), no effect on the abundance of MHC class I was observed except at the 24 h time point. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0039] FIG. 13A is a graph showing BzF dose-dependently affects the overall surface expression of SIINFEKL-bound MHC class I on IMG cells. IMG cells were treated for up to 4 h with 25 g/mL BzF and then pulsed for 1 h with 0.5 nM SIINFEKL. These cells were then stained with the PE-label clone 25-D1.16 antibody specific for the MHC class I/SIINFEKL complex. Examination of the DC2.4 and IMG cells by flow cytometry revealed a significant enhancement (p<0.001 and 0.0001, respectively) of MHC class I/SIINFEKL complexes that increased over time. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0040] FIG. 13B is a graph showing BzF does not affect the overall surface expression of MHC class I on IMG cells. When identically treated IMG cells were stained with the MHC class I H-2Kb specific antibody (AF6-88.5.5.3), no effect on the abundance of MHC class I was observed except at the 24 h time point. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0041] FIG. 14 is a graph showing the effect of BzF is not due to modulation of MHC class I expression. Acid-stripped DC2.4 were treated with BzF or GL during recovery and showed no increase in MHC class I production. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05, * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation. Legend (grey squares=no treatment control; open circle=BzF; triangle=GL).

[0042] FIG. 15 is a graph showing the effect of BzF is not due to modulation of MHC class I expression. BFA treatment of DC2.4 following a 24 h exposure to BzF or GL and then a 1 h pulse with 30 nM SIINFEKL showed no increase in overall stability when compared to the untreated controls at t=0 decay. Legend (black squares=no treatment control; triangle=BzF; triangle=GL). Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05, * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0043] FIG. 16 is a graph showing BzF is able to displace peptide from surface MHC class I. The addition of 25 g/mL (132.2 M) BzF to DC2.4 cells after a 1 h pulse with 30 nM SIINFEKL revealed that BzF was able to significantly increase the rate of peptide turnover as compared to the untreated controls (p<0.0001). Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05, * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation. Legend (black squares=no treatment control; triangle=BzF; triangle=GL).

[0044] FIG. 17A is a graph showing that E.G7-OVA's endogenously produced SIINFEKL can activate B3Z cells. E.G7-OVA produces OVA endogenously and via processing is able to display the SIINFEKL peptide bound on its surface MHC class I H-2Kb. The resulting MHC class I/SIINFEKL complex can be presented to and activate B3Z cells. Coculture of 2-fold dilutions of E.G7-OVA cells with B3Z reveal the ability of E.G7-OVA to dose-dependently activate B3Z. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05, * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0045] FIG. 17B is a graph showing BzF is able to displace peptide from surface MHC class I. Treatment of E.G7-OVA cells with increasing concentrations of BzF resulted in a dose-dependent reduction of surface MHCI/SIINFEKL complexes as detected in the B3Z assay. This apparent displacement of SIINFEKL from the surface of E.G7-OVA was significant at all doses tested. The percent of SIINFEKL decay is based on 100% of bound peptide at t=0. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05, * P<0.05, **P<0.01, *** P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0046] FIG. 18 is a graph showing BzF facilitates binding of peptides toTrp2 H-2Kb. Pretreatment of DC2.4 with 50 g/mL BzF for 1 h, followed by a 1 h pulse with 0, 10, 100, or 1000 nM FITC-labeled melanoma Trp2 H-2Kb peptide, revealed enhancement of the direct binding of an H-2Kb peptide to DC2.4 cells.

[0047] FIG. 19 is a graph showing BzF facilitates binding of a peptide to Trp2 H-2Db. Treatment of DC2.4 with 25 g/mL BzF for 4 or 24 h prior to a 1 h pulse with the H-2Db N-terminal FITC-labeled HPV peptide, RAHYNIVTF, produced a significant increase in peptide binding (p=0.0250 and p=0.0339 respectively). Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, * P<0.05. Error bars represent the standard deviation.

[0048] FIG. 20A is a cartoon showing the peptide exchange assay. The design of the QuickSwitch assay is shown. Image from the user manual supplied by MBL International (MBL Int'l Corp., Woburn, MA).

[0049] FIG. 20B is a graph showing the peptide exchange assay. BzF is significantly better at displacing a pre-bound peptide from the binding groove than when the reaction is performed without an exchange factor (p<0.001) or with the provided exchange molecule (p=0.0013) on H-2Kb QuickSwitch.

[0050] FIG. 20C is a graph showing the peptide exchange assay, demonstrating the ability of BzF to rapidly facilitate peptide exchange on HLA-A*02:01. This response was found to be highly significant (p<0.001).

[0051] FIG. 21A is an image showing molecular docking predicts that BzF's ability to function as a peptide exchange factor is associated with binding within the F pocket of MHC H-2Kb. Molecular docking was performed using AutoDock and the structure coordinates of H-2Kb (PDB id: 39PL). BzF was predicted to bind within the F pocket of H-2Kb by forming hydrogen bonds with Thr80 and Lys146. BzF is shown within the F pocket of class I molecules and forming hydrogen bonds with key amino acids that regulate peptide binding within the pocket.

[0052] FIG. 21B is an image showing molecular docking predicts that BzF's ability to function as a peptide exchange factor is associated with binding within the F pocket of MHC H-2Kb. Molecular docking was performed using AutoDock and the structure coordinates of H-2Kb (PDB id: 39PL). BzF was predicted to bind within the F pocket of H-2Kb by forming hydrogen bonds with Thr80 and Lys146. BzF is shown within the F pocket of class I molecules and forming hydrogen bonds with key amino acids that regulate peptide binding within the pocket.

[0053] FIG. 22A is an image showing molecular docking predicts that BzF's ability to function as a peptide exchange factor is associated with binding within the F pocket of HLA-A*02:01. Molecular docking was performed using AutoDock and the structure coordinates of HLA-A*02:01 (PDB id: 3HLA). BzF was predicted to bind within the F pocket of HLA-A*02:01 by forming hydrogen bonds with just Lys146. BzF is shown within the F pocket of class I molecules and forming hydrogen bonds with key amino acids that regulate peptide binding within the pocket.

[0054] FIG. 22B is an image showing molecular docking predicts that BzF's ability to function as a peptide exchange factor is associated with binding within the F pocket of HLA-A*02:01. Molecular docking was performed using AutoDock and the structure coordinates of HLA-A*02:01 (PDB id: 3HLA). BzF was predicted to bind within the F pocket of HLA-A*02:01 by forming hydrogen bonds with just Lys146. BzF is shown within the F pocket of class I molecules and forming hydrogen bonds with key amino acids that regulate peptide binding within the pocket.

[0055] FIG. 23 is an image showing molecular docking predicts that BzF's ability to function as a peptide exchange factor is associated with binding within the F pocket of MHC H-2Kb and HLA-A*02:01. Molecular docking was performed using AutoDock and the structure coordinates of H-2Kb (PDB id: 3P9L) and HLA-A*02:01 (PDB id: 3HLA). BzF was predicted to bind within the F pocket of both class I molecules, H-2Kb and HLA-A*02:01, by forming hydrogen bonds with key amino acids that regulate peptide binding within the pocket. The predicted average free energy of binding (kcal/mol) for the top 3 poses of BzF and GL for docking with H-2Kb and HLA-A*02:01 is shown. Error bars represent the standard deviation derived from the top 3 poses.

[0056] FIG. 24A is a graph with inset line drawing showing BzF's unique structure is integral for the enhancement of T cell response. DC2.4 cells treated with molecule #2, that is structurally similar to BzF, failed to evoke activation of the B3Z response. Red arrows denote structural variance from BzF. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05.

[0057] FIG. 24B is a graph with inset line drawing showing BzF's unique structure is integral for the enhancement of T cell response. DC2.4 cells treated with molecule #3, that is structurally similar to BzF, failed to evoke activation of the B3Z response. Red arrows denote structural variance from BzF. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05.

[0058] FIG. 24C is a graph with inset line drawing showing BzF's unique structure is integral for the enhancement of T cell response. DC2.4 cells treated with molecule #4, that is structurally similar to BzF, failed to evoke activation of the B3Z response. Red arrows denote structural variance from BzF. Data shown as mean of n3 experiments shown and analyzed using a two-tailed t-test, for curves variance was measured through area under the curve, NS P>0.05.

[0059] FIG. 25 is a graph showing BzF's unique structure is integral for the enhancement of T cell response. None of the BzF analogs, molecules #2 through #4, were found to facilitate binding of SIINFEKL to DC2.4. BzF at 25 and 50 g/mL significantly enhance peptide binding (25 g/mL BzF p=0.0034). Data shown as mean of n3 experiments and analyzed using a two-tailed t-test for curves variance was measured through area under the curve, NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001. Error bars represent the standard deviation.

[0060] FIG. 26 is a graph showing BzF's unique structure is integral for the enhancement of T cell response. When compared to cells pulsed for 1 h with SIINFEKL (black squares), treatment with 25 g/mL of each analog (purple diamonds, green triangles, red circles) did not affect the rate of SIINFEKL loss from the surface of the DC2.4 cells. Data shown as mean of n3 experiments and analyzed using a two-tailed t-test for curves variance was measured through area under the curve, NS P>0.05.

[0061] FIG. 27A is a graph showing the molecular properties predicted by Swiss ADME for BzF and its analogs. All four molecules are predicted to be similarly lipophilic. The consensus Log P.sub.o/w is shown.

[0062] FIG. 27B is a graph showing the molecular properties predicted by Swiss ADME for BzF and its analogs. The molecules differ by the number of sp3 hybrid orbitals they contain. BzF has 0 of its 11 carbons, molecule #2 has 2 of its 11 carbons, molecule #3 has 4 of its 11 carbons, and molecule #4 has 1 of its 10 carbons with sp3 orbitals.

[0063] FIG. 27C is a graph showing the molecular properties predicted by Swiss ADME for BzF and its analogs. The predicted water solubility (expressed in mg of product soluble per mL of water) suggested that BzF and molecule #4 have similar water solubilities, while molecules #2 and #3 should be a bit more soluble.

[0064] FIG. 28A is a graph showing BzF can be detected in the systemic circulation following delivery by oral gavage, iv, and im injections. A representative HPLC chromatogram from the serum of mice receiving 1 mg BzF po (*), compared to the tracing generated by the stock solution of BzF (). The UV/VIS spectra of the BzF peaks at 30.5 and 30.7 min, respectively, is shown in the insert.

[0065] FIG. 28B is a graph showing BzF can be detected in the systemic circulation following delivery by oral gavage, iv, and im injections. Detection of BzF in the serum of mice that had received it by oral gavage, or iv or im injections compared to vehicle only.

[0066] FIG. 28C is a graph showing BzF can be detected in the systemic circulation following delivery by intramuscular injections. The relative concentrations of BzF detected in serum samples 1, 2, or 3 h post-delivery.

[0067] FIG. 29 is a graph showing in vivo administration of BzF and peptide increases peptide binding in vivo. Intravenous administration of 50 g SIINFEKL peptide immediately followed by oral gavage with 1, 2, or 4 mg (50, 100, 200 mg/kg, respectively) of BzF significantly increased the ability of splenocytes to activate B3Z cells ex vivo. Data shown as mean of n3 mice analyzed using a two-tailed t-test CI=95%, NS P>0.05, * P<0.05, ** P<0.01, ***P<0.001. Error bars represent the standard deviation.

[0068] FIG. 30 is a graph showing in vivo administration of BzF and peptide increases peptide binding in vivo. The ability to enhance the in vivo binding of SIINFEKL was demonstrated following iv injection of 50 g peptide and then either oral, iv, or im administration of BzF. Data shown as mean of n3 mice analyzed using a two-tailed t-test CI=95%, NS P>0.05, * P<0.05, ** P<0.01, ***P<0.001. Error bars represent the standard deviation.

[0069] FIG. 31 is a graph showing in vivo administration of BzF and peptide increases peptide binding in vivo. Characterization of the splenocytes by flow cytometry shows that BzF enhances SIINFEKL binding in the CD45+/MHCII+/CD11c+ population in vivo (p=0.0079). Data shown as mean of n3 mice analyzed using a two-tailed t-test CI=95%, NS P>0.05, * P<0.05, ** P<0.01, ***P<0.001. Error bars represent the standard deviation.

[0070] FIG. 32A is a graph showing BzF enhances loading of peptide at a dose compatible with the production of DC vaccines. Histograms of the peptide binding show that pre-treatment with BzF significantly enhanced SIINFEKL binding under conditions used to create peptide pulsed DC vaccines. BzF enhanced the number of SIINFEKL positive cells, but more significantly, it enhanced the amount of SIINFEKL per cell.

[0071] FIG. 32B is a graph showing BzF enhances loading of peptide at a dose compatible with the production of DC vaccines. DC2.4 cells (110.sup.6) were pre-treated for 1 h with 50 g/mL BzF in 200 uL HBSS/2% FCS) at 37 C., washed, and then pulsed for 1 h with 1, 5, or 10 g/mL SIINFEKL peptide. The cells were then stained with PE-labeled 25-D1.16 antibody at 10 C. for 30 min. Pre-treatment with BzF enhanced the binding of all three high doses of peptide commonly used to generate DC vaccines.

[0072] FIG. 33A is a graph showing immunization with DC2.4 cells treated in vitro with BzF and peptide enhances the resulting CD8+ antigen specific T cell response. Mice were vaccinated (primed then 1 week later they were boosted) by subcutaneous injection of 1.210.sup.6 IFN matured DC2.4 cells that had been exposed in vitro to 50 g/mL BzF for 2 h and then pulsed for 1 h with 1 g/mL SIINFEKL peptide. Two weeks later, the percent of SIINFEKL specific CD8+ T cells in the peripheral blood were determined. Data shown as mean of n3 mice per group analyzed using a two-tailed t-test CI=95%, NS P>0.05, * P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0073] FIG. 33B is a graph showing immunization with in vitro treated DC2.4 cells with BzF and peptide enhances the resulting CD8+ antigen specific T cell response. Mice were vaccinated (primed then 1 week later they were boosted) by subcutaneous injection of 1.210.sup.6 IFN matured DC2.4 cells that had been exposed in vitro to 50 g/mL BzF for 2 h and then pulsed for 1 h with 1 g/mL SIINFEKL peptide. Two weeks later, the percent of SIINFEKL specific CD8+ T cells in the splenocyte population were determined. Data shown as mean of n3 mice per group analyzed using a two-tailed t-test CJ=95%, NS P>0.05, * P<0.05, ** P<0.01, ***P<0.001, **** P<0.0001. Error bars represent the standard deviation.

[0074] FIG. 34 is a graph showing PLGA encapsulated BzF enhances development of an anti-tumor response. Immunization of female C57BL/6crl mice with PLGA particles encapsulating whole ovalbumin, a mixture of empty and OVA containing particles (OVA/Empty), or a mixture of particles containing OVA or BzF (OVA/BzF) by intramuscular injection. The mice were then boosted with the same mixtures 10 days later. Ten days post the booster injections, 110.sup.6 E.G7-OVA tumor cells were injected subcutaneously. Tumor growth was monitored every other day, and the width and lengths were measured. The tumor volumes were calculated as the (greatest width{circumflex over ()}2greatest length)/2.

[0075] FIG. 35 is a graph showing PLGA encapsulated BzF enhances development of an anti-tumor response. Immunization of female C57BL/6crl mice with PLGA particles encapsulating whole ovalbumin, a mixture of empty and OVA containing particles (OVA/Empty), or a mixture of particles containing OVA or BzF (OVA/BzF) by intramuscular injection. The mice were then boosted with the same mixtures 10 days later. Ten days post the booster injections, 110.sup.6 E.G7-OVA tumor cells were injected subcutaneously. Tumor volumes obtained at 13 days post tumor cell injection are shown. ns=non-significant; **=p0.01.

DETAILED DESCRIPTION OF THE INVENTION

[0076] As used herein, the singular forms a, an and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a compound includes a mixture of two or more compounds and the like, unless specified to the contrary.

[0077] As used herein, about means approximately or nearly and in the context of a numerical value or range set forth means+/15% of the numerical.

[0078] The term administration, administering, and variants thereof (e.g., administering a compound) is used throughout the specification to describe the delivery or introduction of the compound or a prodrug of the compound into the system of the animal in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents, administration and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents.

[0079] Compounds of the subject invention be administered a number of ways including, but not limited to, oral, parenteral (such term referring to intravenous and intra-arterial as well as other appropriate parenteral routes), intrathecal, intraventricular, intraparenchymal (including into the spinal cord, brainstem or motor cortex), intracisternal, intracranial, intrastriatal, intranigral, and transdermal, among others which term allows a compound of the subject invention to be carried to the ultimate target site where needed. A compound of the subject invention can be administered in the form of active compound, admixtures, or compositions thereof. The compositions according to the present invention may be used without adjuvant, without bio-absorption enhancing agent, without diluent. Alternatively, compositions according to the present invention may include one or more of the adjuvant, bio-absorption enhancing agent, and diluent.

[0080] Administration will often depend upon the type of vaccine, such as dendritic cell vaccines as compared to small molecule antigen vaccines, epitope vaccines, DNA vaccines, recombinant DNA vaccines, messenger RNA (mRNA) vaccines, subunit vaccines, recombinant vaccines, conjugate vaccines, or toxoid vaccines, as well as the disease or condition targeted by the vaccine. For example, administration may preferably be via administration into the cerebral spinal fluid or by direct administration into the affected tissue in the brain, intratumoral for solid cancers/proliferative disorders, or be via a parenteral route, for example, intravenously, for systemic diseases.

[0081] The therapeutic compound is administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight, and other factors known to medical practitioners.

[0082] As used herein animal means a multicellular, eukaryotic organism classified in the kingdom Animalia or Metazoa. The term includes, but is not limited to, mammals. Non-limiting examples include rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms animal or mammal or their plurals are used, it is contemplated that it also applies to any animals.

[0083] The terms comprising, consisting of and consisting essentially of are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.

