Compositions and uses of vasoactive intestinal peptide (VIP) antagonists

Abstract

The present disclosure relates to a VIP antagonist for use in the management of the treatment or prevention of cancer and viral infections. In certain embodiments, the present disclosure relates to chimeric variants of VIP antagonists, such as the peptides disclosed herein, and pharmaceutical compositions comprising the same. In certain embodiments, the present disclosure contemplates methods of stimulating immune cells to target cancer by mixing immune cells with a peptide disclosed herein in vitro and further administering an effective amount of the stimulated immune cells to a subject in need of cancer treatment.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 768,060, filed November 15, 2018. The entire contents of this application are incorporated by reference for all purposes.

[0003] Material submitted electronically through the Office's Electronic Filing System (EFS-WEB) as text file

[0004] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 18223PCT_ST25.txt. The text file is 12 KB, created on November 15, 2019, and is being submitted electronically via EFS-Web. BACKGROUND

[0005] Vasoactive intestinal peptide (VIP) is produced in a variety of cells including immune cells, neurons, and endocrine cells in the central nervous system. Because endogenous VIP is present in nerves of airway smooth muscle and pulmonary vessels within the lung, VIP acts as a bronchodilator. VIP also has the ability to alter cell proliferation and production of inflammatory signals through VIP receptors VPAC1 and VPAC2. A chimeric peptide known as VIPhyb has been developed that has an N-terminal sequence that provides membrane permeability followed by a C-terminal 22 amino acid sequence of VIP. VIPhyb has altered biological activity acting as a VIP antagonist as the six N-terminal amino acids of native VIP are replaced. VIP antagonists are also reported in U.S. Patent Nos. 6,630,124 and 5,217,953.

[0006] Traditionally, cancer treatment has generally utilized surgery, chemotherapy, and radiation therapy. However, alternative methods of fortifying the immune system to attack cancer cells have been reported. These methods include collecting, expanding, and altering T cells in order to target and stimulate the immune system to aggressively eliminate cancer cells. In chimeric antigen receptor (CAR) T cell therapy, isolated T cells are engineered to express a chimeric protein and are then returned to the patient for administration. However, there is a need to identify improved therapies.

[0007] Petersen et al. report that administration of VIPhyb enhances autologous anti-leukemia T cell responses in a murine model of acute leukemia. Oncoimmunology, 2017, 6(5): e1304336. Petersen et al. report that ex vivo treatment with a PI3K5 inhibitor and a VIP antagonist improves T cell expansion and function of adoptive T cell therapy. Blood Adv. 2018, 2(3): 210-223.

[0008] The references cited herein are not an admission that the prior art is prior art with respect to the present disclosure. SUMMARY

[0009] The present disclosure relates to a VIP antagonist for the management of the treatment or prevention of cancer and viral infection. In certain embodiments, the present disclosure relates to chimeric variants of VIP antagonists, such as the peptides disclosed herein, and pharmaceutical compositions comprising the same. In certain embodiments, the present disclosure contemplates methods of stimulating immune cells to target cancer by mixing the immune cells with a peptide disclosed herein in vitro and further administering an effective amount of the stimulated immune cells to a subject in need of cancer treatment.

[0010] In certain embodiments, the VIP antagonist is a peptide comprising K P R R P Y X 1 X 2 N X 3 T X 4 L RK Q X 5 A V X 6 K Y X 7 N X 8 I L N(SEQ ID NO: 11), wherein X 1 is A or any amino acid; X 2 is V or any amino acid; X 3 is C or any amino acid; X 4 is S or any amino acid; X 5 is I or any amino acid; X 6 is N or any amino acid; X 7 is M or any amino acid; X 8 is I or any amino acid; and with the proviso that the peptide is not KPRRPYTDNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 1) or a combination wherein X 1 is T, X 2 is D, X 3 is Y, X 4 is R, X 5 is M, X 6 is K, X 7 is L and X 8 is S.

[0011] In certain embodiments, the peptide comprises or consists of K P R R P Y X 1 X 2 N X 3 T X 4 L RK Q X 5 A V X 6K Y X 7 N X 8 I L N(SEQ ID NO: 11), wherein X 1 is A or T, wherein X 2 is V only if X 3 is C, X 4 is S, X 5 is I, X 6 is N, X 7 is M or X 8 is I, X 1 is T only; wherein X 2 is V or D, wherein X 1 is A only if X 3 is C, X 4 is S, X 5 is I, X 6 is N, X 7 is M or X 8 is I, X 2 is D only; wherein X 3 is C or Y, wherein X 1 is A only if X 2 is V, X 4 is S, X 5 is I, X 6 is N, X 7 is M or X 8 is I, X 3 is Y only; wherein X 4 is S or R, wherein X 1 is A only if X 2 is V, X 3 is C, X 5 is I, X 6 is N, X 7 is M or X 8 is I, X 4 is R only; wherein X 5 is I or M, wherein X 1 is A only if X 2 is V, X 3 is C, X 4 is S, X 6 is N, X 7 is M or X 8 is I, X 5 is M only; wherein X 6 is N or K, wherein X 1 is A only if X 2 is V, X 3 is C, X 4 is S, X 5 is I, X 7 is M or X8 is I, X 6 is K only; wherein X 7 is M or L, wherein X 1 is A, X 2 is V, X 3 is C, X 4 is S, X 5 is I, X 6 is N or X 8 is I, X 7 is L only; and wherein X 8 is I or S, wherein X 1 is A, X 2 is V, X 3 is C, X 5 is I, X 6 is N or X 7 is M, X 8 is S only.

[0012] In certain embodiments, the peptide comprises or consists of:

[0013] KPRRPYADNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 3);

[0014] KPRRPYTVNYTRLRKQMAVKKYLNSILN (SEQ ID NO: 4);

[0015] KPRRPYTDNCTRLRKQMAVKKYLNSILN (SEQ ID NO: 5);

[0016] KPRRPYTDNYTSLRKQMAVKKYLNSILN (SEQ ID NO: 6);

[0017] KPRRPYTDNYTRLRKQIAVKKYLNSILN (SEQ ID NO: 7);

[0018] KPRRPYTDNYTRLRKQMAVNKYLNSILN (SEQ ID NO: 8);

[0019] KPRRPYTDNYTRLRKQMAVKKYMNSILN (SEQ ID NO: 9); or

[0020] KPRRPYTDNYTRLRKQMAVKKYLNLILN (SEQ ID NO: 10).

[0021] In certain embodiments, an amino, carboxyl, hydroxyl, or thiol group in a peptide disclosed herein is substituted. In certain embodiments, the peptide is conjugated to a nanoparticle. In certain embodiments, the present disclosure contemplates a peptide disclosed herein having a fluorescent or radioactive label, for example.

[0022] In certain embodiments, the present disclosure relates to a composition comprising a peptide disclosed herein, such as a pharmaceutical composition and a cell growth medium. In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a peptide disclosed herein and a pharmaceutically 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 salt solution. In certain embodiments, the pharmaceutical composition is in the form of a container configured to spray a liquid with a propellant or a sealed container.

[0023] In certain embodiments, the present disclosure relates to a nucleic acid encoding a peptide as disclosed herein in operable combination with a promoter. In certain embodiments, the present disclosure relates to a recombinant vector comprising a nucleic acid encoding a peptide as disclosed herein in operable combination with a promoter. In certain embodiments, the present disclosure relates to an expression system or cell comprising a recombinant vector disclosed herein.

[0024] In certain embodiments, the present disclosure relates to a method of treating or enhancing an immune response against a cancer or treating a cancer, the method comprising administering to a subject in need thereof an effective amount of a peptide disclosed herein. In certain embodiments, the peptide is administered in combination with another chemotherapeutic agent.

[0025] In certain embodiments, the present disclosure relates to a method of enhancing T cell activation and ex vivo expansion, the method comprising mixing T cells with a peptide disclosed herein. In certain embodiments, the T cells are mixed in combination with an anti-CD3 antibody and / or an anti-CD28 antibody. In certain embodiments, the T cells are mixed in combination with a phosphoinositide 3-kinase delta (PI3Kd) inhibitor.

