Vasoactive intestinal peptide (VIP) receptor antagonists

Abstract

VIP-R antagonists are disclosed for use in managing the treatment or prevention of cancer and viral infection. In certain embodiments, the disclosure relates to chimeric variants of VIP-R antagonists as peptides disclosed herein, and pharmaceutical compositions comprising same. In certain embodiments, the disclosure contemplates a method of treating a subject with cancer or infectious disease with VIP-R antagonists, or a method of stimulating immune cells that target cancer by mixing immune cells with peptides disclosed herein in vitro, and further administering an effective amount of stimulated immune cells to a subject in need of cancer treatment.

Description

[Technical field]

[0001] I. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority benefit of U.S. Provisional Application No. 63 / 189,507, filed May 17, 2021, which is expressly incorporated by reference in its entirety herein. [Background technology]

[0002] II. Background technology Vasoactive intestinal peptide (VIP) is produced in various cells including immune cells, neurons, and endocrine cells in the central nervous system. Endogenous VIP is present in nerves that innervate the smooth muscle of airways and pulmonary blood vessels in the lungs, and VIP functions as a bronchodilator in this organ. VIP can also alter cell proliferation and the production of inflammatory signals through the VIP receptors VPAC1 and VPAC2. A chimeric peptide, designated VIPhyb, was developed in which the six N-terminal amino acids of native VIP were replaced with six highly polar N-terminal 6 amino acids from the neurotensin peptide sequence, followed by the C-terminal 22 amino acid sequence of VIP. Due to the change in the N-terminal amino acids, VIPhyb has altered biological activity compared to VIP, acting as an antagonist to the VIP receptor (VIP-R) by competitively binding to the receptor but not signaling.

[0003] Cancer treatment typically utilizes surgery, chemotherapy, and radiation therapy. However, alternative therapeutic methods have been reported that enhance the immune system to attack cancerous cells. These methods include collecting, expanding, and modifying T cells to target and stimulate the immune system to actively eliminate cancerous cells. In chimeric antigen receptor (CAR) T cell therapy, isolated T cells are engineered to express a chimeric protein and administered back to the patient. However, there is a need to identify therapies that improve the functional properties of T cells. Summary of the Invention

[0004] III. Summary of the Invention Methods and compositions related to vasoactive intestinal peptide (VIP) receptor antagonists for use in the therapeutic or prophylactic management of cancer and viral infections are disclosed. In certain embodiments, the disclosure relates to chimeric variants of VIP-R antagonists as peptides disclosed herein, and pharmaceutical compositions comprising same. In certain embodiments, the disclosure contemplates a method of stimulating immune cells that target cancer by mixing immune cells in vitro with peptides disclosed herein, and further administering an effective amount of the stimulated immune cells to a subject in need of cancer treatment.

[0005] In one aspect, disclosed herein is the amino acid sequence KPRRPYX 1 X 2 X 3 X 4 TX 5 LRKQX 6 AVX 7 X 8 KYLX 9 X 10 ILN (SEQ ID NO: 3), wherein X 1 is T or A, and X 2 is D, V, or S, and X 3 is N or D, and X 4 is Y or C, and X 5 is R or S, and X 6 is M or I, and X 7 is K or N, and X 8 is K and X 9 is N or M, and X 10 is S or L, with the proviso that the peptide is not KPRRPYTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 1) or 1 is T and X 2 is D and X 3 is N and X 4 is Y and X 5 is R and X 6 is M and X 7 is K and X 9 is N and X 10is not S. In one embodiment, disclosed herein is a VIP receptor antagonist having the amino acid sequence KPRRPYX 1 X 2 X 3 X 4 TX 5 LRKQX 6 AVX 7 KYLX 8 X 9 ILN (SEQ ID NO: 21), wherein X 1 is T or A, and X 2 is D, V, or S, and X 3 is N or D, and X 4 is Y or C, and X 5 is R or S, and X 6 is M or I, and X 7 is K or N, and X 8 is N or M, and X 9 is S or L, with the proviso that the peptide is not KPRRPYTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 1) or 1 is T and X 2 is D and X 3 is N and X 4 is Y and X 5 is R and X 6 is M and X 7 is K and X 8 is N and X 9is not a combination where S. For example, disclosed herein are VIP receptor antagonists having the amino acid sequences: KPRRPYADNYTRLRKQMAVNKYLNLILN (SEQ ID NO: 6), KPRRPYAVNYTRLRKQIAVKKYLMSILN (SEQ ID NO: 7), KPRRPYAVNYTRLRKQMAVNKYLMSILN (SEQ ID NO: 8), KPRRPYADNCTRLRKQIAVNKKYLNSILN (SEQ ID NO: 9), KPRRPYTVNYTSLRKQIAVKKYLMLILN (SEQ ID NO: 10), KPRRPYTDNCTSLRKQIAVNKYLN LILN (SEQ ID NO: 11), KPRRPYAVNCTSLRKQIAVNKYLNSILN (SEQ ID NO: 12), KPRRPYAVNCTSLRKQIAVKKYLMSILN (SEQ ID NO: 13), KPRRPYTVNCTSLRKQIAVKKYLMLILN (SEQ ID NO: 14), KPRRPYTSDYTRLRKQMAVKKYLNSILN (SEQ ID NO: 15), KPRRPYTSDYTRLRKQMAVKKYLNLILN (SEQ ID NO: 16), fragments thereof, or analogs thereof.

[0006] Also disclosed herein are VIP-R antagonists of any of the preceding embodiments, wherein an amino, carboxyl, hydroxyl, or thiol group within the VIP-R antagonist is substituted.

[0007] In some embodiments, the VIP-R antagonist is conjugated to and / or encapsulated within the nanoparticles.

[0008] Also disclosed herein is the VIP-R antagonist of any of the preceding embodiments, wherein the VIP-R antagonist further comprises a label, eg, a fluorescent or radioactive label.

[0009] In one aspect, disclosed herein is a pharmaceutical composition comprising a VIP-R antagonist of any of the preceding aspects and a pharma- ceutically acceptable carrier.

[0010] In one aspect, disclosed herein is a nucleic acid encoding a VIP-R antagonist of any of the preceding aspects. Also disclosed herein is a recombinant vector comprising the nucleic acid. In one aspect, disclosed herein is an expression system or cell comprising a recombinant vector of any of the preceding aspects.

[0011] Also disclosed herein are methods of treating, decreasing, inhibiting, reducing, ameliorating, and / or preventing cancer and / or metastasis in a subject, or enhancing an immune response to cancer and / or metastasis in a subject, comprising administering to the subject a therapeutically effective amount of a VIP-R antagonist (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments, or analogs thereof, or a pharmaceutical composition of any of the preceding aspects. In certain embodiments, the VIP-R antagonist or pharmaceutical composition is administered in combination with another anti-cancer agent. In some aspects, the method can further include exposing the subject to radiation and / or transplanting allogeneic hematopoietic stem cells into the subject and / or other adoptive cell therapy (e.g., administering CAR T cells, TCR-modified T cells, CAR NK cells, TILs, TINKs, and / or MILs). In certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., including a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In certain embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent. In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0012] In certain embodiments, the present disclosure relates to a method of enhancing activation and / or proliferation of T cells ex vivo, comprising mixing T cells with a VIP-R antagonist of any of the preceding aspects (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) In certain embodiments, mixing the T cells is performed in combination with an anti-CD3 antibody and / or an anti-CD28 antibody. In certain embodiments, mixing the T cells includes the administration of phosphatidylinositol 3-kinase (PI3K) inhibitors (e.g., fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474, as well as isoform-specific inhibitors, such as PI3K alpha inhibitors (e.g., inavolisib, alpelisib, AZD8835, PWT33597, , taselisib, and / or seravelisib), PI3K beta inhibitors (such as, for example, AZD8186 and / or GSK2636771), PI3K delta inhibitors (such as, for example, AZD8835, AZD8186, nemiralisib, serretalisib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, linperlisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, for example, eganelisib, tenalisib, taselisib, and / or duvelisib). In certain embodiments, mixing the T cells is performed in combination with an immune checkpoint blockade agent. In some embodiments, the immune checkpoint blockade is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0013] Also disclosed herein is a kit comprising a VIP-R antagonist of any preceding embodiment (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments or analogs thereof, etc.) or a pharmaceutical composition of any preceding embodiment, and an anti-CD3 antibody and / or an anti-CD28 antibody. In certain embodiments, the kit includes a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474, as well as an isoform-specific inhibitor, such as a PI3K alpha inhibitor (e.g., inavolisib, alpelisib, AZD8835, PWT 33597, taselisib, and / or seravelisib), PI3K beta inhibitors (such as, for example, AZD8186 and / or GSK2636771), PI3K delta inhibitors (such as, for example, AZD8835, AZD8186, nemiralisib, serretalisib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, limpellisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, for example, eganelisib, tenalisib, taselisib, and / or duvelisib). In certain embodiments, the kit further comprises an immune checkpoint blockade agent. In some embodiments, the immune checkpoint blockade is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0014] Also disclosed herein is an in vitro cell culture composition comprising one or more T cells and a VIP-R antagonist of any preceding aspect (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) or a pharmaceutical composition of any preceding aspect. In some embodiments, the in vitro cell culture composition further comprises an anti-CD3 antibody and / or an anti-CD28 antibody. In certain embodiments, the in vitro cell culture composition includes a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474, as well as an isoform-specific inhibitor, such as a PI3K alpha inhibitor (e.g., inavolisib, alpelisib, AZD8835 , PWT33597, taselisib, and / or seravelisib), PI3K beta inhibitors (such as, for example, AZD8186 and / or GSK2636771), PI3K delta inhibitors (such as, for example, AZD8835, AZD8186, nemiralisib, serretalisib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, limpellisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, for example, eganelisib, tenalisib, taselisib, and / or duvelisib). In certain embodiments, the in vitro cell culture composition further comprises an immune checkpoint blockade agent. In some embodiments, the immune checkpoint blockade is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0015] In certain embodiments, the present disclosure relates to a method of treating or preventing graft-versus-host disease in a subject, comprising administering an effective amount of any of the VIP-R antagonists of any preceding aspect (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, or fragments thereof, etc.) to a subject receiving or that has received transplanted allogeneic tissue or cells.

[0016] In certain embodiments, the present disclosure relates to a method of treating, reducing, inhibiting, decreasing, alleviating, managing, and / or preventing a microbial infection (including, but not limited to, a viral, bacterial, fungal, and / or parasitic infection), comprising administering to a subject infected with or at risk for a microbial infection, a therapeutically effective amount of a VIP-R antagonist of any preceding aspect (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) or a pharmaceutical composition of any preceding aspect.

[0017] Also disclosed herein is a method of treating cancer or a chronic infection in a subject in need thereof, comprising providing one or more T cells; mixing the one or more T cells with a VIP-R antagonist (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments or analogs thereof, etc.) or a pharmaceutical composition of any preceding aspect, thereby expanding the one or more T cells; and administering a therapeutically effective amount of the expanded T cells to the subject.

[0018] In some embodiments, the method of any preceding aspect includes combining one or more T cells in combination with an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the method of any preceding aspect includes combining one or more T cells in combination with an immune checkpoint blockade agent. In some embodiments, the method of any preceding aspect comprises treating one or more T cells with a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474, as well as an isoform-specific inhibitor, such as a PI3K alpha inhibitor (e.g., inavolisib, alpelisib, AZD8835, , PWT33597, taselisib, and / or seravelisib), PI3K beta inhibitors (such as, for example, AZD8186 and / or GSK2636771), PI3K delta inhibitors (such as, for example, AZD8835, AZD8186, nemiralisib, serretalisib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, limpellisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, for example, eganelisib, tenalisib, taselisib, and / or duvelisib).

[0019] In some embodiments, the method of any preceding aspect further comprises administering to the subject a PI3 kinase inhibitor, a VIP receptor antagonist, or an immune checkpoint blockade agent, or a combination thereof, before, during, or after administering the expanded T cells. In some embodiments, the one or more T cells are derived from the subject. In some embodiments, the one or more T cells are engineered T cells. In some embodiments, the one or more receptors comprise a chimeric antigen receptor.

[0020] IV. Brief Description of the Drawings The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments and, together with the description, illustrate the disclosed compositions and methods. [Brief description of the drawings]