[0084] The terms isolated or biologically pure refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state. Preferably, the compound of the invention, (2E)-3-(1-benzofuran-5-yl)prop-2-enoic acid, is administered in an isolated or pure form.

[0085] The term incubation period as used herein refer to a period of time in which a cell is exposed to an external influence, either another cell type or compound. More particularly, the incubation period can refer to exposure of an antigen presenting cell to one or more compounds to elicit a change in the antigen loading or exposure of a leukocyte to an antigen presenting cell.

[0086] As used herein, the term modulation and variations thereof refers to a change of amount of immune response to a stimuli when compared to the amount or quality of activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in chemokine release. As further an example, the modulation includes an increase in t-cell responsiveness to an antigen stimulus, resulting in a change in the absolute or relative amount of a t-cell response.

[0087] As used herein, the term therapeutically effective amount refers to concentrations or amounts of components of the invention that enhance an active vaccine compound or agent, with or without other adjuvants, to elicit the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. In reference to a disease, illness, condition, infection, an effective amount comprises an amount sufficient to prevent or ameliorate the disease. In some embodiments, an effective amount is an amount sufficient to delay development of disease. The therapeutically effective amount can, when used for proliferative disorder therapy, result in the amelioration of cancer or other proliferative disorders or one or more symptoms thereof, prevent advancement of cancer or other proliferative disorder, or cause regression of cancer or other proliferative disorder.

[0088] A therapeutically effective amount of the therapeutic compound or a pharmaceutically acceptable salt, hydrate, or solvate thereof refers to that amount being administered which will enhance, to some extent, a vaccine. Nonlimiting examples of vaccines include dendritic cell vaccines, small molecule antigen vaccines, epitope vaccines, messenger RNA (mRNA) vaccines, DNA vaccines, recombinant DNA vaccines, subunit vaccines, recombinant vaccines, conjugate vaccines, or toxoid vaccines. In reference to the treatment of a proliferative cellular disorder, a therapeutically effective amount refers to the amount which: (1) reduces the size of a tumor, (2) inhibits (i.e. stopping or slowing to some extent) tumor metastasis, (3) inhibits (i.e. stopping or slowing to some extent) tumor growth, or (4) inhibits (i.e. stopping or slowing to some extent) cellular proliferation.

[0089] A prophylactically effective amount refers to concentrations or amounts of components such as the compound(s) of the invention along with an active vaccine compound or agent, with or without other adjuvants, that are sufficient to result in the prevention, recurrence, or spread of disease. The prophylactically effective amount may refer to the amount sufficient to prevent initial disease, recurrence or spread of the disease or the occurrence of the disease in a patient, including, but not limited, to patients particularly susceptible to the disease, or occurrence of disease in another patient, i.e. spread of disease.

[0090] As used herein, treatment refers to obtaining beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of infection, stabilization (i.e., not worsening) of the state of infection, preventing or delaying spread of the disease (such as pathogen growth or replication), preventing or delaying occurrence or recurrence of the disease, delay or slowing of disease progression and amelioration of the disease state. The methods of the invention contemplate any one or more of these aspects of treatment. The term treatment, as used in this definition only, is intended to mean that regiment described is continued until the underlying disease is resolved, whereas therapy requires that the regiment alleviate one or more symptoms of the underlying disease. Prophylaxis means that regiment is undertaken to prevent a possible occurrence, such as where a pre-cancerous lesion is identified.

[0091] The term patient is used herein to describe members of the animal kingdom, such as but not milted to primates including humans, gorillas and monkeys; rodents, such as mice, fish, reptiles and to whom treatment, including prophylactic treatment, with the composition(s) according to the present invention, is provided. The patient may be any animal requiring therapy, treatment, or prophylaxis, or any animal suspected of requiring therapy, treatment, or prophylaxis. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal.

[0092] As used herein, the term proliferative disorders broadly encompasses any neoplastic disease(s) including those which are potentially malignant (pre-cancerous) or malignant (cancerous) and covers the physiological condition in mammals that is typically characterized by unregulated cell growth. The term therefore encompasses the treatment of tumors. Examples of such proliferative disorders include cancers such as carcinoma, lymphoma, blastoma, sarcoma, and leukemia, as well as other cancers disclosed herein. The compositions disclosed herein are useful for treating all types of cancer, and include breast cancer; ovarian cancer, multiple myeloma tumor specimens, pancreatic cancer and blood malignancies, such as acute myelogenous leukemia, (Turkson, et al., U.S. Pat. No. 8,609,639; Jove, et al., WO 00/44774), multiple myeloma, acute myelogenous leukemia (Dalton, et al., PCT/US2000/001845), head and neck cancer, lung cancer, colorectal carcinoma, prostate cancer, melanoma, sarcoma, liver cancer, brain tumors, multiple myeloma, leukemia, cervical cancer, colorectal carcinoma, liver cancer, gastric cancer, prostate cancer, renal cell carcinoma, hepatocellular carcinomas, gastric cancers, and lymphomas (Li, et al., U.S. application Ser. No. 12/677,513), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, a seminoma, an embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, a glioma, an astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma; acute lymphocytic leukemia, acute myelocytic leukemia, chronic leukemia, and polycythemia vera (Jove, et al., U.S. application Ser. No. 10/383,707).

[0093] A safe and effective amount refers to the quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.

[0094] A pharmaceutically acceptable component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

[0095] The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions.

[0096] As used herein, the phrase pharmaceutically acceptable carrier means any of the standard pharmaceutically acceptable carriers, such as a solvent, suspending agent or vehicle, for delivering the compound or compounds in question to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Liposomes, micelles, FDA-approved poly(lactic-co-glycolic acid) (PLGA) microparticles and PLGA nanoparticles are also a pharmaceutical carrier. Examples of carriers include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be a solvent or dispersing medium containing, for example, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Formulations are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Sciences (Martin E W [1995] Easton Pa., Mack Publishing Company, 19.sup.th ed.) describes formulations which can be used in connection with the subject invention. The carrier includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Formulations suitable for parenteral administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question. The pharmaceutical composition can be adapted for various forms of administration. Administration can be continuous or at distinct intervals as can be determined by a person skilled in the art.

[0097] The compounds of the present invention include all hydrates and pharmaceutically acceptable salts of the propanoic acids that can be prepared by those of skill in the art, for example by reacting the inventive compound with a sufficiently basic compound, such as an amine, affording a physiologically acceptable anion. Under conditions where the compounds of the present invention are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Suitable inorganic salts may also be formed by reaching the compound with a basic compound, such as a basic salt of ammonium, calcium magnesium, potassium, or sodium, such as ammonium bicarbonate. When reference is made to a compound or administering a compound, the recitation of the compound includes a pharmaceutically acceptable salt thereof.

[0098] The compounds of the present invention can be formulated as pharmaceutical compositions and administered to a patient, such as a human patient, in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical, or subcutaneous routes.

[0099] Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard- or soft-shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0100] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0101] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water or other suitable solvent, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0102] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0103] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient presenting the previously sterile-filtered solutions.

[0104] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from adsorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0105] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver the compounds of the invention to the skin are disclosed in Jacquet, et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith, et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0106] Useful dosages of the compounds of the present invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art (See, Borch et al., U.S. Pat. No. 4,938,949).

[0107] Accordingly, the invention includes a pharmaceutical composition comprising a compound of the present invention as described above, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount of one or more compounds effective to treat a bacterial infection, are a preferred embodiment of the invention.

[0108] This invention addresses the severe need for new classes of adjuvant compounds to enhance vaccine treatment. The invention is now further described by way of the following examples, which, while illustrative of the invention, are not intended as limitations to the scope. It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

[0109] Two-tailed t-tests were conducted when comparing values against control only ANOVA was utilized when measuring variance between sample groups. Bonferroni's post-test was utilized to test statistical significance. GraphPad Prism 4 and 8 were used for analysis. NS P>0.05, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, error bars represent standard deviation.

[0110] Using the ovalbumin peptide, SIINFEKL.sub.257-264, an antibody recognizing SIINFEKL bound to MHC class I, and SIINFEKL-specific tetramers, the ability of BzF to enhance peptide exchange on both the human HLA-A2 and murine H-2Kb MHC class I complexes is revealed. Molecular docking studies predict that BzF could interact with the F-pocket of MHC class I in a way that would induce H-2Kb and HLA-A*02-01 peptide receptive states. This mechanism of action would allow peptides to bind with increased efficiency and could potentially enhance the efficacy of peptide-pulsed DC vaccines. Herein, BzF's ability to enhance peptide exchange in vitro and in vivo is demonstrated. The unmet need for the identification and development of bioavailable molecules capable of modulating antigen specific immune responses, motivated a search for non-peptide-based compounds proficient at enhancing the in vitro and in vivo presentation of antigen and/or antigen-specific T cell activation.

Example 1: Initial Screening

[0111] The initial screening process utilized the immortalized murine dendritic cell line, DC2.4 as the antigen-presenting cell and was chosen because it closely models primary bone marrow-derived dendritic cell responses and can readily present MHC class I antigens to CD8+ T cells. (Shen, et al., Cloned dendritic cells can present exogenous antigens on both MHC class I and class II molecules. J Immunol. 1997 Mar. 15; 158(6):2723-30; Lin, et al., Soluble CD83 alleviates experimental autoimmune uveitis by inhibiting filamentous actin-dependent calcium release in dendritic cells. Fron Immunol. 2018 Jul. 9; 9:1567). The T cell hybridoma cell line, B3Z, is a CD8+ SIINFEKL peptide specific T-cell hybridoma expressing a beta-galactosidase (-gal) reporter gene driven by NF-AT elements on the interleukin 2 (IL-2) promoter (Karttunen, et al., Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci USA. 1992 Jul. 1; 89(13):6020-4). Activation of B3Z leads to production of 1-gal whose presence can be quantified using the optimized detection assay described by Jendresen et al. (Jendresen, et al., An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells. J Pharmacol Toxicol Methods. 2018 March-April; 90:67-75).

[0112] The DC2.4 cell line, provided by Dr. Kenneth Rock (University of Massachusetts). was routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems, Inc., Minneapolis, MN; cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. The B3Z.A3 cell line, provided by Dr. Nilabh Shastri (University of California Berkeley, Office of Technology Licensing) was cultured in RPMI 1640+GlutaMAX-1 (Gibco; Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol (B-Me) and 1 mM sodium pyruvate (Gibco, cat. no. 11360-070). All culture media included 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals, R&D Systems, Inc., Minneapolis, MN; cat. no. 512450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0113] H-2K.sup.b OVA.sub.257-264 peptide, SIINFEKL (cat. no. AS-60193-1) was purchased from AnaSpec Inc (Fremont, CA). Chlorophenol red--D-galactopyranoside (CPRG, cat. no. 10884308001) was purchased from Sigma Chemical Co (St. Louis, MO). CPRG stock was prepared to 175 g/mL in sterile H.sub.2O and a working solution of 1.75 g/mL was used.

[0114] DC2.4 cells (2.510.sup.4 cells/well) were seeded at 100 L/well in 96 well tissue culture white sided/white bottom plate and incubated overnight to allow for cell adherence. 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. Following pre-treatment of the DC2.4 cells (1-24 h) with escalating doses of the test molecules, the cells were washed, pulsed with 0.5 nM of the MHC class I H-2Kb peptide, SIINFEKL, for 1 h, and then co-cultured with the CD8+ T cell hybridoma, B3Z.

[0115] The level of B3Z cell activation and the effects of the various test molecules on B3Z activation were quantified in cell lysates using the -gal chromogenic substrate, CPRG. The media was removed and 100 L of 250 M CPRG reconstituted in the lysis buffer (1DPBS, 0.2% v/v saponin, 20 mM magnesium chloride) was added directly to each well. Cells were maintained at 37 C. prior to measurement. The absorbance at 560 nm (700 nm for background) was intermittently monitored using a BioTek Synergy HTX multimode microplate reader (Winooski, VT) to detect -gal activity. CPRG lysis buffer and B3Z cells only served as the negative controls. All treatments were performed in triplicate and all experiments were repeated a minimum of three times.

[0116] This screening process (manuscript in preparation) led to the identification of BzF, (3-(1-benzofuran-5-yl)prop-2-enoic acid), seen in FIG. 1, as a promising hit.

Example 2: Toxicity

[0117] BzF was then examined for its effect on the viability of several murine and human cell lines and primary murine splenocytes. The cytotoxicity of BzF was assessed using the CellTiter-Glo colorimetric assay system according to manufacturer's instructions to determine BzF's effect on the viability of several murine and human cell lines and primary murine splenocytes.

[0118] DC2.4 cell line (Dr. Kenneth Rock, University of Massachusetts), RAW 264.7 cell line (American Type Culture Collection (ATCC), Manassas, VA; TIB 71), and IMG cell line (Millipore, Merck KGaA, Burlington, MA; cat. no. SC-C134) were each cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco; cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems, Inc., Minneapolis, MN; cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. HEK293T was cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0119] Six- to ten-week-old female C57BL/6NCrl mice (Charles River Laboratories, Wilmington, MA; cat. no. 027) were maintained in accord with the NIH Guide for the Care and Use of Laboratory Animals. Mice were maintained in an INOVIVE individually ventilated cage system with 3 mice per cage containing 100% virgin wood pulp bedding (Harlan Teklad, Inotiv Inc., West Lafayette, IN; cat. no. 7099). Water and food (Harlan Teklad, Inotiv Inc., West Lafayette, IN; cat. no. 8640) were available ad libitum. Spleens were collected in 4 mL of Hanks Balanced Salt Solution (HBSS) and converted to a single cell suspension using sterile pouches and a Steward Stomacher 80 Biomaster Blender (Fisher Scientific, cat. no. 14-285-27). The resulting cell suspension was pelleted and suspended in 4 mL freshly prepared red blood cell lysis buffer (150 mM NH.sub.4Cl, 10 mM NaHCO.sub.3, 0.1 mM EDTA in sterile distilled H.sub.2O) for 1-3 minutes. The splenocytes were pelleted and filtered through 70 M EASY strainer filters (USA Scientific, cat. no. 5654-2070). The cell count was determined using the MUSE cell viability kit (MCH600103) and MUSE Cell Analyzer (Luminex Corp, Austin TX).

[0120] Murine splenocytes were cultured in complete RPMI (cRPMI) comprised of RPMI 1640+GlutaMAX-1 (Gibco; cat. no. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol, 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals, R&D Systems, Inc., Minneapolis, MN; cat. no. 512450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0121] The cytotoxicity of BzF was assessed using the CellTiter-Glo cell colorimetric viability assay system PI; Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. P3566). according to manufacturer's instructions. Briefly, immortalized cells or murine splenocytes were plated in a volume of 100 L into 96-well white flat-bottom tissue-culture plates at 110.sup.4 or 510.sup.4 cells per well, respectively, and incubated at 37 C., 5% CO.sub.2 overnight for adherence. Two-fold serial dilutions of BzF were prepared in cDMEM and triplicate wells of cells were treated at each dose for 7, 24 or 48 h. CellTiter-Glo was added 1:1 in the media and the level of chemiluminescence was measured using luminescence at wavelengths of 200 nm to 700 nm through a photomultiplier on BioTek's Synergy HTX multimode microplate reader. All microplate viability measurements are reported as a percentage of vehicle only control cells.

[0122] It was found that across all the cell lines examined, except HEK293T, 24 h exposure to BzF at concentrations lower than 50 g/mL (266 M) had small effects on cell viability, as seen in FIG. 2. The viability of primary murine splenocytes was also minimally affected at concentrations below 50 g/mL, as seen in FIG. 2. The IC50 for cell viability was calculated to be 1151 M for DC2.4; 432 M for HEK 293; 316 M for IMG; 783 for RAW 264.7; and 402 M for freshly isolated murine splenocytes. Based on these findings, all subsequent assays utilized doses of BzF less than or equal to 266 M (50 g/mL).

Example 3: BzF Enhances APC Presentation of an Exogenous Peptide to Antigen-Specific T Cells

[0123] DC2.4 cells, provided by Dr. Kenneth Rock (University of Massachusetts), were routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. B3Z.A3 cells, provided by Dr. Nilabh Shastri (University of California Berkeley, Office of Technology Licensing), were cultured in RPMI 1640+GlutaMAX-1 (Gibco, cat. no. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol (B-Me) and 1 mM sodium pyruvate (Gibco, cat. no. 11360-070). Murine splenocytes were cultured in complete RPMI (cRPMI) comprised of RPMI 1640+GlutaMAX-1 (Gibco, cat. no. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol. All culture media included 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals S12450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity. BzF ((2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid) (CAS no. 1210059-73-6, cat. no. CSSB00016286531)(SMILES OC(=O)/C=C\c1ccc2c(c1)cco2) was purchased from Chemspace (Monmouth Junction, NJ), and SIINFEKL (AnaSpec Inc., Fremont, CA; cat. no. AS-60193-1). The compound (2E)-3-(1,3-benzodioxol-5yl)acrylic acid (cat. no. 146242) and chlorophenol red--D-galactopyranoside (CPRG, cat. no. 10884308001) were purchased from Sigma Chemical Co (St. Louis, MO). CPRG stock was prepared to 175 g/mL in sterile H.sub.2O and a working solution of 1.75 g/mL was used.

[0124] Activation of B3Z T cell hybridoma cells was assessed following their exposure to various MHC class I H-2K.sup.b positive cell lines presenting the SIINFEKL peptide. B3Z are a CD8+ SIINFEKL peptide specific T-cell hybridoma expressing a beta-galactosidase (-gal) reporter gene driven by NF-AT elements on the interleukin 2 (IL-2) promoter. (Karttunen, et al., Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci USA. 1992 Jul. 1; 89(13):6020-4). Activation of B3Z leads to production of -gal whose presence can be quantified using the optimized detection assay described by Jendresen et al. (Jendresen, et al., An improved CPRG colorimetric ligand-receptor signal transduction assay based on beta-galactosidase activity in mammalian BWZ-reporter cells. J Pharmacol Toxicol Methods. 2018 March-April; 90:67-75).