[0026] In certain embodiments, the present disclosure relates to a method of treating or preventing host versus graft disease in a subject, the method comprising administering to a subject who is to receive or is receiving an allogeneic tissue or cell transplant an effective amount of a peptide disclosed herein.

[0027] In certain embodiments, the present disclosure relates to a method of treating a cancer, the method comprising: exposing a subject to radiation and / or administering to the subject a chemotherapeutic agent; transplanting allogeneic hematopoietic stem cells into the subject; and administering to the subject a peptide disclosed herein.

[0028] In certain embodiments, the present disclosure relates to methods of managing viral infection, comprising administering to a subject in need thereof an effective amount of a peptide as disclosed herein.

[0029] In certain embodiments, the subject is a mammal, typically a human. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Antagonistic peptides that differ from the VIPhyb internal amino acids of SEQ ID NO: 1 are shown. The native VIP is SEQ ID NO: 2. The sequences are designated ANT-1 through 8 depending on the presence or absence of internal amino acids (labeled "ANT"). Specifically, ANT-1 has the amino acid T substituted for A at seven of the amino acid positions of SEQ ID NO: 3. The ANT-2 sequence has the amino substitution D for V at amino acid position number 8 of SEQ ID NO: 4. The ANT-3 peptide has the amino acid substitution Y for C at amino acid position number 10 of SEQ ID NO: 5. ANT-4 has the amino acid substitution R for S at amino acid position 12 of SEQ ID NO: 6. ANT-5 has the amino acid substitution M for I at amino acid position number 17 of SEQ ID NO: 7. ANT-6 has the amino acid substitution K for N at amino acid position 20 of SEQ ID NO: 8. ANT-7 has the amino acid substitution L for M at amino acid position number 23 of SEQ ID NO: 9. ANT-8 has the amino acid substitution S for L at amino acid position number 25 of SEQ ID NO: 10.

[0031] Figure 2 Data showing T cell proliferation at 24 hours in the presence of original VIPhyb and Ant 1 through Ant 8 are shown. T cells from luciferase + C57 / BL6 mice were harvested and cultured in 96 well plates with 1 μg / ml anti-CD3 antibody and 30 U / ml IL-2 in the presence of 1 μM of original VIPhyb, Ant-1, Ant-2, Ant-3, Ant-4, Ant-5, Ant-6, Ant-7, or Ant-8.

[0032] Figure 3 Data showing T cell proliferation at 24 hours in the presence of original VIPhyb and Ant-8 are shown. T cells from luciferase + C57 / BL6 mice were harvested and cultured in 96 well plates with 0.5 μg / ml or 1 μg / ml anti-CD3 antibody and 30 U / ml IL-2 in the presence of 0.5 μM, 1 μM, or 3 μM of original VIPhyb or Ant-8.

[0033] Figure 4Data showing subcutaneous injection of mice with VIPhyb, Ant8, or PBS daily for 7 days starting the day after intravenous inoculation of leukemia cells.

[0034] Figure 5 Data showing survival of leukemic mice (C1498) treated with ANT-8.

[0035] Figure 6A Data showing KPC luc (i.e., a pancreatic cancer cell line transfected with murine luciferase) injected into the tail of the pancreas of immunocompetent mice treated with Ant-08 and / or anti-PD1. A slower rate of tumor growth was observed in mice treated with Ant-08 + anti-PD1.

[0036] Figure 6B Data showing tumor burden in orthotopic KPC models. Pancreases isolated from the above mice at sacrifice (day 24) were weighed to give tumor burden. A significantly smaller tumor burden was observed in mice treated with Ant-08 + anti-PD1.

[0037] Figure 6C Data showing CD+4 T cell infiltration in orthotopic KPC models. Increased infiltration of CD4 T cells was observed in tumors of mice treated with Ant-08 + anti-PD1.

[0038] Figure 6D Data showing IVIS and MRI imaging of mice at day 26. Tumors from these mice were stained by H&E and CD4.

[0039] Figure 7 Data showing use of ANT8 to demonstrate enhanced T cell yield from chronic lymphocytic leukemia (CLL) donors during ex vivo expansion with anti-CD3 / 28 beads and 30 U / mL IL-2. DETAILED DESCRIPTION

[0040] Before the present disclosure is described in detail, it is to be understood that the disclosure is not limited to the specific embodiments described herein and, as such, can vary. 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 since the scope of the present disclosure will be limited only by the appended claims.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0042] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0043] As will be apparent to those of ordinary skill in the art in the art in light of the present disclosure, each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0044] The embodiments of the present disclosure will employ, unless otherwise indicated, immunological, medical, organic chemical, biochemical, molecular biological, pharmacological, physiological, and like techniques, which are within the skill of the art. Such techniques are explained fully in the literature.

[0045] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow, reference will be made to a number of terms, which will be defined as follows unless a different meaning is expressly provided or is apparent from the context.

[0046] The terms “protein” and “peptide” refer to compounds comprising amino acids joined by peptide bonds, and are used interchangeably. The amino acids can be natural or non-natural. A “chimeric protein” or “fusion protein” is one in which different parts of the protein are derived from different sources such that the entire molecule is not a naturally occurring molecule. A chimeric protein can contain amino acid sequences from different species of the same species, as long as the amino acid sequences are not arranged together in the same way as they exist in their natural state. Examples of chimeric proteins include the sequences disclosed herein that contain one, two, or more amino acids attached to the C-terminus or N-terminus that are not identical to any naturally occurring protein, as in the case of the addition of an amino acid containing an amine side chain group (e.g., lysine), an amino acid containing a carboxylic acid side chain group (e.g., aspartic acid or glutamic acid), a polyhistidine tag (e.g., typically four or more histidine amino acids).

[0047] With respect to a peptide having an amino acid sequence, the term "comprising" means that the peptide can contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to encompass the amino acid sequence within a larger peptide. With respect to a peptide having an amino acid sequence, the term "consisting of means a peptide having an exact number of amino acids in the sequence and no more or no less than the series of amino acids specifically recited in the claim. In certain embodiments, the present disclosure contemplates that "the N-terminus of a peptide can consist of an amino acid sequence" means a peptide having an exact number of amino acids in the sequence and no more or no less than the series of amino acids specified in the claim, however the C-terminus can be attached to additional amino acids, e.g., as part of a larger peptide. Similarly, the present disclosure contemplates that "the C-terminus of a peptide can consist of an amino acid sequence" means a peptide having an exact number of amino acids in the sequence and no more or no less than the series of amino acids specified in the claim, however the N-terminus can be attached to additional amino acids, e.g., as part of a larger peptide.

[0048] In certain embodiments, the present disclosure relates to recombinant peptides comprising a sequence disclosed herein or a fusion thereof, wherein the amino-terminal or carboxy-terminal end of the amino acid sequence is optionally attached to a heterologous amino acid sequence, a label or reporter molecule. A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or protein, to facilitate detection of said molecule. Specific, non-limiting examples of labels include fluorescent tags, enzyme bonds, and radioisotopes. In one example, a "labeled receptor" refers to the incorporation of a heterologous polypeptide into a receptor. Labeling includes the incorporation of radiolabeled amino acids or the covalent attachment of a biotinyl moiety to a polypeptide, which can be detected by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric means). 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.,3H,14C,35S,125I,131I), fluorescent labels (e.g., fluorescein (FITC), rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, b-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes (e.g., c-myc, HA, FLAG, histidine tract, di- leucine motif, binding sites for secondary antibodies, metal binding domains, epitope tags) recognized by a secondary reagent, or magnetic agents (e.g., gadolinium chelates). In some embodiments, labels are attached through various length spacer arms to reduce potential steric hindrance. 35 S or 131 I), fluorescent labels (e.g., fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, b-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags) recognized by a secondary reagent, or magnetic agents (e.g., gadolinium chelates). In some embodiments, labels are attached through various length spacer arms to reduce potential steric hindrance.