[0021] [Figure 1] We show that treatment with a novel peptide derived from VIP extended the survival of mice engrafted with acute myeloid leukemia. B6 (CD45.2, H-2Kb) mice were administered C1498 at 1x106 / mouse via the tail vein. VIP novel peptide was injected subcutaneously at 10 micrograms per mouse daily, starting on day 6 after leukemia challenge, for a total of seven injections. Mice were monitored daily for survival. Data were pooled from three replicate experiments. [Figure 2-1] We show that treatment with a novel peptide derived from VIP extended the survival of mice with leukemia. B6 (CD45.2, H-2Kb) mice were administered C1498 at 1x106 / mouse via the tail vein. VIP novel peptide was injected subcutaneously at 10 micrograms per mouse daily, totaling 7 times, starting 6 days after leukemia challenge. Mice were monitored daily for survival. Mice survival was pooled from 3 replicate experiments and analyzed by log-rank test in comparison to survival of mice treated with scrambled peptide. [Figure 2-2] (As stated above.) [Diagram 3]Figure 1 shows that the predicted affinity and potency of competitive binding VPAC by VIP-derived novel peptides correlated with the increased survival percentage in leukemia-bearing mice treated with VIP-derived novel peptides. For each individual VIP novel peptide, the logarithm of the sum of the absolute predicted binding affinity to human VPAC1 and VPAC2 is generated, and then the logarithm of the absolute binding affinity is plotted against the respective survival percentage. Panel A: Correlation of survival with the logarithm of the absolute predicted binding affinity to human VPAC1 (logarithm of the absolute binding affinity for VPAC1) [survival percentage]. Panel B: Correlation of survival with the logarithm of the absolute value of predicted binding affinity to human VPAC2. (logarithm of absolute value of binding affinity to VPAC2) [survival percentage]. Panel C: The logarithm of the absolute value of the sum of binding affinities for VPAC1 and VPAC2 plotted against the respective survival percentages in leukemic mice treated with each of the novel peptides derived from VIP. [Figure 4]We show that treatment with a novel peptide derived from VIP reduced the levels of leukemic cells in the blood of leukemic mice. B6 SJL (CD45.1, H-2Kb) mice were administered C1498 (CD45.2H-2Kb) at 1x106 / mouse via the tail vein. VIP novel peptide was injected subcutaneously at 10 micrograms per mouse daily for a total of seven doses, beginning on day 6 after leukemic challenge. Mice were bled weekly from the mandibular vein, beginning on day 6 prior to peptide administration. Antibodies against CD45.2 identified myeloid leukemic cells, and antibodies against CD45.1 identified host mouse leukocytes. [Diagram 5] Mice treated with novel peptides derived from VIP show extended survival after rechallenge with myeloid leukemia cells. B6 mice were administered Luc-1498 at 1×106 / mouse for Luc-C198 positive control or C1498 at 1×106 / mouse for C1498 (luciferase) negative control via the tail vein. Mice remaining tumor-free after day 68 were rechallenged via the tail vein with Luc-1498 at 1×106 / mouse. Mice were imaged for luminescence every other week 16 days after leukemia rechallenge. Image exposure time was 15 seconds except for 3 minutes on days 16 and 23. Data are presented as mean luminance [p / s / cm2 / sr]. Dead mice are indicated by white Xs. [Figure 6]Aggregated data from mice treated with novel peptides derived from VIP show extended survival after rechallenge with acute myeloid leukemia. B6 mice were administered Luc-1498 at 1x106 / mouse for the Luc-C198 positive control or C1498 at 1x106 / mouse for the C1498 (luciferase negative) positive control via the tail vein. Tumor-free mice previously inoculated with C1498 and treated with novel VIP-R antagonist peptides and that remained cancer-free for >60 days were rechallenged with Luc-C1498. Survival of mice was measured daily. Eleven mice that survived after initial inoculation with C1498 leukemia and subsequent treatment with novel peptides derived from VIP were pooled as follows: 1 ANT08 mouse, 3 ANT58 mice, 2 ANT107 mice, 2 ANT195 mice, 1 ANT197 mouse, and 2 ANT300 mice. Luciferase-positive C1498 was administered to eight control mice (Luc-C1498 control) that had not been previously exposed to leukemia or VIP-derived novel peptides, and luciferase-negative C1498 was administered to seven control mice (C1498 control) that had not been previously exposed to leukemia or VIP-derived novel peptides. [Figure 7] Shows the reduction in tumor volume upon treatment with ANT308+aPD-1 in C57BL / 6 mice bearing subcutaneously implanted KPC tumors. 22 days after tumor implantation, 3 days after completing the 10-day treatment. *p<0.05 Wilcoxon signed-rank test. [Figure 8A] Figure 1 shows the relative change in volume of Panc02 tumors growing as subcutaneous tumors from the start of treatment on day 10 post-implantation to euthanasia on day 22 post-implantation, following 10 days of treatment with daily injections of ANT308 or scrambled peptide and injections of anti-PD1 monoclonal antibody or isotype-matched antibody every 3 days from day 10 to day 19 post-implantation. Mice were euthanized and tumor volumes were measured on day 22 post-implantation and 3 days after completing the 10-day treatment. [Figure 8B]Actual tumor volumes of subcutaneously grown Panc02 tumors after 10 days of treatment with daily injections of ANT308 or scrambled peptide and injections of anti-PD1 monoclonal or isotype-matched antibodies every 3 days from day 10 to day 19 after implantation are shown. Mice were euthanized and necropsied on day 22 after tumor implantation and 3 days after completing the 10-day treatment. [Figure 9] Tumor size and growth rate are shown 37 days after subcutaneous KPC tumor implantation. Shown are scrambled peptide + IgG, Ant308 + anti-PD1, scrambled peptide + anti-PD-1, Ant308 + IgG, and Ant308 + AMD3100. Tumor volumes were measured with calipers. "Stars" (*) indicate mice that were euthanized due to either large tumor volumes (>500 mm3) or ulceration of the skin overlying the tumor. [Figure 10A] A, B, and C show that treatment of human T cells with VIP-R antagonists (Ant08, Ant308, Ant195) enhances T cell activation as measured by CD69 expression. A shows the total percentage of T cells positive for CD69 expression at 6 hours with each peptide treatment at 3 uM. B shows the total percentage of CD4+ and CD8+ subset T cells positive for CD69 expression at 24 hours with each peptide treatment. "Resting" represents groups maintained in culture for 6 or 24 hours without activation. "Activated" reflects T cells activated on CD3 antibody-coated plates without corresponding peptide treatment. "VS1" represents a group activated in the presence of one VIP-scrambled peptide as a peptide control. Mean values ​​from the "activated" group shown with dotted lines (---) for comparison. Responses from the same healthy donors are shown with dots of the same color. Error bars are calculated as standard error of the mean (SEM) from six healthy donor samples.C shows a representative flow plot demonstrating a higher percentage of CD4+CD69+ T cells in the antagonist-treated group compared to the control group (column 1) at 24 h from one donor (blue dots). [Figure 10B] (As stated above.) [Figure 10C] (As stated above.) [Figure 11] Expression of TIM3 in CD4+ and CD8+ T cells 24 hours after treatment is shown. [Figure 12] Expression of CXCR4 on CD4+ and CD8+ T cells 6 and 24 hours after treatment is shown. [Figure 13A] A, B, C, D, and E show that mouse pancreatic cancer cells (KPC) express VIP receptors, but their growth is not affected by treatment with VIP-R antagonists. A shows Western blots showing the expression of VPAC1, VPAC2, and PAC1 in human and mouse pancreatic cancer cells. B, C, and D show the expression ratios of VPAC1 (B), VPAC2 (C), and PAC1 (D) to GAPDH in each cell line. E shows cell viability compared to controls with increasing concentrations of CIP antagonist. [Figure 13B] (As stated above.) [Figure 13C] (As stated above.) [Figure 13D] (As stated above.) [Figure 13E] (As stated above.) [Figure 14A] A, B, and C show that VIP-R antagonist ANT308 + anti-PD1 antibody treatment promoted infiltration of adoptively transferred T cells into pancreatic tumors. A shows the experimental scheme. B and C show immunohistochemistry images from tumors in untreated (B) and treated (C) mice. [Figure 14B] (As stated above.) [Figure 14C] (As stated above.) [Figure 15] 1 shows survival curves in mice with and without treatment after implantation of pancreatic cancer (KPC) tumors. [Figure 16A]A, B, C, D, E, F, and G show that VIP is overexpressed by PDAC. (A) VIP mRNA expression levels in various solid malignant tumors from TCGA. (B) Representative images of human PDAC tumors stained with antibodies against VIP (green) and CK19 (red), showing higher VIP expression in cancer epithelial cells compared to adjacent normal epithelial cells. Scale bar represents 20 μm. The levels of VIP in culture supernatants from mouse and human PDAC cell lines (n=3 per cell line) cultured for 24 hours were compared with the levels of VIP in culture supernatants from B16F10 and D4M melanoma cells (C), the levels of VIP in the plasma of mice bearing melanoma or PDAC tumors (n=5) were compared with the levels of VIP in the plasma of mice without tumors (D), the levels of VIP in the plasma of PDAC patients (n=19) were compared with the levels of VIP in the plasma of healthy volunteers (n=26) (E), the levels of VIP in the plasma from one C57BL / 6 mouse bearing a subcutaneous KPC.Luc tumor isolated at different tumor volumes (F), the levels of VIP in culture supernatants from primary CAFs isolated from human PDAC tumors (n=9) and the PSCL-12 cell line (n=3) were compared (G). p values ​​in Figures 16C and 16D were calculated using ANOVA and post hoc tests for healthy volunteers. Means were compared to B16F10. p values ​​in e were calculated by Student's t-test. Error bars indicate mean ± SEM. **p<0.01, ***p<0.001, and ****p<0.0001. [Figure 16B] (As stated above.) [Figure 16C] (As stated above.) [Figure 16D] (As stated above.) [Figure 16E] (As stated above.) [Figure 16F] (As stated above.) [Figure 16G] (As stated above.) [Figure 17A]A, B, C, D, E, F, G, H, and I show that inhibition of VIP-R signaling reduces the expression of T cell exhaustion markers in cultured human T cells. (A) Representative Western blots and (B) quantified expression levels of VPAC1 and VPAC2; and (C) PD-1 and CTLA-4 in lysates of healthy human T cells grown with plate-bound human anti-CD3 antibody for 0, 3, 6, 12, 24, 48, and 72 hours. Lower molecular weight band of 25 kD shown for VPAC2 specific expression as confirmed from VPAC2 KO model. (D) CD69 expression at 24 hours post activation in CD4+ and CD8+ T cells normalized to the level of peptide-free control. (E) Percentage of phosphorylation of CREB (phospho-CREB) downstream of VPAC1 / 2 receptor at 6 hours. Peripheral blood T cells from PDAC patients were expanded with plate-bound human anti-CD3 antibody + / - ANT008 for 9 days, and (F) the percentage of Tregs was quantified using the gating strategy shown in (G). The percentages of PD1+, Tim-3+, Lag3+, PD1+Tim-3+, PD1+Lag3+, and PD1+Tim-3+Lag-3+ (triple positive) in (H) CD4+ and (I) CD8+ subsets are shown. Statistical differences in D and E were calculated by repeated measures ANOVA followed by Dunnett's post-hoc test, comparing each sample from the treatment group with a matched sample from the control group (scrambled). Statistical differences in F, H, and I were calculated by paired Student's T-test. Error bars indicate mean ± SEM. *p<0.50, **p<0.01, and ***p<0.001. [Figure 17B] (As stated above.) [Figure 17C] (As stated above.) [Figure 17D] (As stated above.) [Figure 17E] (As stated above.) [Figure 17F] (As stated above.) [Figure 17G] (As stated above.) [Figure 17H] (As stated above.) [Figure 17I] (As stated above.) [Figure 18A] A, B, C, D, and E show improved survival in PDAC-bearing mice treated with a combination of VIP-R antagonist and anti-PD-1. (A) Kaplan-Meier survival plots of C57BL / 6 mice bearing subcutaneously implanted KPC.Luc, MT5, or Panc02 tumors, stratified by treatment. (B) Spider plots for KPC.Luc corresponding to the results in a after subcutaneous tumor implantation measured by caliper in the four different treatment groups. Median tumor volume (----) represented by grey dashed line. In a and b, tumor cells were implanted in female or male mice, with males receiving 20 μg of ANT308 due to their higher body weight compared to female mice. Kaplan-Meier survival plots of (C) C57BL / 6 mice, (D) CD4KO, or (E) CD8KO mice receiving monoclonal CD4 and / or CD8 monoclonal antibodies compared to wild-type CD57BL / 6 mice bearing subcutaneously implanted KPC.Luc tumors, stratified by treatment. Statistical differences in Kaplan-Meier curves were calculated by the Log-rank test. *p<0.05, **p<0.01, and ***p<0.001, ****p<0.0001. [Figure 18B] (As stated above.) [Figure 18C] (As stated above.) [Figure 18D] (As stated above.) [Figure 18E] (As stated above.) [Figure 19A]A, B, C, D, E, F, G, H, and I show that the combination of VIP-R antagonist and anti-PD-1 increased T cell activation and reduced the frequency of Tregs in KPC.Luc tumors. Subcutaneous KPC.Luc tumors in C57BL / 6 mice treated with ANT008 and / or anti-PD-1 (n=5 per treatment group) were analyzed for the percentage of (A) CD4+ and (B) CD8+ T cells expressing Ki67, IFNgamma, IL-4, PD-1, and Tim-3 by flow cytometry 10 days after treatment. (C) Representative flow plot showing the gating strategy used to quantify CD25+FoxP3+Tregs. (D) Percentage of Tregs in tumors of different treatment groups (n=5 per treatment group). Volcano plots showing differential expression of genes in T cells from (E) ANT008+isotype IgG (IgG) vs. scrambled peptide (Scram)+isotype IgG, (F) scrambled peptide+anti-PD-1 vs. scrambled peptide+isotype IgG, and (G) ANT008+anti-PD-1 vs. scrambled peptide+isotype IgG (n=3 mice per treatment group). Horizontal black lines represent false discovery rate (FDR)<0.1. Genes associated with TCR activation and costimulation and at significantly higher levels when compared to Scram+isotype IgG (FDR<0.1) are labeled in red. (H) Heatmap showing gene expression changes of genes associated with TCR activation and costimulation. (I) TCR activation and costimulation pathway scores between T cells in tumors of mice from different treatment groups. Statistical differences in A, 19B, 19D, and 19I were calculated by ANOVA followed by Dunnett's post-hoc test. Error bars represent the mean ± SEM *p<0.05, **p<0.001, ***p<0.0001. [Figure 19B] (As stated above.) [Figure 19C] (As stated above.) [Figure 19D] (As stated above.) [Figure 19E] (As stated above.) [Figure 19F] (As stated above.) [Figure 19G] (As stated above.) [Figure 19H] (As stated above.) [Figure 19I] (As stated above.) [Figure 20A] A, B, C, D, E, F, and G show that combination therapy with VIP-R antagonist and anti-PD-1 increases the frequency of tetramer+, CD8+ T cells in tumors and provides protective immunity against tumor rechallenge. (A) Box plots showing Shannon entropy in T cells from KPC.Luc tumors in each treatment group. (B) Listing of TCR-β amino acid sequences shared between samples in each treatment group, and (C) frequency of shared clones in each treatment group. Sequences are color-coded to represent the number of mice per group (n=4) sharing a particular TCR-β clone. CD8+ in subcutaneous KPC.Luc tumors were stained with MuLV p15E-H2Kb tetramer (E) quantified 10 days after treatment with ANT308 and / or anti-PD-1 (n=3 per treatment group) using (D) a gating strategy. (F) Kaplan-Meier survival curves of subcutaneous KPC.Luc-bearing mice treated with ANT008 / ANT308 and / or anti-PD-1 from days 3–12 after tumor implantation (n=16 per scrambled peptide + isotype IgG, n=20 for ANT008 / ANT308 + isotype IgG and scrambled peptide + anti-PD-1 treatment groups, n=23 for ANT008 / ANT308 + anti-PD-1 treatment group). (G) Kaplan-Meier survival curves of tumor-free mice from F re-challenged with KPC.Luc tumors on the contralateral flank (n=6 for scrambled peptide + anti-PD-1 treatment group; n=8 for ANT008 / ANT308 + anti-PD-1 treatment group). Naïve C57BL / 6 mice were inoculated with tumor cells at the same time as the re-challenge (n=7). Statistical differences in a and e were calculated by ANOVA followed by Dunnett's post-hoc test, and in F and G using the Log-rank test. Error bars indicate mean ± SEM *p<0.05, **p<0.01, ****p<0.0001. [Figure 20B] (As stated above.) [Figure 20C] (As stated above.) [Figure 20D] (As stated above.) [Figure 20E] (As stated above.) [Figure 20F] (As stated above.) [Figure 20G] (As stated above.) [Figure 21A] A, B, C, D, E, F, G, H, and I show that synergy between ANT008 and anti-PD-1 increases T cell infiltration and proliferation and reduces tumor burden in orthotopic KPC.Luc mouse PDAC. KPC.Luc cells were orthotopically implanted into the tail of the pancreas of C57BL / 6 mice and treated with ANT008 and / or anti-PD-1, n=9, 10, 8, and 11 in scrambled+IgG, ANT008+IgG, scrambled+anti-PD-1, and ANT008+anti-PD-1, respectively. (A) Schematic showing orthotopic implantation and treatment strategy of KPC.Luc cells with ANT008 and / or anti-PD-1. (B) Waterfall plot showing % change in tumor flux on day 22 compared to day 7 before treatment initiation. (C) Total flux measured by IVIS bioluminescence imaging in different treatment groups. Isoflurane was used for anesthesia for bioluminescence imaging. Median flux is represented by grey dashed line (...). Cross symbols (+) represent mice euthanized before 25 days due to tumor ulceration, and circle symbols (○) represent mice imaged on day 26 by MRI imaging shown in FIG. 30. (D) Bar graph showing pancreas weight on day 25 when mice were euthanized. Data points with "star" shape (★) represent mice without tumors, and dotted horizontal line (...) represents the average weight of healthy pancreas from naive mice. (E) Representative multiple IHC image showing pancreatic tumors stained for DAPI (blue), CD4 (yellow), CD8 (red) and Ki67 (cyan) (right), and trichrome staining with black arrows showing blue collagen staining within the tissue (left). (F) Bar plot showing the number of CD4+ or (21G)CD8+ T cells / mm2; and (H) Ki67+CD4+ or (I) Ki67+CD8+ T cells / mm2. P values ​​for D were calculated using Student's ANOVA followed by Dunnett's post-hoc test (comparing each treatment group to Scram+IgG). Error bars represent the mean ± SEM. *p<0.05, **p<0.01. [Figure 21B] (As stated above.) [Figure 21C] (As stated above.) [Figure 21D] (As stated above.) [Figure 21E] (As stated above.) [Figure 21F] (As stated above.) [Figure 21G] (As stated above.) [Fig. 21H] (As stated above.) [Figure 21I] (As stated above.) [Figure 22A]A, B, C, E, F show that combination therapy with VIP-R antagonist and anti-PD-1 promotes intratumoral T cell infiltration and reduces CXCR4 expression on T cells in tumor-draining lymph nodes. KPC.Luc tumors were implanted subcutaneously into C57BL / 6 mice. 15 days after tumor implantation, GFP+ T cells were adoptively transferred (via tail vein injection) with ANT308+ / -aPD-1 treatment for 3 days. (A) Schematic showing GFP+ T cell implantation and treatment strategy in mice bearing subcutaneous KPC.Luc tumors. (B) Representative Hoescht (blue indicating nuclei) stained tumor tissue from tumors of each treatment group. Zoom-ins of two regions of interest (RO1), labeled ROI-1 and ROI-2, in the original image of tumor from a mouse treated with ANT308+aPD- are also shown. Percentages of (C) CXCR4+CD69+ and (D) CXCR4+Ki67+ cells in CD4+ (left) and CD8+ (right) subsets of T cells. (E) Tumor growth rates and (F) survival curves generated from mice bearing subcutaneous KPC.Luc tumors treated with scrambled peptide, IgG and PBS, or ANT308 and aPD-1, or AMD3100 and aPD-1, or ANT308 and AMD3100, or a combination of ANT308, aPD-1 and AMD3100. Median tumor volumes (----) represented by grey dashed lines. Statistical differences in c and d were determined by repeated measures ANOVA and Dunnett's post-hoc test with n=4-5 mice per group. Statistical differences in 22E were determined by Log-rank test (n=9-10 mice per group). Lines in c and d indicate the mean. *p<0.05, **p<0.01, **p<0.001, p<0.0001. [Figure 22B] (As stated above.) [Figure 22C] (As stated above.) [Figure 22D] (As stated above.) [Fig. 22E-22F] (As stated above.) [Figure 23A]A, B, C, and D show that PDAC cell lines and human PDAC tissues express VIP and VIP receptors (A). Representative images of one human PDAC tumor stained with antibodies against VIP (green), CK19 (red), and maize (yellow), showing VIP expression in cancer epithelial cells. Scale bars represent 200 μm. (B) Representative Western blots of mouse and human PDAC cell lines probed for lysates from mouse melanoma; and VPAC1, VPAC2, and GAPDH as controls. (C) VPAC1, (D) VPAC2 protein bands from Western blots were analyzed by densitometric analysis and normalized to the intensity of GAPDH. Results are the mean ± SEM of three independent experiments. p values ​​in FIG. 23C were determined by ANOVA followed by Dunnett's post-hoc test. *p<0.05, ***p<0.001. [Figure 23B] (As stated above.) [Figure 23C] (As stated above.) [Figure 23D] (As stated above.) [Figure 24A]A, B, C, D, E, F, G, and H show that the absence of VPAC2 receptor on PDAC cells confers limited autocrine effects on cancer cell growth in vitro and in vivo. (A) Plots of the viability percentage of mouse (MT5, KPC.Luc, Panc02) and human (Capan02, BxPC3) PDAC cell lines cultured for 72 h in the presence of different concentrations of ANT008 ranging from 0 to 5 μM. (B) Confirmation of CRISPR-Cas9 KO of VIPR2-encoded VPAC2 receptor by Western blot, (C) RT-PCR using primers targeting exons 9-12 downstream of the target site, and (D) Sanger sequencing showing validation of an in-del mutation in exon 2. In vitro MTT assay shows (E) proliferation of WT and KO cells over 72 hours; (F) percent viability of wild-type (WT) and VPAC2 KO (KO) Panc02 cells treated with 3 μM ANT008 and ANT308 for 72 hours. (G) Tumor growth curves of WT vs. KO Panc02 cells in C57BL / 6 mice after subcutaneous tumor implantation. Values ​​represent median tumor volume ± 95% confidence interval. (H) Kaplan-Meier survival plot corresponding to the results in G. Median survival is 21 days for WT and 28 days for VPAC2 KO. Error bars represent mean and standard deviation. *p<0.05, **p<0.01. [Figure 24B] (As stated above.) [Figure 24C] (As stated above.) [Figure 24D] (As stated above.) [Figure 24E] (As stated above.) [Figure 24F] (As stated above.) [Figure 24G] (As stated above.) [Fig. 24H] (As stated above.) [Figure 25A]A, B, and C show the gating strategy for flow cytometry analysis of healthy human T cells. (A) Cells were gated as "P1" by plotting forward scatter height (FSC-H) and side scatter height (SSC-H). Singlets from P1 were selected by gating along the diagonal of forward scatter height (FSC-H) versus forward scatter area (FSC-A) plots. Live cells from singlets were selected by plotting live / dead cells versus FSC-A. Live cells were then plotted on a CD4 vs. CD8 plot to identify CD4+ and CD8+ T cells. CD69-expressing T cells in (B) CD4+ and (C) CD8+ subsets were then identified by plotting each subset on a CD4 / CD8 vs. CD69 flow plot. The percentage of CD69+ T cells within each subset is shown in red. [Figure 25B] (As stated above.) [Figure 25C] (As stated above.) [Figure 26A] A, B, C, and D show the gating strategy for flow cytometry analysis of CREB phosphorylation in T cells. (A) Plot of forskolin-treated human T cells used as a positive control to gate phospho-CREB positive cells. T cells were treated with 30 μM forskolin on ice for 30 min and stained for surface expression of CD4 and CD8, followed by intracellular staining with anti-phospho-CREB (S133) antibody. Representative plots of phospho-CREB expression in (B) CD4+ and (C) CD8+ human T cells upon treatment with scrambled peptide (Scram), ANT008, and ANT308 at 3 μM for 6 h. (D) Percentage of CD3+phospho-CREB+ in mouse T cells under similar conditions as in Figure 26C. [Figure 26B] (As stated above.) [Figure 26C] (As stated above.) [Figure 26D] (As stated above.) [Figure 27A]A, B, C, and D show the gating strategy for PD-1, Tim-3, or Lag-3 expression on CD4+ or CD8+ T cells of PDAC patients expanded ex vivo for 9 days. (A) Cells were gated as "P1" by plotting forward scatter area (FSC-A) and side scatter area (SSC-A). Singlets from P1 were selected by gating along the diagonal of forward scatter height (FSC-H) versus forward scatter area (FSC-A) plots. Live cells from singlets were selected by plotting live / dead versus FSC-A. Live cells were then plotted on a CD3 versus FSC-A plot, and cells positive for CD3 were gated as T cells. CD4+ and CD8+ T cells were then identified by plotting T cells on a CD4 versus CD8 plot. (B) PD-1+, (C) Tim-3+, and (D) Lag-3+ cells were gated on CD4+ (top) or CD8+ (bottom) T cells based on FMO controls. [Figure 27B] (As stated above.) [Figure 27C] (As stated above.) [Figure 27D] (As stated above.) [Figure 28A] A, B, C, D, and E show that combination therapy with VIP-R antagonist and anti-PD-1 reduces tumor burden and improves survival in male and female C57BL / 6 mice bearing KPC tumors. Box plots showing tumor volumes of MT5 (A), KPC-Luc (B), and Panc02 (C) measured by caliper at day 22 after subcutaneous tumor implantation for MT5 and day 22 for KPC and Panc02. Kaplan-Meier survival curves of (D) female or (E) male C57BL / 6 mice implanted subcutaneously with KPC.Luc tumors and treated with ANT308 (female: 10 μg, male: 20 μg) and / or anti-PD-1. Statistical differences in a-c were calculated by ANOVA followed by Dunnett's post-hoc test. Solid lines indicate the median values ​​in each treatment group. Statistical differences in d and e were calculated by Log-rank test. *p<0.05, **p<0.01, and ***p<0.001. [Figure 28B] (As stated above.) [Figure 28C] (As stated above.) [Figure 28D] (As stated above.) [Figure 28E] (As stated above.) [Figure 29A] A, B, C, D, E, F, and G show that administration of ANT008 or ANT308 did not result in adverse toxicity in C57BL / 6 mice. C57BL / 6 mice received daily subcutaneous injections of ANT008 or ANT308 for 10 days (n=5 / group) and were analyzed for evidence of toxicity on day 11. (A) Body weight (grams) during the drug administration period; (B) WBC, RBC, and platelet counts in blood (right) are plotted by total blood count (left) percentage of T cells, B cells, NK cells, DCs, and MDSCs in the spleen identified by flow cytometry. Representative H&E stained sections of (C) colon (top), lung (bottom), and (D) liver are shown. Arrows in D indicate focal hepatic lesions in the liver. Focal hepatic necrosis observed in one of five mice in each group is not considered drug-related toxicity as these lesions are commonly observed in several in-house mouse strains at Jackson Laboratory. C57BL / 6 mice received daily subcutaneous injections of 30ug ANT308 (n=6) or a daily combination of 30ug ANT308 with 200ug anti-PD1 (n=6) every 3 days for 4 days. Mice receiving scrambled peptide and isotype IgG served as controls (n=4). (E) Mouse weights, (F) complete blood counts (CBC), and (G) serum chemistries after 4 days of treatment are plotted. p values ​​for B, E, F, and G were calculated by ANOVA followed by Dunnett's post-hoc test. Error bars represent the mean and standard deviation. *p<0.05, **p<0.01. [Figure 29B] (As stated above.) [Figure 29C] (As stated above.) [Figure 29D] (As stated above.) [Figure 29E] (As stated above.) [Figure 29F] (As stated above.) [Figure 29G](As stated above.) [Figure 30A] A, B, C, D, E, F, and G show that bioluminescence signals from orthotopically implanted KPC.Luc tumors positively correlate with tumor burden and indicate desmoplasia. (A) At day 26 after orthotopic KPC.Luc tumor implantation in C57BL / 6 mice, tumor burden in a representative mouse, indicated by the "circle" symbol in FIG. 21C, was compared via bioluminescence imaging, IVIS imaging, and H&E staining of formalin-fixed pancreas isolated after euthanasia. For bioluminescence imaging, isoflurane was used for anesthesia. (B) Total flow (p / s) measured by bioluminescence imaging at day 26 after tumor implantation was plotted against the weight of isolated pancreas after euthanasia. Data points are color-coded to represent mice in different treatment groups, n=9, 10, 8, and 11 in scrambled+IgG, ANT008+IgG, scrambled+anti-PD-1, and ANT008+anti-PD-1, respectively. (C) Trichrome staining shows blue collagen staining within the tissue for orthotopically implanted KPC.Luc tumors in all treatment groups. Representative images of scrambled+IgG, ANT008+IgG, scrambled+anti-PD-1 are shown; ANT008+anti-PD-1 is shown in Figure 21E. XY plot showing correlation between number of (D) CD4+ or (E) CD8+ T cells / mm2 and (F) Ki67+CD4+ or (G) Ki67+CD8+ T cells / mm2 with pancreatic weight with n=4-6 mice per group. [Figure 30B] (As stated above.) [Figure 30C] (As stated above.) [Figure 30D] (As stated above.) [Figure 30E] (As stated above.) [Figure 30F] (As stated above.) [Figure 30G] (As stated above.) [Figure 31A]A, B, and C show increased frequency of GFP+ T cells in tumors of mice treated with a combination of VIP-R antagonist and anti-PD-1, as confirmed by flow cytometry. (A) Singlets from single cell suspensions prepared from tumors of mice in FIG. 22A were gated by plotting forward scatter area (FSC-A) versus forward scatter height (FSC-H). Live CD45+ cells were gated by selecting CD45 positive, followed by gating on CD3 positive cells in a CD3 versus SSC-A plot. GFP+ cells were then selected by gating on GFP positive cells based on a mixed population of unstained splenocytes from unstained C57BL / 6 mice and spleen samples from GFP transgenic mice. (B) Representative plots of CD3+GFP+ cells in the four treatment groups (Scram+IgG, ANT308+IgG, Scram+anti-PD1, ANT308+ant-PD1). (C) Summarized data from b showing the percentage of GFP+ T cells relative to live CD45+ T cells. Percent GFP+ T cells was calculated as the percentage of total CD3+GFP+ events enumerated from FlowJo divided by total live CD45+ events. [Figure 31B] (As stated above.) [Figure 31C] (As stated above.) [Diagram 32] 1 shows the experimental design to test the effect of ANT308 alone or in combination with anti-PD-1 on liver metastases in an intraocular melanoma mouse model. [Diagram 33] Figure 1 shows that ANT308 in combination with anti-PD-1 reduced liver metastases from intraocular melanoma mice at 2 weeks (n=4). [Diagram 34] ANT308 alone or in combination with anti-PD-1 reduced liver metastases from intraocular melanoma mice at 3 weeks (n=6). [Diagram 35] Shown are liver metastases at 2 and 3 weeks after ANT308 / anti-PD-1 (n=10). [Diagram 36] ANT308 inhibited the growth of liver metastases 3 weeks after tumor inoculation (N=6). [Figure 37] ANT308 inhibited angiogenesis (arrows) and the growth of liver metastases (N=10). [Figure 38] 1 shows that intraocular melanoma size was not affected by either ANT308 alone or in combination with anti-PD-1. [Figure 39] 1 shows that the VIP-R antagonist ANT308 induced dose-dependent clearance of C1498 leukemia and long-term survival in AML-bearing mice. [Diagram 40] 1 shows that the VIP-R antagonist ANT308 induced schedule-dependent clearance of C1498 leukemia and long-term survival in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] V. MODE FOR CARRYING OUT THE DISCLOSURE Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to particular synthetic methods, or to particular recombinant biotechnology methods, unless otherwise specified, and are not limited to particular reagents (which may, of course, vary), unless otherwise specified. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0023] A.Definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

[0024] As used herein, ranges may be expressed as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that by using the antecedent "about," the particular value forms another embodiment. It will be further understood that each of the endpoints of a range is significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are several values ​​disclosed herein, and that each value is also disclosed herein as "about" that particular value, in addition to the value itself. For example, when the value "10" is disclosed, "about 10" is also disclosed. As will be appreciated by those of skill in the art, when a value is disclosed to be "less than or equal to," it is also understood that "greater than or equal to" and possible ranges between those values ​​are also disclosed. For example, when the value "10" is disclosed, "less than or equal to 10" as well as "greater than or equal to 10" is also disclosed. It is also understood that throughout this application, data is provided in several different formats, and that this data represents endpoints and starting points, as well as ranges for any combination of data points. For example, when a specific data point "10" and a specific data point "15" are disclosed, it is understood that values ​​between 10 and 15, as well as values ​​greater than 10, greater than 10, less than 10, less than 10, and equal to 10, greater than 15, greater than 15, less than 15, less than 15, and equal to 15, are considered to be disclosed. It is also understood that each unit between two specific units is also disclosed. For example, when 10 to 15 is disclosed, 11, 12, 13, and 14 are also disclosed.

[0025] As used herein and in the claims that follow, reference will be made to a number of terms that are defined to have the following meanings.

[0026] "Administration" or "administering" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including intravenous, intraperitoneal, and the like. Administration includes self-administration and administration by another. "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intraarticular, parenteral, intraarterial, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, or via a transdermal patch, and the like. Administration includes self-administration and administration by another.

[0027] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not occur.

[0028] The term "comprising" with respect to a peptide having an amino acid sequence refers to a peptide that may contain additional N-terminal (amine terminal) or C-terminal (carboxylic acid terminal) amino acids, i.e., the term is intended to include an amino acid sequence within a larger peptide. The term "consisting of" with respect to a peptide having an amino acid sequence refers to a peptide having a precise number of amino acids in the sequence, with no more than or a rage of amino acids explicitly specified in the claims. In certain embodiments, the present disclosure contemplates that "the N-terminus of the peptide may consist of an amino acid sequence," which refers to the N-terminus of a peptide having a precise number of amino acids in the sequence, with no more than or a rage of amino acids explicitly specified in the claims, but the C-terminus may be connected to additional amino acids, for example, as part of a larger peptide. Similarly, the present disclosure contemplates that "the C-terminus of a peptide may consist of an amino acid sequence," which refers to the C-terminus of a peptide having a precise number of amino acids in the sequence, not exceeding or ranging beyond those specified in the claims, but the N-terminus may be joined to additional amino acids, e.g., as part of a larger peptide.

[0029] "Increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a statistically significant amount of a condition, symptom, activity, or composition. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, so long as the increase is statistically significant.

[0030] "Reduction" can refer to any change that results in a lower amount of symptoms, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product containing the substance is less compared to the output of the gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder, such that the symptoms are less than previously observed. A reduction can be any individual, median, or average reduction in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as the reduction is statistically significant.

[0031] "Inhibit," "inhibiting," and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0032] "Inhibitor" or "antagonist" of expression or activity is used to refer to inhibitory molecules identified using in vitro and in vivo assays for the expression or activity of the described target proteins, e.g., ligands, antagonists, and their homologs and mimetics, respectively. Inhibitors are, for example, agents that inhibit the expression of the described target proteins, e.g., antagonists, or bind to them, partially or completely block their stimulatory or enzymatic activity, reduce, prevent, delay activation, inactivate, desensitize, or downregulate their activity. Control samples (not treated with inhibitors) are assigned a relative activity value of 100%. Inhibition of the described target proteins is achieved when the activity value compared to the control is about 80%, optionally 50% or 25, 10%, 5%, or 1%. As used herein, the terms "VIP antagonist" or "VIP receptor antagonist" are used interchangeably.

[0033] "Reduce" or other forms of this term, such as "reducing" or "reduction," refers to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" means reducing the rate of growth of a tumor compared to a standard or control.

[0034] "Prevent" or other forms of this word, such as "preventing" or "prevention," means to stop a particular event or characteristic, stabilize or slow the development or progression of a particular event or characteristic, or minimize the likelihood of a particular event or characteristic occurring. Prevention is typically more absolute than, for example, reduction, and does not require a comparison to a control. As used herein, something can be reduced, but may not be prevented, but something that is reduced can be prevented. Similarly, something can be prevented, but may not be reduced, but something that is prevented can be reduced. It is to be understood that when reduction or prevention is used, the use of other words is expressly disclosed unless specifically specified otherwise.

[0035] The term "subject" refers to any individual who is the target of administration or treatment. The subject may be a vertebrate, e.g., a mammal. In one aspect, the subject may be a human, a non-human primate, a cow, a horse, a pig, a dog, or a cat. The subject may also be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Thus, the subject may be a human or a veterinary patient. The term "patient" refers to a subject under the care of a clinician, e.g., a physician.

[0036] The term "therapeutically effective amount" refers to the amount of the composition used that is sufficient to alleviate one or more causes or symptoms of a disease or disorder. Such alleviation requires only a reduction or alteration, not elimination.

[0037] The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed to ameliorating a disease, condition or disorder, and also includes causal treatment, i.e., treatment directed to eliminating the cause of the associated disease, condition or disorder. In addition, the term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure the disease, condition or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, condition or disorder; and supportive treatment, i.e., treatment used to complement another specific treatment directed to ameliorating the associated disease, condition or disorder.

[0038] "Biocompatible" generally refers to a material and any metabolic or breakdown products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects in the subject.

[0039] "Comprising" is intended to mean that the composition, method, etc. includes the recited elements but does not exclude other elements. When used to define compositions and methods, "consisting essentially of" is intended to mean including the recited elements but excluding other elements of any essential importance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants and pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, etc., from the isolation and purification methods. "Consisting of" is intended to mean excluding more than trace elements of other components and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0040] "Composition" refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, vectors, polynucleotides, cells, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "composition" is used, then, or when a particular composition is specifically identified, it should be understood that the term includes the composition itself, as well as pharmaceutically acceptable, pharmacologically active vectors, polynucleotides, salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0041] A "control" is a substitute control or sample used in an experiment for comparison purposes. Controls may be either "positive" or "negative."

[0042] An "effective amount" of a drug refers to an amount of the drug sufficient to provide a desired effect. The amount of a drug that is "effective" will vary from subject to subject, depending on many factors, such as the age and general condition of the subject, the specific drug, etc. Thus, it is not always possible to specify a quantified "effective amount". However, an appropriate "effective amount" in any subject's case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a drug may refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of a drug required to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0043] A "pharmaceutical acceptable" ingredient may refer to an ingredient that is not biologically or otherwise undesirable, i.e., an ingredient that can be incorporated into a pharmaceutical formulation provided by the present disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is included. When used in relation to human administration, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing or that it is included in the inactive ingredient guide prepared by the US Food and Drug Administration.

[0044] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") generally means a carrier or excipient useful in the preparation of a safe and non-toxic pharmaceutical or therapeutic composition, and includes carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0045] "Pharmacologically active" (or simply "active") can refer to a derivative or analog (e.g., a salt, ester, amide, complex, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound, and to approximately the same extent, in a "pharmacologically active" derivative or analog.

[0046] "Therapeutic agent" refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer). These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0047] The term "prodrug" refers to an agent that is converted to a biologically active form in vivo. Prodrugs are often useful because, in some circumstances, they may be easier to administer than the parent compound. Prodrugs may also have improved solubility in pharmaceutical compositions over the parent drug. Prodrugs may be converted to the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Exemplary prodrugs are pharma-ceutically acceptable esters. Prodrugs include compounds in which a hydroxy, amino, or mercapto (thiol) group is bonded to any group that is cleaved to form a free hydroxy, free amino, or free mercapto group, respectively, when the prodrug of the active compound is administered to a subject. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols, or acetamide, formamide, and benzamide derivatives of amine functional groups in the active compound.

[0048] A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising an agent) refers to an amount effective to achieve a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is control of tumor growth. In some embodiments, the desired therapeutic outcome is control of metastasis. In some embodiments, the desired therapeutic outcome is prevention of recurrence. The therapeutically effective amount of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term can also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect, such as pain relief. The exact desired therapeutic effect will vary according to the condition being treated, the tolerability of the subject, the agent and / or agent formulation being administered (e.g., potency of the therapeutic agent, concentration of the agent in the formulation, etc.), as well as a variety of other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after multiple administrations of the composition over a period of days, weeks, or years.

[0049] As used herein, the term "isolating" refers to isolation from a biological sample, i.e., blood, plasma, tissue, exosomes, or cells. As used herein, the term "isolated," for example, when used in reference to a nucleic acid, refers to a nucleic acid of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the nucleic acid is associated prior to purification.

[0050] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties that result therefrom. Thus, a gene, upon transcription and translation of mRNA, codes for a protein.

[0051] As used herein, the term "engineered" and other grammatical forms of the term can refer to one or more changes in a nucleic acid, such as a nucleic acid in the genome of an organism. The term "engineered" can refer to a genetic change, addition, and / or deletion. An engineered cell can also refer to a cell that contains an added, deleted, and / or altered gene.

[0052] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses that incorporate recombinant polynucleotides (e.g., lentiviruses, retroviruses, adenoviruses, adeno-associated viruses), and the like, all vectors known in the art.

[0053] A "fragment" can include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, whether or not linked to other sequences, provided that the activity of the fragment is not significantly altered or impaired compared to the unmodified peptide or protein. These modifications can provide some additional properties, for example, removing or adding amino acids that allow disulfide bonds, extending its biological lifespan, altering its secretion properties, etc. In any case, the fragment must have a biologically active property, such as modulating the transcription of a target gene.

[0054] The term "gene" or "gene sequence" refers to a coding sequence or a regulatory sequence, or a fragment thereof. A gene may include any combination of coding and regulatory sequences, or fragments thereof. Thus, a "gene" referred to herein may be all or a portion of a naturally occurring gene. A polynucleotide sequence referred to herein may be used interchangeably with the term "gene" or may include any coding, non-coding or regulatory sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, regulatory sequences (e.g., ribosome binding sites) that are upstream of the coding sequence.

[0055] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, when measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website), refer to a specified percentage of amino acid residues or nucleotides that are the same or that are identical (i.e., those that correspond most closely over a window or designated region of comparison). "Substantially identical" refers to two or more sequences or subsequences that contain about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, over a particular region, when compared and aligned. Such sequences are then said to be "substantially identical." This definition can also refer to, or be applied to, the complement of a test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As explained below, preferred algorithms can take into account gaps and the like. Preferably, the identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent nucleotide sequence identity (%) is defined as the percentage of amino acids in a candidate sequence that are identical to nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be accomplished in a variety of ways in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0056] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test and reference sequences are input into computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity for test sequence compared to reference sequence based on program parameters.

[0057] One example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hits in each direction is stopped when the cumulative alignment score falls by a quantity X from its maximum achieved value, when the cumulative score falls below zero, due to the accumulation of one or more negative scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3 and an expectation (E) of 10, with the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) using an alignment (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0058] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0059] The term "nucleic acid" as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides (DNA) or ribonucleotides (RNA). The terms "ribonucleic acid" and "RNA" as used herein means a polymer composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" as used herein means a polymer composed of deoxyribonucleotides. (Used in conjunction with "polynucleotide" and "polypeptide.")

[0060] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Also, the phrase nucleotide sequence encoding a protein or RNA may include introns to the extent that a nucleotide sequence encoding a protein may contain introns in some versions.