[0125] To stimulate -gal production in the B3Z, the H-2Kb murine cell lines, DC2.4, B16F10, and E.G7-OVA, or freshly isolated murine splenocytes, were plated at a density of 2.5 or 510.sup.4 cells/well (100 L/well in 96 well plates) and cultured overnight at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. The cells were washed with DPBS and then treated for 1-24 h with the indicated concentrations of BzF suspended in complete media. Vehicle controls were always included.

[0126] Following the pretreatment with BzF, the media was removed from DC2.4 cells or was removed following pelleting of nonadherent cells in the plate. The cells were then pulsed with various concentrations of SIINFEKL peptide (0.5 nM) for the indicated lengths of time (1 h if not otherwise indicated). The cells were washed and then 100 L of complete DMEM containing 510.sup.4 B3Z cells was added. The co-culture was incubated at 37 C. for 19 h to allow for B3Z activation.

[0127] To detect activation of the B3Z cells, the media was removed and 100 L of 250 M CPRG reconstituted in the lysis buffer (1DPBS, 0.2% v/v saponin, 20 mM magnesium chloride) was added directly to each well. Cells were maintained at 37 C. prior to measurement. The absorbance at 560 nm (700 nm for background) was intermittently monitored using a BioTek Synergy HTX multimode microplate reader (Winooski, VT) to detect -gal activity. CPRG lysis buffer and B3Z cells only served as the negative controls. All treatments were performed in triplicate and all experiments were repeated a minimum of three times.

[0128] When 100 L of DC2.4 cells (510.sup.5/mL) were pretreated in 96 well plates with up to 25 g/mL (133 M) BzF for 6 h and then co-cultured for 19 h with an equal number of B3Z cells, doses greater than 3.13 g/mL (17 M) were found to significantly enhance B3Z cell activation, seen in FIG. 3 (p<0.001). Direct treatment of B3Z cells with BzF alone had no effect on cell viability or cell activation (data not shown). Examining the kinetics of the BzF activity revealed it capable of significantly enhancing binding of SIINFEKL to DC2.4 cells in less than 1 h, seen in FIG. 4, and activating B3Z in as little as 5 minutes, seen in FIG. 5. Such a rapid effect suggests that BzF likely functions by facilitating binding of peptides within the MHC class I complex.

[0129] To visualize the magnitude of effect the BzF has on the B3Z response, DC2.4 cells were co-treated for 1 h with low concentrations (15 pM to 1 nM) of SIINFEKL in the presence of 25 g/mL BzF. After a 19 h coculture with B3Z, the BzF response was found to be significantly greater than that obtained with peptide pulsed DC2.4 alone, seen in FIG. 6 (p<0.001).

[0130] To detect binding of peptides to MHC class I H2Kb molecules on various murine cells, the cells were cultured as previously described and treated for various times as indicated in results and figure legends. Cells were centrifuged and washed using cold FACS buffer (DPBS with 5% FBS, 2 mM EDTA, 0.1% sodium azide). Cells were blocked using Fc block (bioLegend; cat. no. 101320) for 10 min to 15 min to inhibit non-specific binding, followed by staining with fluorophore-conjugated antibody PE-labeled anti-MHC I/SIINFEKL (clone 25-D1.16; Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 12-5743-82), for 30 min at 4 C. After washing, the cells were subjected to flow cytometric analysis with the Attune NxT Flow Cytometer (Invitrogen). Cells were gated based on live single-cell populations and positives were assessed using isotype controls and fluor-minus-one analysis when needed. Data were analyzed using the Attune NxT software or FlowJo versions 10.7 and 10.8.

[0131] To evaluate the potential of BzF to enhance peptide loading under conditions used to prepare DC vaccines, 110.sup.6 DC2.4 cells were pretreated with 50 g/mL BzF for 1 h in a total volume of 200 L OptiMEM, then pulsed for 1 h with concentrations of SIINFEKL (1-10 g/mL) often used for the in vitro production of DC vaccines. The cells were then stained for 30 min in 200 L HBSS/2% FCS containing 0.4 g PE-labeled MHC class I/SIINFEKL specific antibody, 25-D1.16. The cells were then examined using flow cytometry which revealed significant enhancement of peptide binding at all three doses of BzF, seen in FIG. 7. These results demonstrate the significant ability of BzF to enhance binding of peptide to MHC class I on the surface of APC, which leads to enhanced antigen presentation to and activation of the CD8+ T cell hybridoma, B3Z.

Example 4: Effects of BzF on Antigen Cross-Presentation by DC2.4 and Primary Splenocytes

[0132] BzF was analyzed to determine if the compound could be affecting cross-presentation. To this end, DC2.4 cells were analyzed with OVA and SIINFEKL. It has been shown that the whole OVA enters the cell primarily through the mannose receptor and is then digested through proteasome degradation, prepared through the endoplasmic reticulum and Golgi, or via endosomal processing prior to the resulting peptide fragments being presented on the cell surface. (Blum, et al., Pathways of antigen processing. Ann Rev Immunol. 2013; 31:443-73; Belizaire & Unanue, Targeting proteins to distinct subcellular compartments reveals unique requirements for MHC class I and II presentation. Proc Natl Acad Sci USA. 2009 Oct. 13; 106(41):17463-8). In contrast, the exogenously supplied SIINFEKL peptide can passively bind the cell surface MHC class I; therefore, does not need to enter the intracellular processing pathway. However, it has been reported that a small amount of SIINFEKL does enter the cell and is presented through canonical MHC class I presentation. (Day, et al., Direct delivery of exogenous MHC class I molecule-binding oligopeptides to the endoplasmic reticulum of viable cells. Proc Natl Acad Sci USA. 1997 Jul. 22; 94(15):8064-9; Spiliotis, et al., Selective export of MHC class I molecules from the ER after their dissociation from TAP. Immunity. 2000 December; 13(6):841-51).

[0133] DC2.4 cells, provided by Dr. Kenneth Rock (University of Massachusetts) and B16F10 (CRL-6475; American Type Culture Collection (ATCC); cat. no. CRL-6475), were routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. B3Z.A3 cells, provided by Dr. Nilabh Shastri (University of California Berkeley, Office of Technology Licensing), were cultured in RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol (B-Me) and 1 mM sodium pyruvate (Gibco, cat. no. 11360-070). Murine splenocytes were cultured in complete RPMI (cRPMI) comprised of RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol. All culture media included 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals 512450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0134] BzF ((2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid) (CAS no. 1210059-73-6, cat. no. CSSB00016286531)(SMILES OC(=O)/C=C\c1ccc2c(c1)cco2) was purchased from Chemspace (Monmouth Junction, NJ). Chicken ovalbumin (OVA, grade V) was purchased from Sigma (cat. no. A5503). Glycyl-L-leucine (cat. no. G0181) was purchased from TCI America (Portland, OR).

[0135] DC2.4 cells (510.sup.5 cells/ml) were seeded at 100 L/well in 96 well plates and cultured overnight. The cells were washed with DPBS, and co-exposed to 0.5 mg/mL soluble whole OVA, along with increasing concentrations of BzF for 6 h, The cells were then co-cultured with equal number of B3Z cells at 37 C. for 19 h. Co-treatment of DC2.4 with increasing amounts of BzF and 0.5 mg/mL of OVA for 6 h were found to significantly enhance the B3Z response, seen in FIG. 8 (p<0.001 for all doses tested).

[0136] Murine splenocytes were plated at a density of 2.5 or 510.sup.4 cells/well (100 L/well in 96 well plates) and cultured overnight in cRPMI. The cells were washed with DPBS and then treated for 1-24 h with the indicated concentrations of BzF or co-treated with the indicated concentrations of BzF and 0.5 mg/mL OVA, suspended in complete media. Cells treated with BzF only were pulsed with 0.5 nM SIINFEKL pulsed for 1 h. The cells were then co-cultured with equal number of B3Z cells at 37 C. for 19 h. Co-treatment of murine splenocytes with increasing concentrations of BzF and 0.5 mg/mL OVA for 24 h enhanced their ability to activate B3Z, as seen in FIG. 9 (p<0.0001 for doses >6.25 g/mL). Likewise, pre-treatment of murine splenocytes for 24 h with increasing concentrations of BzF, followed by pulsing them with 0.5 nM SIINFEKL for 1 h also enhanced their ability to activate the B3Z, seen in FIG. 9 (p<0.0001 for doses >12.5 g/mL).

[0137] These observations suggest that BzF might either be affecting some aspects of cross-presentation, or more likely, stabilizing the surface expression of the MHC class I/peptide complex. This experiment was repeated using the murine melanoma cell line, B16F10, as the APC.

[0138] B16F10 were plated at a density of 2.5 or 510.sup.4 cells/well (100 L/well in 96 well plates) and cultured overnight in cDMEM. The cells were washed with DPBS and then treated for 1-24 h with the indicated concentrations of BzF or co-treated with the indicated concentrations of BzF and 1.0 mg/mL OVA, suspended in complete media. Cells treated with BzF only were pulsed with 2 nM SIINFEKL pulsed for 1 h. The cells were then co-cultured with equal number of B3Z cells at 37 C. for 19 h.

[0139] Since these cells lack the machinery for cross-presentation (Madden, The three-dimensional structure of peptide-MHC complexes. Ann Rev Immunol. 1995; 13(1):587-622), it was not surprising that co-treatment with BzF and 1 mg/mL OVA failed to activate the B3Z cells. However, since they express adequate levels of surface MHC class I H-2Kb, they were able to significantly activate B3Z following pre-treatment with BzF and a 1 h pulse with 2 nM SIINFEKL, as seen in FIG. 10 (25 g p=0.0190, 50 g p=0.0017).

[0140] In this experiment the activity of the dipeptide, GL, was also examined, as it has been previously reported to function as a peptide exchange factor. (Saini, et al., Dipeptides catalyze rapid peptide exchange on MHC class I molecules. Proc Natl Acad Sci USA. 2015 Jan. 6; 112(1):202-7; Saini, et al., Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells. Sci Immunol. 2019 Jul. 19; 4(37):eaau9039). B16F10 were plated at a density of 2.5 or 510.sup.4 cells/well (100 L/well in 96 well plates) and cultured overnight in cDMEM. The cells were washed with DPBS and then co-treated for 24 h with 10 mM (1.88 mg/mL) of GL and 1.0 mg/mL OVA or were pulsed with 2 nM SIINFEKL for 1 h following treatment with 10 mM GL in complete media. The cells were then co-cultured with an equal number of B3Z cells at 37 C. for 19 h.

[0141] Like BzF, GL had no effect on the uptake or processing of OVA but was able to significantly enhance B3Z activation by SIINFEKL pulsed splenocytes, as seen in FIG. 10 (p=0.0013).

[0142] To further investigate the potential role of cross-presentation in BzF's ability to enhance the B3Z response, cells were exposed to various cellular pathway inhibitor molecules. A list of the small molecule inhibitors used in the assessment of BzF's mechanism of action is summarized in Table 1. The cytotoxicity of all inhibitors was assessed using CellTiter96 Aqueous One (Promega (Madison WI, USA); cat. no. G3580).

[0143] Briefly, doses of inhibitor, found to be nontoxic and within the range of activity, were applied to DC2.4 in 100 L cDMEM 1 h prior to cotreatment with BzF. Mitomycin C (MMC) was applied to cells 30 min prior to BzF addition. The cells were then washed 2 before addition of BzF. Cells exposed to the other inhibitors were incubated with an appropriate concentration of each inhibitor, seen in Table 1 in cDMEM at 37 C., 5% CO.sub.2. After a 1 h pretreatment with the other inhibitors, 100 L of a 2 solution containing 50 g/mL BzF (final concentration of 25 g/mL) or vehicle control, with or without 0.5 mg/mL OVA, was added to each well. Cells were then incubated for 6 h. Before B3Z addition the inhibitor and BzF were removed, and the cells washed with warm DPBS. If testing peptide presentation, the DC2.4 cells were pulsed with 0.5 nM SIINFEKL for 1 h prior to being cocultured with B3Z.

TABLE-US-00001 TABLE 1 List of Small Molecule Inhibitors Inhibitor Compound Target Identifier CAS No. Source Product ID Conc. NF-B Bay11 19542-67-7 Cayman 14795 5-10 M NF-B Dexamethasone 50-02-2 Cayman 20340 2 M NF-B RO106-9920 62645-28-7 Cayman 15373 5-8 M I Kinase TPCA-1 507475-17-4 Fisher Sci NC1561402 50 nM ERK FR180204 865362-74-9 Cayman 15544 5 M ERK SCH722984 942183-80-4 Cayman 19166 1.25 M mTOR Torin 1 1222998-36-8 Cayman 10997 250 nM mTOR Rapamycin 53123-88-9 Sigma 553211 5 nM AP-1 SR11302 160162-42-5 Cayman 16338 25 M Protein Brefeldin A 20350-15-6 Sigma B6542 3 g/mL Transport Endosome Bafilomycin A1 88899-55-2 Cayman 11038 100 nM acidification Endocytosis Cytochalasin D 22144-77-0 Fisher Sci NC9996786 0.5 M Autophagy 3-Methyladenine 5142-23-4 Cayman 13242 2.5 mM Endosome Chloroquine 54-05-7 Sigma C6628 4.32 M acidification Translation Cycloheximide 66-81-9 Sigma C1988 15-30 ng/mL Transcription Doxorubicin 23214-92-8 Santa Clara SC-280681 100 nM Biotech Cell cycle Mitomycin C 50-07-7 Sigma M4287 30 g/mL Eisen, et al., Promiscuous binding of extracellular peptides to cell surface class I MHC protein. Proc Natl Acad Sci USA. 2012 Mar. 20; 109(12): 4580-5. Wu, et al., Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem. 2010 Apr. 2; 285(14): 10850-61.

[0144] DC2.4 cells were pretreated for 1 h with 3 g/mL BFA, a specific inhibitor of exocytosis and the intracellular transport of newly synthesized class I molecules as described above, then treated for 6 h with 25 g/mL BzF and pulsed with 0.5 nM SIINFEKL or 0.5 mg/mL OVA As anticipated, B3Z activation using OVA as the antigen source was significantly suppressed by BFA while B3Z activation using DC2.4 cells treated with BzF and the exogenously supplied SIINFEKL peptide was unaffected, as seen in FIG. 11. The finding that BFA treatment suppressed B3Z activation by DC2.4 that had been pulsed with peptide alone (NoTx), suggests BzF's primary mechanism of action involves an ability to extracellularly stabilize surface expression of peptide bound MHC class I complexes. The response to GL was similar to that produced by BzF.

[0145] Continued interrogation of the effects of BzF on cross-presentation utilized DC2.4 treated with a number of small molecule inhibitors known to disrupt the conventional cross-presentation pathway, seen in Table 1, was undertaken similar to that of BFA. Bafilomycin A1 and chloroquine inhibit endosomal acidification, cytochalasin D inhibits endocytosis, 3-methyladenine inhibits autophagy, cycloheximide inhibits translation, and doxorubicin inhibits transcription. All of these inhibitors failed to inhibit the ability of BzF to enhance B3Z activation by DC2.4 pulsed with the exogenously supplied peptide, SIINFEKL (data not shown). Since these inhibitors work on nearly every part of the cross-presentation pathway, their inability to significantly affect the magnitude of B3Z activation by the DC2.4 cells treated with BzF and SIINFEKL supports the hypothesis that BzF works extracellularly.

[0146] Together these data strongly suggest that BzF's primary mechanism of action is based on its ability to affect peptide binding to the surface expressed MHC class I complex. However, it does not completely rule out any effects on the intracellular mechanisms of cross-presentation.

Example 5: The Effect of BzF is not Due to Modulation of MHC Class I Expression

[0147] To examine the potential effects of BzF on the surface expression of the MHC class I complex, the abundance and stability of MHC I H-2Kb was measured via flow cytometry.

[0148] The DC2.4, provided by Dr. Kenneth Rock (University of Massachusetts) and the IMG cell line (Millipore, Merck KGaA, Burlington, MA; cat. no. SC-C134) were routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0149] The B3Z.A3 cell line was provided by Dr. Nilabh Shastri (University of California Berkeley, Office of Technology Licensing) while E.G7-OVA (CRL-2113) and THP-1 (TIB-202) cells were purchased from ATCC. These cells were cultured in RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol (B-Me) and 1 mM sodium pyruvate (Gibco, cat. no. 11360-070). Murine splenocytes were cultured in complete RPMI (cRPMI) comprised of RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol. All culture media included 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals 512450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0150] BzF ((2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid) (CAS no. 1210059-73-6, cat. no. CSSB00016286531)(SMILES OC(=O)/C=C\c1ccc2c(c1)cco2) was purchased from Chemspace (Monmouth Junction, NJ). Chicken ovalbumin (OVA, grade V) was purchased from Sigma (cat. no. A5503). Glycyl-L-leucine (GL; TCI America, Tokyo Chemical Industry Co., Ltd. (Portland, OR); cat. no. G0181).

[0151] By using an MHC class I H-2K.sup.b specific antibody, a relative comparison of the surface MHC class I was measured before and after BzF treatment of DC2.4 and IMG cells. These cells were treated with 25 g/mL BzF for 0-4 h. Cells were centrifuged and washed using cold FACS buffer (DPBS with 5% FBS, 2 mM EDTA, 0.1% sodium azide). Cells were blocked with Fc block (bioLegend; cat. no. 101320) for 10 min to 15 min to inhibit non-specific binding, followed by staining with fluorophore-conjugated antibodies; one set of cells was stained with the PE-labeled MHC/SIINFEKL specific antibody (clone 25-D1.16, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 12-5743-82), and the other set was stained with the PE-labeled MHC class I H-2Kb specific antibody (AF6-88.5.5.3,Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 12-5958-82) PE-labeled MHC class I H-2kb specific (clone AF6-88.5.5.3; Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 12-5958-82), for 30 min at 4 C. After washing, the cells were subjected to flow cytometric analysis with the Attune NxT Flow Cytometer (Invitrogen). Cells were gated based on live single-cell populations and positives were assessed using isotype controls and fluor-minus-one analysis when needed. Data were analyzed using the Attune NxT software or FlowJo versions 10.7 and 10.8.