[0049] As used herein, the term "derivative" refers to a structurally similar peptide that retains sufficient functional properties of the identified analog. The derivative can be structurally similar because it lacks one or more atoms, for example, replacing an amino group, a hydroxyl group, or a thiol group with hydrogen, a substituted salt in a different hydration / oxidation state, or because one or more atoms within the molecule are exchanged, such as, but not limited to, replacing an oxygen atom with a sulfur atom or replacing an amino group with a hydroxyl group. The derivative can be a prodrug, including a lipid, a polyethylene glycol, a sugar, a polysaccharide. The derivative can be two or more peptides linked together by a linker group. It is contemplated that the linker group can be biodegradable. The derivative can be prepared by synthetic or various synthetic methods presented in organic chemistry textbooks or appropriate adaptations, such as Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley, 6thedition (2007), Michael B. Smith, or Domino Reactions in Organic Synthesis, Wiley (2006), Lutz F. Tietze, incorporated by reference.

[0050] In certain embodiments, the peptides disclosed herein have at least one non-naturally occurring molecular modification, such as attachment of polyethylene glycol, attachment of another peptide that results in a chimeric peptide, attachment of a chelator including aromatic groups, fluorescent peptides, ability to bind a radionuclide (such as 18 F, N-terminal acetyl, propionyl, myristoyl, and palmitoyl groups, or N-terminal methylation or C-terminal alkyl esters). In certain embodiments, the present disclosure contemplates labeling the peptides disclosed herein using commercially available biotinylation reagents. The biotinylated peptides can be used for streptavidin affinity binding, purification, and detection. In certain embodiments, the present disclosure contemplates that the peptides disclosed herein contain azide derivatives of naturally occurring monosaccharides, such as N-azidoacetylglucosamine, N-azidoacetylmannosamine, and N-azidoacetylgalactosamine.

[0051] In certain embodiments, the present disclosure contemplates derivatives of the peptides disclosed herein, in which one or more amino acids are substituted with a chemical group, optionally connected by a linker, to improve pharmacokinetic properties, such as solubility and serum half-life. In certain embodiments, such derivatives can be prodrugs, in which the substituent or linker is biodegradable, or the substituent or linker is non-biodegradable. In certain embodiments, contemplated substituents include sugars, polysaccharides, acetyl groups, fatty acids, lipids, and / or polyethylene glycol. The substituent can be covalently bonded by forming an amide bond on the C- or N-terminus of the peptide, optionally connected by a linker. In certain embodiments, it is contemplated that the substituent can be covalently bonded by an amino acid within the peptide, for example by an amine side chain group (such as lysine) or a carboxylic acid side chain group (such as aspartic acid or glutamic acid) within a peptide comprising a sequence disclosed herein. In certain embodiments, it is contemplated that the substituent can be covalently bonded by a cysteine to a sequence disclosed herein, optionally connected by a linker. In certain embodiments, the substituent is connected by a linker that forms a disulfide with the cysteine amino acid side group.

[0052] The term "substituted" refers to a molecule in which at least one hydrogen atom is replaced with a substituent group. When substituted, one or more of these groups are "substituents." A molecule can be multiply substituted. In the case of an oxo substituent ("=0"), two hydrogen atoms are replaced. Example substituents within this context can include halogen, hydroxyl, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocyclylalkyl, heterocarbocyclyl, heterocarbocyclylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, -NRaRb, -NRaC(=0)Rb, -NRaC(=0)NRaNRb, -NRaC(=0)ORb, -NRaS02Rb, -C(=0)Ra, -C(=0)ORa, -C(=0)NRaRb, -OC(=0)NRaRb, -ORa, -SRa, -SORa, -S(=0)2Ra, -OS(=0)2Ra, and -S(=0)2ORa. Raand Rbwithin this context can be the same or different and independently hydrogen, halogen, hydroxyl, alkyl, alkoxy, alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocyclylalkyl, heterocarbocyclyl, heterocarbocyclylalkyl, aryl, arylalkyl, heteroaryl, and heteroarylalkyl. Substituents can be further optionally substituted.

[0053] As used herein, a "lipid" group refers to a naturally or non-naturally occurring, highly water-insoluble, hydrophobic group. As used herein, when the point of attachment on a lipid is replaced with hydrogen and the solubility of the resulting compound is less than 0.63 x 10 -4A lipid group is considered highly insoluble in water at % w / w (at 25°C) which is the weight percent solubility of octane in water. See Solvent Recovery Handbook, 2ndEdition, Smallwood, 2002 Blackwell Science, page 195. Examples of naturally occurring lipids include saturated or unsaturated hydrocarbon chains found in fatty acids, glycerolipids, cholesterols, steroids, polyketides, and derivatives. Non-naturally occurring lipids include derivatives of naturally occurring lipids, acrylic polymers, aromatic and alkylated compounds, and derivatives thereof.

[0054] The term "prodrug" refers to a pharmaceutical agent that is converted into a biologically active form in vivo. Prodrugs are often useful because, in some situations, they can be easier to administer than the parent compound. Prodrugs can also have improved solubility over the parent drug, making them useful in pharmaceutical compositions. Prodrugs can be converted to parent drugs by a variety of mechanisms, including enzymatic processes and metabolic hydrolysis. A typical prodrug is a pharmaceutically acceptable ester. Prodrugs include compounds wherein a hydroxy, amino, or mercapto (thiol) group is bonded to any group that, when the prodrug of the active compound is administered to a subject, cleaves to form a free hydroxyl, free amino, or free mercapto group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohols or acetamides in the active compound, formamide and benzamide derivatives of amine functional groups, and the like.

[0055] For example, if the disclosed peptide or pharmaceutically acceptable form of a peptide contains a carboxylic acid functional group, the prodrug can include pharmaceutically acceptable esters formed when the hydrogen atom of the acid group is replaced by a (Ci-C8)alkyl, (C2-C 12 alkanoyloxy methyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1- (alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N- (alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-tetrahydroxyphthalidyl, gamma- butyrolacty-4-yl, di-N,N-(Ci-C2)alkylamino(C2-C3)alkyl such as beta-dimethylaminoethyl, carbamoyl-(Ci-C2)alkyl, N,N-di(Ci-C2)alkylcarbamoyl-(Ci-C2)alkyl, and piperidino-, pyrrolidino- or morpholino(C2-C3)alkyl.

[0056] If the disclosed peptide or the pharmaceutically acceptable form of the peptide incorporates an alcohol functional group, a prodrug can be formed by replacing the hydrogen atom of the alcohol group with a group such as (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 wherein each alpha-aminoacyl is independently selected from naturally occurring L-amino acids P(O)(OH)2, -P(O)(O(C1-C6)alkyl)2, and glycosyl (a radical derived by removal of a hydroxyl group from a carbohydrate in hemiacetal form).

[0057] If the disclosed peptide or the pharmaceutically 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, wherein R and R' are each independently (C1-C6)alkyl, (C3-C7)cycloalkyl, benzyl, naturally occurring alpha-aminoacyl, -C(OH)C(O)OY 10 )alkyl, (C3-C7)cycloalkyl, benzyl, naturally occurring alpha-aminoacyl, -C(OH)C(O)OY l , wherein Y 1 is H, (C1-C6)alkyl or benzyl, -C(OY2)Y3, wherein Y2 is (C1-C4)alkyl and Y3 is (C1-C6)alkyl, carboxy(C1-C6)alkyl, amino(C1-C4)alkyl or mono-N or di-N, N-(C1-C6)alkylaminoalkyl, -C(Y4)Y5, wherein Y4 is H or methyl and Y5 is mono-N- or di-N, N-(C1-C6)alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.

[0058] As used herein, "pharmaceutically acceptable ester" 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 boric acids.

[0059] As used herein, "pharmaceutically acceptable enol ether" includes, but is not limited to, derivatives of the formula -C=C(OR), wherein R can be selected from alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl. "Pharmaceutically acceptable enol ester" includes, but is not limited to, derivatives of the formula -C=C(OC(O)R), wherein R can be selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl.

[0060] "Linking group" refers to any kind of molecular arrangement that can be used to bridge molecular moieties together. An example formula can be -Rm - where R is chosen individually and independently for each occurrence: -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)-, can each contain a single, double, or triple bond between the R groups. If R has R... m Branched chains can be capped with groups such as -CH3, -H, -CH=CH2, -CCH, -OH, -SH, -NH2, -N3, -CN, or -Hal, or the R groups of two branches can form a cyclic structure. It is conceivable that, in some cases, the total R or "m" can be less than 100, 50, 25, or 10. Examples of linking groups include bridging alkyl and alkoxyalkyl groups. Linking groups can be substituted with one or more substituents.