[0061] As used herein, "operably linked" can refer to the placement of regulatory sequences useful for the expression of the coding sequence of the nucleic acid in a suitable position relative to the coding sequence in the nucleic acid molecule to effect expression of the coding sequence. This same definition applies, as the case may be, to the placement of coding sequences and / or transcription control elements (e.g., promoters, enhancers, and termination elements) and / or selectable markers in an expression vector. The term "operably linked" can also refer to the placement of polypeptide segments in a single polypeptide chain, where the individual polypeptide segments can be, but are not limited to, proteins, fragments thereof, linking peptides, and / or signal peptides. The term operably linked can refer to the direct fusion of different individual polypeptides in a single polypeptide or fragments thereof, where there are no intervening amino acids between the different segments, and where the individual polypeptides are connected to each other via one or more intervening amino acids.

[0062] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers.

[0063] The term "polypeptide" refers to a compound consisting of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0064] The terms "peptide," "protein," and "polypeptide" are used interchangeably to refer to natural or synthetic molecules containing two or more amino acids linked by the carboxyl group of one amino acid to the alpha-amino group of another amino acid.

[0065] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate the specific transcription of a polynucleotide sequence.

[0066] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0067] The term "variant" as used herein refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. In general, the differences are limited so that the sequences of the reference polypeptide and the variant are overall closely similar (homologous) and identical in many regions. A variant and a reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions).

[0068] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body, and examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0069] As used herein, the term "metastasis" is meant to refer to the process by which cancer cells originating from one organ or part of the body relocate to another part of the body and continue to replicate, with or without transport by bodily fluids. The metastasized cells can then form tumors that can further metastasize. Thus, metastasis refers to the spread of cancer from the part of the body where it first originated to other parts of the body.

[0070] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also discussed in the text by reference and are individually and specifically incorporated by reference herein for the material contained therein.

[0071] B. Composition Disclosed are the components used to prepare the disclosed compositions, as well as the compositions themselves used within the methods disclosed herein. When these and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each is specifically contemplated and described herein, although specific reference to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed. For example, when a specific VIP-R antagonist is disclosed and discussed, and several modifications that may be made to several molecules including VIP-R antagonists are discussed, all combinations and permutations of VIP-R antagonists and possible modifications are expressly contemplated, unless specifically indicated otherwise. Thus, when classes of molecules A, B, and C are disclosed, as well as classes of molecules D, E, and F, and AD as an example of a combination molecule, each is considered to be individually and collectively contemplated, even if each is not individually listed, i.e., AE, AF, BD, BE, BF, CD, CE, and CF are disclosed. Similarly, any subset or combination of these is also disclosed. Thus, for example, the subgroups AE, BF, and CE would be considered disclosed. This concept applies to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, where there are various additional steps that may be performed, it is understood that each of these additional steps may be performed in any particular embodiment or combination of embodiments of the disclosed methods.

[0072] The terms "vasoactive intestinal peptide" and "VIP" refer to HSDAVFTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 2) unless the context suggests otherwise. VIP is a multifunctional endogenous polypeptide that regulates both innate and adaptive immunity at multiple levels of immune cell differentiation and activation. VIP is typically secreted by a variety of cells, such as neurons (in both the central and peripheral nervous systems), B cells, T cells, and accessory cells. VIP and the closely related neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP) bind to three known receptors: VPAC1, VPAC2, and PAC1. T cells and dendritic cells (DCs) are thought to express VPAC1 and VPAC2, but not PAC1. PAC1 is expressed primarily on neurons and endocrine cells in the brain and pituitary and adrenal glands, and in most forms selectively binds PACAP.

[0073] Some cancers are caused by viruses, and conventional vaccines against these viruses, such as HPV vaccines and Hepatitis B vaccines, would prevent these cancers. It is contemplated that the peptides disclosed herein can be administered in combination with these vaccines to improve therapeutic efficacy.

[0074] It is believed that cancer cells arise and are destroyed by the immune system, and cancer forms when the immune system fails to destroy cancer cells. One approach to cancer vaccination is to isolate proteins from cancer cells and immunize cancer patients against those proteins, stimulating an immune response that kills the cancer cells. Cancer vaccines are intended for the treatment of acute myeloid leukemia, multiple myeloma, lymphoma, breast cancer, lung cancer, colon cancer, skin cancer, kidney cancer, prostate cancer, and other cancers.

[0075] In certain embodiments, the present disclosure relates to treating cancer by administering any of the vasoactive intestinal peptide receptor (VIP-R) antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) in combination with a cancer antigen. Other VIP-R or VIP antagonists are also reported in U.S. Patent Nos. 6,630,124 and 5,217,953, which are incorporated herein by reference in their entireties.

[0076] Prevention of the action of microorganisms may be controlled by the addition of any of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, etc. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0077] Any of the peptides disclosed herein can optionally be modified with carbohydrate or polyethylene glycol groups to provide improved properties such as solubility, bioavailability, and / or biological degradation.

[0078] In certain embodiments, the present disclosure relates to a method of coupling any of the vasoactive intestinal peptide receptor (VIP-R) antagonists disclosed herein (such as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, and any of the VIP-R antagonists / VIP antagonists disclosed in U.S. Patent Nos. 6,630,124 and 5,217,953, the entireties of which are incorporated herein by reference) or a nucleic acid encoding a VIP-R antagonist disclosed herein to a nanoparticle. Thus, in some embodiments, the VIP-R antagonists disclosed herein or the nucleic acid encoding a VIP-R antagonist disclosed herein are conjugated to and / or encapsulated within the nanoparticle. In certain embodiments, the nanoparticles are comprised of poloxamer-stabilized polypropylene sulfide. As used herein, the term "nanoparticle" typically refers to a particle or structure ranging in size from about 1 nm to about 1000 nm. In certain embodiments, the nanoparticles have a diameter of about 10 to about 100 nm. In certain embodiments, the nanoparticles have a diameter of about 20 to about 50 nm, preferably about 30 nm. In certain embodiments, the nanoparticles have a diameter of about 50 nm to about 500 nm in size, more preferably about 50 nm to about 350 nm in size, more preferably about 100 nm to about 250 nm in size.

[0079] In certain embodiments, the present disclosure contemplates the use of the particles disclosed herein where the peptide sequence coupled to the nanoparticle contains a C-terminal linker peptide GGGGSC (SEQ ID NO:22) in addition to any of the VIP-R antagonists disclosed herein (such as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, and any of the VIP-R antagonists disclosed in U.S. Patent Nos. 6,630,124 and 5,217,953, the entireties of which are incorporated herein by reference). In certain embodiments, the chemical link between the peptides disclosed herein and the nanoparticles is a disulfide bond.

[0080] In certain embodiments, the present disclosure relates to recombinant peptides or fusions thereof comprising the sequences disclosed herein, where the amino or carbon terminus of the amino acid sequence is optionally linked to a heterologous amino acid sequence, a label, or a reporter molecule. "Label" refers to a detectable compound or composition that is directly or indirectly conjugated to another molecule, such as an antibody or a protein, to facilitate detection of the molecule. Specific and non-limiting examples of labels include fluorescent tags, enzyme linkages, and radioisotopes. In one example, a "labeled receptor" refers to the incorporation of a heterologous polypeptide in a receptor. Labeling includes the incorporation of a radiolabeled amino acid or the covalent attachment of a biotinyl moiety to a polypeptide, which can be detected by a marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 35 S or 131I) Fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide fluorophores, etc.), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, etc.), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.), or magnetic agents such as gadolinium chelates. In some embodiments, the labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0081] 1. Peptides a) Protein variants As disclosed herein, numerous variants of vasoactive intestinal peptide receptor (VIP-R) antagonists are known and contemplated herein. In addition to known functional variants, there are derivatives of VIP-R antagonists that also function in the disclosed methods and compositions. Protein variants and derivatives are well understood by those of skill in the art and can include amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitution variants, insertion variants, or deletion variants. Insertions include amino- and / or carboxyl-terminal fusions, as well as intrasequence insertions of single or multiple amino acid residues. Insertions will usually be smaller than the insertion of amino- or carboxyl-terminal fusions, for example, on the order of 1-4 residues. Deletions are characterized by the removal of one or more amino acid residues from the sequence of a protein. Typically, no more than about 2-6 residues are deleted at any one site within the protein molecule. These variants are usually prepared by site-directed mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, followed by expression of the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of a single residue, but can occur at several different locations at once, insertions are usually on the order of about 1-10 amino acid residues, and deletions range from about 1-30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletion of two residues or insertion of two residues. Substitutions, deletions, insertions, or any combination thereof, can be combined to arrive at the final construct. Mutations must not cause the sequence to be out of the reading frame, and preferably do not generate complementary regions that could produce secondary protein structures. Substitution variants are those in which at least one residue has been removed and in its place a different residue has been inserted. Such substitutions are generally made in accordance with Tables 1 and 2 below, and are referred to as conservative substitutions. [Table 1] [Table 2]

[0082] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., by selecting residues that are significantly different in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains. The substitutions that are generally expected to result in the greatest changes to protein properties will be those that: (a) substitute a hydrophilic residue, e.g., seryl or threonyl, for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) substitute a cysteine ​​or proline for (or by) any other residue; (c) substitute a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, for (or by) an electronegative residue, e.g., glutamyl or aspart; or (d) substitute a residue having a bulky side chain, e.g., phenylalanine, for a residue having no side chain, e.g., glycine in this case; (e) increase the number of sites for sulfylation and / or glycosylation.

[0083] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution is the replacement of one hydrophobic residue with another, or one polar residue with another. Substitutions include, for example, Gly, Ala, Val, Ile, Leu, Asp, Glu, Asn, Gln, Ser, Thr, Lys, Arg, and combinations of Phe, Tyr, etc. Such conservatively substituted variations of each explicitly disclosed sequence are included in the mosaic polypeptides provided herein.

[0084] Substitutional or deletional mutagenesis can be used to insert sites for N-glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletion of cysteines or other labile residues may also be desirable. Deletion or substitution of potential proteolysis sites, e.g., Arg, is accomplished, for example, by deleting one of the basic residues or substituting one by a glutaminyl or histidyl residue.

[0085] Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently deamidated post-translationally to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco pp79-86

[1983] ), acetylation of the N-terminal amine, and, in some cases, amidation of the C-terminal carboxyl.

[0086] It should be understood that one way of defining variants and derivatives of proteins disclosed herein is by defining them in terms of homology / identity to a particular known sequence. For example, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 show particular sequences of VIP-R antagonists. Specifically disclosed are variants of these and other proteins disclosed herein that have at least 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequences. Those skilled in the art can easily understand how to determine the homology of two proteins. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0087] Different methods of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0088] For example, the same types of homology can be obtained for nucleic acids by the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, and Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0089] It is understood that the descriptions of conservative variations and homology can be combined together in any combination, such as, for example, in an embodiment having at least 70% homology to a particular sequence where the variant is a conservative variation.

[0090] Since the present specification discusses various proteins and protein sequences, it is understood that the nucleic acids that can code for those protein sequences are also disclosed. This would include all degenerate sequences related to a particular protein sequence, i.e., all nucleic acids having a sequence that codes for one particular protein sequence, as well as all nucleic acids including degenerate nucleic acids that code for the disclosed variants and derivatives of the protein sequence. Thus, although each specific nucleic acid sequence may not be described herein, it is understood that each and every sequence is actually disclosed and described herein through the disclosed protein sequence. It is also understood that there is no amino acid sequence that indicates the specific DNA sequence that codes for the peptide or protein in the organism in which the specific variant of the disclosed VIP-R antagonist is disclosed herein, but known nucleic acid sequences that code for the peptide are known and disclosed and described herein.

[0091] It is understood that there are numerous amino acids and peptide analogs that can be incorporated into the disclosed compositions. For example, there are numerous D-amino acids or amino acids with different functional substituents than those shown in Tables 1 and 2. The opposite stereoisomers of naturally occurring peptides are disclosed, as well as stereoisomers of peptide analogs. These amino acids can be readily incorporated into a polypeptide chain by engineering a genetic construct that charges a tRNA molecule with the amino acid of choice and inserts the analog amino acid into the peptide chain in a site-specific manner, for example, using an amber codon.

[0092] Molecules can be produced that resemble peptides but are not linked through natural peptide bonds. For example, bonds for amino acids or amino acid analogs include CHNH--, --CHS--, --CH--CH--, --CH=CH-- (cis and trans), --COCH--, --CH(OH)CH--, and --CHHSO-- (these and others are described in Spatola, AF, Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, AF, Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res. 14:177-185(1979)(--CH2NH--, CH2CH2--), Spatola et al.Life Sci 38:1243-1249(1986)(--CH H2--S), Hann J.Chem.Soc Perkin Trans.I 307-314(1982)(--CH--CH--, cis and trans), Almquist et al. al.European Appln,EP 45665 CA (1982): 97:39405 (1982) (--CH(OH)CH--), Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH--), and Hruby Life Sci 31:189-199 (1982) (--CH--S--), each of which is incorporated herein by reference.A particularly preferred non-peptide bond is --CH2NH--. It is understood that peptide analogs can have more than one atom between the bond atoms, such as b-alanine, g-aminobutyric acid, and the like.

[0093] Amino acid analogs and analogs, as well as peptide analogs, often have more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broader spectrum of biological activity), reduced antigenicity, etc.

[0094] D-amino acids can be used to generate more stable peptides because they are not recognized by peptidases and the like. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine instead of L-lysine) can be used to generate more stable peptides. Cysteine ​​residues can be used to cyclize or link two or more peptides together. This can be beneficial to constrain the peptides into a particular conformation.

[0095] In certain embodiments, the present disclosure contemplates derivatives of the VIP-R antagonists disclosed herein in which one or more amino acids are replaced with chemical groups to improve pharmacokinetic properties such as solubility and serum half-life, optionally connected via a linker. In certain embodiments, such derivatives may be prodrugs in which the substituent or linker is biodegradable, or in which the substituent or linker is not biodegradable. In certain embodiments, contemplated substituents include sugars, polysaccharides, acetyl, fatty acids, lipids, and / or polyethylene glycol. The substituents may be covalently attached by forming an amide bond on the C-terminus or N-terminus of the peptide, optionally connected via a linker. In certain embodiments, it is contemplated that the substituents may be covalently attached through amino acids in the peptide, for example, through amino acids containing amine side groups, such as lysine, or carboxylic acid side groups, such as aspartic acid or glutamic acid, in peptides comprising sequences disclosed herein. In certain embodiments, it is contemplated that the substituents may be covalently attached through cysteines in sequences disclosed herein, optionally connected via a linker. In certain embodiments, the substituent is connected via a linker that forms a disulfide with a cysteine ​​amino acid side group.

[0096] The term "substituted" refers to a molecule in which at least one hydrogen atom has been replaced with a substituent. When substituted, one or more of the groups is a "substituent." A molecule may be multiply substituted. In the case of an oxo substituent ("=O"), two hydrogen atoms are replaced. Exemplary substituents in this context may include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=O)Rb, -NRaC(=O)NRaNRb, -NRaC(=O)ORb, -NRaSORb, -C(=O)Ra, -C(=O)ORa, -C(=O)NRaRb, -OC(=O)NRaRb, -ORa, -SRa, -SORa, -S(=O)Ra, -OS(=O)Ra, and -S(=O)ORa. Ra and Rb in this context may be the same or different and may independently be hydrogen, halogen, hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. Substituents may be further optionally substituted.

[0097] As used herein, a "lipid" group refers to a naturally occurring or non-naturally occurring hydrophobic group that is highly insoluble in water. As used herein, a lipid group is considered to be highly insoluble in water when the attachment points on the lipid are replaced with hydrogen, and the resulting compound has a water solubility of 0.63×10 -4 % w / w (at 25°C), which is the weight percent solubility of octane in water. Solvent Recovery Handbook, 2 ndSee Ed, Smallwood, 2002, Blackwell Science, page 195. Examples of naturally occurring lipids include saturated or unsaturated hydrocarbon chains found in fatty acids, glycerolipids, cholesterol, steroids, polyketides, and derivatives. Non-naturally occurring lipids include derivatives of naturally occurring lipids, acrylic polymers, aromatic, and alkylated compounds, and their derivatives.

[0098] For example, where a disclosed peptide or a pharma- ceutically acceptable form of a peptide contains a carboxylic acid functional group, the prodrug may be a (C1-C8) alkyl, (C2-C 12 )alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)aminomethyl having 4 to 10 carbon atoms, These may include pharma- ceutically acceptable esters formed by replacing a hydrogen atom of the acid group with a group such as N,N-(C1-C2)alkylamino)ethyl, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolactone-4-yl, di-N,N-(C1-C2)alkylamino(C2-C3)alkyl (such as beta-dimethylaminoethyl), carbamoyl-(C1-C2)alkyl, N,N-di(C1-C2)alkylcarbamoyl-(C1-C2)alkyl, and piperidino-, pyrrolidino-, or morpholino(C2-C3)alkyl.

[0099] When the disclosed peptides or pharma- ceutically acceptable forms of the peptides contain an alcohol functional group, the prodrugs may be selected from the group consisting of (C1-C6)alkanoyloxymethyl, 1-((C1-C6)alkanoyloxy)ethyl, 1-methyl-1((C1-C6)alkanoyloxy)ethyl(C1-C6)alkoxycarbonyloxymethyl, -N-(C1-C6)alkoxycarbonylaminomethyl, succinoyl, (C1-C6)alkanoyl, alpha-amino(C1-C4 ) alkanoyl, arylacyl, and alpha-aminoacyl, or alpha-aminoacyl-alpha-aminoacyl, groups such as, where each alpha-aminoacyl group is independently selected from the naturally occurring L-amino acids P(O)(OH)2, -P(O)(O(C1-C6)alkyl)2, and glycosyl (the radical resulting from removal of the hydroxyl group of the hemiacetal form of a carbohydrate).

[0100] When a disclosed peptide or a pharma- ceutically acceptable form of the peptide incorporates an amine functional group, a prodrug can be formed by replacing a hydrogen atom in the amine group with a group such as R-carbonyl, RO-carbonyl, NRR′-carbonyl, or the like, where R and R′ are each independently (C1-C 10 )alkyl, (C3-C7)cycloalkyl, benzyl, natural alpha-aminoacyl, -C(OH)C(O)OY1, where Y 1 is H, (C1-C6)alkyl or benzyl, C(OY2)Y3 where Y2 is (C1-C4)alkyl and Y3 is (C1-C6)alkyl, carboxy(C1-C6)alkyl, aminol(C1-C4)alkyl, or mono-N- or di-N,N-(C1-C6)alkylaminoalkyl, -C(Y4)Y5 where Y4 is H or methyl and Y5 is mono-N- or di-N,N-(C1-C6)alkylamino, morphinolino, piperidin-1-yl, or pyrrolidin-1-yl.

[0101] As used herein, "pharmacologically acceptable esters" includes, but is not limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, and cycloalkyl esters of acidic groups, including, but not limited to, carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids, and boronic acids.

[0102] As used herein, "pharmacologically acceptable enol ethers" include, but are not limited to, derivatives of the formula -C=C(OR), where R can be selected from alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of the formula -C=C(OC(O)R), where R can be selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl.

[0103] "Linking group" refers to any of a variety of molecular configurations that can be used to bridge molecular moieties together. An exemplary formula is -R m -, where R is individually and independently selected at each occurrence as follows: -CR m R m -, -CHR m -, -CH-, -C-, -CH2-, -C(OH)R m , -C(OH)(OH)-, -C(OH)H, -C(Hal)R m -, -C(Hal)(Hal)-, -C(Hal)H-, -C(N3)R m -, -C(CN)R m -, -C(CN)(CN)-, -C(CN)H-, -C(N3)(N3)-, -C(N3)H-, -O-, -S-, -N-, -NH-, -NR m -, -(C=O)-, -(C=NH)-, -(C=S)-, -(C=CH2)- (which may individually and independently contain single, double, or triple bonds between the R groups). mWhen branched with, it may terminate with a group such as -CH3, -H, -CH=CH2, -CCH, -OH, -SH, -NH2, -N3, -CN, or -Hal, or two branched Rs may form a cyclic structure. In certain cases, it is contemplated that the total Rs or "m" may be less than 100, less than 50, less than 25, or less than 10. Examples of linking groups include bridging alkyl groups and alkoxyalkyl groups. The linking group may be substituted with one or more substituents.

[0104] 2.Homology / Identity It should be understood that one way of defining any known variants and derivatives or possible variants of genes and proteins disclosed herein is by defining the variants and derivatives in terms of homology to a particular known sequence.For example, any of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 show the particular sequences of VIP-R antagonists disclosed herein.Specifically, disclosed are variants of these and other genes and proteins disclosed herein that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the described sequences. Those of skill in the art will readily understand how to determine the homology of two proteins or nucleic acids, e.g., genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.

[0105] Different ways of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48:443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0106] For example, at least with respect to material related to nucleic acid alignment, the same types of homology can be obtained for nucleic acids by the algorithms disclosed in Zuker, M. Science 244:48-52, 1989; Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989; Jaeger et al. Methods Enzymol. 183:281-306, 1989, which are incorporated herein by reference.

[0107] 3. Pharmaceutical Carriers / Delivery of Pharmaceutical Products In certain embodiments, the present disclosure contemplates a pharmaceutical composition comprising a peptide disclosed herein, or a nanoparticle thereof, or optionally another pharmaceutical agent, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable excipient.

[0108] In certain embodiments, the present disclosure relates to compositions, such as pharmaceutical compositions and cell growth media, comprising the peptides disclosed herein. In certain embodiments, the present disclosure relates to pharmaceutical compositions comprising any vasoactive intestinal peptide receptor (VIP-R) disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, and any VIP-R antagonists disclosed in U.S. Patent Nos. 6,630,124 and 5,217,953, the entireties of which are incorporated herein by reference) and a pharma- ceutically acceptable excipient. In certain embodiments, the pharmaceutical composition is in the form of a capsule, tablet, pill, powder, or granule. In certain embodiments, the pharmaceutical composition is in the form of a sterile pH-buffered aqueous solution. In certain embodiments, the pharmaceutical composition is in the form of a container configured to spray a propellant into a liquid or sealed container.

[0109] As mentioned above, the composition can also be administered in vivo in a pharma- ceutically acceptable carrier. "Pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to a subject together with a nucleic acid or vector, without causing any undesirable biological effects or interacting in a detrimental manner with any of the other components of the pharmaceutical composition with which it comes into contact. The carrier can necessarily be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

[0110] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, transdermally, extracorporeally, topically (including topical intranasal administration or administration by inhalation), and the like. As used herein, "topical intranasal administration" means delivery of the composition to the nose and nasal passages through one or both nostrils, and can include delivery by spray or drop mechanism, or by aerosolization of the nucleic acid or vector. Administration of the composition by inhalation can be through the nose or mouth via delivery by spray or drop mechanism. Delivery can also be delivered directly to any area of ​​the respiratory system (e.g., lungs) via intubation. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight, and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount for every composition. However, appropriate amounts can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0111] Parenteral administration of compositions, when used, is generally characterized by injection.Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid before injection, or as emulsions.Recently revised approaches to parenteral administration involve the use of slow or sustained release, so that a constant dosage is maintained.See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0112] The materials may be in solution, suspension (e.g., incorporated into microparticles, liposomes, or cells), and they may be targeted to specific cell types via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al. al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as "stealth" and other antibody-conjugated liposomes (including lipid-mediated drug targeting to colon cancer), receptor-mediated targeting of DNA via cell-specific ligands, lymphocyte-tropic tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989), and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand-induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through acidified endosomes where the receptors are sorted and then either recycled to the cell surface, stored intracellularly, or degraded in lysosomes.Internalization pathways perform a variety of functions, including nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and regulation of receptor levels. Many receptors follow more than one intracellular pathway, depending on cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

[0113] a) Pharmaceutically acceptable carrier The present compositions comprising antibodies can be used therapeutically in combination with a pharma- ceutically acceptable carrier.

[0114] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharma- ceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of pharma- ceutically acceptable carriers include, but are not limited to, saline, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, the matrices being in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to one skilled in the art that certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered.

[0115] Pharmaceutical carriers are known to those skilled in the art. These are most typically standard carriers for administering drugs to humans, including solutions such as sterile water, saline, and buffers at physiological pH. These compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0116] Pharmaceutical compositions may include, in addition to the molecule of choice, carriers, thickeners, diluents, buffers, preservatives, surface active agents, etc. Pharmaceutical compositions may also include one or more active ingredients, e.g., antibacterial agents, anti-inflammatory agents, anesthetics, etc.

[0117] The pharmaceutical compositions can be administered in a number of ways, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal), oral, inhalation, or parenteral, for example, by intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0118] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0119] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0120] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets.Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders may be desirable.

[0121] Some of the present compositions may potentially be administered as pharma- ceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl-, and arylamines, and substituted ethanolamines.

[0122] In certain embodiments, the present disclosure contemplates a pharmaceutical composition comprising a peptide or nanoparticle thereof disclosed herein, an agent disclosed herein, and a pharma- ceutically acceptable excipient. In certain embodiments, the present disclosure contemplates the manufacture of a medicament comprising a peptide or nanoparticle thereof disclosed herein, or an agent disclosed herein, and the use for the methods disclosed herein.

[0123] b) Therapeutic use Effective dosages and schedules for administering the composition may be determined empirically, and making such determinations is within the skill of one of ordinary skill in the art. The dosage range for administration of the composition is large enough to produce the desired effect in which the symptoms of the disorder are affected. The dosage should not be so large as to cause adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, and the like. In general, dosage will vary according to the age, condition, sex, and extent of disease of the patient, the route of administration, or whether other drugs are included in the regimen, and can be determined by one of ordinary skill in the art. Dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary and can be administered in one or more doses per day for one or several days. Guidance can be found in the literature for appropriate dosages of a given class of pharmaceutical agent. For example, guidance in selecting the appropriate dose for an antibody can be found in the literature on therapeutic use of antibodies, such as Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ, (1985) ch.22 and pp.303-357, Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp.365-389. A typical daily dosage of an antibody used alone may range from about 1 μg / kg body weight up to 100 mg / kg body weight or more per day, depending on the factors mentioned above. For the peptides or nanoparticles thereof, or other agents disclosed herein, the dosage administered to a patient is typically 0.0001 mg / kg to 100 mg / kg of the patient's body weight.Preferably, the dosage administered to a patient is between 0.0001 mg / kg and 20 mg / kg, between 0.0001 mg / kg and 10 mg / kg, between 0.0001 mg / kg and 5 mg / kg, between 0.0001 and 2 mg / kg, between 0.0001 and 1 mg / kg, between 0.0001 mg / kg and 0.75 mg / kg, between 0.0001 mg / kg and 0.5 mg / kg, between 0.0001 mg / kg and 0.25 mg / kg, between 0.0001 and 0.15 mg / kg, between 0.0001 and 0.10 mg / kg, between 0.001 and 0.5 mg / kg, between 0.01 and 0.25 mg / kg, or between 0.01 and 0.10 mg / kg of the patient's body weight. Furthermore, the dosage and frequency of administration of the peptides or nanoparticles thereof, or drugs disclosed herein may be reduced by enhancing uptake and tissue penetration, for example, by modifications such as lipidation and inclusion of natural or artificial pulmonary surfactants.

[0124] 4.Nucleic acid The term "nucleic acid" refers to a polymer of nucleotides, or polynucleotide. The term is used to designate a single molecule or a collection of molecules. A nucleic acid may be single-stranded or double-stranded and may contain a coding region as well as a region of various control elements, as described below.

[0125] For example, there are various molecules disclosed herein that are nucleic acid based, including nucleic acids encoding any of the vasoactive intestinal peptide receptors (VIP-R) shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, as well as various functional nucleic acids or any VIP-R antagonist / VIP antagonists disclosed in U.S. Patent Nos. 6,630,124 and 5,217,953, which are incorporated herein by reference in their entirety. The disclosed nucleic acids are composed of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. For example, when the vector is expressed in a cell, it is understood that the expressed mRNA is typically composed of A, C, G, and U. Similarly, for example, when an antisense molecule is introduced into a cell or cellular environment, e.g., through exogenous delivery, it will be appreciated that it will be advantageous for the antisense molecule to be composed of nucleotide analogues which reduce degradation of the antisense molecule in the cellular environment.