[0152] Cells stained with the PE-labeled MHC/SIINFEKL specific antibody (clone 25-D1.16, (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 12-5743-81) demonstrate the enhancement of peptide binding by BzF, seen in FIGS. 12A and 13A, while the PE-labeled MHC class I H-2Kb specific antibody (AF6-88.5.5.3) determines if BzF, in the absence of exogenous peptide was affecting the levels of surface expressed MHC class I, as seen in FIGS. 12B and 13B. While BzF was able to enhance the binding of SIINFEKL to the surface MHC class I of both DC2.4 and IMG cells, flow cytometric analysis of these cells revealed no significant effect of BzF on the overall surface expression (MFI) of MHC class I between 1-4 h, but a significant increase was observed in cells treated for 24 h, seen in FIGS. 12A and 13A.

[0153] To determine if BzF affects synthesis or cycling of the MHC class I molecules, the class I molecule/peptide complexes were stripped from the surface of DC2.4 cells using a citric acid buffer, then treated with 25 g/mL BzF, 5 mM of the dipeptide GL, or 3 g/mL BFA (see Example 4) as a negative control.

[0154] An acid wash to strip the cell surface of MHC class I was performed as described with modifications. (Montealegre, et al., Dissociation of 2-microglobulin determines the surface quality control of major histocompatibility complex class I molecules. FASEB. 2015 July; 29(7):2780-8). DC2.4 were incubated overnight at 37 C. at 110.sup.5. Cells were washed for 2 min in acidic buffer [(pH 2.5) 465 mM Citric acid, 35 mM sodium citrate, in PBS with 1% v/v FBS] or neutral buffer [(pH 7.4) PBS with 1% v/v FBS]. Afterwards the pH was neutralized by the addition of cDMEM. The cells were washed with PBS and treated with BzF or GL in cDMEM for various times to allow for MHC class I recovery at 37 C. Cells were washed and stained with an H-2Kb-specific antibody (Invitrogen, cat. no. 12-5958-82) and analyzed by flow cytometry as described previously

[0155] MHC I recovery on the cell surface at various time points was detected by flow cytometry using the PE-labeled MHC class I H-2Kb specific antibody (AF6-88.5.5.3). Cells treated with BzF post-stripping did not show an increase in MHC class I recovery as compared to vehicle control when treated for 4-24 h. At 6 hours >60% of the MHC class I molecules recovered, seen in FIG. 14. When DC2.4 cells were exposed to the same acid stripping protocol and then used in the B3Z assay, it was observed that BzF was still able to increase the B3Z response post acid-stripping when the cells were treated during recovery (data not shown). This data suggests that BzF works to stabilize existing MHC class I complexes rather than enhancing the production of new ones, and further confirms that BzF increases peptide binding irrespective of the class I molecule abundance.

[0156] The relative kinetic stability or off-rate of the SIINFEKL peptide was determined using the BFA decay experiment as previously described. (Boulanger, et al., Absence of tapasin alters immunodominance against a lymphocytic choriomeningitis virus polytope. J Immunol. 2010 Jan. 1; 184(1):73-83; Montealegre, et al., Dissociation of 2-microglobulin determines the surface quality control of major histocompatibility complex class I molecules. FASEB. 2015 July; 29(7):2780-8). Briefly, DC2.4 cells were plated at 110.sup.5 cells overnight at 37 C. for adhesion. Cells were treated for one hour with 30 nM SIINFEKL peptide. After washing with media, de novo transport of MHC I to the cell surface was blocked by treatment with 1.5 g/mL BFA. The same assay was repeated without the addition of BFA to evaluate the importance of de novo MHC I synthesis on BzF treated SIINFEKL decay. Cells were incubated at 37 C. for various times to allow the decay of unstable molecules. To measure retained peptide, the cells were stained with anti-MHC I/SIINFEKL (25-D1.16) monoclonal antibody (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 12-5743-81). Samples were analyzed by flow cytometry as described. Positive staining was assessed using cells stained in the absence of peptide to set negative gates.

[0157] This measurement informs relative immunodominance of the peptide, as well as stability of the MHC class I peptide complex due to the ability of BFA to inhibit de novo protein transport to the surface. In order to normalize the confounding increase in SIINFEKL binding when the cells are treated with BzF, comparisons in the decay rate were based on the percent change from the time-zero geometric mean fluorescence for each treatment group. Pre-treatment of DC2.4 for 24 h with BzF did not affect the decay rate of SIINFEKL, as seen in FIG. 15, suggesting the overall stability of the H-2K.sup.b/SIINFEKL complex was not affected by the presence of BzF.

Example 6: BzF is Able to Displace MHC Class I Bound Peptide

[0158] The DC2.4 cell line was provided by Dr. Kenneth Rock (University of Massachusetts). The cell line was routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0159] The B3Z.A3 cell line was provided by Dr. Nilabh Shastri (University of California Berkeley, Office of Technology Licensing) while E.G7-OVA (CRL-2113) and THP-1 (TIB-202) cells were purchased from ATCC. These cells were cultured in RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol (B-Me) and 1 mM sodium pyruvate (Gibco, cat. no. 11360-070). Murine splenocytes were cultured in complete RPMI (cRPMI) comprised of RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol. All culture media included 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals 512450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0160] BzF ((2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid) (CAS no. 1210059-73-6, cat. no. CSSB00016286531)(SMILES OC(=O)/C=C\c1ccc2c(c1)cco2) (Chemspace, Monmouth Junction, NJ), 3 chlorophenol red--D-galactopyranoside (CPRG, cat. no. 10884308001) were purchased from Sigma Chemical Co (St. Louis, MO). CPRG stock was prepared to 175 g/mL in sterile H.sub.2O and a working solution of 1.75 g/mL was used. Vaccine grade high molecular weight polyinosinic-polycytidylic acid (Poly (I:C) (cat. no. VPIC-264) and ultrapure LPS, E. coli 0111:B4 were obtained from InvivoGen (cat. no. tlrl-3pelps).

[0161] Chicken ovalbumin (OVA, grade V) was purchased from Sigma (cat. no. A5503). The H-2D.sup.b binding peptide, HPV E7.sub.49-57 (AnaSpec Inc., Fremont, CA; cat. no.AS-61022) and the H-2K.sup.b OVA.sub.257-264 peptide, SIINFEKL (AnaSpec Inc., Fremont, CA; cat. no. AS-60193-1). The Trp2 (tyrosinase-related protein 2) peptide (SVYDFFVWL.sub.180-188) was modified at AnaSpec by replacing the Trp (W) with a lysine (K) containing an attached molecule of FITC (SVYDFFVK(FITC)L). The HPV E7.sub.49-57 peptide RAHYNIVTF, modified at the N-terminus by labeling with FITC, was purchased from AnaSpec (cat. no. SQ-ASPE-77874). Glycyl-L-leucine (GL; TCI America, Tokyo Chemical Industry Co., Ltd. (Portland, OR); cat. no. G0181).

[0162] Activation of B3Z T cell hybridoma cells was assessed following their exposure to various MHC class I H-2K.sup.b positive cell lines presenting the SIINFEKL peptide. To stimulate -gal production in the B3Z, the H-2Kb murine cell lines, DC2.4 and E.G7-OVA were plated at a density of 2.5 or 510.sup.4 cells/well (100 L/well in 96 well plates) and cultured overnight at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity. The cells were washed with DPBS and then treated for 1-24 h with the indicated concentrations of BzF suspended in complete media. Vehicle controls were always included. Cells being examined for the uptake and processing of soluble OVA were co-treated with BzF and 0.5 mg/mL grade V chicken ovalbumin for 6-24 h, and then washed and cocultured with B3Z as indicated.

[0163] Following the pretreatment with BzF, the media was removed from adherent cells (DC2.4 or B16F10) or was removed following pelleting of nonadherent cells in the plate. The cells were then pulsed with various concentrations of SIINFEKL peptide (typically 0.5 nM was used) for the indicated lengths of time (typically 1 h). The cells were washed and then 100 L of complete DMEM containing 510.sup.4 B3Z cells was added. The co-culture was incubated at 37 C. for 19 h to allow for B3Z activation. To detect activation of the B3Z cells, the media was removed and 100 L of 250 M CPRG reconstituted in the lysis buffer (1DPBS, 0.2% v/v saponin, 20 mM magnesium chloride) was added directly to each well. Cells were maintained at 37 C. prior to measurement. The absorbance at 560 nm (700 nm for background) was intermittently monitored using a BioTek Synergy HTX multimode microplate reader (Winooski, VT) to detect -gal activity. CPRG lysis buffer and B3Z cells only served as the negative controls. All treatments were performed in triplicate and all experiments were repeated a minimum of three times.

[0164] For peptide loading studies, cultured cells were treated for various times as indicated in results and figure legends. Cells were centrifuged and washed using cold FACS buffer (DPBS with 5% FBS, 2 mM EDTA, 0.1% sodium azide). Cells were blocked using Fc block (bioLegend; cat. no. 101320) for 10 min to 15 min to inhibit non-specific binding, followed by staining with fluorophore-conjugated antibody PE-labeled anti-MHC I/SIINFEKL (clone 25-D1.16, Invitrogen 12-5743-82), for 30 min at 4 C. For N-terminal FITC labeled H-2Db peptide RAHYNIVTF (AnaSpec Inc., Fremont, CA; cat. no.) cells were incubated with peptide for 1-4 h before washing and analysis. After washing, the cells were subjected to flow cytometric analysis with the Attune NxT Flow Cytometer (Invitrogen). Cells were gated based on live single-cell populations and positives were assessed using isotype controls and fluor-minus-one analysis when needed. Data were analyzed using the Attune NxT software or FlowJo versions 10.7 and 10.8.

[0165] Interestingly, while pre-treatment with BzF showed no effects on kinetic stability of the SIINFEKL-MHC class I complex, when BzF was provided post-peptide addition, a significantly greater loss of the surface complex was observed. Treatment of DC2.4 cells with 25 g/mL BzF or 5 mM GL following a 1 h peptide pulse (30 nM SIINFEKL) significantly reduced the MHCI/SIINFEKL complex staining when compared to the untreated cells, as seen in FIG. 16 (p<0.0001, p=0.0027, respectively).

[0166] The apparent ability of BzF to enhance turnover of preexisting, high affinity, MHC class I bound peptides could allow for the targeted binding of more appropriate or clinically desirable peptides to a variety of cells, including immune cells or tumor cells. Evidence supporting this hypothesis was obtained by examining BzF's ability to affect E.G7-OVA cells activation of B3Z cells. E.G7-OVA is a murine H-2Kb+ cell line expressing OVA endogenously and able to generate and present bound to MHC class I, the SIINFEKL peptide, as seen in FIG. 17A. Treatment of these cells with increasing concentrations of BzF (0-100 g/mL), dose-dependently reduced the ability of these cells to activate B3Z, as seen in FIG. 17B. This effect was significant at a dose as low as 6.25 g/mL.

Example 7: BzF Enhances Peptide Binding to H-2D.SUP.b .MHC Class I and HLA-A*02:01

[0167] DC2.4 are a C57Bl/6 lineage murine cell line and as such they express MHC class I H-2Kb and H-2Db. These two alleles exhibit slightly different requirements for peptide binding. H-2Kb favors a bulkier hydrophobic residue at position five (Tyr, Phe), while H-2Db favors a hydrophilic residue at position five (Asn). Tyrosine or proline is preferred by H-2Db at position three. Both alleles prefer a hydrophobic residue (Met, Ile, Leu or Val) at the C-terminus to interact with the F pocket of the MHC class I binding groove. The H-2Db requires the peptide to arch out between residues 6 and 7 for interaction with the T cell receptor, and as such requires peptides to have a length of at least nine residues. In H-2Kb this region is deeper and favors binding to peptides that are 8 residues in length. (Reynisson, eta al., NetMHCpan-4.1 and NetMHCIIpan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data. Nucleic Acids Res. 2020 Jul. 2; 48(W1):W449-W5422).

[0168] DC2.4 cells (Dr. Kenneth Rock, University of Massachusetts) and IMG cell line (Millipore, Merck KGaA, Burlington, MA; cat. no. SC-C134) were cultured in cDMEM containing 4500 mg/mL glucose supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin, and FBS, as outlined in more detail in Example 3.

[0169] Cells were cultured as previously described and treated for various times as indicated in results and figure legends. Cells were centrifuged and washed using cold FACS buffer (DPBS with 5% FBS, 2 mM EDTA, 0.1% sodium azide). Cells were stained with N-terminal fluorophore-conjugated antibodies FITC labeled Trp2.sub.180-188 peptide (SVYDFFVWL) and N-terminal FITC labeled H-2Db peptide RAHYNIVTF (Anaspec) cells were incubated with Fc block (bioLegend; cat. no. 101320) for 10 min to 15 min to inhibit non-specific binding, followed by peptide for 1-4 h, before washing and analysis. After washing, the cells were subjected to flow cytometric analysis with the Attune NxT Flow Cytometer (Invitrogen). Cells were gated based on live single-cell populations and positives were assessed using isotype controls and fluor-minus-one analysis when needed. Data were analyzed using the Attune NxT software or FlowJo versions 10.7 and 10.8.

[0170] While H-2Kb is the dominant haplotype, BzF was investigated to determine if the compound could assist peptide binding on H-2Kb and H-2Db. Using the N-terminal FITC labeled Trp2.sub.180-188 peptide (SVYDFFVWL) and the N-terminal FITC labeled HPV.sub.49-57 peptide (RAHYNIVTF) that bind with high affinity to H-2Kb and H-2Db, respectively, BzF was found to enhance peptide binding on both haplotypes, seen in FIGS. 18 and 19. This demonstrates that the phenomenon is not solely restricted to binding of the SIINFEKL peptide.

[0171] A tetramer quick switch assay was performed according to manufacturer's instructions for flow cytometric analysis using both the QuickSwitch Quant HLA-A*02:01 tetramer Kit (MBL International Corporation TB-7300-K1) and the H-2Kb kit (TB-7400-K2). The abilities of MBL's proprietary peptide exchange factor (unknown concentration), 1 mM of the glycl-leucine dipeptide, was compared to 1 mM of BzF to promote peptide exchange. The QuickSwitch assay utilizes an antibody specific for an irrelevant exchangeable low affinity exiting peptide to quantify its removal from an MHC class I tetramer and replacement with a higher affinity peptide of choice. All volumes and concentrations were as instructed in the protocol provided. Briefly, the given reference peptide was mixed with the QuickSwitch tetramer, then the kit's peptide exchange factor, BzF, or GL were added in the volumes described in the manufacturer's protocol. With the aim of comparing the molecule's peptide exchange rate, peptide exchange was measured at various timepoints. The tetramer-peptide-molecule mix was maintained at 25 C. in the dark, and 5 L of the sample was removed at each time point. This mixture was added to the supplied magnetic capture beads and shaken in the dark for 45 min at 25 C. After this, the sample was washed with a 1 assay buffer and FITC-labeled exiting peptide antibody was added to the mixture and the incubation was repeated. After washing, each sample was analyzed by flow cytometry. The percent of peptide exchange was calculated using the online QuickSwitch Quant-Peptide-Exchange-Calculator (MBL Int'l Corp., Woburn, MA) to compare peptide exchange over time.

[0172] This peptide exchange reaction is catalyzed by the presence of an exchange factor which they supply with the kit, as seen in FIG. 20A. When BzF and GL were used in place of their proprietary exchange factor, it was discovered that nearly all (99%) of the exiting peptide bound to the MHC class I H-2Kb tetramer had been displaced by 30 min. The kit supplied exchange factor required 2 h to exchange >90% of the peptide and 4 h to produce the same level of exchange achieved with BzF, seen in FIG. 20B.

[0173] When 1 mM BzF and GL were used in place of the kit's exchange factor, along with the HLA-A*02:01 tetramer, >90% exchange was achieved within 30 min, but 4 h was required to exchange 96% of the test peptide. The proprietary exchange factor was able to exchange 99% of the test peptide within 2 h, as seen in FIG. 20C. Compared to exchange in the absence of exchange factor, BzF was significantly able to increase peptide turnover on both murine and human class I complexes (p<0.001), seen in FIGS. 20A through 20C, but like GL, it appeared to be more efficient with HLA A*02:01. These results demonstrate that BzF is not only facilitating rapid peptide turnover, but rather peptide exchange on MHC class I complexes already bound with peptide.

Example 8: Molecular Docking Predicts BzF Interaction with the MHC Class I F Pocket

[0174] The crystal structure of H-2Kb in complex with high affinity peptide SIINFEKL (PDB ID:3P9L) and the HLA A*02-01 with octomeric tax peptide (PDB ID:31-HLA) were obtained from the Protein Data Bank and served as starting structures for molecular docking analysis. Ligand structures were drawn using PubChem Sketcher v2.4, saved in the MDL molfile (sdf) format, and converted into the Sybyl Mol2 (mol2) PDB format using Open Babel GUI v3.1.1. The mol2 file was opened in AutoDock Tools v1.5.6 and converted into a ligand by adding gasteiger charges, merging nonpolar hydrogen atoms with their attached carbon atoms to form united atoms, and setting rotatable bonds. It was then saved in the PDBQT format. The grid box for each receptor was created to include the F-pocket with a spacing of 0.375 , followed by the docking of BzF to each receptor was initiated. The structural models were collected from the lowest-energy docking solutions of each receptor. The inhibition constant was calculated in AutoDock using the formula:

[00001]$\mathrm{Ki}=\exp \left(G1000\right)/\left(\mathrm{Rcal}\mathrm{TK}\right)$

where G is the docking energy, Rcal is 1.98719, and TK is 298 (Iman, et al., Molecular docking analysis and molecular dynamics simulation study of ameltolide analogous as a sodium channel blocker. Turk J Chem. 2015; 39(2):306-316).