[0061] The term "nucleic acid" refers to a polymer of nucleotides or polynucleotides. This term is used to specify a single molecule or a collection of molecules. Nucleic acids can be single-stranded or double-stranded and can contain coding regions and regions of various control elements, as described below.

[0062] The term "nucleic acid sequence encoding a specified peptide" refers to a nucleic acid sequence that includes the coding region of the peptide, or in other words, a nucleic acid sequence encoding a peptide product. The coding region can exist in the form of cDNA, genomic DNA, or RNA. When present in DNA form, the oligonucleotide, polynucleotide, or nucleic acid can be single-stranded (i.e., sense strand) or double-stranded. If it is necessary to allow proper initiation of transcription and / or correct processing of the primary RNA transcript, suitable control elements (such as enhancers / promoters, splice sites, polyadenylation signals, etc.) can be placed near the coding region. Alternatively, the coding region utilized in the expression vector can contain endogenous enhancers / promoters, splice sites, insertion sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous control elements.

[0063] The term "vector" or "expression vector" refers to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in, for example, a cellular or cell-free, specific host organism or expression system. Nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, operator (optional), and ribosome binding site, often together with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, as well as termination and polyadenylation signals.

[0064] A protein "expression system" refers to in vivo and in vitro (cell-free) systems. Systems for reconstituted protein expression typically utilize cells transfected with a DNA expression vector containing a template. Cells are cultured under conditions such that the cells translate the desired protein. The expressed protein is extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Likewise, some proteins are recovered using denaturants and protein refolding procedures. In vitro (cell-free) protein expression systems typically use whole cell or a composition's translation-compatible extract containing components sufficient 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 a protein of interest. Cell-free systems typically do not contain proteases and are capable of labeling proteins with modified amino acids. Some cell-free systems incorporate encoded components to translate an expression vector. See, for example, Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotectmol., 19, 751-755 and Asahara and Chong, Nucleic Acids Research, 2010, 38(13): el41, both of which are incorporated by reference in their entireties.

[0065] "Cancer" refers to any of various cellular diseases characterized by the uncontrolled proliferation of cells that are malignant neoplasms. The cells of the disease do not necessarily have to invade surrounding tissues and metastasize to new body sites in practice. Cancer can involve any tissue of the body and has many different forms at each body site. In the context of certain embodiments, "whether cancer is reduced" can be identified by a variety of diagnostic means known to one of skill in the art, including but not limited to observing a reduction in the size or number of tumor masses or if an increase in cancer cell apoptosis is observed, for example, if an increase in cancer cell apoptosis of more than 5% is observed for a sample compound compared to a control without the compound. It can also be identified by changes in relevant biomarkers or gene expression profiles, such as PSA for prostate cancer, HER2 for breast cancer, and the like.

[0066] The cancer to be treated in the context of the present disclosure can be any type of cancer or tumor. These tumors or cancers include, but are not limited to, tumors of hematopoietic and lymphoid tissues or hematopoietic and lymphoid malignancies, tumors affecting the blood, bone marrow, lymph, and lymphatic system. Hematological malignancies can arise from either of two major blood cell lineages: the myeloid lineage and the lymphoid lineage. The myeloid lineage generally gives rise to granulocytes, red blood cells, platelets, macrophages, and mast cells; the lymphoid lineage gives rise to B cells, T cells, NK cells, and plasma cells. Lymphomas, lymphocytic leukemias, and myelomas are of lymphoid origin, while acute and chronic myelogenous leukemias, myelodysplastic syndrome, and myeloproliferative disorders are of myeloid origin.

[0067] It also envisions malignant tumors located in the colon, abdomen, bones, breasts, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eyes, head and neck, (central and peripheral) nervous system, lymphatic system, pelvis, skin, soft tissues, spleen, chest, and genitourinary organs, and more specifically, childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult (primary) hepatocellular carcinoma, adult (primary) liver cancer, adult acute lymphoblastic leukemia, adult acute myeloid leukemia, and adult Hodgkin's disease. 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 tumors, anal cancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer, brainstem 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, pediatric (primary) liver cancer, pediatric acute lymphoblastic leukemia, pediatric acute myeloid leukemia, pediatric brainstem glioma, pediatric cerebrospinal leukemia, pediatric cerebrospinal glioma, pediatric cerebrospinal leukemia, pediatric cerebrospinal leuk ... Astrocytoma of the brain, pediatric astrocytoma, pediatric extracranial germ cell tumors, pediatric Hodgkin's disease, pediatric Hodgkin's lymphoma, pediatric visual pathway and hypothalamic glioma, pediatric lymphoblastic leukemia, pediatric medulloblastoma, pediatric non-Hodgkin's lymphoma, pediatric supratentorial primitive neuroectodermal and pineal tumors, pediatric primary liver cancer, pediatric rhabdomyosarcoma, pediatric soft tissue sarcoma, pediatric visual pathway and hypothalamic glioma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, cutaneous T-cell lymphoma, endocrine pancreatic islet cell carcinoma, endometrial cancer, ependymoma, epithelial carcinoma, esophageal cancer, Ewing's sarcoma. Sarcoma and related tumors, exocrine pancreatic cancer, extracranial germ cell tumors, gonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, female breast cancer, Gaucher's disease, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal tumors, germ cell tumors, gestational trophoblastic tumors, head and neck cancer, hepatocellular carcinoma, Hodgkin's disease, Hodgkin's lymphoma, hypergammaglobulinemia, hypopharyngeal cancer, colorectal cancer, intraocular melanoma, islet cell carcinoma, islet cell pancreatic carcinoma, Kaposi's sarcoma.sarcoma), kidney cancer, laryngeal and lip and oral cavity cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer with occult primary, multiple myeloma, multiple myeloma / plasmacytoma, myelodysplastic syndrome, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma during pregnancy, non-melanoma skin cancer, non-small cell lung cancer, primary squamous neck cancer with occult primary, oropharyngeal cancer, malignant fibrous histiocytoma, malignant fibrous osteosarcoma / osteocytoma, epithelial ovarian cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, paraproteinemia, purpura, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, plasmacytoma / 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 neck cancer, stomach cancer, pineal and supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, transitional renal pelvis and ureter cancer, trophoblastic tumor, renal pelvis and ureter cell cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and any other hyperproliferative disease and neoplasia located in any of the previously mentioned organ systems.

[0068] "Chemotherapy agents," "chemotherapy," and "anticancer agents" refer to molecules believed to be helpful in cancer treatment. Envisioned examples include molecules or derivatives such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, temozolomide, ado-trastuzumab emtansine, denileukin diftitox, blinatumomab, and interferon-alpha. Alpha, interleukin, carmustine, bevacizumab, procarbazine, lomustine, vincristine, gefitinib, erlotinib, cisplatin, carboplatin, oxaliplatin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine (The following are listed as unrelated to the previous sentence and are likely separate entries): arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mitramycin, vinblastine, vindesine, vinorelbine, paclitaxel, taxol, docetaxel, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene.raloxifene, droloxifene, fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorozole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, azacitidine, azathioprine, capecitabine, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, doxifluridine, epothilone, irinotecan, mechlorethamine, mercaptopurine, mitoxantrone, pemetrexed, tioguanine, valrubicin, rituximab and / or lenalidomide or combinations thereof, such as cyclophosphamide, methotrexate, 5-fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); adriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone (RCHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); methotrexate, vincristine,Doxorubicin, cisplatin (MVAC).