[0126] a) Nucleotides and related molecules A nucleotide is a molecule that contains a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate and sugar moieties to form an internucleoside bond. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is ribose or deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. Non-limiting examples of nucleotides would be 3'-AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are a wide variety of these types of molecules available in the art and available herein.

[0127] The term "nucleic acid sequence encoding" a particular peptide refers to a nucleic acid sequence that includes a coding region of the peptide, or in other words, a nucleic acid sequence that encodes a peptide product. The coding region may be present in either the form of cDNA, genomic DNA, or RNA. If present in DNA form, the oligonucleotide, polynucleotide, or nucleic acid may be single-stranded (i.e., the sense strand) or double-stranded. Suitable control elements, such as enhancers / promoters, splice junctions, polyadenylation signals, etc., may be positioned adjacent to the coding region as necessary to allow proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in an expression vector may include endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0128] A nucleotide analog is a nucleotide that contains some type of modification in either the base, sugar, or phosphate moieties. Modifications to nucleotides are known in the art and would include, for example, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, and 2-aminoadenine, as well as modifications in the sugar or phosphate moieties. There are a wide variety of these types of molecules available in the art and available herein.

[0129] Nucleotide substitutes are molecules that have similar functional properties as nucleotides, but do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through moieties other than the phosphate moiety. Nucleotide substitutes can conform to a double helix type structure when interacting with an appropriate target nucleic acid. There are a wide variety of these types of molecules available in the art and available herein.

[0130] Other types of molecules (conjugates) can be linked to nucleotides or nucleotide analogs, for example, to enhance cellular uptake. Conjugates can be chemically linked to nucleotides or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties, such as cholesterol moieties. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are a wide variety of these types of molecules available in the art and available herein.

[0131] A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, N1, and C6 positions of a purine-based nucleotide, nucleotide analog, or nucleotide substitute, and the C2, N3, and C4 positions of a pyrimidine-based nucleotide, nucleotide analog, or nucleotide substitute.

[0132] A Hoogsteen interaction is an interaction that occurs on the Hoogsteen face of a nucleotide or nucleotide analogue that is exposed in the major groove of duplex DNA. The Hoogsteen face includes the reactive groups (NH2 or O) at the N7 and C6 positions of purine nucleotides.

[0133] b) Array There are various sequences related to the VIP-R antagonists disclosed herein, all of which are encoded by or are nucleic acids. The sequences of the human analogs of these genes, as well as other analogs, and alleles of these genes, as well as splice variants and other types of variants, are available in various protein and gene databases, including Genbank. Those skilled in the art will understand how to analyze sequence discrepancies and differences, and how to adjust compositions and methods related to a particular sequence to other related sequences. Primers and / or probes can be designed for any given sequence given the information disclosed herein and known in the art.

[0134] 5. Nucleic Acid Delivery In the above methods involving administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transfer or transfection), the disclosed nucleic acids can be in the form of naked DNA or RNA, or the nucleic acid can be within a vector for delivery of the nucleic acid to a cell whereby the DNA fragment encoding the antibody is under the transcriptional control of a promoter, as will be well understood by one of skill in the art. The vector can be a commercially available preparation such as an adenoviral vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector into a cell can be via a variety of mechanisms. By way of example, delivery can be via liposomes using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany), and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed following standard procedures in the art. Additionally, the disclosed nucleic acids or vectors can be delivered in vivo by electroporation, technology for which is available from Genetronics, Inc. (San Diego, CA), as well as by the SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

[0135] As an example, delivery of the vector can be via a viral system, such as a retroviral vector system, which can package a recombinant retroviral genome (see, e.g., Pastan et al., Proc. Natl. Acad. Sci. USA 85:4486, 1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinant retrovirus can then be used to infect and thereby deliver a nucleic acid encoding a broadly neutralizing antibody (or an active fragment thereof) to the infected cell. The exact method of introducing the altered nucleic acid into the mammalian cell is, of course, not limited to the use of a retroviral vector. Other techniques for this procedure are widely available, including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors (Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidini et al., Science 272:263-267, 1996), pseudotyped retroviral vectors (Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physical transduction techniques such as liposomal delivery and receptor-mediated and other endocytosis mechanisms can also be used (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996). The compositions and methods of the present disclosure can be used in conjunction with any of these or other commonly used gene transfer methods.

[0136] As an example, when an antibody-encoding nucleic acid is delivered to a subject's cells in an adenovirus vector, the dosage for administering the adenovirus to a human is about 10 per injection. 7 ~10 9 plaque forming units (pfu) per injection, but may range from 10 12pfu can be achieved (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997). Subjects can receive a single injection, or if additional injections are required, they can be repeated at six month intervals (or other appropriate time intervals as determined by one of skill in the art) indefinitely and / or until efficacy of the treatment is established.

[0137] Parenteral administration of nucleic acids or vectors, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. Recently revised approaches to parenteral administration involve the use of slow or sustained release such that a constant dosage is maintained. For further discussion of suitable formulations and various routes of administration of therapeutic compounds, see, for example, Remington: The Science and Practice of Pharmacy (19th ed.) ed. AR Gennaro, Mack Publishing Company, Easton, PA 1995.

[0138] 6. Expression System The nucleic acid delivered to cells typically contains an expression control system. For example, the inserted gene in viral and retroviral systems usually contains a promoter and / or enhancer that helps control the expression of the desired gene product. A promoter is generally a sequence of DNA that functions when it is in a relatively fixed position with respect to the transcription start site. A promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.

[0139] Protein "expression systems" refer to in vivo and in vitro (cell-free) systems. Systems for recombinant protein expression typically utilize cells that are transfected with a DNA expression vector containing a template. The cells are cultured under conditions that result in translation of the desired protein. The expressed protein is extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Some proteins are also recovered using denaturants and protein refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain sufficient components for transcription, translation, and optionally post-translational modification, such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids and nucleotides. In the presence of an expression vector, these extracts and components can synthesize the protein of interest. Cell-free systems typically do not contain proteases, allowing for labeling of proteins with modified amino acids. Some cell-free systems have incorporated components coded for translation into expression vectors. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755, and Asahara & Chong, Nucleic Acids Research, 2010, 38(13):e141, both of which are incorporated by reference in their entireties.

[0140] a) Viral promoters and enhancers Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from various sources, such as the genomes of viruses such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably cytomegalovirus, or from heterologous mammalian promoters, such as the beta-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication (Fiers et al., Nature, 273:113 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment (Greenway, PJ et al., Gene 18:355-360 (1982)). Of course, promoters from host cells or related species are also useful herein.

[0141] Enhancers generally refer to sequences of DNA that function at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78:993 (1981)) or 3' (Lusky, ML, et al., Mol. Cell Bio. 3:1108 (1983)). In addition, enhancers can be found within introns (Banerji, J Let al., Cell 33:729 (1983)) and within the coding sequence itself (Osborne, TF, et al., Mol. Cell Bio. 4:1293 (1984)). They are usually 10-300 bp in length and function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of gene expression. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but typically, for gene expression, enhancers from eukaryotic cell viruses will be used for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0142] The promoters and / or enhancers can be specifically activated either by light or by specific chemical events that trigger their function. The system can be regulated by agents such as tetracycline and dexamethasone. There are also ways to enhance gene expression of viral vectors by exposure to irradiation, such as gamma irradiation, or by alkylating chemotherapy drugs.

[0143] In certain embodiments, the promoter and / or enhancer region can act as a constitutive promoter and / or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain constructs, the promoter and / or enhancer region is active in all eukaryotic cell types, even if it is only expressed in certain types of cells at certain times. A preferred promoter of this type is the CMV promoter (650 bases). Other preferred promoters are the SV40 promoter, the cytomegalovirus (full-length promoter), and the LTR of retroviral vectors.

[0144] It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types, such as melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.

[0145] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells) may also contain sequences necessary for the termination of transcription that may affect the expression of the mRNA. These regions are transcribed as polyadenylation segments in the untranslated portion of the mRNA encoding the tissue factor protein. The 3' untranslated region also includes the transcription termination site. The transcription unit preferably also contains a polyadenylation region. One advantage of this region is that it increases the likelihood that the transcription unit will be processed and transported like an mRNA. The identification and use of polyadenylation signals in expression constructs is well established. It is preferred that a homologous polyadenylation signal is used in the transgene construct. In certain transcription units, the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcription unit contains other standard sequences that, alone or in combination with the above sequences, improve the expression or stability from the construct.

[0146] b) Marker Viral vectors can contain nucleic acid sequences that encode a marker product that is used to determine whether the gene has been delivered to a cell and is being expressed upon delivery. Preferred marker genes are the E. coli lacZ gene, which encodes β-galactosidase, and green fluorescent protein.

[0147] In some embodiments, the marker may be a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin. When such selectable markers are successfully transferred to a mammalian host cell, the transformed mammalian host cell can survive if placed under selection pressure. There are two different categories of selection regimes that are widely used. The first category is based on the metabolism of the cell and the use of mutant cell lines that lack the ability to grow independently of a supplemented medium. Two examples are CHO DHFR-cells and mouse LTK-cells. These cells lack the ability to grow without the addition of nutrients such as thymidine or hypoxanthine. These cells lack certain genes necessary for a complete nucleotide synthesis pathway and therefore cannot survive unless the missing nucleotides are provided in a supplemented medium. An alternative to supplementing the medium is to alter the growth requirements of cells lacking the respective genes by introducing an intact DHFR or TK gene: individual cells not transformed with the DHFR or TK gene will not be able to survive in unsupplemented medium.

[0148] The second category is dominant selection, which refers to selection schemes that can be used with any cell type and does not require the use of mutant cell lines. These schemes typically use a drug to stop the growth of the host cells. Those cells with the new gene will express a protein that conveys drug resistance and will survive the selection. Examples of such dominant selection use the drugs neomycin (Southern P. and Berg, P., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid, (Mulligan, RC and Berg, P. Science 209:1422 (1980)), or hygromycin (Sugden, B. et al., Mol. Cell. Biol. 5:410-413 (1985)). Three examples use bacterial genes under the control of eukaryotic cells to convey resistance to the appropriate drugs G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively. Others include the neomycin analog G418 and puramycin.

[0149] C.Kit In some aspects, disclosed herein is a kit comprising a VIP-R antagonist disclosed herein (e.g., any of the VIP-R antagonists set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, as well as any VIP-R antagonist / VIP antagonist disclosed in U.S. Pat. Nos. 6,630,124 and 5,217,953, the entireties of which are incorporated herein by reference). In some embodiments, the kit further comprises an anti-CD3 antibody and / or an anti-CD28 antibody. In some embodiments, the kit further comprises a phosphatidylinositol 3 kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor).

[0150] Phosphatidylinositol-3-kinase (PI3K) / AKT / mammalian target of rapamycin (mTOR) signaling is one of the most important intracellular pathways regulating cell growth, motility, survival, metabolism, and angiogenesis. PI3Ks are a group of plasma membrane-associated lipid kinases that consist of three subunits: p85 regulatory subunit, p55 regulatory subunit, and p110 catalytic subunit. PI3Ks can be divided into three classes: class I, II, and III. Class I PI3Ks consist of class IA and class IB PI3Ks. Class IA PI3Ks are heterodimers of p58 regulatory subunit and p110 catalytic subunit. Class IA PI3Ks contain p110α, p110β, and p110δ catalytic subunits that are produced from distinct genes PIK3CA, PIK3CB, and PIK3CD, respectively. The subunit p110γ produced by PIK3CG represents a catalytic subunit in class IB PI3K. A PI3K inhibitor can inhibit one or more p110 isoforms of class I PI3K. In some embodiments, the PI3K inhibitors described herein are PI3Kα inhibitors, PI3Kβ inhibitors, PI3Kδ inhibitors, or PI3Kγ inhibitors. In some embodiments, the PI3K inhibitors described herein are Pan-PI3K inhibitors, isoform-specific inhibitors, or dual PI3K inhibitors.Examples of PI3K inhibitors described herein include fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474, as well as isoform-specific inhibitors, such as PI3K alpha inhibitors (e.g., inavolisib, alpelisib, AZD8835, PWT33597, taselisib , and / or seravelisib), PI3K beta inhibitors (such as, for example, AZD8186 and / or GSK2636771), PI3K delta inhibitors (such as, for example, AZD8835, AZD8186, nemiralisib, seralysib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, limpellisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, for example, eganelisib, tenalisib, taselisib, and / or duvelisib). In some embodiments, the PI3K inhibitor described herein is a PI3K delta inhibitor (such as, for example, idelalisib).

[0151] In some embodiments, a kit disclosed herein comprising a VIP-R antagonist disclosed herein (e.g., any of the VIP-R antagonists set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, a fragment thereof, or an analog thereof) further comprises an immune checkpoint blockade. In some embodiments, the immune checkpoint blockade is a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor.

[0152] As used herein, the term "PD-1 inhibitor" refers to a composition that binds to PD-1 and reduces or inhibits the interaction between bound PD-1 and PD-L1. In some embodiments, the PD-1 inhibitor is a monoclonal antibody that is specific for PD-1 and reduces or inhibits the interaction between bound PD-1 and PD-L1. Non-limiting examples of PD-1 inhibitors are pembrolizumab, nivolumab, and cemiplimab. In some embodiments, the pembrolizumab is KEYTRUDA or a bioequivalent. In some embodiments, the pembrolizumab is as described in U.S. Pat. No. 8,952,136, U.S. Pat. No. 8,354,509, or U.S. Pat. No. 8,900,587, all of which are incorporated by reference in their entirety. In some embodiments, the pembrolizumab has a U.S. Food and Drug Administration Unique Ingredient Identifier (UNII) of DPT0O3T46P. In some embodiments, the nivolumab is OPDIVO or a bioequivalent. In some embodiments, nivolumab has a United States Food and Drug Administration Unique Component Identifier (UNII) of 31YO63LBSN. In some embodiments, nivolumab is as described in U.S. Patent No. 7,595,048, U.S. Patent No. 8,738,474, U.S. Patent No. 9,073,994, U.S. Patent No. 9,067,999, U.S. Patent No. 8,008,449, or U.S. Patent No. 8,779,105, all of which are incorporated by reference in their entirety. In some embodiments, cemiplimab is LIBTAYO or a biological equivalent. In some embodiments, cemiplimab has a United States Food and Drug Administration Unique Component Identifier (UNII) of 6QVL057INT. In some embodiments, cemiplimab is as described in U.S. Patent No. 10,844,137, all of which are incorporated by reference in their entirety. In some embodiments, the PD-1 inhibitor described herein is spartalizumab, JTX-4014, camrelizumab, sintilimab, pembrolizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, or AMP-514 (MEDI0680).

[0153] The term "PD-L1 inhibitor" refers to a composition that binds to PDL-1 and reduces or inhibits the interaction between bound PD-L1 and PD-1. In some embodiments, the PD-L1 inhibitor is a monoclonal antibody that is specific for PD-L1 and reduces or inhibits the interaction between bound PD-L1 and PD-1. Examples of PD-L1 inhibitors include atezolizumab, avelumab, and durvalumab. In some embodiments, atezolizumab is TECENTRIQ or a bioequivalent. In some embodiments, atezolizumab has a U.S. Food and Drug Administration Unique Ingredient Identifier (UNII) of 52CMI0WC3Y. In some embodiments, atezolizumab is as described in U.S. Patent No. 8,217,149, which is incorporated by reference in its entirety. In some embodiments, avelumab is BAVENCIO or a bioequivalent. In some embodiments, avelumab has a U.S. Food and Drug Administration Unique Ingredient Identifier (UNII) of KXG2PJ551I. In some embodiments, the avelumab is as described in U.S. Patent Application Publication No. 2014321917, which is incorporated by reference in its entirety. In some embodiments, the durvalumab is IMFINZI or a bioequivalent. In some embodiments, the durvalumab has a U.S. Food and Drug Administration Unique Component Identifier (UNII) of 28X28X9OKV. In some embodiments, the durvalumab is as described in U.S. Patent No. 8,779,108, which is incorporated by reference in its entirety. In some embodiments, the PD-L1 inhibitor described herein is atezolizumab, avelumab, durvalumab, CK-301, or BMS-986189.

[0154] In some embodiments, the immune checkpoint blockade comprises a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, cemiplimab, dostallimab, spartalizumab, JTX-4014, camrelizumab, sintilimab, tislelizumab, toripalimab, INCMGA00012 (MGA012), AMP-224, or AMP-514 (MEDI0680)), a PD-L1 inhibitor (e.g., atezolizumab, avelumab, durvalumab, CK-301, or BMS-986189), or a CTLA-4 inhibitor (e.g., ipilimumab or tremelimumab). In some embodiments, immune checkpoint blockade comprises pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, or ipilimumab.

[0155] In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an anti-TIM3 inhibitor, an anti-LAG3 inhibitor, an anti-CD47 inhibitor, imiquimod, polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), pexidartinib, an anti-TIGIT inhibitor, an anti-B7-H3 inhibitor, an anti-B7-H4 inhibitor, an anti-A2aR inhibitor, an anti-CD73 inhibitor, an anti-NKG2A inhibitor, an anti-PVRIG / PVRL2 inhibitor, an anti-CEACAM1 inhibitor. , anti-CEACAM5 inhibitors, anti-CEACAM6 inhibitors, focal adhesion kinase (FAK) inhibitors, CCL2 / CCR2 inhibitors, anti-leukemia inhibitory factor (LIF) inhibitors, anti-CD47 / SIRPα inhibitors, anti-colony stimulating factor (CSF)-1 inhibitors, anti-IL-1 inhibitors, anti-IL-1R3 inhibitors, anti-IL-8 inhibitors, anti-semaphorin 4D (Sema4D) inhibitors, angiopoietin (Ang)-2 inhibitors, CLEVER-1 inhibitors, Axl-targeted enapotamab vedotin (EnaV), or anti-phosphatidylserine inhibitors.

[0156] In some embodiments, disclosed herein are any of the VIP-R antagonists disclosed herein (e.g., any of the VIP-R antagonists set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof), immune checkpoint blockade (e.g., pembrolizumab, nivolumab, , cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, or ipilimumab), and / or PI3K inhibitors (fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bilirubin, Miralisib, paxalisib, voxalisib, GSK1059615, MEN1611, ZSTK474, as well as isoform-specific inhibitors, such as PI3K alpha inhibitors (e.g., inavolisib, alpelisib, AZD8835, PWT33597, taselisib, and / or seravelisib), PI3K beta inhibitors (e.g., AZD8186 and / or GSK2636771), PI3K delta inhibitors (e.g., AZD8835, AZD 8186, nemiralisib, seratisib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, limpellisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, but not limited to, eganelisib, tenalisib, taselisib, and / or duvelisib).

[0157] D.How to use In certain embodiments, the disclosure relates to the proliferation of T cells, activation of T cells, expansion or reversal of senescence in T cells, or reversal of exhaustion in T cells with natural reactivity to cancer that may be found to infiltrate tumors in a subject. Tumors can be harvested and these tumor infiltrating lymphocytes (TILs) can be isolated from the tumor and then expanded using the methods disclosed herein.

[0158] In certain embodiments, the present disclosure relates to compositions and methods for reversing senescence or reversing exhaustion in T cells by interrupting vasoactive intestinal peptide (VIP) signaling and / or inhibiting phosphatidylinositol-3-kinase (PI3 kinase) inhibitor signaling, and uses in managing cancer and chronic viral infections. In certain embodiments, the present disclosure contemplates a method of reversing senescence or reversing exhaustion in T cells by mixing T cells in vitro with any VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.), or nanoparticles comprising any VIP-R antagonist disclosed herein that prevent VIP from interacting with the VIP receptor, and / or adding a PI3 kinase inhibitor. In some embodiments, the method further comprises mixing the T cells with an immune checkpoint blockade (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor). In certain embodiments, the present disclosure contemplates expanding senescent T cells by mixing with a PI3 kinase inhibitor, a nanoparticle, or a polypeptide disclosed herein, a VIP-degrading enzyme, an immune checkpoint blockade, and combinations thereof.

[0159] In certain embodiments, the present disclosure contemplates a method of stimulating isolated T cells or expanding senescent T cells by exposing the T cells in vitro to antibodies that bind CD3 and / or CD28 in combination with a PI3 kinase inhibitor (e.g., idelalisib), any VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof), or nanoparticles disclosed herein, VIP-degrading enzymes, and combinations thereof. In certain embodiments, the present disclosure contemplates the use of anti-CD3 and anti-CD28 antibodies or binding agents, optionally linked to a solid substrate such as magnetic beads.

[0160] In certain embodiments, the present disclosure contemplates methods of expanding T cells that are negative for CD28 and / or CD27 using the in vitro cell cultures disclosed herein, providing replicated T cells with increased expression of CD28 and / or CD27 compared to pre-replication levels.

[0161] In certain embodiments, the present disclosure contemplates a method of expanding T cells, where before, during, or after expanding the T cells, the T cells are mixed with a vector having a nucleic acid sequence encoding a chimeric antigen receptor, the chimeric antigen receptor comprising a cancer targeting sequence of a T cell antigen receptor domain, a transmembrane domain, a T cell costimulatory molecule domain, and a signaling component, under conditions such that the cells express the chimeric antigen receptor on the surface of the cell.

[0162] In certain embodiments, the present disclosure relates to an in vitro cell culture composition comprising a minimum essential medium and T cells, and any VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.), or nanoparticles comprising any of the VIP-R antagonists disclosed herein, and a phosphatidylinositol-3-kinase inhibitor, a VIP-degrading enzyme, and combinations thereof, and optionally further comprising anti-CD3 and anti-CD28 antibodies immobilized on a solid substrate, such as beads. In certain embodiments, the T cells are purified from bone marrow cells or blood cells, peripheral blood.

[0163] In some embodiments, the PI3K inhibitors described herein are PI3K alpha inhibitors, PI3K beta inhibitors, PI3K delta inhibitors, or PI3K gamma inhibitors. In some embodiments, the PI3K inhibitors described herein are Pan-PI3K inhibitors, isoform-specific inhibitors, or dual PI3K inhibitors. Examples of PI3K inhibitors described herein include fimepinostat, rigosertib, buparlisib, CH5132799, piralalisib, ZSTK474, sonolisib, pictilisib, copanlisib, B591, TG-100-115, RIDR-PI-103, dactolisib, apitolisib, gedatolisib, SF1126, omipalisib, samotricisib, bimiralisib, paxalisib, voxtalisib, GSK1059615, MEN1611, ZSTK474, as well as isoform-specific inhibitors, such as PI3K alpha inhibitors (e.g., inavolisib, alpelisib, AZD8835, PWT33597, taselisib , and / or seravelisib), PI3K beta inhibitors (such as, for example, AZD8186 and / or GSK2636771), PI3K delta inhibitors (such as, for example, AZD8835, AZD8186, nemiralisib, seralithib, acalisib, CAL263, TG100-115, duvelisib, idelalisib, tenalisib, taselisib, zandelisib, AMG319, limpellisib, palsaclisib, umbralisib, and / or renolisib), and / or PI3K gamma inhibitors (such as, for example, eganelisib, tenalisib, taselisib, and / or duvelisib). In certain embodiments, the phosphatidylinositol-3-kinase inhibitor is selected from idelalisib, wortmannin, demethoxyviridin, perifosine, buparlisib, duvelisib, copanlisib, and alpelisib. In certain embodiments, the phosphatidylinositol-3-kinase inhibitor is present in the culture at a concentration of about 0.001 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or about 10 nM to about 10 micromolar, or about 10 nM to about 500 nM, or about 10 nM to about 1 micromolar.In certain embodiments, the phosphatidylinositol-3-kinase inhibitor is selected from idelalisib in the culture at a concentration of about 0.001 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or about 10 nM to about 10 micromolar, or about 10 nM to about 500 nM, or about 10 nM to about 1 micromolar.

[0164] In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an anti-TIM3 inhibitor, an anti-LAG3 inhibitor, an anti-CD47 inhibitor, imiquimod, polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), pexidartinib, an anti-TIGIT inhibitor, an anti-B7-H3 inhibitor, an anti-B7-H4 inhibitor, an anti-A2aR inhibitor, an anti-CD73 inhibitor, an anti-NKG2A inhibitor, an anti-PVRIG / PVRL2 inhibitor, an anti-CEACAM1 inhibitor. , anti-CEACAM5 inhibitors, anti-CEACAM6 inhibitors, focal adhesion kinase (FAK) inhibitors, CCL2 / CCR2 inhibitors, anti-leukemia inhibitory factor (LIF) inhibitors, anti-CD47 / SIRPα inhibitors, anti-colony stimulating factor (CSF)-1 inhibitors, anti-IL-1 inhibitors, anti-IL-1R3 inhibitors, anti-IL-8 inhibitors, anti-semaphorin 4D (Sema4D) inhibitors, angiopoietin (Ang)-2 inhibitors, CLEVER-1 inhibitors, Axl-targeted enapotamab vedotin (EnaV), or anti-phosphatidylserine inhibitors.

[0165] In certain embodiments, the culture comprises an enzyme that hydrolyzes VIP. In certain embodiments, the culture comprises a VIP-degrading enzyme, such as a peptidase, a serine peptidase, a tryptase, a chymase, or human chymase 1 (CMA1). In certain embodiments, the culture has at least about 0.001 micrograms / mL, about 0.01 micrograms / mL, about 0.1 micrograms / mL, or about 1 micrograms / mL of a VIP-degrading enzyme, such as mast cell chymase. In certain embodiments, the present disclosure contemplates a T cell culture comprising a minimal essential medium and isolated cells expressing CD3 and / or CD4 and / or CD8 and negative for CD27 and / or CD28, and a PI3 kinase inhibitor, any VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof), or nanoparticles comprising any of the VIP-R antagonists disclosed herein, and combinations thereof. Cells may be isolated by negative or positive selection using binding agents bound to a solid support, such as beads, magnetic beads, or particles of fluorescent binding agents.

[0166] In certain embodiments, the anti-CD3 and anti-CD28 antibodies are immobilized on beads, magnetic beads, or solid surfaces. In certain embodiments, more than 5.0%, 10%, or 15% of the total cells in the culture express CD3 and / or CD4 and / or CD8. In certain embodiments, more than 20%, 25%, or 50% of the total cells express CD3 and / or CD4 and / or CD8. In certain embodiments, more than 15%, 20%, or 30% of the T cells in the culture are negative for CD28 and / or CD27. In certain embodiments, more than 20%, 25%, or 50% of the T cells are negative for CD28 and / or CD27.

[0167] In certain embodiments, purified T cells are obtained by centrifugation of blood under conditions such that plasma and red blood cells separate, providing purified T cells in a mixture of white blood cells between the plasma and red blood cells, hi certain embodiments, purified T cells are obtained by bone marrow aspirate or bone marrow biopsy.

[0168] In certain embodiments, purified T cells are obtained by mixing cells with a fluorescent marker that binds CD3 and purifying the cells by fluorescence activated cell sorting. In certain embodiments, purified T cells are obtained by mixing cells with a magnetic marker that binds CD3 and purifying the cells by magnetic sorting. In certain embodiments, purified T cells are obtained by mixing cells with a fluorescent marker that binds CD3 and / or CD4 and / or CD8 and purifying the cells by fluorescence activated cell sorting. In certain embodiments, purified T cells are obtained by mixing cells with a magnetic marker that binds CD3 and / or CD4 and / or CD8 and purifying the cells by magnetic sorting.

[0169] In certain embodiments, the present disclosure contemplates a solid substrate, such as a bead, having anti-CD3 and anti-CD28 antibodies and having a VIP degrading enzyme coupled to its surface. In certain embodiments, it is contemplated that the beads are placed in a medium and T cells are grown on top of the medium such that the beads are intracellular.

[0170] In certain embodiments, the VIP degrading enzyme comprises human CMA1 Accession Number GenBank: AAI03975.1:MLLKLKEKASLTLAVGTLPFPSQFNFVPPGRMCRVAGWGRTGVLKPGSDTLQEVKLRLMDPQACSHFRDFDHNLQLCVGNPRKTKSAFKGDSGGPLLCAGVAQGIVSYGRSDAKPPAVFTRISHYRPWINQILQAN (SEQ ID NO: 19).