[0175] Based on the observations that BzF's activity is similar to that described for dipeptide exchange factors like GL (Saini, et al., Dipeptides catalyze rapid peptide exchange on MHC class I molecules. Proc Natl Acad Sci USA. 2015 Jan. 6; 112(1):202-7; Saini, et al., Empty peptide-receptive MHC class I molecules for efficient detection of antigen-specific T cells. Sci Immunol. 2019 Jul. 19; 4(37):eaau9039), computational molecular docking was utilized to determine if BzF is predicted to interact with the MHC class I and HLA-A peptide binding grooves in a manner described for the dipeptides. Using the AutoDock Suite to perform the docking studies (Morris, et al., AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009 December; 30(16):2785-91), it was predicted that BzF interacts with the F pocket of the H-2K.sup.b MHC class I and HLA-A*02:01 peptide binding grooves in a manner similar to that described for the SIINFEKL peptide and the GL dipeptide. (Madden, The three-dimensional structure of peptide-MHC complexes. Ann Rev Immunol. 1995; 13(1):587-622; Hein, et al., Peptide-independent stabilization of MHC class I molecules breaches cellular quality control. J Cell Sci. 2014 Jul. 1; 127(13):2885-97). In the top three predicted poses, the benzofuran portion of BzF is observed deep within the hydrophobic F pocket, while its carboxylic acid group is hydrogen-bonded to a key amino acid, Lys146, affecting conformation of the F pocket, as seen in FIGS. 21A through 22B.

[0176] Lys146 and Tyr84 are two amino acids that are conserved across all MHC class I and HLA-A, B, and C haplotypes and are believed to be universally involved in peptide binding and F pocket opening. (Remesh, et al., Unconventional peptide presentation by major histocompatibility complex (MHC) class I allele HLA-A* 02: 01: BREAKING CONFINEMENT. J Biol Chem. 2017 Mar. 31; 292(13):5262-70). Lys146 forms a partial lid above the F pocket that is held in position by the hydrogen bond interaction with the carboxyl terminus of the peptide. Lifting of Lys146 opens the F pocket, especially in the presence of extended peptides with negatively charged residues near their carboxyl end. Tyr84 has been found to swing out in the presence of extended peptides with positive charges near their carboxyl end. This also allows for the opening of the F pocket. (Remesh, et al., Unconventional peptide presentation by major histocompatibility complex (MHC) class I allele HLA-A* 02: 01: BREAKING CONFINEMENT. J Biol Chem. 2017 Mar. 31; 292(13):5262-70). Therefore, the finding that BzF interacts with key amino acids on H-2Kb and HLA-A02:01 could explain its ability to stabilize the open conformation and displace previously bound peptides. Similar to BzF, the GL dipeptide and the C-terminal residue of SIINFEKL also form hydrogen bonds with Lys146, as well as with Tyr84. (Hein, et al., Peptide-independent stabilization of MHC class I molecules breaches cellular quality control. J Cell Sci. 2014 Jul. 1; 127(13):2885-97).

[0177] When compared with the free energy of binding (binding affinity) predicted for GL binding in the F pocket of H-2K.sup.b (6.20 kcal/mol) and HLA-A*02:01 (6.71 kcal/mol), AutoDock predicted that BzF required similar free energy for binding to H-2K.sup.b (5.54 kcal/mol) and HLA-A*02:01 (5.36 kcal/mol), as seen in FIG. 23.

[0178] In the same simulations it can be seen that GL binds in a similar manner as BzF, forming a hydrogen bond with Lys146 and burying its hydrophobic region deep into the F pocket. These results show that like the GL dipeptide and the C-terminal amino acids of MHC class I peptides, BzF is predicted to interact with the F pocket of H-2K.sup.b and HLA-A*02:01 and potentially induce a change in conformation that is more receptive to exogenous full-length peptides.

Example 9: The Unique Structure of BzF is Critical to its Enhancement of the CD8+ T Cell Response

[0179] The DC2.4 cell line was provided by Dr. Kenneth Rock (University of Massachusetts). These cell lines were routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0180] The B3Z.A3 cell line was provided by Dr. Nilabh Shastri (University of California Berkeley, Office of Technology Licensing) while E.G7-OVA (CRL-2113). These cells were cultured in RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol (B-Me) and 1 mM sodium pyruvate (Gibco, cat. no. 11360-070). Murine splenocytes were cultured in complete RPMI (cRPMI) comprised of RPMI 1640+GlutaMAX-1 (Gibco, Cat. No. 61870-036) containing 2000 mg/mL glucose and supplemented with 40 M 2-mercaptoethanol. All culture media included 50 units/mL penicillin and 50 g/mL streptomycin and 10% by volume heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals S12450H). Cells were cultured at 37 C., in an atmosphere containing 5% CO.sub.2 and 95% humidity.

TABLE-US-00002 TABLE 2 BzF analog molecules examined. Molecular Molecule Name Abbreviation Structure Weight (2E)-3-(1-benzofuran-5-yl)prop-2- enoic acid BzF [00001] 188.18 3-(2,3-dihydro-1-benzofuran-5- yl)acrylic acid Analog #2 [00002] 190.20 3-(2,3-dihydrobenzofuran0-5- yl)propanoic acid Analog #3 [00003] 192.22 (2E)-3-(1,3-benzodioxol-5yl)acrylic acid Analog #4 [00004] 192.17 4-(1-benzofuran-5-yl)butan-2-one Analog #5 [00005] 190.19 Glycine-leucine dipeptide GL [00006] 188.22 [00007]

[0181] BzF ((2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid) (CAS no. 1210059-73-6, cat. no. CSSB00016286531)(SMILES OC(=O)/C=C\c1ccc2c(c1)cco2) (Chemspace, Monmouth Junction, NJ), 3-(2,3-dihydro-1-benzofuran-5-yl)acrylic acid (Matrix Scientific, Columbia, SC; cat. no. 007326) and 3-(2,3-dihydrobenzofuran0-5-yl)propanoic acid (Matrix Scientific, Columbia, SC; cat. no. 007325), (2E)-3-(1,3-benzodioxol-5yl) acrylic acid (Sigma Chemical Co., St. Louis, MO; cat. no. 146242) and chlorophenol red-P3-D-galactopyranoside (CPRG, Sigma Chemical Co., St. Louis, MO; cat. no. 10884308001) were purchased acquired as indicated. CPRG stock was prepared to 175 g/mL in sterile H.sub.2O and a working solution of 1.75 g/mL was used.

[0182] DC2.4 were plated at a density of 2.5 or 510.sup.4 cells/well (100 L/well in 96 well plates) and cultured overnight in cDMEM. The cells were washed with DPBS and then treated for 1-24 h with the indicated concentrations of BzF or an analog, seen in Table 2, suspended in complete media. Following the pretreatment with BzF or an analog, seen in Table 2, the media was removed from adherent cells. The cells were then pulsed with various concentrations of SIINFEKL peptide (typically 0.5 nM was used) for the indicated lengths of time (typically 1 h). The cells were washed and then 100 L of complete DMEM containing 510.sup.4 B3Z cells was added. The co-culture was incubated at 37 C. for 19 h to allow for B3Z activation.

[0183] To detect activation of the B3Z cells, the media was removed and 100 L of 250 M CPRG reconstituted in the lysis buffer (1DPBS, 0.2% v/v saponin, 20 mM magnesium chloride) was added directly to each well. Cells were maintained at 37 C. prior to measurement. The absorbance at 560 nm (700 nm for background) was intermittently monitored using a BioTek Synergy HTX multimode microplate reader (Winooski, VT) to detect -gal activity. CPRG lysis buffer and B3Z cells only served as the negative controls. All treatments were performed in triplicate and all experiments were repeated a minimum of three times.

[0184] To illuminate how the structure of BzF informs its ability to enhance peptide binding, a series of molecules differing from BzF by one or two structural modifications were obtained, as seen in Table 2. All of the analogs were tested at doses up to 50 g/mL (265 M) and pulsed with 0.5 nM SIINFEKL. When compared to BzF, they all failed to enhance B3Z activation, as seen in FIGS. 24A through 24C. These analogs were further analyzed for their ability to increase SIINFEKL binding, as well as altering surface MHC class I abundance. Treatment of DC2.4 cells with 25 or 50 g/mL of each analog for up to 24 h failed to enhance SIINFEKL peptide binding, seen in FIG. 25, and did not increase MHC class I abundance (data not shown). These molecules were then tested for the ability to displace the SIINFEKL peptide from the H-2K.sup.b complex on DC2.4 cells. Using the same conditions that had been used to test BzF, treatment with 25 g/mL of each analog after a 1 h pulse with SIINFEKL did not enhance peptide displacement at any time point tested, as seen in FIG. 26. This limited examination of the structure activity relationship revealed the importance of the double bond in the furan for BzF activity.

Example 10: In Silico Assessment of BzF Shows Drug-Like Features

[0185] SwissADME, a component of a web-based suite of tools that utilizes a pool of predictive models for examining the physicochemical properties, pharmacokinetics, and drug-likeness of target molecules provided by the Swiss Institute of Bioinformatics (Ndombera, et al., Pharmacokinetic, physicochemical and medicinal properties of n-glycoside anti-cancer agent more potent than 2-deoxy-d-glucose in lung cancer cells. J Pharm Pharamcol. 2019; 7(4): 165-76; Daina, et al., SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017 Mar. 3; 7(1):1-13), was used to predict the ADME profile of BzF. This database assesses the structure of the input molecule to inform the behavior of the molecule in vivo for characteristics such as: physicochemical properties, lipophilicity, water solubility, pharmacokinetics, synthetic accessibility, and drug-likeness. The structure of BzF was drawn using Chem Axons Marvin JS (Chemaxon Ltd., Budapest, Hungary) with 2D features.

[0186] Evaluation of BzF indicated it to be drug-like in four of the five rule-based filters, with the only violation being that its molecular weight is lower than 200 daltons. In addition, the software was used to evaluate a combination of molecular properties, determining if BzF is similar to already known drugs in electronic distribution, hydrophobicity, lipophilicity, solubility, polarity, level of conjugation, hydrogen bonding characteristics, molecular flexibility, and size. (Anjanappa, et al., Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection. Nat Commun. 2020 Mar. 11; 11(1):1314). One of the most interesting predictions is that BzF has a high probability of being able to cross the blood brain barrier and/or be absorbed through the GI tract. If this is the case, examining BzF's therapeutic potential could be quite beneficial.

[0187] When structurally similar analogs of BzF were examined for physicochemical properties, lipophilicity, water solubility, pharmacokinetics, synthetic accessibility, and drug-likeness, BzF's lipophilicity (consensus Log P.sub.o/w), seen in FIG. 27A, the ratio of sp.sup.3 hybridized carbons over the total carbon count of the molecule (Fraction Csp3), seen in FIG. 27B, and water solubility, seen in FIG. 27C, separated BzF from the non-active analogs that were tested. BzF, like the other 3 molecules is predicted to be approximately twice as soluble in n-octanol as it is in water (2.00, 1.81, 1.83, and 1.68 respectively). The distinguishing characteristic of BzF is its fraction of sp3 hybridized carbons. BzF possesses no saturated carbons (0 out of its 11 total carbons) while the analogs contain 0.18, 0.36, and 0.1 of their carbons possessing sp3 orbitals. This rigidness in structure and lipophilic nature of BzF could help explain its enhanced affinity for the hydrophobic region of the F pocket and the inability of the structurally similar analogs to affect peptide binding. Further work needs to be done to verify the importance of these properties.

Example 11: BzF can be Detected in the Blood for at Least 3 h Post-IV, -IM, or -Oral Administration

[0188] Six- to ten-week-old male and female C57BL/6NCrl mice (Charles River Laboratories, Wilmington MA; cat. no. 027) were used for all animal studies. The mice were maintained in accord with the NIH Guide for the Care and Use of Laboratory Animals. Experimental procedures were performed in accord with research protocols approved by the Tampa Bay Research Institute's Institutional Animal Care and Use Committee. Mice were maintained in an INOVIVE individually ventilated cage system with 3 mice per cage containing 100% virgin wood pulp bedding (Harlan Teklad, Harlan Sprague Dawley Inc., Indianapolis, IA; cat. no. 7099). Water and food (Harlan Teklad, cat. no. 8640) were available ad libitum. The mice were maintained on a cycle of 12 h of light and 12 h of dark in an environment with a temperature of 74-76 C. and 40-60% humidity. All cages underwent 40 air changes hourly in the negative pressure mode, in accordance with manufacturer's recommendations. Both the intake and exhaust air from the system were filtered through HEPA filters to maintain a clean environment. Stock solutions of BzF were solubilized in alkaline water (pH 10) and then sterile filtered through 0.22 m syringe filters. BzF used for iv or im injections was diluted in 0.9% saline (normal saline). The stock solutions of the SIINFEKL peptide were prepared in 1 calcium/magnesium free Dulbecco's phosphate-buffered saline (DPBS; ThermoFisher Scientific, cat. no. 14190144).

[0189] BzF ((2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid) (CAS no. 1210059-73-6, cat. no. CSSB00016286531)(SMILES OC(=O)/C=C\c1ccc2c(c1)cco2) was purchased from Chemspace (Monmouth Junction, NJ), 3-(2,3-dihydro-1-benzofuran-5-yl)acrylic acid (cat. no. 007326) and 3-(2,3-dihydrobenzofuran0-5-yl)propanoic acid (cat. no. 007325) were purchased from Matrix Scientific (Columbia, SC).

[0190] After overnight fasting, six-to ten-week-old mice were gavaged with 1-2 mg of BzF solubilized in 200 L alkaline water or were injected intramuscular (IM) or intravenous with 100 g diluted in normal saline. After various times mice were anesthetized using 2.0% isoflurane with O.sub.2 at 1 L/min, and 200-500 L of blood was collected in a microfuge tube containing 700 L of 50 mM EDTA in DPBS as an anticoagulant. The blood samples were centrifuged at 4500g for 20 min to separate the plasma from the cells. The resulting plasma (200 L) was removed and extracted with 4 mL methanol. This mixture was vortexed for 30 s and centrifuged at 4000g for 10 min at 4 C. The supernatant was collected and dried using a SpeedVac (SC210 ) under a vacuum of 0.4 torr at 65 C. When dry, the sample was removed and resuspended in 100 L methanol and then re-centrifuged to remove any precipitates. A 20 L sample of this mixture was analyzed by HPLC.

[0191] BzF was detected in the serum using a Shimadzu high performance liquid chromatography (HPLC) system. The Shimadzu system consisted of two LC-20AR pumps, a SPD-M20A diode array UV-Vis detector, and a 20 L injection loop onto a Phenomenex Luna 5 m Analytical PFP column 100 A (cat. no. OOF-4448-E0). Analysis and data collection were conducted using Shimadzu LabSolutions software. Samples (20 L) were injected onto the column and separated using a gradient of acetonitrile (ACN) and water as the mobile phase. The gradient started with 10% ACN and increased stepwise to 80% ACN for peak clarity. Buffer B was water adjusted to pH 2.5 using phosphoric acid. The column was kept at a constant temperature of 30 C. (Chai, et al., Comprehensive study of the intestinal absorption of four phenolic compounds after oral administration of Ananas comosus leaf extract in vivo and in vitro. Afr J Pharm Pharmacol. 2013 Jul. 15; 7(26):1781-92).

[0192] Following up on the prediction that BzF should be able to be absorbed through the GI tract and gain access to the systemic circulation, C57Bl/6crl mice were gavaged (po) with either 200 L of alkaline water (negative control) or with 2 mg of BzF solubilized in 200 L alkaline water. Fifteen minutes later, peripheral blood was obtained. The serum was extracted with methanol, lyophilized, and then examined by HPLC for the presence of BzF. Analysis of the HPLC chromatogram revealed the presence of BzF, as seen in FIG. 28A. BzF was then administered to another group of mice iv (100 g), im (100 g), and po (1 mg). After 15 min, the serum was collected, extracted with methanol, and then analyzed by HPLC. HPLC analysis revealed the presence of the appropriate peak in mice receiving BzF by each of the different routes of administration, as seen in FIG. 28B. The persistence of BzF within the peripheral blood was examined by sampling the serum 1, 2, and 3 h post im injection of 1 mg BzF that had been diluted in 50 L normal saline. HPLC analysis revealed that while the serum levels of BzF were declining over the 3 h period, it was still detectable, as seen in FIG. 28C.

Example 12: Oral, Intravenous, or Intramuscular Administration of BzF Significantly Enhances the In Vivo Binding of an MHC I Peptide

[0193] Having detected the presence of BzF in the serum of mice following administration via po, iv, and im delivery, BzF was analyzed to determine if the compound could enhance peptide loading in vivo.

[0194] Six- to ten-week-old male and female C57BL/6NCrl mice (Charles River Laboratories, Wilmington MA; cat. no. 027) were used for all animal studies. The mice were maintained in accord with the NIH Guide for the Care and Use of Laboratory Animals. Experimental procedures were performed in accord with research protocols approved by the Tampa Bay Research Institute's Institutional Animal Care and Use Committee. Mice were maintained in an INOVIVE individually ventilated cage system with 3 mice per cage containing 100% virgin wood pulp bedding (Harlan Teklad, Harlan Sprague Dawley Inc., Indianapolis, IA; cat. n.o 7099). Water and food (Harlan Teklad; cat. no. 8640) were available ad libitum. The mice were maintained on a cycle of 12 h of light and 12 h of dark in an environment with a temperature of 74-76 C. and 40-60% humidity. All cages underwent 40 air changes hourly in the negative pressure mode, in accordance with manufacturer's recommendations. Both the intake and exhaust air from the system were filtered through HEPA filters to maintain a clean environment. Stock solutions of BzF were solubilized in alkaline water (pH 10) and then sterile filtered through 0.22 m syringe filters. BzF used for iv or im injections was diluted in 0.9% saline (normal saline). The stock solutions of the SIINFEKL peptide were prepared in 1 calcium/magnesium free Dulbecco's phosphate-buffered saline (DPBS; ThermoFisher Scientific, cat. no. 14190144).