[0069] In certain embodiments, the treatment of cancer can be combined with another anti-cancer agent. In certain embodiments, the anti-cancer agent is selected from the group consisting of abemaciclib, abiraterone acetate, methotrexate, paclitaxel, doxorubicin, acalabrutinib, bendamustine, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant, palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexed disodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine, aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronate disodium, exemestane, nelarabine, arsenic trioxide, ofatumumab, atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib, azacitidine, carmustine, belinostat, bendamustine, inotuzumab, bevacizumab, bosutinib, bortezomib, brentuximab vedotin, busulfan, cabozantinib, capecitabine, carfilzomib, cetuximab, cladribine, crizotinib, cytarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daratumumab, darbepoetin alfa, denileukin diftitox, denosumab, docetaxel, doxorubicin, epirubicin, epoetin alfa, erlotinib, estramustine, etoposide, everolimus, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin, goserelin, histrelin, ibritumomab tiuxetan, idarucicin, ifosfamide, imatinib, interferon alfa, interferon alfa-n3, interferon alfa-n1, interferon alfa-2b, interferon alfa-2a, interferon alfacon-1, interferon alfa-n2, interferon alfa-n3, interferon alfa-n1, interferon alfa-2b, interferon alfa-2a, interferon alfacon-1, interferon alfa-n2, interferon alfa-n3, interferon alfa-n1, interferon alfa-2b, interferon alfa-2a, interferon alfacon-1, interferon alfa-n2, interferon alfa-n3, interferon alfa-n1, interferon alfa-2b, interferon alfa-2a, interferon alfacon-1, interferon alfa-n2, interferon alfa-n3, interferon alfa-n1, interferon alfa-2b, interferon alfa-2a, interferon alfacon-1, interferon alfa-n2,ozogamicin), bevacizumab, bexarotene, bicalutamide, bleomycin, blinatumomab, bortezomib, bosutinib, brentuximab vedotin, (brigatinib, busulfan, irinotecan, capecitabine, fluorouracil, carboplatin, carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine, cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate, dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine, dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib, defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone, dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab, rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine, enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide, everolimus, raloxifene, toremifene, panobinostat, fulvestrant, letrozole, filgrastim, fludarabine, flutamide, pralatrexate, obinutuzumab, gefitinib, gemcitabine, gemtuzumabozogamicin), glucarpidase, goserelin, propranolol, trastuzumab, topotecan, palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin, idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin, ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin, pembrolizumab, ribociclib, tisagenlecleucel, lanreotide, lapatinib, olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine, trifluridine, olaparib, vincristine, procarbazine, mechlorethamine, megestrol, trametinib, temozolomide, methylnaltrexone bromide, naltrexone, nalmefene, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefene hydrochloride, nalmefenebromide), midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine, necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib, nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase, ondansetron, osimertinib, panitumumab, pazopanib, interferon alpha-2b, pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab, rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus, thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine, vemurafenib, vorinostat, zoledronic acid, or combinations thereof.

[0070] In certain embodiments, the method of administration is in a subject having a lymphoid-depleted environment. In certain embodiments, the lymphodepleting agent is cyclophosphamide and fludarabine.

[0071] As used herein, the term "idelalisib" refers to the compound (S)-2-(l-(9H-purin-6- ylamino)propyl)-5-fluoro-3-phenylquinazolin-4(3H)-one or an alternative salt thereof.

[0072] As used herein, "T cells negative for CD28 and / or CD27" refers to a relative low expression or lack of expression of these markers when compared to normal T cells expressing CD3 surface antigen markers in healthy subjects.

[0073] The term“fluorescence activated cell sorting” or“FACS” refers to a method of sorting a mixture of cells into two or more regions (usually one cell at a time) based on the fluorescence properties of each cell, an applied charge, and movement through an electrostatic field. Typically, a vibrating mechanism causes the cell stream to split into individual droplets. Before the droplets form, the cells in the fluid pass through a region for measuring the fluorescence of the cells. A charging mechanism is configured at the point where the stream splits into droplets. Based on the fluorescence intensity measurement, a corresponding charge is applied to the droplet as it breaks off from the stream. The charged droplets then move through an electrostatic deflection system that diverts the droplets into individual regions based on their relative charge. In some systems, the charge is applied directly to the stream and the interrupted droplets retain the same sign of charge as the stream. After the droplet is interrupted, the stream then returns to neutral. In other systems, the charge is provided on a conduit, inducing an opposite charge on the droplet. The cells are typically made to fluoresce by mixing them with antibodies specific to a marker that fluoresces by conjugation to a fluorescent molecule. However, other methods of making cells fluoresce are contemplated, such as by using molecular beacons.

[0074] “Minimum essential medium” refers to a medium containing salts of calcium, magnesium, potassium, sodium, phosphate, and bicarbonate, vitamins, and essential amino acids. The twelve essential amino acids are: L-arginine; L-cystine; L-glutamine; L-histidine; L-isoleucine; L-leucine; L-methionine; L-phenylalanine; L-threonine; L-tryptophan; L-tyrosine; and L-valine. MEM is often supplemented with components such as bicarbonate or glutamine. In certain embodiments, the present disclosure contemplates a minimum essential medium supplemented with the following non-essential amino acids: L-ala; L-asn; L-asp; L-glu; L-gly; L-pro; and L-ser. In certain embodiments, the present disclosure contemplates a minimum essential medium supplemented with nucleosides (ribonucleosides and / or deoxyribonucleosides).

[0075] The term“recombinant” when referring to a nucleic acid molecule refers to a nucleic acid molecule comprising segments of nucleic acids joined together by means of molecular biological techniques. The term“recombinant” when referring to a protein or polypeptide refers to a protein molecule expressed using a recombinant nucleic acid molecule. The term recombinant nucleic acid is distinct from a natural recombinant produced by a crossover between homologous chromosomes. As used herein, a recombinant nucleic acid is an unnatural combination of nucleic acids from non-homologous sources, typically from different organisms.

[0076] The term "vector" or "expression vector" refers to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in, for example, a cellular or cell-free, specific host organism or expression system. Nucleic acid sequences necessary for expression in prokaryotes typically include a promoter, an operator (optional), and a ribosome-binding site, often with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, as well as termination and polyadenylation signals.

[0077] The terms "vasoactive intestinal peptide" and "VIP" refer to (SEQ ID NO: 2) HSDAVFTDNYTRLRKQMAVKKYLNSILN, unless context dictates otherwise. VIP is a multifunctional endogenous polypeptide that modulates both innate and adaptive immunity at multiple levels of immune cell differentiation and activation. VIP is commonly secreted by a variety of cells such as neurons (in both the central nervous system and the peripheral nervous system), B cells, T cells, and helper cells. VIP and the closely related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) bind to three known receptors, VPAC1, VPAC2, and PAC1. T cells and dendritic cells (DCs) are believed to express VPAC1 and VPAC2, but not PAC1. PAC1 is primarily expressed on neurons and endocrine cells of the brain and pituitary, and adrenal glands, and selectively binds PACAP in most forms.

[0078] "Subject" refers to any animal, preferably a human patient, a farm animal, or a domestic pet.

[0079] As used herein, the terms "prevent," "preventing," and "prevention" include prophylaxis of recurrence, transmission, or onset. The disclosure is not intended to be limited to complete prevention. In some embodiments, onset is delayed, or the severity of the disease is reduced.

[0080] As used herein, the terms "treat," "treating," and "treatment" are not limited to situations when a subject (e.g., a patient) is cured and the disease is eradicated. Rather, embodiments of the disclosure also contemplate treatment where symptoms are merely alleviated and / or the progression of a disease is slowed.

[0081] As used herein, the term "in combination with" when used to describe administration with another therapy means that the agent can be administered prior to, with, or after the other therapy, or a combination thereof.

[0082] Compositions

[0083] In certain embodiments, the present disclosure contemplates a pharmaceutical composition comprising a peptide disclosed herein or nanoparticles thereof, or optionally other agents, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0084] In certain embodiments, the present disclosure relates to compositions, such as pharmaceutical compositions and cell growth media, comprising a peptide disclosed herein. In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a peptide disclosed herein and a pharmaceutically 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 salt solution. In certain embodiments, the pharmaceutical composition is in the form of a container configured to spray a liquid with a propellant or a sealed container.

[0085] Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage administered to a patient typically is in the range from 0.0001 mg / kg to 100 mg / kg of patient body weight for a peptide disclosed herein or nanoparticles thereof or other agents. Preferably, the dosage administered to a patient is between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 mg / kg and 2 mg / kg, 0.0001 mg / kg and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 mg / kg to 0.15 mg / kg, 0.0001 mg / kg to 0.10 mg / kg, 0.001 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.25 mg / kg, or 0.01 mg / kg to 0.10 mg / kg of patient body weight. Further, the dosage and frequency of administration of a peptide disclosed herein or nanoparticles thereof or agents can be reduced by modifications that enhance uptake and tissue penetration, such as lipidization and inclusion of natural or artificial lung surfactants.