[0171] In one particular embodiment, the VIP degrading enzyme is human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) having the following sequence: (SEQ ID NO:20).

[0172] In certain embodiments, the cell cultures and methods described herein further comprise IL-12. In certain embodiments, it is contemplated that IL-12 enhances the effect of the peptides disclosed herein or nanoparticles thereof on T cell proliferation stimulated in vitro with antibodies against CD3 and CD28.

[0173] In certain embodiments, the present disclosure relates to a method of enhancing immune response to cell therapy, comprising administering to a subject any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments or analogs thereof) in combination with cells. In certain embodiments, the subject has been diagnosed with leukemia or lymphoma. In certain embodiments, the cells are blood cells, bone marrow cells, white blood cells, T cells, natural killer cells, hematopoietic stem cells, G-CSF mobilized or non-mobilized blood mononuclear cells.

[0174] In certain embodiments, the cells are selected from the group consisting of autologous T cells, allogeneic cells from an HLA-matched donor, or allogeneic cells from an HLA-mismatched donor. In certain embodiments, the cells are bone marrow cells. In certain embodiments, the cells are blood mononuclear cells that contain / express granulocyte colony-stimulating factor. Cell therapy may be performed using non-mobilized blood mononuclear cells.

[0175] In certain embodiments, it is contemplated that any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) may be administered to a subject before, during, or after cell-based immunotherapy, including the recipient or donor. Immunotherapy may be performed in combination with chemotherapy and / or radiation therapy. It is contemplated that the peptides may be used in combination with other immune stimulants, including, but not limited to, CpG oligonucleotides, granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, interferon alpha, pegylated interferon, interleukin-12, interleukin-2, and pegfilgrastim.

[0176] In certain embodiments, the present disclosure relates to a method for treating or preventing graft-versus-host disease in a subject, comprising administering an effective amount of a VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments, or analogs thereof, etc.) to a subject after hematopoietic stem cell transplantation or to a subject receiving or having received transplanted allogeneic tissue or cells. In certain embodiments, the subject has received transplanted allogeneic hematopoietic stem cells. In certain embodiments, the subject has received transplanted allogeneic hematopoietic stem cells separated from peripheral blood. In certain embodiments, the subject has received chemotherapy to radiotherapy before receiving transplanted allogeneic hematopoietic stem cells.

[0177] 1. How to treat an infection The disclosed VIP-R antagonists can be used to treat infectious diseases. In one aspect, disclosed herein is a method of treating, reducing, inhibiting, reducing, alleviating, and / or preventing a microbial infection, wherein the microbial infection is a viral infection, and the viral infection is selected from the group consisting of herpes simplex virus-1, herpes simplex virus-2, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes virus-6, herpes lymphotropic virus, roseolovirus, Kaposi's sarcoma associated herpes virus, smallpox virus, vesicular stomatitis virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rhinovirus, coronavirus (avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV), and the like.HKU15), porcine epidemic diarrhea virus (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV), influenza A virus (including but not limited to H1N1), influenza B virus, influenza C virus, measles virus, polyomavirus, human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, parvovirus B19, molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, respiratory syncytial virus, adenovirus, coxsackievirus, chikungunya virus, dengue virus, mumps virus, poliovirus, rabies virus ... The infection is caused by a virus selected from the group consisting of encephalitis virus, reovirus, yellow fever virus, human adenovirus types (HAdV-1 to 55), norovirus, rinderpest virus, California encephalitis virus, Friend spleen focus forming virus (SFFV) or xenotropic MuLV-related virus (XMRV), Ebola virus, Marburg virus, Lassa fever virus, Eastern equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, rotavirus A, rotavirus B, rotavirus C, rotavirus D, rotavirus E, Sindbis virus, simian immunodeficiency virus, human T-cell leukemia virus type 1, hantavirus, rubella virus, simian immunodeficiency virus, human immunodeficiency virus type 1, and human immunodeficiency virus type 2. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor).

[0178] In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an anti-TIM3 inhibitor, an anti-LAG3 inhibitor, an anti-CD47 inhibitor, imiquimod, polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), pexidartinib, an anti-TIGIT inhibitor, an anti-B7-H3 inhibitor, an anti-B7-H4 inhibitor, an anti-A2aR inhibitor, an anti-CD73 inhibitor, an anti-NKG2A inhibitor, an anti-PVRIG / PVRL2 inhibitor, an anti-CEACAM1 inhibitor. , anti-CEACAM5 inhibitors, anti-CEACAM6 inhibitors, focal adhesion kinase (FAK) inhibitors, CCL2 / CCR2 inhibitors, anti-leukemia inhibitory factor (LIF) inhibitors, anti-CD47 / SIRPα inhibitors, anti-colony stimulating factor (CSF)-1 inhibitors, anti-IL-1 inhibitors, anti-IL-1R3 inhibitors, anti-IL-8 inhibitors, anti-semaphorin 4D (Sema4D) inhibitors, angiopoietin (Ang)-2 inhibitors, CLEVER-1 inhibitors, Axl-targeted enapotamab vedotin (EnaV), or anti-phosphatidylserine inhibitors. In some embodiments, the immune checkpoint blockade comprises pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, or ipilimumab.

[0179] In some embodiments, the disclosure relates to the use of a VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) in the manufacture of an antiviral drug for the treatment of a viral infection. In some embodiments, the antiviral drug further comprises a phosphatidylinositol 3 kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the antiviral drug further comprises an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor). In some embodiments, the subject has been diagnosed with a chronic viral infection. In certain embodiments, the subject undergoes serological monitoring. In some embodiments, the administration is performed under conditions such that the viral infection is no longer detectable. In some embodiments, the subject has been diagnosed with an RNA virus, a DNA virus, or a retroviral infection. In some embodiments, the subject is diagnosed with a virus, i.e., a double-stranded DNA virus, a sense single-stranded DNA virus, a double-stranded RNA virus, a sense single-stranded RNA virus, an antisense single-stranded RNA virus, a sense single-stranded RNA retrovirus, or a double-stranded DNA retrovirus.In some embodiments, the subject is diagnosed with a rotavirus, an influenza virus, a herpes virus, a hepatitis virus, or a lentivirus.In some embodiments, the titer of the virus in the subject is reduced after treatment compared to before treatment.

[0180] In certain embodiments, the disclosure relates to a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing a viral infection, comprising administering to a subject at risk for, exhibiting symptoms of, or diagnosed with a viral infection any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, etc.). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor). In certain embodiments, the subject is immunocompromised or the subject is an allogeneic bone marrow transplant donor or recipient. In typical embodiments, the subject is an organ transplant recipient, undergoes hemodialysis, is diagnosed with cancer, is receiving immunosuppressive drugs, and / or is diagnosed with HIV infection. In certain embodiments, the present disclosure relates to preventing viral infection in an immunocompromised subject at risk of infection by administering any of the VIP-R antagonists disclosed herein and, optionally, one or more antiviral agents.

[0181] In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an anti-TIM3 inhibitor, an anti-LAG3 inhibitor, an anti-CD47 inhibitor, imiquimod, polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), pexidartinib, an anti-TIGIT inhibitor, an anti-B7-H3 inhibitor, an anti-B7-H4 inhibitor, an anti-A2aR inhibitor, an anti-CD73 inhibitor, an anti-NKG2A inhibitor, an anti-PVRIG / PVRL2 inhibitor, an anti-CEACAM1 inhibitor. , anti-CEACAM5 inhibitors, anti-CEACAM6 inhibitors, focal adhesion kinase (FAK) inhibitors, CCL2 / CCR2 inhibitors, anti-leukemia inhibitory factor (LIF) inhibitors, anti-CD47 / SIRPα inhibitors, anti-colony stimulating factor (CSF)-1 inhibitors, anti-IL-1 inhibitors, anti-IL-1R3 inhibitors, anti-IL-8 inhibitors, anti-semaphorin 4D (Sema4D) inhibitors, angiopoietin (Ang)-2 inhibitors, CLEVER-1 inhibitors, Axl-targeted enapotamab vedotin (EnaV), or anti-phosphatidylserine inhibitors. In some embodiments, the immune checkpoint blockade comprises pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, or ipilimumab.

[0182] The disclosed VIP-R antagonists (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, etc.) are useful for the treatment of not only viral infections, but also other microbial infections, including but not limited to bacterial, fungal, and parasitic infections. Accordingly, also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing a microbial infection, wherein the microbial infection is a bacterial infection, and the bacterial infection is selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroide, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetellaavium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii and other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species.

[0183] In one aspect, disclosed herein is a method of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing a microbial infection, wherein the microbial infection is a fungal infection, and the fungal infection is an infection by a fungus selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata.

[0184] Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing a microbial infection, wherein the microbial infection is a parasitic infection, and the parasitic infection is selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species,The infection is caused by a parasite selected from the group consisting of Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica.Despite the ability of the disclosed VIP-R antagonists (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments, or analogs thereof, etc.) to inhibit virulence of and achieve clearance of microorganisms in tissues without the addition of antimicrobial agents, it is understood and contemplated herein that the addition of an antimicrobial agent (to the composition itself or as a separate administration) may be desirable in some cases. Thus, disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and / or preventing microbial infections, autoimmune diseases, autoinflammatory diseases, or cancer, further comprising administering an antimicrobial agent to a subject. Antimicrobial agents can include any antibiotic, antibody, small molecule, and functional nucleic acid (siRNA, RNAi, antisense oligonucleotides) that directly attacks an infectious microorganism or alters a host condition that renders the host system unfit for the microorganism. Such agents include abacavir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, baravir, beta-D-N4-hydroxycytidine (NHC, EIDD-1931), cidofovir, combivir, dolutegravir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, enteric acid, erythropoietin ... Cavir, Ecoliever, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Phosphonet, Ganciclovir, Hydroxy-chloroquine, Ibacitabine, Immunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide, Norvir,Oseltamivir, peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir, sovosbuvir, stavudine, telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, clofazimine; dapsone; caprevir; mycin;cycloserine;ethambutol (Bs);ethionamide;isoniazid;pyrazinamide;rifampicin;rifabutin;rifapentine;streptomycin;arsphenamine;chloramphenicol (Bs);fosfomycin;fusidic acid;metronidazole;mupirocin;platensimycin;quinupristin / dalfopristin;thiamphenicol;tigecycline (Bs);tinidazole;trimethoprim (Bs);aminoglycosides, e.g., amikacin, gentamicin, kanamycin, meropenem, neomycin, netilmi Cin, tobramycin, paromomycin, streptomycin, spectinomycin, nitazoxanide, melarsoprol, eflornithine, metronidazole, tinidazole, miltefosine, mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin, niclosamide, praziquantel, albendazole, praziquantel, rifampin, amphotericin B, fumagillin, amphotericin B, candicidin, filipino, hamycin, natamycin, nystatin, rimocidin, bifonazole, butoconazole , Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Efinaconazole, Epoxiconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Propiconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Anidulafungin, Caspofungin, Micafungin, Oron, Benzoic Acid, Ciclopirox,Flucytosine, Griseofulvin, Haloprogin, Tolnaftate, Undecylenic Acid, Crystal Violet, Balsam of Peru, Orotomid, Miltefosine, etc.; Ansamycins, such as Geldanamycin, Rifaximin, Herbimycin, etc.; Carbapenems, such as Ertapenem, Doripenem, Imipenem / Cilastatin, and Meropenem, etc.; Cephalosporins, such as Cefadroxil, Cefazolin, Cephradine, Cephapirin, Cephalothin, Cephalexin, Cefaclor, Cefoxitin, Cefotetan, Cefama such as cefotaxime, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, ceftaroline fosamil, and ceftobiprole; glycopeptides such as teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin; lincosamides (Bs) such as clindamycin and lincomycin; Lipopeptides, such as daptomycin; macrolides (Bs), such as azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, and spiramycin; monobactams, such as aztreonam; nitrofurans, such as furazolidone and nitrofurantoin (Bs); oxazolidinones (Bs), such as linezolid, pocizolid, radezolid, and torezolid; penicillins, such as amoxicillin, ampicillin, azlocillin, dicloxacillin, flucloxacillin, such as mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, penicillin G, temocillin, and ticarcillin; polypeptides such as bacitracin, colistin, and polymyxin B; quinolones / fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,Sparfloxacin and temafloxacin; Sulfonamides (Bs), such as mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilamide (archaic), sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (cotrimoxazole) (TMP-SMX), and sulfonamide chrysoidine (archaic); Tetracyclines (Bs), such as demeclocycline, doxycycline, methacycline, minocycline, oxytetracycline, and tetracycline; Monoclonal antibodies, such as actoxumab, atidoluxumab, and the like; and checkpoint inhibitors; pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, pidilizumab, AMP-224, AMP-514, PDR001, cemiplimab, and ipilimumab. In certain embodiments, the subject is administered a pharmaceutical composition comprising a VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) and a second antiviral agent.

[0185] In certain embodiments, the present disclosure relates to treating a subject having a post-infection viral infection by administering a VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof) and immunoglobulin.

[0186] In certain embodiments, the present disclosure relates to treating or preventing a viral infection by administering a VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments or analogs thereof, etc.) and a viral vaccine, or in the absence of a viral vaccine.

[0187] In certain embodiments, the present disclosure relates to enhancing an immune response to a vaccine, comprising administering a VIP-R antagonist disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) to a subject in need thereof. Vaccines are usually classified as follows: shingles vaccine, smallpox vaccine, polio vaccine, whooping cough vaccine, influenza vaccine, diphtheria vaccine, tetanus vaccine, meningococcal vaccine, influenza A vaccine including subtype H1N1 vaccine, influenza B vaccine, influenza C vaccine, rotavirus A vaccine, rotavirus B vaccine, rotavirus C vaccine, rotavirus D vaccine, rotavirus E vaccine, SARS coronavirus vaccine, human adenovirus types (HAdV-1 to 55) vaccine, human papillomavirus (HPV) vaccine, parvovirus B19 vaccine, molluscum contagiosum vaccine, JC vaccine, BK vaccine, Merkel cell polyomavirus vaccine, Coxsackie A vaccine, norovirus vaccine, rubella vaccine, lymphocytic choriomeningitis vaccine, yellow fever vaccine, measles vaccine, mumps vaccine, respiratory syncytial vaccine , rinderpest vaccine, California encephalitis vaccine, Hantavirus vaccine, rabies vaccine, Ebola vaccine, Marburg vaccine, herpes simplex virus-1 (HSV-1) vaccine, herpes simplex virus-2 (HSV-2) vaccine, varicella zoster vaccine, Epstein-Barr virus (EBV) vaccine, cytomegalovirus (CMV) vaccine, herpes lymphotropic vaccine, roseolovirus vaccine, Kaposi's sarcoma associated herpes virus vaccine, hepatitis A (HAV) vaccine, hepatitis B (HBV) vaccine, hepatitis C (HCV) vaccine, hepatitis D (HDV) vaccine, hepatitis E (HEV) vaccine, human immunodeficiency virus (HIV) vaccine, human T-lymphotropic virus type I (HTLV-1) vaccine, Friend splenic focus forming virus (SFFV) vaccine, and xenotropic MuLV-related virus (XMRV) vaccine.In certain embodiments, the subject has been diagnosed with a chronic viral infection.

[0188] In certain embodiments, the vaccine comprises a protein or peptide, a carbohydrate, a sugar, a polysaccharide, or a nucleic acid. Typically, the vaccine is an attenuated replication competent virus or an inactivated virus. In certain embodiments, the vaccine comprises live or killed or inactivated prokaryotic or eukaryotic cells.

[0189] In certain embodiments, human T cells are activated in vitro by co-incubation with viral antigen.In certain embodiments, viral antigen is presented on microvesicles.In certain embodiments, viral antigen is presented on dendritic cells.

[0190] Nucleic acid vaccines, typically DNA plasmids or RNA vaccines, are genetically engineered to encode and / or produce one or more antigens from a pathogen. The nucleic acid is transfected or infected into a host cell, where the cell's internal machinery expresses proteins. These proteins are recognized as foreign and therefore processed by the host cells, triggering an immune response when displayed on their surface. Cytotoxic T lymphocyte responses can also be enhanced by co-inoculation with costimulatory molecules such as GM-CSF, B7-1, or B7-2. In certain embodiments, the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) may be administered in combination with a nucleic acid vaccine or other costimulatory molecules.

[0191] In certain embodiments, the present disclosure relates to vaccine compositions comprising the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.), as well as methods of administering the VIP-R antagonists disclosed herein in combination with a vaccine. In certain embodiments, the vaccine contains an antigen from a pathogen, presented to the immune system from a weakened or killed form of the microorganism or its toxin. The antigen stimulates the immune system. The vaccine can be prophylactic (e.g., to prevent or reduce the effects of future infections by any pathogen) or therapeutic by being administered after diagnosis of an infection or disease.

[0192] Some vaccines contain killed, but previously virulent, microorganisms that have been destroyed with chemicals or heat. Influenza vaccines, cholera vaccines, bubonic plague vaccines, polio vaccines, Hepatitis A vaccines, and rabies vaccines are examples of killed vaccines contemplated by the present disclosure.

[0193] Some vaccines contain live attenuated microorganisms. Typically these are live viruses cultivated under conditions that neutralize certain virulence properties, or live viruses that use closely related but less dangerous organisms to generate a broad immune response. However, some are bacterial in nature.

[0194] In certain embodiments, the cells are protein subunits. Rather than introducing inactivated or attenuated microorganisms into the immune system, fragments thereof can be used to generate an immune response. Examples include subunit vaccines against Hepatitis B virus, which are composed only of the surface proteins of the virus, virus-like particle (VLP) vaccines against human papillomavirus (HPV), which are composed of the major capsid proteins of the virus, and hemagglutinin and neuraminidase subunits of influenza virus.

[0195] In certain embodiments, the vaccine comprises a polysaccharide. Certain bacteria have polysaccharide coats that are typically immunogenic. By linking these polysaccharides to proteins (e.g., toxins), the immune system can be directed to recognize the polysaccharide as if it were a protein antigen.

[0196] Toxin vaccines are made from inactivated toxic compounds. Examples of toxoid-based vaccines include diphtheria and tetanus toxoids. In certain embodiments, the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments, or analogs thereof, etc.) are administered in combination with DPT. DPT (also including DTP and DTwP) refers to a class of combination vaccines against three infectious diseases in humans: diphtheria, whooping cough, and tetanus. The vaccine components include diphtheria, tetanus toxoid, and killed whole cells of the organism that causes whooping cough (wP). DTaP (also known as Tdap, DTPa, and TdaP) refers to similar combination vaccines in which the whooping cough component is acellular. Also contemplated are DT or TD vaccines lacking the whooping cough component.

[0197] Other specific vaccines contemplated by the present disclosure include anthrax vaccines, e.g., the avirulent non-encapsulated strain known as V770-NP1-R, Bacille Calmette-Guerin (BCG), e.g., a live attenuated strain of bovine tuberculosis bacillus, haemophilus influenzae type B vaccines, e.g., a Hib polysaccharide-protein conjugate vaccine, hepatitis A vaccines, e.g., inactivated hepatitis A virus, hepatitis B vaccines, e.g., hepatitis B surface antigen, human papillomavirus (HPV) vaccines, e.g., non-infectious virus-like particles assembled from the L1 proteins of HPV types 6, 11, 16 and 18, meningococcal vaccines, e.g., the capsular polysaccharide antigens of Neisseria meningitides serogroups A, C, Y and W-135 strains individually conjugated to diphtheria toxoid protein.

[0198] In certain embodiments, the present disclosure relates to a method of treating an active cytomegalovirus infection, comprising administering to a subject diagnosed with and exhibiting signs or symptoms of an active cytomegalovirus infection an effective amount of a vasoactive intestinal peptide antagonist disclosed herein, wherein the vasoactive intestinal peptide antagonist comprises a peptide having a C-terminal amide, and is optionally modified with a carbohydrate or polyethylene glycol group. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor).

[0199] In certain embodiments, the present disclosure relates to a method of reducing an active cytomegalovirus infection, comprising administering to a subject suffering from an active cytomegalovirus infection an effective amount of a vasoactive intestinal peptide antagonist disclosed herein, wherein the vasoactive intestinal peptide antagonist comprises a peptide having a C-terminal amide, and is optionally modified with a carbohydrate or polyethylene glycol group. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor). In certain embodiments, the titer of cytomegalovirus in the subject is reduced after administration of the vasoactive intestinal peptide antagonist compared to pretreatment.

[0200] In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an anti-TIM3 inhibitor, an anti-LAG3 inhibitor, an anti-CD47 inhibitor, imiquimod, polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), pexidartinib, an anti-TIGIT inhibitor, an anti-B7-H3 inhibitor, an anti-B7-H4 inhibitor, an anti-A2aR inhibitor, an anti-CD73 inhibitor, an anti-NKG2A inhibitor, an anti-PVRIG / PVRL2 inhibitor, an anti-CEACAM1 inhibitor. , anti-CEACAM5 inhibitors, anti-CEACAM6 inhibitors, focal adhesion kinase (FAK) inhibitors, CCL2 / CCR2 inhibitors, anti-leukemia inhibitory factor (LIF) inhibitors, anti-CD47 / SIRPα inhibitors, anti-colony stimulating factor (CSF)-1 inhibitors, anti-IL-1 inhibitors, anti-IL-1R3 inhibitors, anti-IL-8 inhibitors, anti-semaphorin 4D (Sema4D) inhibitors, angiopoietin (Ang)-2 inhibitors, CLEVER-1 inhibitors, Axl-targeted enapotamab vedotin (EnaV), or anti-phosphatidylserine inhibitors. In some embodiments, the immune checkpoint blockade comprises pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, or ipilimumab.

[0201] 2. How to treat cancer In certain embodiments, any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) are contemplated to be used in certain cellular immunotherapies effective in the treatment of cancer, such as lymphocyte infusion or allogeneic bone marrow transplantation. Donor immune cells, particularly NK cells and T cells, have anti-cancer cytotoxic activity. VIP antagonism of peptides enhances cellular immune responses in vivo. VIP antagonism increases the cytotoxic activity of antigen-specific T cells and NK cells. VIP antagonism is predicted to increase the anti-cancer activity of NK cells or antigen-specific T cells. VIP antagonism in combination with cellular immunotherapy is predicted to increase the efficacy of the therapy. It is believed that the absence of VIP does not increase the "off-target" graft-versus-host disease activity of donor lymphocytes in the recipient of an allogeneic bone marrow transplant. Thus, administering a VIP-R antagonist to a subject with cancer undergoing cell therapy, such as donor lymphocyte infusion or allogeneic bone marrow transplant, will increase the anti-cancer activity of the therapy. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3Kα inhibitor, a PI3Kβ inhibitor, a PI3Kδ inhibitor, or a PI3Kγ inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor).

[0202] In some embodiments, the immune checkpoint blockade agent is a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an anti-TIM3 inhibitor, an anti-LAG3 inhibitor, an anti-CD47 inhibitor, imiquimod, polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), pexidartinib, an anti-TIGIT inhibitor, an anti-B7-H3 inhibitor, an anti-B7-H4 inhibitor, an anti-A2aR inhibitor, an anti-CD73 inhibitor, an anti-NKG2A inhibitor, an anti-PVRIG / PVRL2 inhibitor, an anti-CEACAM1 inhibitor. , anti-CEACAM5 inhibitors, anti-CEACAM6 inhibitors, focal adhesion kinase (FAK) inhibitors, CCL2 / CCR2 inhibitors, anti-leukemia inhibitory factor (LIF) inhibitors, anti-CD47 / SIRPα inhibitors, anti-colony stimulating factor (CSF)-1 inhibitors, anti-IL-1 inhibitors, anti-IL-1R3 inhibitors, anti-IL-8 inhibitors, anti-semaphorin 4D (Sema4D) inhibitors, angiopoietin (Ang)-2 inhibitors, CLEVER-1 inhibitors, Axl-targeted enapotamab vedotin (EnaV), or anti-phosphatidylserine inhibitors. In some embodiments, the immune checkpoint blockade comprises pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, or ipilimumab.

[0203] "Cancer" refers to any of a variety of cellular diseases with malignant tumors characterized by cell proliferation. It is not intended that diseased cells must actually invade surrounding tissues and metastasize to new body sites. Cancer can involve any tissue of the body and has many different forms in each body area. In the context of certain embodiments, "whether cancer is reduced" can be identified by various diagnostic methods known to those skilled in the art. This includes, but is not limited to, observing a reduction in the size or number of tumor masses, or observing an increase in apoptosis of cancer cells, for example, observing a greater than 5% increase in apoptosis of cancer cells of the sample compound compared to a control without the compound. It can also be identified by changes in associated biomarkers or gene expression profiles, such as PSA for prostate cancer, HER2 for breast cancer, serum levels of VIP in patients with pancreatic cancer.

[0204] The disclosed compositions can be used to treat, inhibit, reduce, reduce, alleviate, and / or prevent any disease in which uncontrolled cell proliferation occurs, such as cancer. A representative, but non-limiting list of cancers that can be treated using the disclosed compositions is as follows: malignant tumors located in the colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid gland, pituitary gland, testes, ovaries, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, breast, and urinary system, more specifically, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, adrenal cortical carcinoma, adult ( Primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphocytic leukemia, adult acute myeloid leukemia, adult Hodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia, adult non-Hodgkin's lymphoma, adult primary liver cancer, adult soft tissue sarcoma, AIDS-related lymphoma, AIDS-related malignant tumor, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, renal pelvis and ureter cancer, primary central nervous system lymphoma, central nervous system lymphoma, cerebellar astrocytoma, brain astrocytoma, cervical cancer, pediatric (primary) Hepatocellular carcinoma, childhood (primary) liver cancer, childhood acute lymphoblastic leukemia, childhood acute myeloid leukemia, childhood brain stem glioma, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, childhood extracranial germ cell tumor, childhood Hodgkin's disease, childhood Hodgkin's lymphoma, childhood optic nerve hypothalamic glioma, childhood lymphoblastic leukemia, childhood medulloblastoma, childhood non-Hodgkin's lymphoma, childhood supratentorial primitive neuroectodermal tumor and pineal tumor, childhood primary liver cancer, childhood rhabdomyosarcoma, childhood soft tissue sarcoma, childhood optic nerve hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia disease, colon cancer, cutaneous T-cell lymphoma, endocrine islet cell carcinoma, endometrial cancer, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal tumors, germ cell tumors, gestational trophoblastic neoplasms, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinal cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic cancercancer), Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, Merkel cell carcinoma, occult primary metastatic squamous neck cancer, primary metastatic squamous neck cancer cancer), metastatic squamous cell neck cancer, multiple myeloma, multiple myeloma / plasma cell neoplasms, myelodysplastic syndromes, myeloid leukemia, myeloproliferative disorders, paranasal sinus and nasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma during pregnancy, non-melanoma skin cancer, non-small cell lung cancer, metastatic squamous cell neck cancer of unknown primary, buccopharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / histiocytoma of bone, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemia, purpura, parathyroid Cancer, penile cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm / multiple myeloma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoidosis, sarcoma, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell neck cancer, gastric cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional carcinoma of the renal pelvis and ureter renal pelvis and ureter cancer, trophoblastic tumor, renal pelvis and ureter cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic nerve hypothalamic glioma, vulvar cancer, Waldenstrom's hypergammaglobulinemia, Wilms' tumor and any other hyperproliferative disease, as well as neoplasms located in the aforementioned organ systems.