[0195] Since the oral route of drug delivery is preferred, various doses of BzF (1, 2, 4 mg) were administered by oral gavage, immediately following an iv injection of 50 g SIINFEKL peptide. One-hour post-treatment the blood and spleens from each mouse was collected. Blood samples were processed as described above. Spleens were collected in 4 mL of Hanks Balanced Salt Solution (HBSS) and converted to a single cell suspension using sterile pouches and a Steward Stomacher 80 Biomaster Blender (Fisher Scientific, cat. no. 14-285-27). The resulting cell suspension was pelleted and suspended in 4 mL freshly prepared red blood cell lysis buffer (150 mM NH.sub.4Cl, 10 mM NaHCO.sub.3, 0.1 mM EDTA in sterile distilled H.sub.2O) for 1-3 minutes. The splenocytes were pelleted and filtered through 70 M EASY strainer filters (USA Scientific, cat. no. 5654-2070). The cell count was determined using the MUSE cell viability kit (MCH600103) and MUSE Cell Analyzer (Luminex Corp, Austin TX).

[0196] One hour later the splenocytes were isolated and immediately tested for peptide loading or used to activate B3Z cells. For T-cell activation, splenocytes were prepared to 510.sup.5/mL in cRPMI, then 100 L of cells was added to wells of clear round-bottom 96 well tissue culture plates. The cells were incubated with 100 L of B3Z cells, at a concentration of 510.sup.5/mL, were added to each well. The cells were cocultured for 19 h. After 19 h, the media was removed and 100 L of freshly prepared CPRG lysis buffer was added. The colorimetric response was allowed to develop for 15-240 min and the OD 560 nm was measured at various time points using a Synergy HTX plate reader. This assay was performed in replicates with both male and female C57BL/6 mice.

[0197] For peptide loading, splenocytes were prepared to 510.sup.5/mL in cRPMI, then 100 L of cells was added to wells of clear round-bottom 96 well tissue culture plates. The cells were incubated at 37 C. and treated for various times as indicated in results and figure legends. Cells were centrifuged and washed using cold FACS buffer (DPBS with 5% FBS, 2 mM EDTA, 0.1% sodium azide). Cells were blocked using Fc block (bioLegend; cat. no. 101320) for 10 min to 15 min to inhibit non-specific binding, followed by staining with fluorophore-conjugated antibodies as specified: Super Bright 600-labeled MHC class II (I-A/I-E) (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA; cat. no. 63-5321-82) FITC-labeled CD54 (Invitrogen; cat. no. 11-0541-81), PE-labeled MHC class I H-2kb specific (clone AF6-88.5.5.3; Invitrogen; cat. no. 12-5958-82), PE-labeled anti-MHC I/SIINFEKL (clone 25-D1.16, Invitrogen; cat. no. 12-5743-82), FITC-labeled CD45.2 (Invitrogen; cat. no. 11-0454-82), APC-labeled CD11c (Invitrogen; cat. no. 17-0114-81) for 30 min at 4 C. After washing, the cells were subjected to flow cytometric analysis with the Attune NxT Flow Cytometer (Invitrogen). Cells were gated based on live single-cell populations and positives were assessed using isotype controls and fluor-minus-one analysis when needed. Data were analyzed using the Attune NxT software or FlowJo versions 10.7 and 10.8.

[0198] If BzF had facilitated the in vivo loading of SIINFEKL it was expected that the peptide-bearing splenocytes would be able to activate B3Z. Since the oral route of drug delivery is preferred, various doses of BzF (1, 2, 4 mg) were administered by oral gavage, immediately following an iv injection of 50 g SIINFEKL peptide. One hour later the splenocytes were isolated and immediately used to activate B3Z cells. If BzF had enhanced the in vivo loading of SIINFEKL it was expected that the peptide-bearing splenocytes would be able to enhance B3Z activation. As shown in FIG. 29, the splenocytes indeed activated the B3Z cells and did so in a manner that was dependent on the dose of BzF that had been administered. These splenocytes elicited a significantly greater response from the B3Z than those from the mice administered peptide alone.

[0199] To determine if BzF could facilitate peptide exchange when delivered by other routes, mice were injected iv with 50 g of SIINFEKL, then immediately either gavaged with 1 mg BzF, iv injected with 50 g BzF, or im injected with 100 g of BzF. One hour later, the splenocytes from these mice were used ex vivo to stimulate B3Z activation. All three routes of administration were successful at enhancing the ability of the splenocytes to activate B3Z. As anticipated, the delivery of peptide and BzF via iv injection produced the greatest response, as seen in FIG. 30.

[0200] When splenocytes within the CD45+/MHC II/CD11c+ population were examined by flow cytometry, it was discovered that significantly more of these cells from the BzF treated mice co-expressed MHCI bound SIINFEKL, as seen in FIG. 31 (p<0.001). Since this phenotype is most often associated with classic dendritic cells, these results suggest that administration of BzF could potentially enhance the targeting of therapeutically relevant peptides to dendritic cells, which could lead to enhancement of a clinically relevant immune response.

Example 13: BzF is Nontoxic Following Oral Gavage or Intramuscular Injection

[0201] Six- to ten-week-old male and female C57BL/6NCrl mice (cat. no. 027, Charles River Laboratories, Wilmington MA) were used for all animal studies. The mice were maintained in accord with the NIH Guide for the Care and Use of Laboratory Animals. Experimental procedures were performed in accord with research protocols approved by the Tampa Bay Research Institute's Institutional Animal Care and Use Committee. Mice were maintained in an INOVIVE individually ventilated cage system with 3 mice per cage containing 100% virgin wood pulp bedding (Harlan Teklad, Harlan Sprague Dawley Inc., Indianapolis, IA; cat. no. 7099). Water and food (Harland Teklad; cat. no. 8640) were available ad libitum. The mice were maintained on a cycle of 12 h of light and 12 h of dark in an environment with a temperature of 74-76 C. and 40-60% humidity. All cages underwent 40 air changes hourly in the negative pressure mode, in accordance with manufacturer's recommendations. Both the intake and exhaust air from the system were filtered through HEPA filters to maintain a clean environment. Stock solutions of BzF were solubilized in alkaline water (pH 10) and then sterile filtered through 0.22 m syringe filters. BzF used for iv or im injections was diluted in 0.9% saline (normal saline).

[0202] Mice were gavaged with BzF at doses ranging from 25 mg/kg body to 100 mg/kg body weight, injected intravenously (iv) with 50 g BzF or injected intramuscularly with BzF at 25 to 40 mg/kg body weight. For two weeks post-treatment, water intake and weight were monitored. At the end of the two-week period, the body and organ weights were measured to evaluate health outcomes, but no differences were measured. The limit to the solubility of BzF in alkaline water is 4 mg, therefore no further doses were tested. These results demonstrate that at the doses tested, in vivo administration of BzF is non-toxic.

Example 14: BzF Enhances the In Vitro Peptide Loading of DC Under Conditions Used to Generate DC Vaccines

[0203] The DC2.4 (Dr. Kenneth Rock, University of Massachusetts) were routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0204] Cultured cells were treated for various times as indicated in results and figure legends. Cells were centrifuged and washed using cold FACS buffer (DPBS with 5% FBS, 2 mM EDTA, 0.1% sodium azide). Cells were blocked using Fc block (bioLegend; cat. no. 101320) for 10 min to 15 min to inhibit non-specific binding, followed by staining with fluorophore-conjugated antibody PE-labeled anti-MHC I/SIINFEKL (clone 25-D1.16, Invitrogen 12-5743-82), for 30 min at 4 C. with Fc block (bioLegend; cat. no. 101320) to inhibit non-specific binding. After washing, the cells were subjected to flow cytometric analysis with the Attune NxT Flow Cytometer (Invitrogen). Cells were gated based on live single-cell populations and positives were assessed using isotype controls and fluor-minus-one analysis when needed. Data were analyzed using the Attune NxT software or FlowJo versions 10.7 and 10.8.

[0205] Previous experiments demonstrated that BzF can significantly enhance the binding of low concentrations (nM) of SIINFEKL. When dendritic cells are pulsed in vitro with peptide for the generation of DC vaccines, higher concentrations (1-10 M) of peptide are often used. Since a pulse with 1 g/mL of peptide is most commonly reported, the effects of a 2 h pretreatment with 50 g/mL BzF prior to pulsing DC2.4 cells for 1 h with 1, 5, or 10 g/mL SIINFEKL were examined. Surface levels of MHC class I/SIINFEKL complexes were detected by flow cytometry following staining with the PE-labeled 25-D1.16 antibody. While peptide alone was able to bind to nearly all of the DC2.4 cells, as seen in FIG. 32A, significantly more peptide was detected per cell if they had been pre-treated with BzF, seen in FIG. 32B.

Example 15: Immunization Using DC2.4 Cells Matured by Exposure to IFN and then Pre-Treated In Vitro with BzF and then with an MHC I Peptide, Enhances Generation of Antigen Specific CD8+ T Cells

[0206] The DC2.4 cells (Kenneth Rock, University of Massachusetts) were routinely cultured in complete Dulbecco's Modified Eagle Medium (cDMEM) containing 4500 mg/mL glucose (Gibco, cat. no. 10566-016) and supplemented with GlutaMAX, 50 U/mL penicillin and 50 g/mL streptomycin (Gibco, cat. no. 15070-063) and 10% heat inactivated fetal bovine serum (FBS) (R&D Systems cat. no. 11150H) at 37 C. in an atmosphere containing 5% CO.sub.2 and 95% humidity.

[0207] DC2.4 cells that had been matured by an overnight treatment with 20 ng/mL murine IFN (He, et al., Interferon gamma stimulates cellular maturation of dendritic cell line DC2.4 leading to induction of efficient cytotoxic T cell responses and antitumor immunity. Cell Mol Immunol. 2007; 4(2):105-11) were treated with 30 g/mL mitomycin C to block replication and were then pulsed for 1 h with 1 g/mL SIINFEKL peptide (Group 2), or with 50 g/mL BzF for 1 h and then with 1 g/mL SIINFEKL peptide for 1 h (Group 3).

[0208] Six- to ten-week-old male and female C57BL/6NCrl mice (cat. no. 027, Charles River Laboratories, Wilmington MA) were used for all animal studies. The mice were maintained in accord with the NIH Guide for the Care and Use of Laboratory Animals. Experimental procedures were performed in accord with research protocols approved by the Tampa Bay Research Institute's Institutional Animal Care and Use Committee. Mice were maintained in an INOVIVE individually ventilated cage system with 3 mice per cage containing 100% virgin wood pulp bedding (Harlan Teklad, Harlan Sprague Dawley Inc., Indianapolis, IA; cat. no. 7099). Water and food (Harland Teklad; cat. no. 8640) were available ad libitum. The mice were maintained on a cycle of 12 h of light and 12 h of dark in an environment with a temperature of 74-76 C. and 40-60% humidity. All cages underwent 40 air changes hourly in the negative pressure mode, in accordance with manufacturer's recommendations. Both the intake and exhaust air from the system were filtered through HEPA filters to maintain a clean environment. Stock solutions of BzF were solubilized in alkaline water (pH 10) and then sterile filtered through 0.22 m syringe filters. BzF used for iv or im injections was diluted in 0.9% saline (normal saline). The stock solutions of the SIINFEKL peptide were prepared in 1 calcium/magnesium free Dulbecco's phosphate-buffered saline (DPBS; ThermoFisher Scientific, cat. no. 14190144).

[0209] Female C57BL/6crl mice were separated into 3 groups with 6 mice per group. The mice in Group 1 served as the nave, no treatment group, Group 2 treated with by subcutaneous injection of 1.210.sup.6 DC2.4 cells primed with IFN, mitomycin C and SIINFEKL, and Group 3 treated by subcutaneous injection of 1.210.sup.6 DC2.4 cells primed with BzF and SIINFEKL as outlined above. One week later, the same mice were boosted with 1.210.sup.6 DC2.4 cells that had been treated as described. Fourteen days later, the number of MHC class I/SIINFEKL tetramer positive CD8+ cells in the peripheral blood and spleen were determined.

[0210] On day 21 (14 days after the last immunization) blood was collected from the retro-orbital plexus. The red blood cells were lysed using RBC lysis buffer and the white blood cells (WBC) were pelleted at 500g for 2 min. The WBC were washed in FACS buffer (DPBS containing 2% FBS and 0.1% sodium azide), pelleted, and suspended in 50 L FACS buffer containing 10 L of the iTAG APC-labeled class I H-2K.sup.b OVA (SIINFEKL) tetramer (MBL International, cat. no. TB-5001-2). The cells were incubated for 30 min at room temperature while being protected from the light. Next, 40 L of FACS buffer containing 0.4 L of FITC-labeled anti-CD8 (clone KT15) (Fisher Scientific, cat. no. MA5-16759) and 1 L of eFluor450-labeled anti-CD19 (clone eBio1D3 (1D3)) (Fisher Scientific, cat. no. 48-0193-80) was added and incubated at 10 C. for 30 min. The cells were washed with 3 mL FACS buffer, pelleted, and fixed for 1 h at 10 C. in 1 mL of FACS buffer containing 0.5% paraformaldehyde. The number of CD19 negative.sup./CD8 positive/SIINFEKL tetramer positive cells was determined by flow cytometric analysis following collection of positive events detected in the lymphocyte gate.

[0211] Spleens were isolated and processed as previously described and then stained with antibodies specific for CD19, CD8, and tetramers specific for TCR's recognizing H2Kb presenting the SIINFEKL peptide.

[0212] The results show an enhanced SIINFEKL-specific CD8+ T cell response in mice immunized with BzF treated DC2.4, as seen in FIGS. 33A and 33B.

Example 16: PLGA Encapsulated BzF Enhances the Anti-Tumor Immune Response Generated by PLGA Encapsulated Whole Ovalbumin

[0213] Six- to ten-week-old male and female C57BL/6NCrl mice (cat. no. 027, Charles River Laboratories, Wilmington MA) were used for all animal studies. The mice were maintained in accord with the NIH Guide for the Care and Use of Laboratory Aanimals. Experimental procedures were performed in accord with research protocols approved by the Tampa Bay Research Institute's Institutional Animal Care and Use Committee. Mice were maintained in an INOVIVE individually ventilated cage system with 3 mice per cage containing 100% virgin wood pulp bedding (Harland Teklad, cat. no. 7099). Water and food (Harland Teklad cat. no. 8640) were available ad libitum. The mice were maintained on a cycle of 12 h of light and 12 h of dark in an environment with a temperature of 74-76 C. and 40-60% humidity. All cages underwent 40 air changes hourly in the negative pressure mode, in accordance with manufacturer's recommendations. Both the intake and exhaust air from the system were filtered through HEPA filters to maintain a clean environment. Stock solutions of BzF were solubilized in alkaline water (pH 10) and then sterile filtered through 0.22 m syringe filters. BzF used for iv or im injections was diluted in 0.9% saline (normal saline). The stock solutions of the SIINFEKL peptide were prepared in 1 calcium/magnesium free Dulbecco's phosphate-buffered saline (DPBS; ThermoFisher Scientific, cat. no. 14190144).

[0214] PLGA microparticles were prepared by dissolving 200 mg PLGA (poly(D,L-lactide-co-glycolide, Resomer RG 752 H, acid terminated, lactide:glycolide 75:25, M.sub.w 4,000-15,000 from Sigma) in 2 mL solvent (ethyl acetate, EtAc) in a 15 mL polypropylene centrifuge tube. Whole ovalbumin (40 mg, Fraction V, Sigma) was solubilized in 400 L 1% polyvinyl alcohol (PVA, average mw of 30,000-70,000; Sigma) in a 5 mL conical centrifuge tube by vortexing for several minutes. The solubilized ovalbumin was then added, dropwise into the 2 mL PLGA mixture with continuous vortexing. To create an O/W emulsion, the mixture was placed into an ice bath and subjected to sonication (40% power, 50% pulses for 1 minute). The resulting emulsion was mixed with 8 mL of 1% PVA and sonicated as described above for 1 minute. The emulsion was then transferred to a 100 mL beaker containing 50 mL of 0.1% PVA. This was stirred with a magnetic stirrer (700 rpm) for a minimum of 3 h to evaporate the ethyl acetate and harden the particles.

[0215] The PLGA/BzF particles were prepared by adding 190 mg of PLGA and 20 mg BzF to a 15 mL polypropylene test tube. One milliliter of EtAc was added to the tube and then vortexed on high until the powders had completely dissolved (10 min). A solution of 0.1% PVA was prepared and 2 mL placed of into a separate 15 mL tube and vortexed on high. While this was being vortexed, the 1 mL of the PLGA/BzF/EtAc solution was added dropwise into the 0.1% PVA. Once the polymer solution had been added to the PVA, the mixture was vortexed for an additional 15 sec. The emulsified polymers were immediately placed in an ice bath and the probe ( diameter) of a Branson Sonifier 450 ultrasonicator was placed into the tube. The mixture was sonicated in three 10 sec bursts using 30% amplitude. The emulsion was move up and down the probe to ensure even sonication, being careful to avoid touching the probe to the sides or bottom of the test tube. Sonication was paused between each ten second pulse to allow the solution to cool before proceeding. Next, 50 ml of 0.1% PVA was added to a 200 ml glass beaker which was placed on a magnetic stir plate and stirred at 600 rpm. The emulsion was added to the beaker and stirred for a minimum of 3 hours. This allowed the solvent to evaporate and the particles to harden.