[0086] Compositions include drug substance compositions that can be used to manufacture pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions that can be used to prepare unit dosage forms (i.e., compositions suitable for administration to a subject or patient). Such compositions include a prophylactically or therapeutically effective amount of a prophylactic and / or therapeutic agent disclosed herein or those agents in combination with a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition contains a pharmaceutically acceptable excipient that is a solubilizing agent such as a lipid, cholesterol, a fatty acid, a fatty acid alkyl ester, linoleic acid, oleic acid, arachidonic acid, a sugar, a polysaccharide, a cyclodextrin, 2-hydroxypropyl (cyclodextrin), or combinations thereof.

[0087] In certain embodiments, the pharmaceutically acceptable excipient is selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium croscarmellose, polyvinyl N-pyrrolidone, cross-linked polyplasdone, ethyl cellulose, povidone, methyl acrylate and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, dodecyl sulfates, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, ferric oxide, glyceryl triacetate, gum acacia, esters or salts thereof.

[0088] In certain embodiments, the pharmaceutical composition is in a solid form, surrounded by an enteric coating (i.e., a polymeric barrier applied to orally administered drugs to prevent their dissolution or disintegration in the stomach environment). Compounds typically found in enteric coatings include methyl acrylate-methacrylic acid copolymer, cellulose acetate phthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, and combinations thereof.

[0089] In particular embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant) (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such a pharmaceutical carrier can be both a naturally occurring and a synthetic lung surfactant, a sterile liquid such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0090] Generally, the ingredients of the compositions are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed by a infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0091] The compositions can be formulated in neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0092] One embodiment provides a pharmaceutical pack or kit comprising one or more containers filled with a peptide disclosed herein or nanoparticles thereof or an agent disclosed herein. Additionally, one or more other prophylactic or therapeutic agents useful for treating a disease can also be included in the pharmaceutical pack or kit. One embodiment provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of a pharmaceutical composition. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the same.

[0093] In certain embodiments, the present disclosure contemplates a pharmaceutical composition comprising a peptide disclosed herein or nanoparticles thereof and an agent disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the present disclosure contemplates the production of a medicament comprising a peptide disclosed herein or nanoparticles thereof or an agent disclosed herein and the use for the methods disclosed herein.

[0094] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a peptide disclosed herein or nanoparticles thereof and an agent disclosed herein and a pharmaceutically acceptable excipient. In certain embodiments, the composition is in the form of a pill or capsule, or the composition is an aqueous buffer, for example, with a pH between 6 and 8. In certain embodiments, the pharmaceutically acceptable excipient is selected from a filler, a glidant, a binder, a disintegrant, a lubricant, and a sugar.

[0095] Compositions suitable for parenteral injection can include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable (such as olive oil) and sesame oils, formulations in lung surfactants (both natural and artificial), and injectable organic esters such as ethyl oleate.

[0096] Prevention of the action of microorganisms can be achieved by the addition of any of the usual antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It can also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0097] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the peptides disclosed herein or nanoparticles or agents thereof can be mixed with at least one inert, pharmaceutically-acceptable excipient (or carrier) such as sodium citrate or dicalcium phosphate or: (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) humectants such as glycerol; (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solution retarders such as paraffin; (f) absorption accelerators such as quaternary ammonium compounds; (g) moisturizing agents such as glycerol and sorbitol; (h) respiration agents such as kaolin and bentonite; and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms can also comprise buffering agents.

[0098] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the peptides disclosed herein or nanoparticles and agents thereof, the liquid dosage forms can contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, oils, in particular cottonseed, groundnut, corn, germ, olive, castor, and sesame oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances, and the like.

[0099] In certain embodiments, manufacturing processes are envisioned in which two components of the peptides disclosed herein or nanoparticles and agents disclosed herein have been provided in a combined dry form ready to be reconstituted together. In other embodiments, it is envisioned that the peptides disclosed herein or nanoparticles and optionally agents disclosed herein can be mixed with a pharmaceutical carrier to provide a pharmaceutical composition.

[0100] A pharmaceutical composition can be provided in a one-step process simply by adding a suitable pharmaceutically-acceptable diluent to the composition in a container. In certain embodiments, the container is preferably a syringe to administer the reconstituted pharmaceutical composition upon contact with the diluent. In certain embodiments, the peptides disclosed herein or nanoparticles or agents thereof can be filled into a syringe and the syringe can then be closed with a stopper. The diluent should be in an amount to achieve the desired final concentration. The pharmaceutical composition can contain other useful components such as ions, buffers, excipients, stabilizers, and the like.

[0101] A "dry" pharmaceutical composition typically has only a residual moisture content, which can correspond roughly to the moisture content of a comparable commercial product, e.g., with about 12% moisture as a dry product. Typically, the residual moisture content of a dry pharmaceutical composition according to the present application is preferably below 10% moisture, more preferably below 5% moisture, in particular below 1% moisture. The pharmaceutical composition can also have a lower moisture content, e.g., 0.1% or even lower. In certain embodiments, the pharmaceutical composition should be provided in dry form to prevent degradation and to achieve storage stability.

[0102] The container can be any container suitable for containing (and storing) the pharmaceutical composition, such as an inhaler, a syringe, a vial, a tube, etc. The pharmaceutical composition can then be applied by actuation or a specific needle of a syringe or through a suitable catheter. Typical diluents include water for injection and NaCI (preferably 50 to 150 mM, in particular 110 mM), CaCI2(preferably 10 to 80 mM, in particular 40 mM), sodium acetate (preferably 0 to 50 mM, in particular 20 mM), and mannitol (preferably up to 10% w / w, in particular 2% w / w). Preferably, the diluent can also comprise a buffer or buffer system in order to buffer the pH of the reconstituted dry composition, preferably at a pH of 6.2 to 7.5, in particular at a pH of 6.9 to 7.1.

[0103] In certain embodiments, the present disclosure contemplates a kit comprising a pharmaceutical composition as disclosed herein (such as a peptide as disclosed herein) or a nanoparticle or medicament thereof and, optionally, a container with a suitable diluent. Further components of the kit can be instructions for use, an application device (such as an inhaler, a syringe, a catheter, a brush, etc. (if not already provided with the composition in the application device)) or other components necessary for use in a medical (surgical) practice, such as a replacement needle or catheter, an additional vial or a further wound covering device. In certain embodiments, the kit comprises a syringe containing the dry and stable hemostatic composition and a syringe containing a diluent (or provided to draw the diluent from another diluent container).

[0104] In certain embodiments, the diluent is provided in a separate container. This can preferably be a syringe. The diluent in the syringe can then be easily applied to the container to reconstitute the dry composition. If the container is also a syringe, the two syringes can be packaged together in a pack. Thus, it is preferred to provide the dry composition in a syringe packaged with a diluent syringe having a pharmaceutically acceptable diluent for reconstituting the dry and stable composition.

[0105] It is contemplated that any of the peptides disclosed herein can be modified with a hydrocarbon or polyethylene glycol group in order to provide improved properties such as solubility, bioavailability, and / or biodegradation.

[0106] In certain embodiments, the present disclosure relates to methods of coupling a peptide disclosed herein to a nanoparticle. In certain embodiments, the nanoparticle comprises a poloxamer stabilized sulfurized polypropylene. In certain embodiments, the nanoparticle has a diameter between 10 and 100 nm. In certain embodiments, the nanoparticle has a diameter between 20 and 50 nm, preferably 30 nm.

[0107] In certain embodiments, the present disclosure contemplates the use of a particle disclosed herein when the peptide sequence coupled to the nanoparticle contains a peptide disclosed herein plus a C-terminal linker peptide GGGGSC (SEQ ID NO: 15). In certain embodiments, the particle contains a peptide sequence such as KPRRPYTDNYTRLRKQMAVKKYLNLILNGGGGSC (SEQ ID NO: 12). In certain embodiments, the chemical linkage between the peptide disclosed herein and the nanoparticle is a disulfide bond.