[0205] In one aspect, it is understood that the treatment of cancer need not be limited to the administration of a VIP-R antagonist, but can include the further administration of an anti-cancer agent to treat, inhibit, reduce, decrease, ameliorate, and / or prevent cancer or metastasis. Anti-cancer therapeutics (such as checkpoint inhibitors, chemotherapeutic agents, immunotoxins, peptides, and antibodies) that can be used in combination with any of the disclosed VIP-R antagonists in methods of treating, inhibiting, reducing, diminishing, ameliorating, and / or preventing cancer and / or metastasis can include any anti-cancer therapeutic known in the art, including, but not limited to, abemaciclib, abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib dimaleate, Afinitor (everolimus), Akynzeo (Netupitant and Palonosetron hydrochloride), Hydrochloride), Aldara (Imiquimod), Aldesleukin, Alecensa (Alectinib), Alectinib, Alemtuzumab, Alimta (Pemetrexed Disodium), Aliqopa (Copanlisib Hydrochloride), Alkeran for Injection (Melphalan Hydrochloride), Alkeran Tablets (Melphalan), Aloxi (Palonosetron Hydrochloride), Alunbrig (Brigatinib), Ambochlorin (Chlorambucil), Amifostine, Aminolevulinic Acid, Anastrozole, Aprepitant, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arsenic Trioxide, Arzerra (Ofatumumab), Asparaginase Erwinia chrysanthemi, atezolizumab, Avastin (bevacizumab), avelumab, axitinib, azacitidine, Bavencio (avelumab), BEACOPP, Becenum (carmustine),Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, Besponsa (inotuzumab ozogamicin), bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigatinib, bumel, busulfan, Busulfex (busulfan), cabazitaxel, Cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF, Campath (alemtuzumab), Camptosar, (I Rinotecan hydrochloride), Capecitabine, CAPOX, Carac (fluorouracil-topical), Carboplatin, Carboplatin-Taxol, Carfilzomib, Carmubris (carmustine), Carmustine, Carmustine implant, Casodex (bicalutamide), CEM, Ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), Cetuximab, CEV, Chlorambucil, Chlorambucil-prednisone, CHOP, Cisplatin, Cladribine, Clafen (cyclophosphamide), Clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cobimetinib, Cometriq (cabozantinib-S-malate), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), Crizotinib, CVP, Cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab) lumab), cytarabine, cytarabine liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, Darzalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposomal, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin diftitox,Denosumab, DepoCyt (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, duvelisib, Efudex (fluorouracil-topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), Empliciti (elotuzumab), enasidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib salt, Erwinaze (asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (Doxorubicin hydrochloride liposome), Everolimus, Evista, (Raloxifene hydrochloride), Evomela (Melphalan hydrochloride), Exemestane, 5-FU (Fluorouracil injection), 5-FU (Fluorouracil - topical), Fareston (Toremifene), Farydak (Panobinostat), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine phosphate), Fludarabine phosphate, Fluoroplex (Fluorouracil - topical), Fluorouracil injection, Fluorouracil - topical, Flutamide, Folex (Methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin,Gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV monovalent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper CVAD, Ibrance (palbociclib), Ibritumomab tiusetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), Idarubicin hydrochloride, Idelalisib, Idhifa (Enasidenib Mesylate), Ifex (Ifosfamide), Ifosfamide, Ifosfamidum (Ifosfamide), IL-2 (Aldesleukin), Imatinib Mesylate, Imbruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Talimogene-Laherparepvec), Inlyta (Axitinib), Inotuzumab-Ozogamicin, Interferon Alpha-2b, Recombinant, Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha-2b), Iodine I 131 Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposome, Istodax (romidepsin), ixabepilone, ixazomib citrate, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trasulfamethasone), tuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, Lartruvo (olaratumab), lenalidomide,Lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, Leustatin (cladribine), Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposomal), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposomal), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megestrol Acetate, Mekinist (Trametinib), Melphalan, Melphalan Hydrochloride, Mercaptopurine, Mesna, Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate LPF (methotrexate), Methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), Midostaurin, Mitomycin C, Mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plelixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozogamicin), Nanoparticle Paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), necitumumab, nelarabine, Neosar (cyclophosphamide), neratinib maleate, Nerlynx (neratinib maleate), netupitant-palonosetron hydrochloride, Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (nilutamide), nilotinib, nilutamide, Ninlaro (ixazomib citrate), niraparibut tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab,Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olaratumab, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposomal), Ontak (denileukin diftitox), Opdivo (nivolumab, oppa, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride-netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargase, pegfilgra Stim, peginterferon alfa-2b, PEG-Intron (peginterferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), plerixafor, pomalidomide, Pomalyst (pomalidomide), ponatinib hydrochloride, Portrazza (necitumumab), pralatrexate , prednisone, procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag olamine), propranolol hydrochloride, Provenge (sipuleucel-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), radium-223 dichloride, raloxifene hydrochloride, ramucirumab, rasburicase, R-CHOP, R-CVP, recombinant human papillomavirus (HIV) Human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alpha-2b, regorafenib, Relistor (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), RituxanHycela (rituximab and hyaluronidase human), rituximab, rituximab and hyaluronidase human, rolapitant hydrochloride, romidepsin, romiplostim, Rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparib camsylate), rucaparib camsylate, ruxolitinib phosphate, Rydapt (midostaurin), Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steritalc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon alfa-2b), Sylvant (Siltuximab), Synribo (Omacetaxine Mepesuccinate), Tabloid (Thioguanine), TAC, Tafinlar (Dabrafenib), Tagrisso (Osimertinib), Talc, Talimogene Laherparepvec, Tamoxifen Citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq, (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, Thalomid, thioguanine, thiotepa, tisagenlecleucel, Tolak (fluorouracil-topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I131 Tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Varubi (rorapitamide) Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Venclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, VincasarPFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposomal, vinorelbine tartrate, VIP, vismodegib, Vistogard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Vyxeos (daunorubicin hydrochloride and cytarabine liposomal), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondel Anticancer and immunomodulatory agents include, but are not limited to, cyclosporine (trabectedin), Zaltrap (Ziv-aflibercept), Zarxio (filgrastim), Zejula (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelarib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate). Anticancer and immunomodulatory agents can also include checkpoint inhibitors. Checkpoint inhibitors include PD-1 (JTX-4014, cemiplimab, camrelizumab, dostallimab, toripalimab, tislelizumab, spartalizumab, sintilimab, nivolumab (BMS-936558 or MDX1106), CT-011, pembrolizumab (MK-3475)), PD-L1 (atezolizumab, avelumab, durvalumab, CK-301, MDX-11 05 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHIgM12B7), CTLA-4 (ipilimumab (MDX-010) and tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (relatimab, BMS-986016).

[0206] In certain embodiments, the method of administration is in a subject having a lymphodepleted environment, hi certain embodiments, the lymphodepleting agents are cyclophosphamide and fludarabine.

[0207] As used herein, the term "idelalisib" refers to the compound (S)-2-(1-(9H-purin-6-ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one or an alternative salt thereof.

[0208] The VIP-R antagonists of the present disclosure can be further combined with radiation therapy and / or cellular transplantation therapy, including but not limited to administration of expanded, modified, or cultured tumor infiltrating lymphocytes (TILs), administration of expanded, modified, or cultured bone marrow infiltrating lymphocytes (MILs), administration of expanded, modified, or cultured tumor infiltrating natural killer cells (TINKs), administration of chimeric antigen receptor (CAR) T cells, administration of TCR-modified T cells, and / or administration of CAR NK cells. In certain embodiments, the present disclosure relates to a method of treating a subject diagnosed with cancer, comprising administering to a subject in need thereof a cell in combination with any of the VIP-R antagonists disclosed herein. In certain embodiments, the subject has been diagnosed with leukemia. In certain embodiments, the subject has been diagnosed with lymphoma. In certain embodiments, the cell is a blood mononuclear cell. In certain embodiments, the cell is a bone marrow cell. In certain embodiments, the cell is a white blood cell. In certain embodiments, the cell is a T cell. In certain embodiments, the cell is a natural killer cell. In certain embodiments, the cell is a hematopoietic stem cell. In certain embodiments, the cell is a G-CSF mobilized blood mononuclear cell. In certain embodiments, the cell is an HLA matched or mismatched allogeneic cell. In certain embodiments, the cell is a syngeneic cell. In certain embodiments, the cell is an autologous cell. In certain embodiments, the peptide has a C-terminal amide and / or is optionally modified with a carbohydrate or polyethylene glycol group. In one aspect, disclosed herein relates to a method of treating, inhibiting, reducing, decreasing, ameliorating, and / or preventing leukemia comprising administering to a subject any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments or analogs thereof) in combination with hematopoietic stem cell transplantation.In certain embodiments, the present disclosure relates to a method comprising expanding lymphocytes in vitro and providing expanded cells, and exposing the expanded cells with any of the VIP-R antagonists disclosed herein.

[0209] In certain embodiments, the present disclosure relates to a method of treating cancer by performing stem cell transplantation, comprising administering to a subject any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments, or analogs thereof) in combination with a transplant of pluripotent hematopoietic stem cells from the subject (autologous) or donor. Stem cells may be harvested from peripheral blood, such as umbilical cord blood or placenta-derived stem cells, or from bone marrow. To limit the risk of transplanted stem cell rejection or severe graft-versus-host disease, the donor will typically have substantially the same human leukocyte antigens (HLA) as the recipient. However, the donor may have a mismatch for certain antigens. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor).

[0210] In certain embodiments, the present disclosure relates to methods of providing lymphocyte infusion following hematopoietic progenitor cell transplantation to treat hematologic malignancies (e.g., blood or bone marrow cancers such as leukemia or lymphoma). Transplant recipients are typically infused with lymphocytes obtained in a leukocyte replacement procedure from the original allogeneic stem cell (hematopoietic progenitor cell) donor.

[0211] In certain embodiments, the present disclosure relates to extraction of lymphocytes from blood and in vitro proliferation against tumor antigens, and optionally exposing the cells to appropriate stimulatory cytokines and / or any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.).

[0212] In certain embodiments, the present disclosure relates to a method of enhancing local immunotherapy, comprising administering any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof, etc.) in combination with the provision of an immune enhancing cream, such as imiquimod, that includes an interferon-producing drug that causes T cell activation. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor).

[0213] In certain embodiments, it is contemplated that the peptides disclosed herein may be used in conjunction with adoptive cell therapy. For example, T cells with naturally occurring reactivity against cancer may be found infiltrating the tumor of a subject. The tumor may be harvested, and these tumor-infiltrating lymphocytes (TILs) may be expanded or made more effective in vitro using interleukin-2 (IL-2), anti-CD3, and alloreactive feeders. These T cells may then be returned to the subject along with administration of a VIP-R antagonist. Prior to reinfusion, lymphodepletion of the recipient is typically performed to remove regulatory T cells as well as normal endogenous lymphocytes that compete with the transplanted cells. It is also contemplated that adoptive cell transfer of lymphocytes may be transduced with a vector encoding a T cell receptor (TCR) that recognizes a cancer antigen. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor (e.g., a PI3K alpha inhibitor, a PI3K beta inhibitor, a PI3K delta inhibitor, or a PI3K gamma inhibitor). In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint blockade agent (e.g., a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor).

[0214] In certain embodiments, the present disclosure relates to a method for enhancing T cell activation and ex vivo proliferation by co-incubation of human T cells with nanoparticles containing small molecule antagonists of VIP signaling. In certain embodiments, human T cells are activated with plate-bound anti-CD3 antibodies. In certain embodiments, human T cells are activated in a mixed lymphocyte reaction. In certain embodiments, human T cells are activated in vitro by co-incubation with tumor-associated antigens. In certain embodiments, tumor-associated antigens are presented on tumor microvesicles. In certain embodiments, activated human T cells are infused into human patients with cancer.

[0215] In certain embodiments, the activated human T cells are infused into a human patient with cancer. In certain embodiments, the human patient with cancer has leukemia. In certain embodiments, the human patient with cancer has lymphoma. In certain embodiments, the human patient with cancer has multiple myeloma. In certain embodiments, the human patient with cancer has epithelial cancer. In certain embodiments, the human patient has lung cancer. In certain embodiments, the human patient has breast cancer. In certain embodiments, the human patient has colon cancer. In certain embodiments, the human patient has prostate cancer. In certain embodiments, the human patient has malignant melanoma. In certain embodiments, the human patient has brain cancer.

[0216] In certain embodiments, the present disclosure relates to a method of enhancing anti-cancer immune response by injecting any of the VIP-R antagonists disclosed herein (e.g., SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and / or SEQ ID NO:16, fragments thereof, or analogs thereof) or nanoparticles expressing the VIP-R antagonists disclosed herein. In certain embodiments, the activated T cells are infused into a patient with a chronic CMV infection. In certain embodiments, the activated T cells are infused into a patient with a chronic EBV infection. In certain embodiments, the activated T cells are infused into a patient with a chronic BK virus infection. In certain embodiments, the activated T cells are infused into a patient with a chronic adenovirus infection. EXAMPLES

[0217] VI. Working Examples The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices, and / or methods claimed herein, and are intended to be purely illustrative and not limiting of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise specified, parts are parts by weight, temperature is ° C. or is ambient temperature, and pressure is at or near atmospheric pressure.

[0218] 1. Example 1 We evaluated whether tumor-specific expression of vasoactive intestinal polypeptide represents a mechanism of tumor-mediated immune escape. There is a spectrum of VIP expression across tumors, with the highest expression seen in pancreatic exocrine cancer and the lowest expression seen in melanoma. In general, the level of VIP expression by tumors is inversely proportional to the expression of other co-inhibitory pathway molecules, such as PDL1. Some tumors that express and secrete VIP may have mutations in the VIP coding sequence, resulting in peptide molecules secreted by the cancer having improved pharmacokinetics or pharmacodynamics in the tumor microenvironment. The pharmacokinetic advantage to cancer cell-secreted VIP may be that the mutations make the mutant VIP less susceptible to proteases and improve its half-life. The pharmacodynamic advantage to cancer in more potently inhibiting anti-cancer immunity may be the result of mutations in VIP that enhance binding affinity to receptors and therefore enhance inhibitory signaling on T cells expressing VAPC1 and / or VPAC2.

[0219] Experiments were performed to determine whether mutated VIP produced by tumors results in more sustained suppression of anticancer T cells in the tumor microenvironment. Analysis of mutations in specific genes curated from deposited tumor sequences revealed the presence of multiple cancers with mutations in the coding sequence of VIP. Notably, 140 missense mutations and 17 truncation mutations were present within the VIP gene cluster listed in the Cancer Genome Atlas. Mutations in the VIP coding sequence present in breast cancer, prostate adenocarcinoma, esophageal adenocarcinoma, cutaneous melanoma, small cell lung carcinoma, gastric adenocarcinoma, endometrial carcinoma, cutaneous melanoma, esophageal adenocarcinoma, colorectal adenocarcinoma, uterine carcinosarcoma, hepatocellular adenoma, lung adenocarcinoma, and gastric adenocarcinoma were identified within the coding sequence of the 28 amino acid VIP peptide. Eight specific mutations were present in the C-terminal amino acids that comprise the alpha helix of VIP that binds to the receptor.

[0220] VIP-related peptides tested in silico for predicted binding affinity to human VPAC1 and VPAC2 (Creative Biolabs). Docking scores indicate the predicted free energy change associated with peptide binding. Note that larger negative scores are predicted to be associated with higher binding affinity. Based on an initial screening of 300 peptides for binding affinity to VPAC1 and a subset of 100 peptides for binding affinity to VPAC2, a selected group of peptides was synthesized and tested for their ability to stimulate proliferation of luciferase+ mouse T cells in vitro. The lowest concentration of peptide that resulted in maximum luminescence is shown. Proliferation data represents the relative level of luminescence from quadruplicate wells containing 1×10E5 T cells / well in a 96-well tissue culture plate stimulated with plate-bound anti-CD3 antibody for 96 hours. Confirmatory in vivo testing of selected peptides was then performed based on their ability to induce an autologous anti-leukemia response in C57Bl / 6 mice previously injected with 1×10E6 C1498 acute myeloid leukemia cells. Leukemia cells were injected on day 0 and 10ug of peptide was injected sc daily on days 6-12. Predicted binding affinity to VPAC1 and / or VPAC2 was associated with enhanced activity in stimulating mouse T cell proliferation and anti-leukemia activity in mice. Sequence differences between the VIP peptide and the test peptides are shown in red font (Table 3).

[0221] To test the ability of the disclosed VIP-R antagonists to confer survival benefits on acute myeloid leukemia patients, we used B6 (CD45.2, H-2K b ) Mice were inoculated with 1×10 6 VIP novel peptide was administered via tail vein to 10 mice / mouse. VIP novel peptide was subcutaneously injected at 10 micrograms per mouse daily for a total of 7 times starting from day 6 after leukemia challenge. Mice were monitored daily for survival. As shown in Figures 1 and 2, in each case, survival was increased compared to the negative control and VIP (SEQ ID NO: 1).

[0222] We next correlated the increase in survival with the affinity and potency of competitive binding of VPAC by each VIP-R antagonist. As shown in Figure 3, there is a direct correlation between the affinity and potency of competitive binding to both human VPAC1 (Panel A) and VPAC2 (Panel B) and the combined binding affinity to VPAC1 and VPAC2 (Panel C).

[0223] Figure 4 shows the C1498 at 1×10 6 Treatment with a novel peptide derived from VIP reduced the levels of leukemia cells in the blood of leukemic mice for up to 20 days after administration via the tail vein in mice. VIP novel peptide was injected subcutaneously at 10 micrograms per mouse daily for a total of seven injections, beginning on day 6 after leukemia challenge.

[0224] In a leukemia rechallenge model, mice previously inoculated with C1498 leukemia cells achieved complete clearance of leukemia and prolonged survival without detectable leukemia after treatment with a novel peptide derived from VIP. 6 The mice were rechallenged with 100-fold increased leukemia cells (100 / mouse) per day. Sixteen days after leukemia rechallenge, the mice were imaged weekly for luminescence (Figure 5). Furthermore, as shown in Figure 6, mice previously treated with novel peptides derived from VIP had extended survival when rechallenged with acute myeloid leukemia, indicating that previous treatment with novel peptides derived from VIP had developed an immune memory that enabled the mice to reject newly injected leukemia cells.

[0225] Furthermore, we tested the ANT308 peptide in two models of pancreatic cancer, as a single agent and in combination with anti-PD1 MoAb, and found that ANT308 had single-agent activity and synergistic effects in controlling tumor growth when combined with anti-PD1 therapy. As shown in Figure 7, ANT308 treatment reduced tumor volume in C57BL / 6 mice bearing subcutaneously implanted KPC tumors. This reduction in tumor volume was more pronounced in combination with anti-PD1 treatment. We also examined the effect of ANT308 in combination with Iso IgG or anti-PD1 antibody on tumor volume, measured as a percentage change in volume (Figure 8A) and measured volume (Figure 8B). Interestingly, treatment with a combination of VIP-R antagonist and anti-PD1 had a synergistic effect on reducing measured tumor volume and controlling the growth of subcutaneous pancreatic tumors in mice (Figure 8C). To investigate this effect more fully, it was observed that combined treatment with both anti-PD1 and VIP-R antagonists was able to slow tumor growth compared to control mice treated with scrambled peptide and isotype-matched IgG, showing a statistically significant improvement in tumor growth rate with the combination (Figure 8D). Further examination of the survival and regression of each of the various treatment groups showed that 20% of mice treated with ANT308 and anti-PD1 were tumor-free, and an additional 50% of the tumors showed regression. In comparison, only 10% of anti-PD1-treated mice were tumor-free, 20% showed tumor regression, the remaining 30% had advanced tumors, and 40% of the mice were euthanized due to the growth of large tumors. Tumor growth in mice treated with ANT308 alone was not statistically different from tumor growth in control mice that received scrambled peptide and isotype-matched IgG (Figure 9).

[0226] 2. Example 2: Treatment of human T cells with VIP-R antagonists (Ant08, Ant308, Ant195) This study employed an ex vivo system using human T cells isolated from healthy donors to screen for novel VIP-R antagonist peptides. Human T cells were cultured for 6 or 24 hours under activation in the presence or absence of peptides and assessed for percent CD69 surface expression by flow cytometry to measure the state of T cell activation. Peptide activity was determined by the percent increase in CD69 expression compared to the non-peptide control. VIP-scrambled 1 (VS1) was used as a peptide control. The activity of two new peptides, Ant308 and Ant195, was tested in comparison to Ant08, which showed high activity in previous screening. Thus far, all screenings have been performed using mouse T cells, but this study reports an alternative screening method using human T cells, which allows for robust results within 24 hours, informing the activity of peptides against different human T cell subtypes. Using this method, this study shows that treatment of human T cells activated in vitro in the presence of VIP-R antagonists, Ant08, Ant308, and Ant195, demonstrates increased activation as early as 6 hours after treatment, with an approximately 10-15% increase in mean CD69 expression compared to T cells in non-peptide control cultures (---) (Figure 10A). All donors, represented by dots of the same color, show increased CD69 expression at 6 hours in the antagonist-treated group, despite donor-to-donor variability. Of the three antagonists, Ant195 shows the highest activity across multiple donors. The activity of the antagonists to stimulate T cells is further shown to be maintained at 24 hours, with this effect being more pronounced in CD4+ T cells compared to CD8+ T cells (Figure 10B). However, the results also show that the VS1 peptide stimulates T cells by approximately 3% compared to the non-peptide control, but less than the three antagonists in question. It was also observed that among donors, most were classified as average responders, with measurements of CD69 expression falling within the error bars and two other measurements falling outside the error bars, one sample reflecting a super-responder (purple dot) and one sample reflecting a slow responder (green dot) (Figures 10A and 10B).Representative flow plots from one mean responder (blue dots) showing a higher percentage of CD4+CD69+ in the presence of VIP-R antagonists compared to controls at 24 hours, as indicated by increased intensity of the red signal in the gated population (Figure 10C). Overall, this study shows that all VIP-R antagonists are able to enhance human T cell activation, and the data confirm that Ant195 had the highest activity compared to Ant08 and Ant308.

[0227] The same T cells were used to evaluate the effect of treatment on T cell activation, as indicated by TIM3 expression (Figure 11). Similar to CD69, the percentage of CD4+TIM3+ and CD8+TIM3+ cells increased at 24 hours in the treated group. In contrast, CXCR4 expression decreased at 24 hours in treated mice (Figure 12).

[0228] This study also investigated the expression of VIP receptors in pancreatic cancer models using various pancreatic cancer cells (KPC, Panc02, PANC01, Capan02, BxPC3, and MT5). As measured by Western blot, VPAC1, VPAC2, and PAC1 were all expressed in cancer cells (Figures 13A-13D). In addition, cell viability and growth were not affected by treatment with VIP-R antagonists.

[0229] The next experiment evaluated the ability of ANT308 and anti-PD-1 treatment to effect adoptively transferred T cell homing and infiltration into established pancreatic tumors. Mice were injected with 5×10 5KPC cells were injected subcutaneously and treated 15 days after inoculation with immunologically naive GFP+ T cells along with ANT308 and anti-PD-1 antibodies. Mock-treated control mice had minimal infiltration of GFP+ T cells, whereas mice treated with ANT308 and anti-PD-1 showed significant infiltration of GFP+ T cells (Figures 14A-14C). Finally, this study measured the effect of the combination of ANT308 and anti-PD-1 on survival. Mock-treated animals inoculated with all pancreatic cancer cells died by day 30, whereas the treatment group had 90% survival until at least day 35 after inoculation (Figure 15). Unlike many solid malignancies, pancreatic ductal adenocarcinoma (PDAC) generally does not respond to immune checkpoint blockade (ICB) therapy, which targets molecules such as programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte antigen-4 (CTLA-4). The therapeutic resistance of PDAC to ICB may be partially attributed to low tumor mutational burden, except for a small proportion of PDCA patients with tumors with high microsatellite instability. In addition, the tumor microenvironment (TME) in PDAC is characterized by cancer-associated fibroblasts (CAFs) that secrete immunosuppressive proteins and metabolites, abundant regulatory T cells (Tregs), immunosuppressive tumor-associated macrophages (TAMs) and dendritic cells, and limited numbers of functional T cells. Although several clinical strategies have targeted immunosuppressive cells in some cancers, there have been limited improvements in the clinical management of PDAC over the past few years. Recently, preclinical and translational clinical studies have shown that the combination of treatment with an agnostic CD40 antibody and gemcitabine / nab-paclitaxel can induce potent immune-mediated control of PDAC, but these promising preclinical results were not replicated in early-phase clinical trials. This study identified overexpression of VIP, an immunosuppressive neuropeptide, as a novel target for immune checkpoint therapy in PDAC.

[0230] VIP is a 28 amino acid long neuropeptide present in the brain, pancreas, colon and lung. Overexpression of VIP and its receptor has been previously reported in breast, prostate and lung cancers and has been noted to promote tumor growth and metastasis. Immune cells, including T cells, have VIP receptors that are upregulated upon T cell activation and respond to VIP receptor signaling by inhibiting activation and proliferation, as well as promoting the generation of Treg and Th2 cells. Inhibition of VIP-R signaling by treating mice with a VIP-R antagonist improved T cell-dependent antitumor responses and enhanced adaptive antiviral immunity in a preclinical model of acute leukemia. This study explores VIP-R antagonists in a solid tumor cancer model.

[0231] This study shows that VIP is robustly expressed in murine and human PDAC compared to most other solid malignancies. Paracrine production of VIP by tumor cells within the PDAC TME constitutes an immune checkpoint pathway that limits the antitumor activity of VIP receptor-expressing T cells. Inhibition of VIP receptor signaling can improve T cell-dependent responses to checkpoint therapy and improve survival in preclinical models of PDAC. This study investigated these concepts by treating tumor-bearing mice with a more potent VIP-R antagonist designed to have a higher binding affinity to the VIP receptor compared to VIPhyb. Shown herein is that the combination of a VIP-R antagonist with anti-PD-1 significantly enhances activation, reduces attrition, and recruits tumor-infiltrating T cells in murine PDAC (TIL). Furthermore, the combination drug therapy reduces tumor growth rates and results in tumor regression.

[0232] VIP is overexpressed in human and mouse pancreatic cancer. Expression levels of VIP mRNA across different human tumors were compared using the Cancer Genome Atlas (TCGA). Pancreatic and gastrointestinal cancers had the highest VIP mRNA expression compared to other solid malignancies (Figure 16a). Immunofluorescence (IF) staining of human PDAC tumors showed increased expression of VIP in pancreatic ductal carcinoma cells with co-expression of cytokeratin-19 (CK19) compared to adjacent normal tissues (Figures 16b and 23a). Furthermore, analysis of culture supernatants obtained from human and mouse PDAC cell lines showed that most PDAC cell lines secrete VIP (Figure 16c). On the other hand, supernatants from mouse melanoma cell lines B16F10 and D4M had low to undetectable VIP (Figure 16c). The possibility that tumor-secreted VIP has systemic effects on the immune system is supported by the observation that immune-competent C57BL / 6 mice implanted with murine PDAC tumors had significantly elevated levels of plasma VIP compared to mice bearing B16F10 melanomas of comparable tumor volume (Figure 16d). These findings are consistent with human VIP mRNA expression data, where melanomas had low expression of VIP and PDACs had high levels of VIP mRNA. Similarly, human PDAC patients with pancreatic cancer had significantly higher plasma VIP levels than healthy volunteers (Figure 16e), indicating that plasma VIP is a biomarker for PDAC. In support of this notion, plasma VIP increased linearly with increasing volume of KPC-Luc tumors in mice (Figure 16f).

[0233] Orthotopic implantation of KPC-Luc cells resulted in higher plasma VIP levels than both KPC-Luc culture supernatants and plasma from mice bearing subcutaneous KPC-Luc tumors, suggesting that the desmoplastic TME created in the orthotopic model contributes to higher circulating VIP levels (Fig. 16d). In support of this, primary CAFs from PDAC patients and supernatants from the human pancreatic CAF cell line, h-iPSC-PDAC-1, secreted high levels of VIP (Fig. 16g). These data confirm the expression of high levels of VIP by tumor and stromal cells within the TME of human and mouse PDAC.