[0216] The number of OVA encapsulated in the PLGA particles was determined using the micro-bicinchoninic acid (BCA) assay kit (Pierce, Rockford, IL) following the manufacturer's instructions. Briefly, a standard curve of 2-fold serial dilutions of OVA (50-0 g/mL) was prepared in particle lysis buffer (0.1% SDS and 0.1 N NaOH). Then, for each lot of OVA particles, 2-fold serial dilutions were prepared in particle lysis buffer (50% down to 7.8%). One hundred and fifty microliters of each dilution were mixed with 150 L of the Micro BCA assay solution, incubated at 37 C.2h, cooled to room temperature, and the next day the absorbance was read at 562 nm. The standard curve was used to determine the concentration of OVA in each of the particle dilutions.

[0217] FITC-labeled ovalbumin was encapsulated in PLGA as follows. Dissolved 200 mg PLGA in 2 mL ethyl acetate (75:25) in a 5 mL polypropylene centrifuge tube. Dissolved 40 mg of ovalbumin and 0.5 mg FITC-labeled ovalbumin (ThermoFisher cat #023020) in 400 L H2O. To create an O/W emulsion, the PLGA solution was added dropwise into the OVA solution while vigorously vortexing. The mixture was then homogenized with a handheld homogenizer (TISSUEMISER, Fisher Scientific) at top speed for 3 min on ice. To form the W/O/W mixture, the resulting emulsion was mixed with 8 mL of 5% PVA in a 50 mL glass beaker and stirred with a magnetic stirrer for 5 min. The emulsion was then transferred to a 250 mL beaker containing 200 mL of 0.1% PVA and was stirred with a magnetic stirrer for 3 h to evaporate the ethyl acetate and harden the particles. The resulting mixture was transferred into four Oakridge centrifuge tubes and the particles were pelleted at 20,000g for 15 min. Each pellet was suspended in 30 mL DPBS and pelleted 3 more times. After the last wash, the particles were suspended in a total of 4 mL H2O and 1 mL aliquots were transferred into four 5 mL polypropylene conical centrifuge tubes. The particles were lyophilized overnight. One of the lyophilized pellets was suspended in 1 mL H2O.

[0218] PLGA particles were collected as follows. The resulting solution was transferred into a 50 mL polypropylene centrifuge tube and very large particles were pelleted at 50g for 2 min. The smaller particles remained in suspension and were transferred into two Oak Ridge tubes that were then placed in a Beckman JA-20 fixed angle rotor and pelleted at 20,000g for 15 min. The pellets were each washed with 20 mL H2O, combined in a single tube and re-pelleted. The single pellet was washed in a total of 30 mL H2O and pelleted (20,000g for 15 min). The particles were suspended in a total of 4 mL water, sonicated in a Vevor, Model JPS-30A, 180 W ultrasonic water bath for 3 min to break up any clumps of particles that occurred during centrifugation. The particles were transferred in 1 mL aliquots into 5 mL pre-weighed polypropylene tubes, lyophilized overnight in a system composed of a Savant SC210A SpeedVac concentrator, a Savant RVT 5105 refrigerated vapor trap, and a VLP 120 vacuum pump. After lyophilization, the tubes were weighed to determine the mass of PLGA particles. The particles in one of the tubes were suspended in 1 mL water and the amount of ovalbumin or BzF that had been encapsulated was determined. The remaining tubes were kept at 10 C.

[0219] BzF encapsulated PLGA particles were quantified as follows. The lyophilized pellet in one of the tubes was suspended in 1 mL sterile H2O by vigorous vortexing. Dilutions of the PLGA/BzF particles were prepared in triplicate wells of a 96 well UV-transparent plate (UV-STAR ultraviolet transparent plate, USA Scientific, Ocala, FL). To the first set of wells was added 175 L particle lysis buffer (0.1% SDS and 0.1 N NaOH) and 25 L of BzF particles (makes a 12.5% solution). Starting with the 12.5% dilution, two more 2-fold dilutions were prepared in particle lysis buffer (making solutions containing 6.25% and 3.13% of the particles). The absorbance at 280 nm was obtained and the concentration of BzF determined from the standard curve (after subtracting the absorbance obtained from the lysis buffer only).

[0220] FITC-OVA PLGA particles were uptaken by DC2.4 cells as follows. DC2.4 cells (1 mL at 410.sup.4/mL) were seeded into wells of a 24 well plate and allowed to adhere overnight. The next day, various dilutions of the FITC-OVA particles were added to the cells for 2 h. The wells were washed with HBSS and then the cells were released by incubating them for 10 min in 100 L DPBS containing 3% EDTA. The cells were suspended in 1 mL FACS buffer (DPBS containing 5% FBS, 2 mM EDTA, and 0.1% sodium azide) and examined for particle uptake using ThemoFisher Scientific's ATTUNE NxT flow cytometer.

[0221] In triplicate wells of a 96 well flat bottom plate, 100 L of complete DMEM (cDMEM, 10% FCS, 100 U/mL penicillin, and 100 g/mL streptomycin) containing DC2.4 at 210.sup.5/mL were plated and allowed to adhere for 4 hours. The cells were then treated by adding 100 L of media containing enough particles to deliver 320, 160, 80, 40, 20, 10, or 5 g/mL of ovalbumin. The cells were treated overnight. The next day, the cells in each well were washed with HBSS3 and then a total of 210.sup.4 B3Z cells were added and co-incubated overnight. The next day, the media was removed from the cells and 100 L of CPRG lysis buffer (DPBS containing 0.2% saponin, 20 mM MgCl2, 100 mM 2ME, and 100 mM chlorophenol red--D-galactopyranoside) was added. The plate was incubated at 37 C. The absorbance at 560 nm was measured at various time intervals (1-20 h) to detect activation of the NFAT-driven lacZ gene.

[0222] To test the effects of PLGA encapsulated BzF on the uptake, processing, and presentation of separately encapsulated whole ovalbumin by DC2.4, cells were plated at 210.sup.4 DC2.4 cells in 100 L cDMEM per well of a flat bottom 96 well clear plate. Allowed the cells to adhere for 1 h and then the media and non-adherent cells were flicked off. The media in triplicate wells was replaced with the following, 100 L of cDMEM only, 50 L of 160 g/mL whole OVA in PLGA particles plus 50 L cDMEM (OVA NP only), 50 L of 80 g/mL whole OVA and then 50 L of 100 g/mL BzF-NP, or 50 L of 40 g/mL whole OVA and then 50 L of 100 g/mL BzF-NP. The cells were then incubated overnight at 37 C. in an atmosphere of 5% CO.sub.2. The next day, the media was flicked off the cells and the cells were washed 3 times with 100 L DPBS. Then, 210.sup.4 B3Z cells, suspended in 100 L cRPMI, were added to all wells and incubated for 19 h. The media was flicked off the cells (B3Z cells adhere to the DC2.4 cells) and 200 L of CPRG lysis buffer was added. Four hours later, the absorbance at 560 nM was measured and plotted.

[0223] Immunization using PLGA particles containing ovalbumin or BzF was performed as follows. Tubes containing empty lyophilized PLGA particles or particles encapsulating ovalbumin or BzF, were freshly suspended in DPBS. Ten-week-old female C57Bl/6crl mice were randomly segregated into 4 groups with 5 mice per group. On days 0 and 10 the mice in Group 1 received an intramuscular (i.m., right rear leg) injection of 50 L DPBS, the mice in Group 2 received the i.m. injection with enough particles to produce a dose of 50 g ovalbumin (680 g/40 L of particles+10 L DPBS), the mice in Group 3 received the injection of particles containing a mixture of empty particles (280 g/9.7 L of particles)+particles containing 50 g ovalbumin (680 g/40 L of particles). The mice in Group 4 were injected i.m. with a mixture of particles containing 50 g ovalbumin (680 g/40 L of particles) and 50 g BzF (280 g/10 L particles).

[0224] On day 20, 10 days post the booster injection, the mice in all groups were shaved on their right flank and then injected subcutaneously with 110.sup.6 live E.G7-OVA cells in 50 L of 0.9% saline. Palpable tumors began to appear within 5-7 days. The width and length of the tumors were measured using digital calipers and the tumor volumes were calculated using the formula,

[00002]$\mathrm{Volume}=\left(\mathrm{greatest}\mathrm{width}\hat{}2\mathrm{greatest}\mathrm{length}\right)/2$

[0225] When the tumors became >15-20 mm in any dimension the mice were euthanized by CO.sub.2 asphyxiation in their home cage. The flow rate of CO.sub.2 was controlled by an AVMA approved EP-2200 Dual Preset Tank Regulator with Restrictors (EZ Systems, Palmer, PA).

[0226] Female C57BL/6crl (5 per group) were immunized by an intramuscular injection of 50 L DPBS only (NoTx), 50 L of OVA PLGA particles encapsulating 50 g of OVA (OVA), and 50 L of a mixture of OVA PLGA particles (50 g OVA) and BzF PLGA particles encapsulating 50 g of BzF (OVA/BzF). The mice were boosted with the same mixtures 10 days later. Ten days post-booster, the mice were injected with 110.sup.6 E.G7-OVA tumor cells subcutaneously into the right flank. The greatest width and greatest length of the resulting tumors were measured every other day once palpable tumors had appeared, as seen in FIGS. 34 and 35. In FIGS. 8 through 11, it was demonstrated that BzF could significantly enhance the cross-presentation of whole ovalbumin by the dendritic cell line, DC2.4. To enhance the targeting of OVA to phagocytic APC in vivo, the protein was encapsulated in PLGA particles. BzF was encapsulated in a separate preparation of PLGA particles. When mice were immunized with OVA particles, a mixture of OVA and empty particles, or a mixture of OVA and BzF particles, it was found that only the mix of OVA and BzF was able to significantly (p<0.01) suppress tumor growth, as seen in FIGS. 34 and 35. Neither OVA particles or the mix of OVA plus empty particles induced a tumor suppressive response. Therefore, the independent delivery of PLGA encapsulated BzF is able to enhance the presentation of Class I peptides generated from exogenously delivered whole proteins

Example 17

[0227] Herein, the mechanism by which BzF [(2E)-3-(1-benzofuran-5-yl)prop-2-eonic acid)]interacts with dendritic cells to subsequently enhance their ability to activate CD8+ T cells was examined. BzF is able to support peptide loading and displacement on H-2K.sup.b, H-2D.sup.b, and HLA-A*02:01 MHC class I structures. Through the use of both peptides and whole proteins, the mechanism by which BzF is capable of enhancing cell surface display of peptides presented through both direct and cross-presentation was elucidated. Although there has been some debate of whether CD8+ T cell functional response is dependent on cross-presentation directly, or if direct presentation plays an underlying role (Wu, et al., Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J Biol Chem. 2010 Apr. 2; 285(14):10850-61), data suggests that both modes of peptide presentation are enhanced via treatment with BzF. Enhancing peptide loading speed and capacity is critical for the development of practical DC-based vaccines, as current DC vaccine protocols are expensive and time consuming. (Wang, et al., Direct loading of CTL epitopes onto MHC class I complexes on dendritic cell surface in vivo. Biomaterials. 2018 November; 182:92-103). BzF allows for quick peptide loading within a span of hours, and more importantly, it functions in vivo, even after oral delivery, to enhance the loading of exogenous antigens.

[0228] The ability for BzF to exchange peptides was examined using a variety of methods. Not only was BzF able to rapidly exchange peptide in a cell-free setting for the creation of both A*02:01 and H-2K.sup.b tetramers, but it also facilitated peptide exchange on a variety of cell types (dendritic cells, microglial cells, and melanoma cells) that resulted in activation of a reporter CD8.sup.+ T cell hybridoma, as seen in FIGS. 3 through 11. The versatility of BzF is also highlighted in its ability to displace both high and lower affinity peptides. When applied after the high affinity SIINFEKL peptide, BzF was able to displace it, as seen in FIGS. 16 through 17B. The same activity was detected when HLA-A*02:01 tetramers were used in the QuickSwitch Quant assay, as seen in FIGS. 20A through 20C. This ability to facilitate rapid peptide exchange will allow BzF to be used in the rapid creation of MHC multimers for various immunodiagnostics. Furthermore, the in vivo data shows that this ability is translatable to use as a vaccine adjuvant allowing increased peptide loading in vitro and in vivo. This activity could be used to increase selection of peptide specific CD8+ T cells and increase their functional competence in a manner found in high peptide-dose vaccination protocols, but without the need for further peptide dose escalation. (Carretero-Iglesia, et al., High peptide dose vaccination promotes the early selection of tumor antigen-specific CD8 T-cells of enhanced functional competence. Front Immunol. 2020 Jan. 8; 10:3016).

[0229] Although the possibility remains that BzF could be affecting peptide binding by interacting with other regions of the MHC class I complex, the molecular docking studies support the experimental data that BzF binds with high affinity in the F pocket of the MHC class I and HLA-A*02:01 peptide binding grooves, as seen in FIGS. 21A through 22B. Among the binding pockets, the F pocket is the most important for the stability of the MHC class I peptide and accommodates the carboxyl terminal residue of the peptide. The stability of peptide binding is provided by hydrogen bonding between the peptide and specific residues in the vicinity of the F pocket. (Abualrous, et al., F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins. Eur J Immunol. 2015 April; 45(4):1248-57). According to docking studies, BzF is predicted to facilitate MHC class I stability and peptide binding though hydrogen bonding with the same residues previously reported to be critical for the binding of H-2K.sup.b peptides and the dipeptide, GL. (Saini, et al., Dipeptides promote folding and peptide binding of MHC class I molecules. Proc Natl Acad Sci USA. 2013 Sep. 17; 110(38):15383-8). This binding motif could possibly facilitate not only exchange of bound peptides, but also result in an increased binding efficiency while the F pocket remains in the peptide receptive state. (Anjanappa, et al., Structures of peptide-free and partially loaded MHC class I molecules reveal mechanisms of peptide selection. Nat Commun. 2020 Mar. 11; 11(1):1314). Data support both of these actions. BzF not only works to increase turnover, as seen in FIGS. 16 through 17B, but when cells are treated with BzF for prolonged periods of time the density of MHC class I complexes increases on the cell's surface, as seen in FIGS. 12B and 13B.

[0230] Like BzF, dipeptides, including GL, have been described as being able to increase MHC class I stability and facilitate peptide exchange. (Saini, et al., Dipeptides catalyze rapid peptide exchange on MHC class I molecules. Proc Natl Acad Sci USA. 2015 Jan. 6; 112(1):202-7; Hein, et al., Peptide-independent stabilization of MHC class I molecules breaches cellular quality control. J Cell Sci. 2014 Jul. 1; 127(13):2885-97). However, even though both BzF and GL interact with the MHC class I F pocket, the concentration of dipeptides required to either displace or enhance peptide binding is significantly higher (3-fold) than that required by BzF, as seen in FIG. 16. Also, unlike dipeptides which are rapidly destroyed in vivo (Newey H, Smyth D. The intestinal absorption of some dipeptides. J Physiol. 1959 Jan. 28; 145(1):48-56; Adibi, et al., Influence of molecular structure on half-life and hydrolysis of dipeptides in plasma: importance of glycine as N-terminal amino acid residue. Metabolism. 1986 September; 35(9):830-6), BzF is much more stable in the GI tract and bloodstream, providing more time to interact with cells and facilitate a functional peptide exchange, as seen in FIG. 29 through 31.

[0231] Detection of BzF in the blood plasma of mice, as quickly as 30 minutes and for as long as 3 h following oral administration, confirmed the predictions of the SwissADME program (Daina, et al., SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Sci Rep. 2017 Mar. 3; 7(1):1-13) that BzF could be absorbed into the bloodstream from the GI tract. The BzF absorbed through the GI tract was intact and functional, seen in FIGS. 28A, 28B and 29. Although in vitro kinetic stability has been found to correlate with in vivo CD8+ T cell response in C57BL/6 mice, the true applicability in vivo immunological action has been debated. (Thirdborough, et al., Tapasin shapes immunodominance hierarchies according to the kinetic stability of peptide-MHC class I complexes. Eur J Immunol. 2008 February; 38(2):364-9; Celli, et al., Decoding the dynamics of T cell-dendritic cell interactions in vivo. Immunol Rev. 2008 February; 221(1):182-7). As such studies were undertaken in a murine model to determine the translational ability of BzF. In these studies, mice were injected intravenously with SIINFEKL peptide and then immediately thereafter, received BzF by gavage, intravenous, or intramuscular injections. One hour later, the splenocytes were isolated and co-cultured with reporter CD8+ T cell hybridoma cells (B3Z). The results demonstrated that the splenocytes from mice receiving both SIINFEKL and BzF, regardless of the route of BzF delivery, were able to activate the CD8+B3Z cells to an extent significantly greater than that produced by splenocytes from mice receiving peptide alone, as seen in FIGS. 29 through 31. Upon further analysis of these splenocytes it was found that BzF was able to increase SIINFEKL binding in the dendritic cell population, as seen in FIG. 31. These findings suggest that BzF has the potential to be used in vivo to facilitate therapeutically and immunologically relevant peptide exchange.

[0232] In a cursory examination of the structure-activity relationship, it became clear that BzF's structure, particularly its state of conjugation, is critical for its function. When molecules that differed from BzF by their level of saturation, and hence their loss of aromaticity and reduction in hydrophobicity, were tested in the B3Z activation assay, SIINFEKL binding assay, and the peptide displacement assay, they all failed to achieve the same activity as BzF, as seen in FIGS. 24A through 26. These findings implicate the specific BzF structure in its ability to modulate MHC class I peptide binding.