[0108] In certain embodiments, the pharmaceutically acceptable excipient is a propellant or a phospholipid. In certain embodiments, the propellant is a hydrofluorocarbon, 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane, propane, n-butane, isobutene, carbon dioxide, air, nitrogen, nitrous oxide, dimethyl ether, trans 1,3,3,3-tetrafluoroprop-1-ene, or a combination thereof. In certain embodiments, the phospholipid is dipalmitoyl phosphatidylcholine, palmitoyl-oleoyl phosphatidylglycerol, phosphatidylglycerol, or a combination thereof.

[0109] In certain embodiments, the pharmaceutical composition can be stored in a nebulizer, inhaler, or other container optionally sealed or under pressure for propelling one or more pharmaceutical agents. The container can contain a hand-actuated or pressurized spray device. Metered dose inhalers (MDIs) generally have a hand-held aerosol canister that releases a metered amount of a drug for inhalation once it is propelled. Dry powder inhalers (DPIs) do not use a propellant to release the drug. Rather, the peptide or nanoparticles thereof or agents in dry powder form are inhaled into the lungs after a breath. In certain configurations, the container comprising a peptide disclosed herein or nanoparticles thereof is inserted into a device. Pressing a button or portion on the device punctures the container. The powder contained in the container can be inhaled through a mouthpiece on the device.

[0110] In certain embodiments, the pharmaceutical composition can contain a naturally or non-naturally occurring pulmonary surfactant composition. Envisioned natural pulmonary surfactant compositions generally include 70-90% phospholipids (PCs) such as dipalmitoyl phosphatidylcholine (DPPC), phosphatidylcholine, and phosphatidylglycerol (PG); and 1-10% surfactant-associated proteins, apolipoprotein SP-A (SFTPA1), B (SFTPB), C (SFTPC), and D (SFTPD) (SP stands for "surfactant-associated protein"); and 1-10% cholesterol (neutral lipid). Artificial pulmonary surfactants include colfosceril palmitate, a mixture of DPPC added as a dispersing agent with cetanol and tyloxapol; pumactant (artificial lung expansion compound or ALEC), a mixture of DPPC and PG; KL-4, which consists of DPPC, palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined with a 21-amino acid synthetic peptide that mimics structural properties of SP-B; and a composition consisting of DPPC, PG, palmitic acid, and recombinant SP-C, which shares nearly identical sequence with human SP-C except for the absence of a palmitoylated cysteine and has been replaced by phenylalanine to eliminate protein oligomerization. Envisioned animal-derived surfactants include: beractant (Alveofact TM ) extracted from bovine lung lavage fluid and (Survanta TM ) extracted from minced bovine lung along with additional DPPC, palmitic acid, and tripalmitin; calfactant (Infasurf TM ) extracted from calf lung lavage fluid; and poractant alfa (Curosurf TM ) extracted from material derived from minced pig lung.

[0111] In certain embodiments, the pharmaceutical compositions disclosed herein further include a respiratory agent selected from the group consisting of glucocorticoid receptor agonists (steroids and non-steroids) such as triamcinolone, triamcinolone acetonide, prednisone, mometasone furoate, loteprednol etabonate, fluticasone propionate, fluticasone furoate, fluocinolone acetonide, dexamethasone cipecilate, disisobutyryl ciclesonide, clobetasol propionate, ciclesonide, budesonide, beclomethasone dipropionate, alclometasone dipropionate; p38 antagonists such as losmapimod; phosphodiesterase (PDE) inhibitors such as methylxanthines, theophylline, and aminophylline; selective PDE isozyme inhibitors, PDE4 inhibitors, and isoform PDE4D such as tetomilast, roflumilast, oglemilast, ibudilast; modulators of chemokine receptor function such as maraviroc, cenicriviroc, navarixin; leukotriene biosynthesis inhibitors, 5-lipoxygenase (5-LO) inhibitors, and 5-lipoxygenase activating protein (FLAP) antagonists such as TA270 (4-hydroxy-1-methyl-3-octyloxy-7-senecioylamino-2(1H)-quinolinone), such as licofelone, zileuton, zafirlukast, or montelukast; and myeloperoxidase antagonists such as resveratrol and piceatannol.

[0112] In certain embodiments, the present disclosure relates to an in vitro cell culture composition comprising a minimal essential medium and a peptide disclosed herein or a nanoparticle thereof.

[0113] Methods of use

[0114] In certain embodiments, the present disclosure relates to a peptide disclosed herein, e.g., Ant-8, KPRRPYTDNYTRLRKQMAVKKYLNLILN (SEQ ID NO: 10), for use in a method of treating cancer. In certain embodiments, the present disclosure contemplates a method of treating cancer comprising administering an effective amount of a peptide disclosed herein, optionally in combination with a chemotherapeutic agent. In certain embodiments, the present disclosure relates to a method of using a peptide disclosed herein, optionally conjugated to a nanoparticle, in combination with an antibody to an immune checkpoint molecule or enhancing an immune response to a cancer in a subject in need thereof.

[0115] In certain embodiments, the subject is diagnosed with a cancer selected from the group consisting of melanoma, lung cancer, kidney cancer, leukemia, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, myeloma, bladder cancer, pancreatic cancer, gastric cancer, esophageal cancer, glioblastoma, colon cancer, breast cancer, and prostate cancer.

[0116] In certain embodiments, the antibody to an immune checkpoint molecule is selected from the group of anti-PD1 antibodies comprising pembrolizumab (Keytruda TM ) and nivolumab (Opdivo TM ). In certain embodiments, the antibody to an immune checkpoint molecule is selected from the group of anti-PDL1 antibodies comprising atezolizumab (Tecentriq TM ), avelumab (Bavencio), and durvalumab (Imfinzi TM ). In certain embodiments, the antibody to an immune checkpoint molecule is ipilimumab (Yervoy TM ).

[0117] In certain embodiments, the peptide therapeutic is administered by intravenous or subcutaneous injection. In certain embodiments, the peptide therapeutic is administered by inhalation to deliver the drug to the alveoli. In certain embodiments, the peptide therapeutic is administered to a hand-held delivery device driven by compressed gas. In certain embodiments, the peptide therapeutic is dissolved in a sterile saline solution and administered as an aerosol.

[0118] In certain embodiments, it is contemplated that the peptides disclosed herein can be used in certain cellular immunotherapies effective for treating cancer, such as lymphocyte infusions or allogeneic bone marrow transplantation. Donor immune cells, specifically NK cells and T cells, have anticancer cytotoxic activity. VIP antagonism of the peptides enhances the cellular immune response in vivo. VIP antagonism increases the cytotoxic activity of antigen-specific T cells and NK cells. It is contemplated that VIP antagonism increases the anticancer activity of NK cells or antigen-specific T cells. It is contemplated that VIP antagonism in combination with cellular immunotherapy increases the efficacy of the therapy. It is believed that the absence of VIP in recipients of allogeneic bone marrow transplantation does not increase the activity of "off-target" graft versus host disease of donor lymphocytes. Thus, administration of a VIP antagonist to a subject having cancer who is receiving a cell therapy (e.g., donor lymphocyte infusion or allogeneic bone marrow transplantation) will increase the anticancer activity of the therapy.

[0119] In certain embodiments, the present disclosure relates to methods of enhancing an immune response to a cell therapy, the methods comprising administering to a subject a peptide disclosed herein in combination with a cell. In certain embodiments, the subject is diagnosed with leukemia or lymphoma. In certain embodiments, the cell is a blood cell, a bone marrow cell, a white blood cell, a T cell, a natural killer cell, a hematopoietic stem cell, a G-CSF mobilized or non-mobilized blood mononuclear cell.

[0120] In certain embodiments, the cell is 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 cell is a bone marrow cell. In certain embodiments, the cell is a blood mononuclear cell comprising / expressing granulocyte colony stimulating factor. The cell therapy can be performed with non-mobilized blood mononuclear cells.

[0121] In certain embodiments, it is contemplated that the peptides disclosed herein can be administered to a subject prior to, during, or after a cell-based immunotherapy comprising a recipient or a donor. The immunotherapy can be performed in combination with chemotherapy and / or radiation therapy. It is contemplated that the peptides can 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.