[0234] Inhibiting VIP-R signaling promoted T cell activation while reducing attrition in vitro. Expression of VIP by PDAC and TME indicated that VIP may function as a paracrine and / or autocrine factor for cancer cell survival. Human PDAC tissue expresses both VPAC1 and VPAC2, the two VIP receptors that appear to be most relevant to VIP-R signaling on immune cells. Furthermore, expression of VPAC1 and VPAC2 is confirmed in both mouse and human PDAC cell lines by Western blot (Figures 23b-23d). Thus, to test the autocrine effect of VIP made by tumor cells on VIP receptor-expressing tumor cells, we first assessed whether inhibiting VIP-R signaling affected PDAC cell growth in vitro. These and subsequent studies used peptide VIP-R antagonists predicted to have higher receptor affinity for human VPAC1 and VPAC2 than VIPhyb, based on in silico modeling. Treatment of PDAC cells with increasing concentrations of ANT008 did not affect the viability of PDAC cell lines in vitro, except for a transient effect in MT5 (Figure 24a). Notably, the growth of KPC-Luc, Panc02, Capan02 and BxPC3 was not affected by the addition of VIP-R antagonist (Figure 24a). To test whether VIP produced by PDACs can have an autocrine effect on PDAC growth via VIP-R, VPAC2 was knocked out from the PDAC cell line Panc02 (Figures 24b-24d). VPAC2 knockout Panc02 cells had a similar growth rate in vitro compared to the wild-type parenteral cell line (Figure 24e). Treatment of VPAC2 knockout or wild-type Panc02 cells with the VIP-R antagonist ANT308 did not inhibit in vitro proliferation (Figure 24f). VPAC2 KO cells had slight in vivo growth retardation (FIG. 24g) and improved survival (FIG. 24h) compared to wild-type cells, suggesting a modest direct effect of VIP-R signaling on tumor growth mediated by the VPAC2 receptor in the Panc02 cell line.

[0235] It has been previously reported that inhibiting VIP-R signaling with VIPhyb reduces phosphorylation of CREB (phospho-CREB) and enhances T cell proliferation. This study evaluated the effect of ANT008 or ANT308 on downstream phospho-CREB signaling resulting in higher T cell activation and proliferation. Human T cells activated with anti-CD3 antibody upregulated VPAC1 and VPAC2 expression within 48 hours of activation (Fig. 17a and 17b), with kinetics that are slightly delayed compared to those of PD-1 and CTLA-4 (Fig. 17a and 17c), two targetable immune checkpoint molecules. To determine the effect of VIP-R antagonists on T cell activation, this study measured CD69 expression after treatment with scrambled VIP sequence control peptide (Scram), ANT008, or ANT308 using the gating strategy shown in Fig. 25. Inhibition of VIP-R signaling by the addition of VIP-R antagonist significantly increased the expression of CD69 (Fig. 17d). Notably, ANT308, which has a higher predicted binding affinity for VPAC1 and VPAC2 compared to ANT008, significantly increased the levels of CD69 in both CD4 and CD8 human T cells (Figure 17d). Similarly, addition of ANT308 to cultures more potently inhibited activation-induced phosphorylation of CREB and increased T cell activation compared to treatment with a control scrambled peptide sequence (Figure 17e, Figure 26).

[0236] This study next investigated the effect of VIP-R antagonists on ex vivo expansion of human T cells isolated from peripheral blood of PDAC patients. In vitro treatment with ANT008 significantly reduced the percentage of human Tregs (CD4+CD25+FoxP3+) assessed after 9 days of T cell expansion (Figures 17f and 17g). Furthermore, ANT008 also significantly reduced T cells with an "exhausted" phenotype, as measured by the percentage of T cells co-expressing PD1 and Tim-3, or PD1 and Lag-3, or PD1, Tim-3 and Lag-3 in both CD4+ and CD8+ T cell subsets (Figures 17h and 17i, Figure 27).

[0237] The antitumor effect of combined blockade of VIP receptor with anti-PD-1 inhibition is T cell dependent in murine PDAC. To evaluate the effect of VIP-R-mediated signaling inhibition on PDAC tumor growth in vivo, three different murine PDAC tumors (KPC-Luc, MT5 and Panc02) were implanted into syngeneic immunocompetent C57BL / 6 mice. After tumors were palpable, mice were randomly assigned to treatment with VIP-R antagonist and / or anti-PD-1 monoclonal antibody, or to control groups that received scrambled peptide or isotype-matched IgG. Single-agent VIP-R antagonist treatment in MT5 cells resulted in a modest reduction in growth in vitro (Figure 28a), and mice bearing MT5 tumors had significantly improved survival and reduced tumor burden after monotherapy with VIP-R antagonist (Figures 18a and 28a), indicating direct inhibition of autocrine signaling of VIP through VIP-R in MT5. In all mice bearing either MT5, KPC-Luc, or Panc02 tumors, the combination of VIP-R antagonist and anti-PD-1 significantly reduced tumor burden (Figures 28a-28c) and improved survival (Figures 18a and 18b) compared to mice treated with scrambled peptide and isotype-matched IgG. Notably, in all three tumor models, the combination therapy led to tumor eradication in a significant proportion of mice (40% in KPC and MT5 tumor-bearing mice and 30% in Panc02 tumor-bearing mice), with no difference in outcome based on the sex of the mice (Figures 28d and 28e). In addition to the antitumor efficacy, the safety of VIP-R antagonist in immunologically naive mice was confirmed (Figures 29a-29g). Specifically, daily subcutaneous administration of ANT008 or ANT308 at the same doses and frequencies used in anticancer treatment protocols did not affect overall survival, activity levels, or body weight of mice (Figure 29a). Analysis of blood samples showed a modest reduction in total white blood cells with treatment (Figure 29b), while analysis of splenocytes showed no significant differences in the frequencies of T, B, NK, dendritic cells (DC), or myeloid-derived suppressor cells (MDSC) (Figure 29b).Furthermore, H&E staining of sections of colon and lung tissue in ANT008- and ANT308-treated mice did not show lymphocytic infiltration or histopathology suggestive of autoimmunity (FIGS. 29c-29d).

[0238] This study next tested whether the enhanced survival in VIP-R antagonist and anti-PD-1 treated KPC-Luc tumors was T cell dependent. The enhanced survival seen with VIP-R antagonist and anti-PD-1 combination therapy was blocked by depletion of either CD4+ or CD8+ T cells (Figure 18c). Combination therapy failed to improve survival in both CD4- / - and CD8- / - tumor-bearing mice (Figures 18d and 18e, respectively), indicating that the enhanced anti-tumor response seen with combination therapy was both CD4+ and CD8+ T cell dependent.

[0239] Increased T cell activation in tumors of mice treated with a combination of VIP-R antagonist and anti-PD-1. To examine whether inhibiting VIP-R signaling promotes T cell activation in vivo, this study analyzed tumor-infiltrating T cells in subcutaneous KPC-Luc tumors for differences in activation markers. There was no difference in the percentage of Ki67-, IFN-γ-, or IL-4-expressing CD4+ or CD8+ T cells, but monotherapy with ANT008 or anti-PD-1 significantly altered the levels of PD-1 and / or Tim-3-expressing T cells. ANT008 monotherapy increased the levels of PD-1+Tim-3- T cells in CD4+ and CD8+ T cell subsets, indicating enhanced T cell activation (Figures 19a and 19b). On the other hand, anti-PD-1 monotherapy increased the frequency of PD-1+Tim-3+CD4+ T cells, consistent with T cell exhaustion (Figures 19a and 19b). Furthermore, ANT008 or anti-PD-1 monotherapy, as well as the combination therapy, significantly reduced the frequency of Tregs in PDAC tumors (Figs. 19c and 19d). These findings are consistent with the effect of VIP-R antagonists on Tregs in vitro (Fig. 17f). The experiment further confirmed the effect of ANT008 and anti-PD-1 on T cell activation by analyzing mRNA expression in TILs with Nanostring. No genes were significantly upregulated in TILs from mice treated with single agent ANT008 (Fig. 19e) or anti-PD-1 (Fig. 19f), whereas the combination therapy significantly upregulated the expression of genes related to TCR activation and costimulation (>4-fold change, FDR<0.1) (Fig. 19g). Notably, several markers of T cell activation and costimulation, such as CD27, CD28, CD247, ICOS, TIGIT, and CTLA4, were upregulated in the combination group. Cytokines such as IFN-γ, TNF-α, and IL2 expressed by activated T cells were also expressed at significantly higher levels in TILs from the combination group (Figure 19h). Overall, TCR activation and costimulatory pathway scores were significantly higher in T cells in tumors from mice treated with combination therapy compared to control mice treated with scrambled peptide + isotype IgG (Figure 19i).

[0240] Combination therapy with VIP-R antagonist and anti-PD-1 induces tumor-specific T cell responses and confers protective immunity against tumor rechallenge. In the next experiment, we evaluated whether combination therapy increases the frequency of specific T cell clones and / or antigen-specific T cells within the tumor. DNA was extracted from the tumors and the TCRβ gene was subsequently amplified. Deep sequencing of the TCRβ gene showed increased TCR diversity in tumors from the ANT008 and anti-PD-1 treated group (n=4) compared to control treated mice (scrambled peptide and isotype IgG; n=4), as shown by Shannon's entropy (Figure 20a), indicating more unique TCR responses in the combination group. Analysis of the top 50 most frequent clones in each group showed that the combination treatment group had the greatest number of clones shared by at least two samples compared to all other treatment groups (ANT008 and anti-PD-1:12, control peptide and anti-PD-1:8, ANT008 and isotype IgG:6, control peptide and isotype IgG:6) (Figure 20b). No significant differences were observed in the frequency of shared clones in each treatment group (Figure 20c).

[0241] We next examined whether combination therapy with VIP-R antagonist and anti-PD-1 promotes tumor-specific T cell responses. MuLV p15E is an antigen expressed on KPC-Luc, Panc02, and MC38 tumors, and is considered a tumor-specific antigen due to the lack of expression of MuLVp15E in C57BL / 6 mice. Antigen-specific CD8+ TILs were enumerated by flow cytometry using MuLV p15E-H2Kb tetramer reagent. When tumors were analyzed after 10 days of treatment, tumors from ANT308 and anti-PD-1 treated mice had significantly increased frequencies of tumor antigen-specific tetramer+CD8+ T cells compared to control treated tumors (2.85% vs. 0.72%, p<0.01; Figure 20d and Figure 20e). Taken together, these data indicate that combination therapy with VIP-R antagonist promotes tumor antigen-specific T cell responses in KPC-Luc tumors.

[0242] The KPC-Luc model was shown to be partially responsive to single-agent anti-PD1 antibodies, with some mice observed without obvious KPC-Luc tumors after single-agent anti-PD1 treatment (Figure 18a). To test whether treatment with a VIP-R antagonist in combination with anti-PD1 antibodies enhanced anti-cancer immune memory more than treatment with anti-PD1 antibodies alone, tumor-free mice that had no detectable tumors by palpation or BLI after treatment with anti-PD1 monotherapy or anti-PD1 in combination with ANT008 or Ant308 were rechallenged with a second inoculation of KPC-Luc. These mice were 80-100 days after initial treatment with anti-PD1 alone (n=6) or in combination with either anti-PD1 antibodies and ANT008 (n=5) or ANT308 (n=3) (Figure 20f). Mice previously treated with the VIP-R antagonist and anti-PD1 combination had 100% survival after tumor rechallenge, whereas mice initially treated with single-agent anti-PD1 antibody had 0% long-term survival (Figure 20g). These results demonstrate that protective anti-cancer immune memory was generated long-term after treatment with the VIP-R antagonist peptide and anti-PD-1 combination alone, but not with anti-PD1 monotherapy. Synergy between ANT008 / ANT308 and anti-PD-1 reduced tumor burden and increased intratumoral T cell frequency in orthotopic murine PDAC.

[0243] The next experiment tested the efficacy of combining ANT008 with anti-PD-1 therapy in a more clinically relevant orthotopic KPC-Luc model, in which PDAC cells are directly transplanted into the pancreas, recapitulating some elements of the TME in clinical PDAC. Bioluminescence imaging (BLI) confirmed successful engraftment of KPC-Luc cells into the tail of the pancreas in wild-type mice 6–7 days after transplantation. At day 7 after isotopic transplantation, tumor-bearing mice were randomly assigned to treatment with a combination of control peptide, isotype control antibody, ANT008, or anti-PD-1 MoAb (Figure 21a). The anti-tumor response was greatest in the ANT008 + anti-PD-1 combination, resulting in tumor regression in 7 of 11 mice (63%) compared to 5 / 9 mice (55%) receiving anti-PD-1 monotherapy (55%) and 4 / 10 mice (40%) receiving ANT008 monotherapy (Figure 21b). In addition, the combination of ANT008 and anti-PD-1 had a synergistic effect (Table 4) with slower tumor growth and the lowest tumor burden (measured by pancreas weight at day 25) compared to control mice (Figures 21c and 21d). Also, 1 of 10 mice each in the anti-PD-1 monotherapy and ANT008 / anti-PD-1 combination treatment groups were tumor-free as judged by histological analysis of serial H&E stained tissue sections. The accuracy of the bioluminescence signal from the tumor (tumor flux) was verified by comparing IVIS and MRI images with cross-sectional images of H&E stained paraffin embedded pancreatic tissue obtained at autopsy (Fig. 30a) and by correlating tumor BLI flux with pancreatic weight (Fig. 30b).

[0244] Finally, immunohistochemistry analysis of pancreases on day 25 of treatment assessed collagen and infiltrating T cells across tumors from mice in the different treatment groups (Fig. 21e). Collagen bands were visualized in all tumors (Fig. 21e, Fig. 30c), consistent with evidence of the characteristic desmoplastic TME of human PDAC. Tumors from mice receiving combination therapy had significantly higher intratumoral levels of CD4+ and CD8+ T cells (Fig. 21f and 21g), as well as a higher percentage of Ki67+ CD4+ and CD8+ T cells (Fig. 21h and 21i). Furthermore, there was an inverse correlation between T cell density and pancreatic weight, such that the smallest tumors in the combination group had higher numbers of proliferating T cells (Fig. 30d-30g). These findings indicated that combination therapy with ANT008 and anti-PD-1 not only resulted in enhanced T cell activation, but also promoted T cell infiltration in the collagen-rich TME of orthotopic mouse PDAC tumors.

[0245] Combination therapy with VIP-R antagonist and anti-PD-1 promotes T cell homing into tumors and reduces CXCR4 expression on T cells in tumor-draining lymph nodes. To test whether the enhanced anti-tumor response with combination therapy is due to increased infiltration of T cells into the TME, immunologically naive GFP+ T cells were adoptively transferred from C57Bl / 6 EGFP transgenic mice into wild-type C57BL / 6 mice bearing subcutaneously implanted KPC-Luc tumors, and infiltration of GFP+ T cells into tumors was measured (Fig. 22a). After 3 days of treatment with ANT308 and / or anti-PD1 MoAb, mice treated with combination therapy had increased numbers of GFP+ T cells in tumors compared to all other treatment groups (Figs. 31a-31c). Fluorescence microscopy of DAPI-stained frozen tumor sections further confirmed the significant increase in GFP+ T cell infiltration with combination therapy that infiltrated throughout the tumor (Fig. 22b).

[0246] Preclinical and clinical studies have previously shown that downregulation of CXCR4 promotes recruitment and increases intratumoral T cell infiltration in human and mouse PDAC tumors. The next experiment tested whether combination therapy with VIP-R antagonist and anti-PD1 modulates the expression level of CXCR4 on T cells. Anti-PD-1 monotherapy increased the percentage of Ki67 or CD69 expressing CD4+ and CD8+ T cells, but a significant percentage of activated or proliferating cells also expressed CXCR4 (Figures 22c and 22d). On the other hand, treatment with the combination of anti-PD-1 and VIP-R antagonist increased the percentage of activated CD69+CD4+ or CD8+ cells accompanied by a decrease in CXCR4 expression (Figures 22c and 22d). CXCR4 antagonists, such as AMD3100, have been clinically evaluated as a strategy to recruit CD8+ T cells to PDAC tumors, and the next experiment tested treatment with AMD3100 in combination with anti-PD-1 and / or VIP-R antagonists. Combination therapy with VIP-R antagonist and anti-PD-1 was superior to combination therapy with AMD3100 and anti-PD-1 (Fig. 22e), resulting in complete tumor regression in 20% and 10% of mice, respectively (p=NS) (Fig. 22f). When all three drugs (VIP-R antagonist, anti-PD-1, and AMD3100) were used in combination, survival was not significantly better than that of control mice receiving scrambled peptide, isotype-matched IgG, and PBS (Fig. 22e). These findings indicate that downregulation of CXCR4, but not complete CXCR4 blockade, may be a superior therapeutic strategy to promote T cell trafficking and cytotoxicity within the PDAC TME.

[0247] The clinical efficacy of immune checkpoint blockade in pancreatic cancer targeting PD-1 and CTLA-4 has not been very high, despite the remarkable success of ICB in treating patients with other solid malignancies. Clinical and preclinical studies have shown that the poor responsiveness of PDAC to ICB is mainly due to an immunologically cold TME, characterized by a limited number of T cells in the tumor parenchyma and multiple mechanisms that limit intratumoral T cell activation. Thus, strategies to "boost" T cell priming or activation could promote enhanced T cell-mediated antitumor responses and improve responsiveness to anti-PD-1 or anti-CTLA-4 ICB. Using a murine preclinical model of PDAC, studies have tested whether treatment with VIP-R antagonists promotes the activation and proliferation of antitumor T cells, and whether these drugs synergize with anti-PD-1.

[0248] The results herein, involving selective depletion of CD4+ or CD8+ T cells (Figs. 18c-18e) and generation of cancer antigen-specific immune memory (Figs. 19e and 19g), indicate that the superior effect of VIP-R antagonists is exerted via inhibition of paracrine signaling of VIP produced by tumor cells on T cells expressing VIP-R. Although knockout of VPAC2 in Panc02 cells showed a modest direct effect of VIP signaling on cancer cell growth in vivo, mice injected with VPAC2 knockout Panc02 tumors have a 7-day median survival benefit compared to mice fed with VPAC2 wild-type tumors (Fig. 24h). In contrast, the combination of VIP-R antagonists and anti-PD-1 synergistically improved T cell-dependent antitumor responses in PDAC-bearing mice, resulting in tumor elimination in up to 40% of treated tumor-bearing mice. Moreover, rechallenge of tumor-free mice resulted in complete tumor rejection in mice receiving the VIP-R antagonist and anti-PD-1 combination, further highlighting the role of enhanced, adaptive, long-lasting anti-tumor immunity generated in response to inhibition of VIP-R signaling. Recipients of VIP-R antagonist peptide / anti-PD-1 combination therapy had increased homing, activation, and proliferation of intratumoral CD4+ and CD8+ T cells, as well as a marked increase in tumor antigen-specific T cells within the TME. Induction of robust T cell responses via CD40 signaling overcomes the refractoriness of PDAC tumors to ICB.

[0249] Exclusion of T cells from the TME is a key feature of PDAC tumors, which is likely the result of a dense desmoplastic stroma that contains robust immunosuppressive cells and soluble factors that limit T cell activation. In multiple studies, components of the stroma, such as cancer-associated fibroblasts (and the cytokines and chemokines they secrete), suppress T cell effector functions, resulting in immunologically "cold" tumors. The role of the CXCR4 / CXCL12 axis in T cell infiltration in PDAC tumors is complex. CXCR4 expression promotes homing of naive T cells to lymph nodes with high CXCL12 levels, where priming to tumor antigens can occur. However, CXCR4+ T cells can be "trapped" within the peritumoral extracellular matrix by binding to CXCL12, which is expressed by CAFs and linked to KRT19. Thus, high expression of CXCR4 is a predictive marker of poor survival in PDAC patients, and treatment with CXCR4 antagonists increases CD8+ T cell infiltration in the TME. Consistent with these data, the current findings of synergy between VIP-R antagonist and anti-PD1 are consistent with the modulation of CXCR4 levels. Treatment with anti-PD-1 increases the expression of CXCR4 on intratumoral T cells, while the addition of VIP-R antagonist to anti-PD-1 significantly reduces the expression of CXCR4 on T cells, potentially preventing T cells from becoming "trapped" in the extracellular matrix. Treatment with a triple combination of VIP-R antagonist, anti-PD-1, and CXCR4 antagonist resulted in tumor growth rates and survival similar to control mice that did not receive drugs (Figure 22e), indicating that down-regulation of CXCR4, although not a complete blockade, may be a good therapeutic strategy to promote T cell trafficking and cytotoxicity within the TME.

[0250] In mice receiving the combined drug therapy, TILs expressed significantly higher levels of transcripts associated with TCR signaling and activation and showed increased mRNA levels of the Th1 cytokines TNF-α, IFN-γ, and IL-2. Furthermore, upregulation of mRNA levels of the chemokines CCL2, CCL4, and CCL5 on T cells and the chemokine receptor CXCR6 indicates that intratumoral T cells activated by combined VIP-R antagonist / anti-PD1 therapy can promote the recruitment of additional T cells from the blood to the TME. This is supported by the increased accumulation of immunologically naive GFP+ T cells in tumors in mice treated with the combined therapy.

[0251] These data indicate that VIP-R antagonists act by blocking inhibitory signaling pathways that limit immune cell activation, proliferation, and survival. The reduction in CREB phosphorylation following in vitro treatment of human T cells with VIP-R antagonists suggests that enhanced NF-κB signaling is responsible for the enhanced T cell activation seen in mouse PDAC models. One of the safety concerns in the use of any ICB is the induction of autoimmunity. Notably, treatment of wild-type mice with daily subcutaneous injections of ANT008 or ANT308 VIP-R antagonists for 10 days was not associated with histopathological evidence of autoimmunity and had no apparent effect on behavior, consistent with the lack of autoimmune disease in VIP knockout mice. Thus, the beneficial effects of VIP-R antagonist treatment on anti-cancer immunity in PDAC models are likely due to local effects of VIP in the TME, where VIP is pathologically overexpressed.

[0252] This study shows that mouse T cells penetrate deeply into orthotopically implanted tumors treated with a combination of VIP-R antagonist and anti-PD1, apparently crossing dense stromal bands of collagen. Although the current study focuses on the treatment of PDAC, several other cancers may also be potential targets for VIP-R antagonists. Published studies have demonstrated the antitumor activity of VIP-R antagonists in mouse models of myeloid leukemia and lymphoma, as well as other cancers that overexpress VIP. The focus and conclusions of the current study are orthogonal to previous studies that have examined the suppression of autoimmunity by natural VIP or the activity of VIP-R antagonists as tumor cell suppressors.

[0253] Finally, why has VIP signaling not been previously identified as a targetable ICB pathway? Pharmacological antagonism of the VIP receptor was explored as a strategy to block autocrine signaling of VIP expressed by tumor cells that stimulates tumor growth. The human pancreatic cancer cell line CAPAN-2 expresses the VIP receptor and its growth is stimulated by VIP. VIP-R antagonists inhibit the induction of c-fos mRNA by VIP and delay the growth of CAPAN-2 cells in nude mice, indicating that VIP receptor antagonists have tumor-intrinsic cytostatic effects. Preclinical studies have explored VIP-R antagonists as cytostatic anticancer drugs, but no clinical trials of VIP-R antagonists in humans or studies testing the potential of VIP-R antagonists to enhance adaptive immunity have been conducted. Although VIP was identified more than 40 years ago, the effects of VIP on immunity have been studied. Although the focus of current research is the effect of anti-PD1 antibodies and VIP-R antagonists on antitumor T cells, this therapy may also affect immunosuppressive myeloid cells in the TME, block the generation of tolerogenic dendritic cells, and affect antigen presentation by tumor-infiltrating macrophages and dendritic cells in tertiary lymphoid structures within the tumor.

[0254] The current study shows that VIP-R antagonists represent a novel and tractable approach in the treatment of PDAC. Notably, the VIP amino acid sequence is identical between humans and mice, and the VIP-R sequence is highly conserved, indicating that VIP-R antagonists with immunological activity in tumor-bearing mice may have comparable properties in human patients. In support of this concept, ex vivo treatment of human T cells isolated from the blood of PDAC patients with VIP-R antagonists promoted downregulation of PD-1, Tim-3, and Lag-3 immune checkpoint molecules associated with T cell activation, immunological senescence, and reduced the frequency of regulatory T cells (Figure 17f). Furthermore, VIP overexpression may be a useful biomarker to identify patients with PDAC and other cancers susceptible to the ICB activity of VIP-R antagonists, similar to the use of PD-L1 staining in cancers as a predictive biomarker for response to anti-PD1 ICB (Figures 16b and 16e).

[0255] 3. Example 3: Materials and Methods Cell lines and reagents. MT5 and KPC-Luc cells were generously gifted by Dr. Tuveson (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and Dr. Logsdon (MD Anderson Cancer Center, Houston, Texas), respectively, and Panc02 cells were provided by Dr. Pilon-Thomas (H. Lee Moffitt Cancer Center, Tampa, FL). BXPC3, Panc1, and B15F10 were from ATCC (Manassass, VA), and SM1 was from Dr. Antoni Ribas (UCLA, Los Angeles, CA). MT5 and BXPC3 cells were cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 5% and 10% fetal bovine serum (FBS), respectively, in addition to 10 mM L-glutamine and antibiotics. KPC-Luc, Panc02, and Panc1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 10 mM L-glutamine, and antibiotics. Synthego (Redwood City, CA) provided the CRISPR / Cas9 VPAC2 knockout pool of Panc02 cell lines. Single clones of VPAC2 KO Panc02 cells were selected by limiting dilution and grown in the same medium as wild-type Panc02 cells. B16F10 cells were cultured in DMEM with L-glutamine and sodium pyruvate supplemented with 10% FBS, 100ug / mL streptomycin, and 1500mg / L sodium bicarbonate. The human pancreatic cancer-associated stellate (PSC) cell line h-iPSC-PDAC-1 was generated and maintained as previously described.

[0256] Pharmaceutical grade mouse antibodies against PD-1 (clone RMP1-14) or isotype control (clone 2A3) were purchased from BioXcell (West Lebanon, NH). Pharmaceutical grade AMD3100 was purchased from Selleck Chemicals LLC (Houston, Texas). Scrambled peptides, ANT008 and ANT308, were purchased from RS Synthesis (Louisville, KY) at >95% purity. Stock solutions of 200 μM were prepared in DEPC-treated pyrogen-free water from IBI Scientific (Dubuque, IA) and stored at -80°C until use. Creative Biolabs (Shirley, NY) performed in silico modeling to predict the binding affinity of ANT008 and ANT308 to the VPAC1 and VPAC2 receptors.

[0257] Patients and Samples. Primary human PSCs / CAFs were isolated from resected pancreatic tumors according to an Institutional Review Board (IRB)-approved protocol at the Winship Cancer Institute of Emory University for deidentified tumor tissue. Briefly, freshly resected pancreatic tissue was dissected into 1 mm3 sections, placed in culture dishes, and incubated in DMEM+10% FBS+antibiotics for 2-3 weeks until binding was observed. Cell-free supernatants were then collected to quantify levels of VIP via a VIP-specific enzyme immunoassay. Blood samples from consenting pancreatic cancer patients and healthy volunteers were collected in EDTA-coated vacutainer tubes from Becton Dickinson and Company (Franklin Lakes, NJ). Plasma was isolated as previously described, and the EDTA vacutainer tubes were centrifuged at 2000 × g for 15 min and stored at -80 °C until used for analysis of VIP levels. Patient demographics are shown in Table 3. Peripheral blood mononuclear cells (PBMCs) were isolated from consenting patients with PDAC or healthy volunteers (IRB 00087397 and IRB 00046063, respectively) by Ficoll-Hypaque gradient centrifugation as previously described and cryopreserved in CryoStor cell cryopreservation medium CS10 (STEMCELL Technologies, Vancouver, Canada) until required for T cell isolation and ex vivo expansion.