[0233] BzF specifically targets MHC class I structures on the cell surface without interfering with common metabolic or inflammatory pathways (data not shown). This specificity allows BzF to be used in combination with known vaccine adjuvants and has the potential to increase efficacy of a wide range of modalities. Targeting extracellular surface receptors that are functionally used in the immune response has been shown to have the most potent effect in vivo when compared to other vaccine mechanisms (Kastenmuller, et al., Dendritic cell-targeted vaccineshope or hype? Nat Rev Immunol. 2014 October; 14(10):705-11). It has been well established that immunotherapies in cancer are most effective when a multi-faceted approach is taken, BzF's targeted effect would allow for the use of other adjuvants, such as PD-1/PD-Li checkpoint blockade or toll-ligand activators, in combination with BzF for a robust response. As previously mentioned, nearly all nucleated cells possess MHC class I on their cell surface, used for self-recognition, MHC class I peptides are nearly ubiquitous in the body. (Davis, et al., Natural killer cell adoptive transfer therapy: exploiting the first line of defense against cancer. Cancer J (Sudbury, Mass). 2015 November-December; 21(6):486-91). As such, the binding of BzF on the MHC class I molecule allows for its use on non-antigen presenting cells and opens the possibility for its use in other disease mechanisms that are exacerbated by diminished MHC class I functionality. BzF might also be utilized to change the peptide profile on tumors that have induced a state of tolerance.

[0234] To test the hypothesis that BzF can enhance the generation of Class I antigen-specific anti-tumor responses, two clinically relevant vaccine models were examined. The first utilized the in vitro generation of a dendritic cell vaccine and monitored the in vivo development of antigen specific CD8+ T cells. As shown in FIG. 33A, immunization of mice with IFN matured DC cells that had been treated for 2 hours with BzF and then pulsed for 1 hour with SIINFEKL peptide, generated significantly more SIINFEKL specific tetramer positive CD8+ T cells in the peripheral blood and the spleen. DC that had been pulsed with SIINFEKL, but not pre-treated with BzF, generated much weaker responses. Vaccines aimed at inducing CD8+ T cell responses have seen promise in targeting and clearing pathogens. (Sei, et al., Peptide-MHC-I from endogenous antigen outnumber those from exogenous antigen, irrespective of APC phenotype or activation. PLoS Pathog. 2015 Jun. 24; 11(6):e1004941).

[0235] The second model utilized whole OVA and BzF that had been separately encapsulated in PLGA microparticles to induce an anti-tumor immune response. Immunization with OVA particles failed to produce a response capable of suppressing the growth of E.G7-OVA tumor cells. However, when the mice were immunized with a mixture of OVA and BzF particles, a significant suppression of tumor growth was observed, as seen in FIGS. 34 and 35. These findings strongly suggest that whether it is used in vitro to generate dendritic cell vaccines, or delivered orally, intravenously, intramuscularly, or via PLGA particles, BzF is capable of significantly enhancing the in vivo generation of a Class I CD8+ T cell vaccine response. The findings support BzF's therapeutic potential to be examined for use in nanoparticle, mRNA, DNA, peptide, dendritic cell vaccines, and to synergize with chemo- or immunotherapeutics to generate in situ anti-tumor vaccines.

[0236] In conclusion, the presentation of class I peptides is central to the generation of CD8+ T cell responses. CD8.sup.+ T cell responses are vital for tumor surveillance and immunological protection against intracellular pathogens. As such, the discovery of an orally available, non-peptide-based, small molecule, capable of modulating peptide exchange in vivo, is an important addition to the therapeutic arsenal. The ability of BzF to enhance peptide loading in vivo is currently being examined for the ability to enhance antitumor and antiviral immune responses generated by mRNA-, peptide-, CAR-T therapies, dendritic cell-based, and micro/nano particle vaccines. Furthermore, as demonstrated by its ability to alter peptide display on tumor cells (B16F10 melanoma and E.G7-OVA T cell lymphoma), as seen in FIGS. 10 and 16 through 17B, the potential of BzF to enhance tumor targeting by CTL and to alter peptide display on targets of cell-mediated autoimmune responses are being examined.

Example 18: BzF Niosome Microvesicle Carriers

[0237] Niosomes have shown benefits over liposomes in that the synthetic niosomes appear more chemically stable as vesicles, are easier to transport and store, are less expensive to manufacture, and possess increased permeability to the blood brain barrier. It is composed of synthetic amphiphilic surfactants and cholesterol that make up a bilayer membrane that is able to entrap hydrophilic solutions in the aqueous core of the vesicle.

[0238] Exemplary surfactants include, without limiting the scope of the invention, crown ether amphiphiles bearing a steroidal moiety, 1,2-dialkyl glycerol polyoxyethylene ether, hexadecyl poly-5-oxyethylene ether, hexadecyl poly-5-oxyethylene ether (C.sub.16EO.sub.5); octadecyl poly-5-oxyethylene ether (C.sub.18EO.sub.5); hexadecyl diglycerol ether (C.sub.16G.sub.2); sorbitan monopalmitate (Span 40) and sorbitan monostearate (Span 60), Solulan C24 (poly-24-oxyethylene cholesteryl ether), polysorbate 20, Span detergents, Brij detergents, such as Brij-35, and polyoxyethylene. Crown ethers are known in the art (Montserrat, et al., Light-induced charge injection in functional crown ether vesicles. J Am Chem Soc. 1980 Aug. 1; 102(17):5527-5529; Darwish & Uchegbu, The evaluation of crown ether based niosomes as cation containing and cation sensitive drug delivery systems. Int J Pharm. 1997 Dec. 15; 159(2):207-213; Uchegbu & Duncan, Niosomes containing N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin (PK1): effect of method of preparation and choice of surfactant on niosome characteristics and a preliminary study of body distribution. Int J Pharm. 1997 Sep. 12; 155(1):7-17). Exemplary solvents involved in noisome formation may include glycerol, oil, water, and combinations thereof.

[0239] Cholesterol stabilizes the vesicles by decreasing the permeability and enhancing solute retention (Uchegbu & Florence, Non-ionic surfactant vesicles (niosomes): physical and pharmaceutical chemistry. Adv Colloid Interface Sci. 1995 Jun. 27; 58(1):1-55; Nasseri, Effect of cholesterol and temperature in the elastic properties of niosomal membranes. Int J Pharm. 2005 Aug. 26; 300(1-2):95-101). In addition, negative charged molecules may be added to the bilayer-producing compounds, such as dicetyl phosphate, cetyl sulphate, phosphatidic acid, phosphatidyl serine, oleic acid, palmitic acid, which provide electrostatic stabilization to the vesicles and prevents vesicle aggregation (Uchegbu & Vyas, Non-ionic Surfactant Based Vesicles (Niosomes) in Drug Delivery. Int J Pharm., 1998 Oct. 15; 172(1-2):33-70; Manosroi, et al., Characterization of vesicles prepared with various non-ionic surfactants mixed with cholesterol. Colloids Surf B. 2003 Jul. 1; 30(1-2):129-138). The ability of the surfactant to form a vesicle depends on two factors, the Hydrophobic Lipophilic Balance (HLB) and the Critical Packing Parameter (CPP). The HLB is calculated using

[00003]$HLB=20Mh/M$

where Mh is the molecular mass of the hydrophilic portion of the surfactant, and M is the molecular mass of the whole niosome, giving a result on an arbitrary scale of 0 to 20. For the surfactant sorbitan monostearate, an HLB number between 4 and 8 was found to be compatible with vesicle formation (Uchegbu & Vyas, Non-ionic Surfactant Based Vesicles (Niosomes) in Drug Delivery. Int J Pharm., 1998 Oct. 15; 172(1-2):33-70).

[0240] The CPP is a dimensionless number that predicts the ability of the amphiphile to form aggregates, with values of 0.5-1.0 predicting that the amphiphile will form a vesicle. (Israelachvili, Intermolecular and Surface Forces: With Applications to Colloidal and Biological Systems. 3d Ed. 2011, Orlando: Academic Press). CPP is calculated using

[00004]$CPP=/l_c{a}_o$

where =hydrocarbon chain volume, l.sub.c=critical hydrophobic chain length (the length above which the chain fluidity of the hydrocarbon may no longer exist), and a.sub.o=area of hydrophilic head (Uchegbu & Florence, Non-ionic surfactant vesicles (niosomes): physical and pharmaceutical chemistry. Adv Colloid Interface Sci. 1995 Jun. 27; 58(1):1-55).

[0241] The niosomes are formed from the self-assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer structures (Uchegbu & Vyas, Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int J Pharm., 1998 Oct. 15; 172(1-2):33-70). The assembly into bilayers is rarely spontaneous and usually involves some input of energy such as physical agitation or heat. Span 60, a surfactant, a cholesterol, and, optionally, dicetyl phosphate and/or sorbitan monosterate, a negatively charged molecule, are added to a flask in an organic solvent at a ratio of 1:1:(0.1), surfactant to cholesterol to dicetyl phosphate. The solution is mixed by agitation over a 600 C bath until the solids dissolve and the solution transferred to an evaporator, such as a buch rotary evaporator with nitrogen gas until a film forms on the flask. The flask is allowed to further dry for at least 12 hours, and the film hydrated using a solution of BzF. The solution is further dried in the rotary evaporator until the thin film dissolves and the niosomes extruded at 600 C, resulting in size-limited niosomes containing BzF. The niosomes are separated from unincorporated BzF by centrifugation at 60000 rpm for 40 minutes.

[0242] These niosomes have been found to exhibit a linear behavior in their size distribution as the concentration of the hydrophilic component increases from 5 millimolar to 15 millimolar. (Alcantar, et al., U.S. Pat. No. 9,522,114).

Example 19: Protein Niosome Microvesicle Carriers

[0243] Protein-loaded niosomes are useful in combination with systemic administration of BzF. The protein-loaded niosomes are formed from the self-assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer structures (Uchegbu & Vyas, Non-ionic surfactant based vesicles (niosomes) in drug delivery. Int J Pharm., 1998 Oct. 15; 172(1-2):33-70). A solution of Span 60, a surfactant, a cholesterol, and, optionally, dicetyl phosphate and/or sorbitan monosterate, a negatively charged molecule, are added to a flask in an organic solvent at a ratio of 1:1:(0.1), surfactant to cholesterol to dicetyl phosphate. The solution is mixed by agitation over a 60 C. bath until the solids dissolve and the solution transferred to an evaporator, such as a buch rotary evaporator with nitrogen gas until a film forms on the flask. The flask is allowed to further dry for at least 12 hours, and the film hydrated using a solution containing the antigen protein is mixed by agitation over a 60 C. bath until the solids dissolve and the solution transferred to an evaporator, such as a buch rotary evaporator with nitrogen gas until a film forms on the flask. The flask is allowed to further dry for at least 12 hours, and the film hydrated using the compound in solution, as described in Example 18. The solution is further dried in the rotary evaporator until the thin film dissolves and the niosomes extruded at 60 C., resulting in size-limited niosomes containing BzF. The niosomes are separated from unincorporated BzF by centrifugation at 60000 rpm for 40 minutes.

Example 20: Combined BzF-Protein Niosome Microvesicle Carriers

[0244] The niosomes are formed from the self-assembly of non-ionic amphiphiles in aqueous media resulting in closed bilayer structures (Uchegbu & Vyas, Non-ionic surfactant-based vesicles (niosomes) in drug delivery. Int J Pharm., 1998 Oct. 15; 172(1-2):33-70). The assembly into bilayers is rarely spontaneous and usually involves some input of energy such as physical agitation or heat. Span 60, a surfactant, a cholesterol, and, optionally, dicetyl phosphate and/or sorbitan monosterate, a negatively charged molecule, are added to a flask in an organic solvent at a ratio of 1:1:(0.1), surfactant to cholesterol to dicetyl phosphate. The solution is mixed by agitation over a 60 C. bath until the solids dissolve and the solution transferred to an evaporator, such as a buch rotary evaporator with nitrogen gas until a film forms on the flask. The flask is allowed to further dry for at least 12 hours, and the film hydrated using the compound in solution, as described in Example 1. The solution is further dried in the rotary evaporator until the thin film dissolves and the niosomes extruded at 600 C, resulting in size-limited niosomes containing BzF. The niosomes are separated from unincorporated BzF by centrifugation at 60000 rpm for 40 minutes.

Example 21: Niosome Target Microvesicle Carriers

[0245] Molecules are loaded onto the niosomes to permit the niosome delivery system to target cancer cells. Examples of methods to incorporate target molecules into the vesicle membrane are known in the art, but can include by way of non-limiting example, anti-integrin molecules specific to antigen presenting cells (APCs). Examples of integrin molecules specific to APCs include BDCA-1, CD1a, CD1c, CD11b, CD11c, CD123, CD141, CD207, and CD303, alone or in combination, as seen in Table 3. (Collin & Bigley, Human dendritic cell subsets: an update. Immunology. 2018 May; 154(1): 3-20). Accordingly, anti-BDCA-1, anti-CD1a, anti-CD1c, anti-CD11b, anti-CD11c, anti-CD123, anti-CD141, anti-CD207, and anti-CD303, alone or in combination are loaded onto the niosomes. While the epitopes listed are for conventional dendritic cells (myeloid dendritic cells, it is within the skill of one of ordinary skill in the art to use the disclosed information to target other APC cell types.

TABLE-US-00003 TABLE 3 List of common integrins on human dendritic cells. Cell type Conventional surface markers Extended markers Plasmacytoid DC CD123 FCER1 CD303, CLEC4c, BDCA-2 ILT3, ILT7 CD304, NRP1, BDCA-4 ILT3, ILT7 Myeloid cDC1 CD141, BDCA-1 CLEC9A CADM1 XCR1 BTLA CD26 DNAM-1, CD226 Myeloid cDC2 CD1c, BDCA-1 CD-2 CD-11c FCER1 CD-11b SIRPA ILT1 DCI1, CLEC4A CLEC10A Langerhans cell CD207 EpCAM CD1a TROP2 E-Caderin

[0246] Niosomes are prepared, as described in any of Examples 17 through 19, using PEG molecules that are modified to introduce an amino group. (Hong, et al., Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin. J Control Release. 2009 Jan. 19; 133(2):96-102). Target molecules are conjugated to the PEG-amino group using an oxidation reaction, by oxidizing the target molecule and introducing the oxidized target molecule to the amino-PEG-niosomes. (Hong, et al., Efficient tumor targeting of hydroxycamptothecin loaded PEGylated niosomes modified with transferrin. J Control Release. 2009 Jan. 19; 133(2):96-102).

Example 22: Liposome Microvesicle Carriers

[0247] Lipid nanoparticles have been shown as an effective transport vehicle to deliver the compounds to biologic target cells. Such lipid nanoparticles are made by mixing a cationic lipid, non-cationic lipid, a polyethyleneglycol (PEG) conjugated lipid, and a structural lipid in a method similar to Example 16, and loaded with BzF, protein, or a combination of BzF and protein, as provided in Examples 15 through 19. In some variations, the lipid nanoparticles can be synthesized using a mixture of ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid at a ratio of 50:10:38.5:1.5 mol/mol (cationic lipid: phosphatidylcholine: cholesterol: PEG-lipid), and encapsulate the BzF. The interaction between the liposomes and the cargo usually relies on hydrophobic interactions or charge attractions, particularly in the case of cationic lipid delivery systems (Zelphati, et al., Intracellular delivery of proteins with a new lipid-mediated delivery system. J Biol Chem. 2001 Sep. 14, 276(37):35103-35110). Modifications to the liposomes, by adding ionizable amino lipids as cationic lipid moieties, such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, increase stability of liposome carriers.

Example 23: Microspheres

[0248] Alternatively, BzF, protein, or a combination of BzF and protein were incorporated into microspheres. (Harris, et al., A synthetic peptide CTL vaccine targeting nucleocapsid confers protection from SARS-CoV2 challenge in rhesus macaques. are separated from unincorporated. Vaccines (Basel). 2021 May 18; 9(5):520; Herst, et al., An effective CTL peptide vaccine from Ebola Zaire based on survivors' CD8+ targeting of a particular nucleocapsid protein epitope with potential implications for COVIS-19 vaccine design. Vaccine. 2020 Jun. 9; 38(28):4464-4475). Briefly, mannose, CpG oligonucleotide, and SINFEKL or other peptide were mixed with a solution of BzF in acetone/water (50:50 v:v). The solution was sonicated and extruded through a Bchi rotovaporator with extruder.

[0249] The BzF can be loaded into microvesicles or microparticle, such as niosomes or liposomes or microsphere, as disclosed in Examples 18 through 21, and the vesicles can optionally possess surface ligands to permit specific targeting to cancer cells or a cell type, allowing the carrier to interface with a cell expressing the conjugate ligand or molecule.

[0250] As shown herein, BzF can enhance the generation of Class I antigen-specific responses. Immunization of mice through matured DC cells treated with BzF and exposed to an antigen peptide, generated significantly higher antigen-specific tetramer positive CD8+ T cells in the peripheral blood and the spleen. Vaccines aimed at inducing CD8+ T cell response have seen promise in targeting and clearing pathogens (Sei, et al., Peptide-MHC-I from endogenous antigen outnumber those from exogenous antigen, irrespective of APC phenotype or activation. PLoS Pathog. 2015 Jun. 24; 11(6):e1004941). This opens the door for BzF to be tested for use in various mRNA, peptide, as well as dendritic cell-based vaccines.

[0251] The presentation of class I peptides is central to the generation of CD8.sup.+ T cell responses. CD8.sup.+ T cell responses are vital for tumor surveillance and immunological protection against intracellular pathogens. As such, the discovery of an orally available, small molecule, capable of modulating peptide exchange in vivo, is an important addition to the therapeutic arsenal. The ability of BzF to enhance peptide loading in vivo is currently being examined for the ability to enhance antitumor and antiviral immune responses generated by mRNA-, peptide-, CAR-T therapies, and dendritic cell-based vaccines. Furthermore, as demonstrated by its ability to alter peptide display on tumor cells (B16F10 melanoma and E.G7-OVA T cell lymphoma), BzF can enhance tumor targeting by CTL and alter peptide display on targets of cell-mediated autoimmune responses.

[0252] In the preceding specification, all documents, acts, or information disclosed does not constitute an admission that the document, act, or information of any combination thereof was publicly available, known to the public, part of the general knowledge in the art, or was known to be relevant to solve any problem at the time of priority.

[0253] The disclosure of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.

[0254] It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.