[0258] Antibodies. For Western blot analysis of VPAC1, VPAC2, PD1 and CTLA4 expression, anti-VPAC1 (1:500), anti-VPAC2 (1:500) monoclonal antibodies from Sigma Aldrich (St. Louis, MO) and anti-PD1 (1:1000) and anti-CTLA-4 (1:500) monoclonal antibodies from Cell Signaling Technology (Danvers, MA) were used. For IF, anti-VIP monoclonal antibody from OriGene (clone OT15B5) at 1:50 and anti-CK18 monoclonal antibody from Abcam (clone EP1580Y) at 1:400 were used. Details of fluorochrome-conjugated antibodies for flow cytometry analysis are provided in Table 4. Fixable Aqua Live / Dead stain from Thermo Fisher Scientific (Waltham, MA) was used to detect and gate live cells in all samples analyzed via flow cytometry analysis. To identify tumor-specific T cells, APC-conjugated MHC Tetramer H-2kb MuLV p15E from MBL International Corporation (Woburn, MA) was used.

[0259] Mice. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University. Female or male C57BL / 6, CD4KO (B6.129S2-Cd4 tm1Mak / J) and CD8KO (B6.129S2-Cd8a tm1MakMice (A, B, C, D, E, F, H, H, I, H, I, J) were obtained at 6–8 weeks of age from Jackson Laboratory (Bar Harbor, ME) and housed in microisolator cages. Transgenic mice expressing enhanced green fluorescent protein (EGFP) on a C57BL / 6 background (strain name: C57BL / 6-Tg(Act-EGFP)C14-Y01-FM131 Osb) were kindly provided by Dr. Masaru Okabe (Osaka University, Osaka, Japan) and were bred and maintained at the Emory University Animal Care Facility (Atlanta, GA). Experiments were performed when the mice were 8–10 weeks of age, and animal care and maintenance were performed in accordance with The Guide for Care and Use of Laboratory Animals (National Research Council). For CD4+ and / or CD8+ T cell depletion studies, antibodies depleting CD4+ T cells (clone GK1.5) or CD8+ T cells (clone 2.43) from BioXcell (West Lebanon, NH) were injected intraperitoneally at 200 μg per mouse on days −3, −1, +1, +3, +7 relative to tumor implantation, twice weekly until completion of the experiment.

[0260] Preparation of single cell suspension. Tumor tissues or tumor-draining lymph nodes (TDLN) harvested from mice bearing KPC-Luc or Panc02 were cut into small pieces using a scalpel and treated with triple enzyme digestion cocktail containing 10 mg / ml collagenase, 1 mg / ml hyaluronidase, and 200 mg / ml DNase in HBSS at 37°C for 20 min (TDLN) or 1 h (tumor), vortexing every 15 min. Tissue pieces were then mechanically dissociated, washed, centrifuged, and passed through a 70 μm nylon mesh filter to obtain a single cell suspension for staining and analysis by flow cytometry. For spleen samples, the single cell suspension obtained by mechanical dissociation was passed through a 70 μm nylon mesh filter, depleted of red blood cells using ammonium chloride lysis buffer, and washed twice. Blood samples were collected into tubes containing 0.1 ml diluted heparin (500 USP units / ml) followed by red blood cell depletion using ammonium chloride lysis buffer and two washes.

[0261] VIP-specific enzyme immunoassay. 3×10 5 B16F10, KPC-Luc, MT5, Panc02, BXPC3, and Panc1 cells were cultured in 6-well plates with 3 ml of medium each. Cell-free medium was collected after 24 hours of culture and stored at -80°C until tested for VIP levels. Peripheral blood collected from consenting PDAC patients and healthy volunteers in EDTA tubes was centrifuged at 2000g for 10 minutes to isolate plasma and stored at -80°C until analysis. VIP levels in cell-free supernatants and plasma were quantified by a VIP-specific enzyme immunoassay (EIA) kit according to the manufacturer's protocol (RayBiotech, Peachtree Corners, Georgia). Absorbance was measured at 450 nm using a Synergy plate reader (BioTek, Winooski, Vermont) and a standard curve was generated and used to determine the concentration of VIP in the samples.

[0262] Cell viability assay. To determine the effect of Ant-08 on proliferation of PDAC cell lines in vitro, MT5, KPC-Luc, Panc02, BXPC3, Capan-02 and cells were plated on 96-well plates and treated with various concentrations of Ant-08 (0-5 μM) in their respective media for 24-72 h. Cell viability was assessed using the Cell Proliferation Kit I from Roche (Basel, Switzerland) according to the manufacturer's instructions. Briefly, at the end of the incubation time, 10 ul of 0.5 mg / ml of MTT labeling reagent was added and the plates were incubated for 4 h in a humidified CO2 incubator. This was followed by overnight incubation in solubilization buffer and absorbance reading at 570 nm. The percentage of cell viability relative to the control (0 μM ANT008) was plotted. Similar assays were performed on wild-type and VPAC2 KO Panc02 cells after treatment with Ant-08 or Ant-308 (3 μ m ) for 72 h.

[0263] In vivo efficacy study. For subcutaneous model, 5 × 10 5 KPC-Luc cells were injected subcutaneously near the right flank of female or male C57BL / 6 mice. For the MT5 or Panc02 models, 5 × 10 51 were injected subcutaneously near the right flank of female C57BL / 6 mice. For the orthotopic KPC-Luc model, mice were anesthetized, and KPC-Luc cells were suspended in Matrigel and injected into the tail of the pancreas after laparotomy. Six to seven days after tumor implantation, mice were randomized into four treatment groups and treated with VIP-R antagonist and / or anti-PD-1. The scram+IgG control mice received scrambled peptide and isotype IgG, whereas the VIP-R antagonist group, anti-PD-1 and VIP-R antagonist group, and anti-PD-1 group received VIP-R antagonist and IgG, scrambled peptide and anti-PD-1, and VIP-R antagonist and anti-PD-1, respectively. The treatment regimen consisted of 10 μg of scrambled or VIP-R antagonist: ANT008 or ANT308 administered subcutaneously daily and 200 μg of IgG or anti-PD-1 administered intraperitoneally once every 3 days for a total of 10 days. For experiments with male mice, 20 μg of VIP-R antagonist was used due to their higher body weight compared to female mice. For experiments, mice receiving AMD 3100 were administered 5 mg / kg of AMD3100 in PBS subcutaneously daily for 10 days. In the KPC-Luc model, tumor growth rates were plotted using tumor flow measurements quantified using IVIS bioluminescence imaging. For the orthotopic KPC-Luc model, one mouse per group with the largest non-ulcerated tumor was sacrificed on day 28, placed in a supine position in a custom-built cradle, and imaged on a 9.4 Tesla MRI scanner. Slice thickness 0.4 mm, total of 40 slices per mouse (RARE factor = 8, average = 25, field of view = 30.7 × 30.7 mm 2 ) using a rapid acquisition with relaxation enhancement (RARE) imaging sequence. In experiments with subcutaneous tumors, tumor dimensions were measured using calipers, and tumor volume was calculated using the formula tumor volume = 1 / 2 (length × width × height).

[0264] Nanostring analysis of tumor-infiltrating T cells. Single cell suspensions of tumors were subjected to magnetic T cell isolation using the EasySep™ Mouse CD90.2 Positive Selection Kit II from STEMCELL Technologies (Vancouver, Canada). RNA was extracted using the Qiagen RNeasy Micro kit and quantity and quality were assessed using the Nanodrop and Agilent 2100. RNA analyzed using the nCounter Metabolic Pathways Panel (Nanostring Technologies, Seattle, WA).

[0265] TCR Deep Sequencing Subcutaneously implanted KPC-Luc tumors treated with ANT008 and / or anti-PD-1 were harvested 21 days after tumor implantation. RNA was extracted using Qiagen RNeasy Micro kit and quantity and quality were assessed using Nanodrop and Agilent 2100. Sequencing of TCR-β CDR3 V and J sequences was performed by Adaptive Biotechnologies (Seattle, WA).

[0266] Human T cell activation and expansion. One day prior to in vitro T cell expansion, cryopreserved PBMCs were thawed and rested by overnight culture at 37°C in a 5% CO2 humidified incubator in complete RPMI medium supplemented with 10% FBS, 100U / mL penicillin and 100ug / mL streptomycin, MEM non-essential amino acids, 20mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and 50uM 2-mercaptoethanol. T cells were isolated by negative magnetic isolation using the EasySep human T cell isolation kit from STEMCELL Technologies (Vancouver, Canada). 50,000-1 million T cells were coated with 10ug / ml of Ultra-LEAF purified anti-human CD3 antibody (clone: ​​UCHT1) from Biolegend (San Diego, CA) and cultured with 30U / ml of recombinant human IL-2 (Peprotech, Inc., Cranbury, NJ) in the presence or absence of 3μM of scrambled peptide, ANT008 or ANT308. Cells were counted using Trypan blue dye and phenotyped by flow cytometry on day 9. Every 3 or 4 days, cells were split into 6 or 48 well or anti-human CD3 coated plates.

[0267] Peptides. ANT008 has the peptide sequence of VIPhyb modified by replacing the serine at amino acid position 25 with leucine. ANT308 is a further modification of the ANT008 sequence, replacing the aspartic acid and asparagine residues at positions 8 and 9 with serine and aspartic acid, respectively. (In silico analysis reveals free binding energies of ANT008: -60.17 kcal / mol for VPAC1, -51.07 for VPAC2, ANT308: -71.56 for VPAC1, and -56.27 for VPAC2).

[0268] Adoptive transfer of GFP+ T cells One million KPC-Luc cells were subcutaneously implanted into C57BL / 6 mice. 15 days after tumor implantation, spleens were harvested from EGFP transgenic mice, processed as described above, and T cells were magnetically isolated using the Pan T cell isolation kit II from Miltenyi Biotech (Auburn, CA). Ten million GFP+ T cells were then injected intravenously into C57BL / 6 mice bearing KPC-Luc tumors. Mice were randomized into four treatment groups: scram+IgG, ANT308+IgG, scram+aPD-1, ANT308+aPD-1, and treated subcutaneously with 10ug of scrambled peptide / ANT308 on days 1, 2, and 3 after T cell transfer, and / or 200ug of IgG or anti-PD-1 on day 1. Mice were sacrificed 18 days after tumor implantation, and harvested tumors were flash frozen in OCT compound (Sakura Finetek, Torrance, CA) for embedding and cryosectioning. Tissue slides were then stained with 2ug / ml Hoescht 33342 (Abcam, Cambridge, MA) and imaged on a BZ-X810 epifluorescence microscope (Keyence Corp, Itasca, IL) using DAPI (359nm / 461nm) and GFP (488nm / 510nm) filter sets (Chroma Technology Corp, Bellows Falls, VT). A Nikon Inc. (Melville, NY) Plan Fluor 40x 1.3NA oil immersion objective was used with 100% of the light from the excitation source reaching the sample, a camera exposure of 1 / 1.2 seconds, and an image aspect ratio of 1920x1440. Multiple images were acquired across the entire tissue section and then stitched using the image stitcher function in Keyence and merged into a composite image using Fiji image analysis software.

[0269] Immunofluorescence. Paraffin-embedded PDAC tissues and adjacent normal tissues were deparaffinized, hydrated, and antigen retrieved by boiling in 1× Trilogy for 15 min (Cell Marque-Trilogy Buffer) followed by washing with distilled water. Permeabilization was performed using 0.3% Triton-X-100, followed by a blocking step with eBioscience™ low protein blocking buffer for 1 h at room temperature. Mouse anti-VIP (OriGene Technologies, Inc. Rockville, MD) diluted 1:50, and rabbit anti-cytokeratin-19 (Abcam, Cambridge, MA) diluted 1:400 were applied and incubated overnight at 4° C. Secondary antibodies were applied: anti-mouse IgG (H+L) conjugated with Alexa Fluor 647 and anti-rabbit IgG (H+L) conjugated with TRITC, and incubated for 1 h at room temperature. Tissue slides were then stained with 2ug / ml Hoescht 33342 (Abcam, Cambridge, MA) and imaged on a BZ-X810 epifluorescence microscope (Keyence Corp, Itasca, IL) using DAPI (359nm / 461nm) and Alexa Fluor 647 (594nm / 633nm) and TRITC (579nm / 599nm) filter sets (Chroma Technology Corp, Bellows Falls, VT). A Plan Fluor 40x 1.3NA oil immersion objective from Nikon Inc. (Melville, NY) was used with 100% of the light from the excitation source reaching the sample, a camera exposure of 1 / 1.7 seconds, and an image aspect ratio of 1920x1440. Multiple images were acquired across the entire tissue section and then stitched using Keyence's image stitcher function and merged into a composite image using Fiji image analysis software.

[0270] Histology. All tissues for histology were fixed in 4% paraformaldehyde in PBS for 2-3 days at 4 °C, embedded in paraffin, and cut into 5 μm thick sections. Slides were deparaffinized with EZ-Prep (#05279771001, Ventana, Tucson, AZ) and then antigen retrieved with CC1 reagent (#950-500, Ventana, Tucson, AZ) for 64 min. Mouse anti-VPAC1 and anti-VPAC2 from Sigma Aldrich (St. Louis, MO) diluted 1:500, or rabbit anti-cytokeratin-19 (Abcam, Cambridge, MA) diluted 1:500 were applied and incubated for 40 min. DISCOVERY OmniMap anti-mouse or anti-rabbit HRP were applied and incubated for 12 min. Detection was completed in conjunction with the DISCOVERY ChromoMap DAB kit according to the manufacturer's recommendations. Pancreata harvested from mice bearing orthotopically implanted KPC-Luc tumors or colons, livers, and expanded lungs harvested from naive C57BL / 6 mice receiving ANT008 or ANT308 were formalin fixed and paraffin embedded before being stained with H&E (Leica 560MX, Wetzlar, Germany) or Masson's Trichrome (Polyscientific Inc., Bay Shore, NY). All slides were dehydrated, coverslipped, scanned at 40x with a Hamamatsu Nanozoomer 2.0 HT, and reviewed by a pathologist.

[0271] Multiplex immunohistochemistry Multiplex IHC staining was performed on a Roche Ventana DISCOVERY automated immunostainer from Ventana Medical Systems (Tucson, AZ). The Ventana DISCOVERY uses a sequential staining procedure with a denaturing step between each staining sequence. Slides were deparaffinized with EZ-Prep (#05279771001, Ventana) and then antigen retrieval was performed with CC1 reagent (#950-500, Ventana) for 64 min. Cell conditioning 2 buffer (CC2, #950-123, Ventana) was used for inactivation of bound primary antibodies and secondary anti-horseradish peroxidase between each staining sequence. Four pre-diluted primary antibodies were applied sequentially at dilutions of 1:300, 1:500, 1:500, and 1:250, respectively, using chromogenic detection as indicated, in the following order: rabbit anti-Ki67 with Opal 570 (#ab833, Abcam, Cambridge, MA), rabbit anti-Foxp3 with Opal 480 (#NB100-39002, Novus Biologicals, Littleton, CO), rabbit monoclonal CD4 with Opal 620 (#ab133616, Abcam), and rabbit anti-CD8 with Opal 690 (#ab4055, Abcam). Slides were covered with VECTASHIELD Antifade mounting medium (Vector Laboratories) and stained slides were stored at 4°C. The numbers of CD4+, CD8+, Ki67+CD4+ and Ki67+CD8+ T cells were quantified using QuPath, an open source software for digital pathology image analysis.

[0272] Statistics. For survival data, the Kaplan-Meier method with log-rank test was performed to determine statistical differences between treatment groups. For comparison of differences in tumor volume and immune cell subsets comparing the four treatment groups, one-way analysis of variance (one-way ANOVA) followed by Dunnett's multiple comparison post-hoc test was used. For data from ex vivo expansion of T cells from healthy volunteers, repeated measures ANOVA was used followed by Dunnett's post-hoc test. For data from expansion of T cells from PDAC patients, T cell phenotype and characteristics between scrambled and ANT008 treated were compared utilizing pairwise Student's t-test. In Figures 30d-30g, R-squared values ​​were generated from linear regression models. P values ​​less than 0.05 were considered significant. Statistical analysis was performed using GraphPad Prism software version 8.2 (GraphPad Software, Inc., San Diego, CA, USA). [Table 3] [Table 4] [Table 5-1] [Table 5-2]

[0273] References: 1. Kabacaoglu, D., et al., Immune Checkpoint Inhibition for Pancreatic Ductal Adenocarcinoma: Current Limitations and Future Options. Front Immunol, 2018.9:p.1878. 2.Saung,M.T.and L.Zheng,Current Standards of Chemotherapy for Pancreatic Cancer.Clin Ther,2017.39(11):p.2125-2134. 3.Le,D.T.,et al.,Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade.Science,2017.357(6349):p.409-413. 4.Young,K.,et al.,Immunotherapy and pancreatic cancer:unique challenges and potential opportunities.Ther Adv Med Oncol,2018.10:p.1758835918816281. 5.Hosein,A.N.,R.A.Brekken,and A.Maitra,Pancreatic cancer stroma:an update on therapeutic targeting strategies.Nat Rev Gastroenterol Hepatol,2020.17(8):p.487-505. 6.Lin,J.H.and R.H.Vonderheide,Dendritic cell dysfunction precedes tumor formation during pancreatic oncogenesis.Cancer Research,2020.80(16). 7.Johnson,B.A.,3rd,et al.,Strategies for Increasing Pancreatic Tumor Immunogenicity.Clin Cancer Res,2017.23(7):p.1656-1669. 8.Vonderheide,R.H.,The Immune Revolution:A Case for Priming,Not Checkpoint.Cancer Cell,2018.33(4):p.563-569. 9.Khalil,M.and R.H.Vonderheide,Anti-CD40 agonist antibodies:preclinical and clinical experience.Update Cancer Ther,2007.2(2):p.61-65. 10.Vonderheide,R.H.,CD40 Agonist Antibodies in Cancer Immunotherapy.Annu Rev Med,2020.71:p.47-58. 11.O’Hara,M.H.,O’Reilly,E.M.,Wolff,R.A.,Wainberg,Z.A.,Ko,A.H.,Rahma,O.E.,Fisher,G.A.,Lyman,J.P,Cabanski,C.R.,Karakunnel,J.J.,Gherardini,P.F.,Kitch,L.J.,Bucktrout,S.,Christopher,E.,Mick,R.,Chen,R.,Trifan,O.C.,Salvador,L.,O’Donnell-Tormey,J.and Vonderheide,R.H.,Gemcitabine(Gem)and nab-paclitaxel(NP)±nivolumab(nivo)±CD40 agonistic monoclonal antibody APX005M(sotigalimab),in patients(Pts)with untreated metastatic pancreatic adenocarcinoma(mPDAC):Phase(Ph)2 final results.Journal of Clinical Oncology 2021. 12.Delgado,M.and D.Ganea,Vasoactive intestinal peptide:a neuropeptide with pleiotropic immune functions.Amino Acids,2013.45(1):p.25-39. 13.Fernandez-Martinez,A.B.,et al.,Multifunctional role of VIP in prostate cancer progression in a xenograft model:suppression by curcumin and COX-2 inhibitor NS-398.Peptides,2009.30(12):p.2357-64. 14.Moody,T.W.and I.Gozes,Vasoactive intestinal peptide receptors:a molecular target in breast and lung cancer.Curr Pharm Des,2007.13(11):p.1099-104. 15.Moody,T.W.,et al.,VIP receptor antagonists and chemotherapeutic drugs inhibit the growth of breast cancer cells.Breast Cancer Res Treat,2001.68(1):p.55-64. 16.Moody,T.W.,B.Nuche-Berenguer,and R.T.Jensen,Vasoactive intestinal peptide / pituitary adenylate cyclase activating polypeptide,and their receptors and cancer.Curr Opin Endocrinol Diabetes Obes,2016.23(1):p.38-47. 17.Moody,T.W.,et al.,A Vasoactive-Intestinal-Peptide Antagonist Inhibits Nonsmall Cell Lung-Cancer Growth.Proceedings of the National Academy of Sciences of the United States of America,1993.90(10):p.4345-4349. 18.Anderson,P.and E.Gonzalez-Rey,Vasoactive intestinal peptide induces cell cycle arrest and regulatory functions in human T cells at multiple levels.Mol Cell Biol,2010.30(10):p.2537-51. 19.Gonzalez-Rey,E.and M.Delgado,Vasoactive intestinal peptide and regulatory T-cell induction:a new mechanism and therapeutic potential for immune homeostasis.Trends Mol Med,2007.13(6):p.241-51. 20.Delgado,M.and D.Ganea,Cutting edge:is vasoactive intestinal peptide a type 2 cytokine? J Immunol,2001.166(5):p.2907-12. 21.Petersen,C.T.,J.M.Li,and E.K.Waller,Inhibition of CREB signaling through antagonism of vasoactive intestinal peptide enhances CD8 T cell function in a murine model of AML.Journal of Immunology,2016.196. 22.Petersen,C.T.,J.M.Li,and E.K.Waller,Administration of a vasoactive intestinal peptide antagonist enhances the autologous anti-leukemia T cell response in murine models of acute leukemia.Oncoimmunology,2017.6(5):p.e1304336. 23.Li,J.M.,et al.,VIPhyb,an antagonist of vasoactive intestinal peptide receptor,enhances cellular antiviral immunity in murine cytomegalovirus infected mice.PLoS One,2013.8(5):p.e63381. 24.Li,J.M.,et al.,Pharmacological inhibition of VIP signaling enhances antiviral immunity and improves survival in murine cytomegalovirus-infected allogeneic bone marrow transplant recipients.Blood,2013.121(12):p.2347-51. 25.Zia,H.,et al.,Breast cancer growth is inhibited by vasoactive intestinal peptide(VIP)hybrid,a synthetic VIP receptor antagonist.Cancer Res,1996.56(15):p.3486-9. 26.Reubi,J.C.,et al.,Vasoactive intestinal peptide / pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin.Cancer Res,2000.60(11):p.3105-12. 27.Schulz,S.,et al.,Immunocytochemical identification of VPAC1,VPAC2,and PAC1 receptors in normal and neoplastic human tissues with subtype-specific antibodies.Clin Cancer Res,2004.10(24):p.8235-42. 28.Schulz,S.,et al.,VPA...

Claims

1. KPRRPYX １ X ２ X ３ X ４ TX ５ LRKQX ６ AVX ７ X ８ KYLX ９ X １０ A vasoactive intestinal peptide receptor (VIP-R) antagonist comprising ILN (SEQ ID NO: 3) or a fragment thereof, wherein, X １ is T or A, X ２ is D, V, or S, X ３ is N or D, X ４ is Y or C, X ５ is R or S, X ６ is M or I, X ７ is K or N, X ８ is K, and X ９ is N or M, X １０ is S or L, However, the peptide is not KPRRPYTDNYTRLRKQMAVKKEYLNSILN (SEQ ID NO: 1) nor X １ where X is T, and X ２ is D, and X ３ is N, and X ４ is Y, and X ５ is R, and X ６ is M, and X ７ is K, and X ９ is N, and X １０ is not a combination where X is S, a VIP-R antagonist.

2. KPRRPYX １ X ２ X ３ X ４ TX ５ LRKQX ６ AVX ７ KYLX ８ X ９ A vasoactive intestinal peptide receptor (VIP-R) antagonist comprising ILN (SEQ ID NO: 21) or a fragment thereof, wherein, X １ is T or A, X ２ is D, V, or S, X ３ is N or D, X ４ is Y or C, X ５ is R or S, X ６ is M or I, X ７ is K or N, X ８ is N or M, X ９ is S or L, However, the peptide is not KPRRPYTDNYTRLRKQMAVKKEYLNSILN (SEQ ID NO: 1) nor X １ where X ２ is T, X ３ is D, X ４ is N, X ５ is Y, X ６ is R, X ７ is M, X ８ is K, X ９ is N, nor is it a combination where X is S, a VIP-R antagonist.

3. The VIP-R antagonist is an amino acid sequence: KPRRPYADNYTRLRKQMAVNKYNLILN (SEQ ID NO: 6), KPRRPYAVNYTRLRKQIAVKKEYLMSILN (SEQ ID NO: 7), KPRRPYAVNYTRLRKQMAVNKYLMSILN (SEQ ID NO: 8), KPRRPYADNCTRLRKQIAVNKKYLNSILN (SEQ ID NO: 9), KPRRPYTVNYTSLRKQIAVKKEYLMLILN (SEQ ID NO: 10), KPRRPYTDNCTSLRKQIAVNKYNLILN (SEQ ID NO: 11), KPRRPYAVNCTSLRKQIAVNKYNLNSILN (SEQ ID NO: 12), KPRRPYAVNCTSLRKQIAVKKEYLMSILN (SEQ ID NO: 13), KPRRPYTVNCTSLRKQIAVKKEYLMLILN (SEQ ID NO: 14), KPRRPYTSDYTRLRKQMAVKKEYLNSILN (SEQ ID NO: 15), The VIP-R antagonist according to claim 1, comprising KPRRPYTSDYTRLRKQMAVKKEYLNLILN (SEQ ID NO: 16) or a fragment thereof.

4. The VIP-R antagonist according to claim 1, wherein the amino, carboxyl, hydroxyl, or thiol group in the peptide is substituted.

5. The VIP-R antagonist according to claim 1, wherein the peptide is conjugated to nanoparticles and / or encapsulated within nanoparticles.

6. A pharmaceutical composition comprising the vasoactive intestinal peptide receptor (VIP-R) antagonist according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. A nucleic acid encoding the amino acid sequence of the vasoactive intestinal peptide receptor (VIP-R) antagonist according to any one of claims 1 to 5.

8. The nucleic acid encoding the amino acid sequence according to claim 7, wherein the nucleic acid is operably combined with a promoter.

9. An expression vector comprising the nucleic acid according to claim 7.

10. A cell comprising the expression vector according to claim 9.

11. A method for enhancing the activation and / or proliferation of T cells ex vivo, comprising mixing one or more T cells with the vasoactive intestinal peptide receptor (VIP-R) antagonist according to any one of claims 1 to 5.

12. The method according to claim 11, wherein mixing the one or more T cells is performed in combination with an anti-CD3 antibody and / or an anti-CD28 antibody.

13. One or more T cells, An in vitro cell culture composition comprising a vasoactive intestinal peptide receptor (VIP-R) antagonist according to any one of claims 1 to 5.

14. The in vitro cell culture composition according to claim 13, further comprising an anti-CD3 antibody, an anti-CD28 antibody, and / or a phosphatidylinositol 3-kinase (PI3K) inhibitor.

15. A method for treating a microbial infection, cancer, or metastasis in a subject infected with a microorganism, having cancer, and / or at risk of microbial infection, and / or a method for enhancing an immune response against cancer and / or metastasis in a subject having cancer, for use in the method according to claim 6.

16. The pharmaceutical composition according to claim 15, wherein the microbial infection is a viral infection, a bacterial infection, a fungal infection, and / or a parasitic infection, and the cancer includes pancreatic cancer, colon cancer, leukemia, liver cancer, lung cancer, or melanoma.

17. The method according to claim 15, further comprising administering to the subject a therapeutically effective amount of a phosphatidylinositol 3-kinase (PI3K) inhibitor.

18. The method according to claim 15, further comprising administering to the subject a therapeutically effective amount of an immune checkpoint blocker.

19. A pharmaceutical composition according to claim 6 for use in a method for treating cancer or a chronic infection in a subject in need thereof, wherein the method comprises: providing one or more T cells; mixing the one or more T cells in vitro or ex vivo with the pharmaceutical composition to thereby proliferate the one or more T cells; administering a therapeutically effective amount of the proliferated T cells to the subject; comprising the pharmaceutical composition.

20. The pharmaceutical composition according to claim 19, wherein mixing the one or more T cells is performed in combination with an anti-CD3 antibody, an anti-CD28 antibody, and / or a phosphatidylinositol 3-kinase (PI3K) inhibitor.

21. The pharmaceutical composition according to claim 19, wherein mixing the one or more T cells is performed in combination with an immune checkpoint blocker.

22. The pharmaceutical composition according to claim 19, wherein the one or more T cells are derived from the subject.

23. The pharmaceutical composition according to claim 19, wherein the one or more T cells comprise a chimeric antigen receptor.

24. The pharmaceutical composition according to claim 19, wherein the expanded T cells have increased levels of CD28 and / or CD27 compared to pre-expansion levels.

25. The pharmaceutical composition according to claim 19, wherein the expanded T cells have decreased levels of PD-1, TIM-3, and / or Lag3 compared to pre-expansion levels.

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