[0122] In certain embodiments, the present disclosure relates to a method of treating or preventing graft versus host disease in a subject, the method comprising administering to a subject who has undergone hematopoietic stem cell transplantation or to a subject who is about to receive or has received a transplant of allogeneic tissue or cells, an effective amount of a peptide disclosed herein. In certain embodiments, the subject has received a transplant of allogeneic hematopoietic stem cells. In certain embodiments, the subject has received a transplant of allogeneic hematopoietic stem cells isolated from peripheral blood. In certain embodiments, the subject has received chemotherapy to be treated with radiation prior to receiving a transplant of allogeneic hematopoietic stem cells.

[0123] In certain embodiments, the present disclosure relates to a method of treating cancer by performing stem cell transplantation, the method comprising administering to a subject a peptide disclosed herein in combination with a transplant of pluripotent hematopoietic stem cells derived from the subject (autologous) or a donor. Stem cells can be collected from peripheral blood, such as umbilical cord blood or placental derived stem cells, or from bone marrow. To limit the risk of rejection of the transplanted stem cells or severe graft versus host disease, the donor will typically have substantially the same human leukocyte antigens (HLA) as the recipient; however, the donor can be mismatched for certain antigens.

[0124] In certain embodiments, the present disclosure relates to a method of providing a lymphocyte infusion after hematopoietic progenitor cell transplantation to treat a hematological malignancy (e.g., a blood or bone marrow cancer, such as leukemia or lymphoma). Typically, the transplant recipient is infused with lymphocytes obtained from the original allogeneic stem cell (hematopoietic progenitor cell) donor in leukocyte-depletion.

[0125] In certain embodiments, the present disclosure relates to extracting lymphocytes from blood and expanding in vitro against one or more tumor antigens, and optionally exposing the cells with appropriate stimulatory cytokines and / or a peptide disclosed herein.

[0126] In certain embodiments, the present disclosure relates to a method of enhancing a local immunotherapy, the method comprising administering a peptide disclosed herein in combination with providing an immune-enhancing cream that includes a drug that causes the production of interferon T cell activation, such as imiquimod.

[0127] In certain embodiments, it is contemplated that the peptides disclosed herein can be used in combination with adoptive cell therapy. For example, T cells that have a naturally occurring reactivity to a cancer can be found to infiltrate a tumor in a subject. The tumor can be harvested and these tumor infiltrating lymphocytes (TILs) can be expanded or made more potent in vitro using interleukin-2 (IL-2), anti-CD3, and allogeneic reactive feeder layers. These T cells can then be transferred back into the subject along with administration of a VIP antagonist. Prior to reinfusion, the recipient is typically subjected to lymphodepletion to eliminate regulatory T cells as well as normal endogenous lymphocytes that compete with the transferred cells. It is also contemplated that adoptive cell transfer of lymphocytes can be transduced with vectors encoding T cell receptors (TCRs) that recognize cancer antigens.

[0128] In certain embodiments, the present disclosure relates to methods of potentiating T cell activation and ex vivo expansion by co-incubating human T cells with nanoparticles containing small molecule antagonists of VIP signaling. In certain embodiments, the human T cells are activated with anti-CD3 antibodies bound to a plate. In certain embodiments, the human T cells are activated in a mixed lymphocyte reaction. In certain embodiments, the human T cells are activated in vitro by co-incubation with a tumor associated antigen. In certain embodiments, the tumor associated antigen is presented on a tumor microvesicle. In certain embodiments, the activated human T cells are infused into a human patient with cancer.

[0129] 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 an 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.

[0130] In certain embodiments, the present disclosure relates to a method of treating a subject diagnosed with cancer, the method comprising administering to a subject in need thereof a cell in combination with a peptide disclosed herein. In certain embodiments, the subject is diagnosed with leukemia. In certain embodiments, the subject is 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 hydrocarbon or polyethylene glycol group.

[0131] In certain embodiments, the present disclosure relates to a method of treating leukemia, the method comprising administering a peptide disclosed herein to a subject in combination with transplanting hematopoietic stem cells. In certain embodiments, the present disclosure relates to a method comprising expanding lymphocytes in vitro to provide expanded cells and exposing the expanded cells with a peptide disclosed herein.

[0132] In certain embodiments, the lymphocytes are extracted from blood or obtained by leukapheresis. In certain embodiments, the expanded cells are further exposed to a stimulatory cytokine or interferon.

[0133] In certain embodiments, the present disclosure relates to a method of boosting an anti-cancer immune response by infusing a peptide disclosed herein or a nanoparticle expressing a peptide disclosed herein. In certain embodiments, the activated T cells are infused into a patient with chronic CMV infection. In certain embodiments, the activated T cells are infused into a patient with chronic EBV infection. In certain embodiments, the activated T cells are infused into a patient with chronic BK virus infection. In certain embodiments, the activated T cells are infused into a patient with chronic adenovirus infection.

[0134] In certain embodiments, the present disclosure relates to compositions and methods to reverse T cell senescence by interrupting vasoactive intestinal peptide (VIP) signaling and / or inhibiting phosphoinositide-3-kinase (PI3 kinase) inhibitor signaling and uses to manage cancer and chronic viral infections. In certain embodiments, the present disclosure contemplates methods to reverse T cell senescence by mixing T cells in vitro with a peptide or nanoparticle comprising a peptide disclosed herein that prevents VIP interaction with VIP receptors and / or adds a PI3 kinase inhibitor. In certain embodiments, the present disclosure contemplates expanding senescent T cells by mixing with a PI3 kinase inhibitor, a nanoparticle or peptide disclosed herein, a VIP-degrading enzyme, and combinations thereof.

[0135] In certain embodiments, the present disclosure contemplates methods to stimulate isolated T cells or expand senescent T cells by exposing T cells in vitro to a combination of antibodies that bind CD3 and / or CD28 in combination with a PI3 kinase inhibitor, an idelalisib, a peptide disclosed herein, or a nanoparticle disclosed herein, a VIP-degrading enzyme, and combinations thereof. In certain embodiments, the present disclosure contemplates the use of anti-CD3 antibodies and anti-CD28 antibodies or binding agents optionally linked to a solid substrate such as a magnetic bead.

[0136] In certain embodiments, the present disclosure contemplates methods of proliferating T cells that are negative for CD28 and / or CD27 using in vitro cell cultures as disclosed herein that provide replicated T cells with increased expression of CD28 and / or CD27 compared to the level prior to replication.

[0137] In certain embodiments, the present disclosure contemplates methods of proliferating T cells, wherein prior to, during, or after proliferating the T cells, the T cells are mixed with a vector having a nucleic acid sequence encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a cancer targeting sequence, a transmembrane domain, a T cell costimulatory molecule domain, and a signaling component of a T cell antigen receptor domain, such that the cell expresses the chimeric antigen receptor on the cell surface.

[0138] In certain embodiments, the present disclosure relates to in vitro cell culture compositions comprising minimal essential media and T cells and peptides disclosed herein or nanoparticles comprising peptides disclosed herein and phosphoinositide-3-kinase inhibitors, VIP-degrading enzymes, and combinations thereof, and optionally further comprising anti-CD3 antibodies and anti-CD28 antibodies optionally immobilized on a solid substrate such as a bead. In certain embodiments, T cells are purified from bone marrow cells or blood cells, peripheral blood.

[0139] 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 selected from idelalisib in culture at a concentration greater than 0.001, 0.1, 1, 10, 100 nM, or between 10 nM and 10 micromolar, or between 10 nM and 500 nM or between 10 nM and 1 micromolar.

[0140] In certain embodiments, the culture includes an enzyme that hydrolyzes VIP. In certain embodiments, the culture includes a VIP-degrading enzyme, such as a peptidase, a serine peptidase, a trypsin-like enzyme, a chymase, or human chymase 1 (CMA1). In certain embodiments, the culture has at least 0.001, 0.01, 0.1, or 1 microgram per mL of VIP-degrading enzyme, such as mast cell chymase. In certain embodiments, the present disclosure contemplates a T cell culture that includes a minimal essential medium and isolated cells that express CD3 and / or CD4 and / or CD8 and are negative for CD27 and / or CD28, and a PI3 kinase inhibitor, a peptide disclosed herein, or a nanoparticle including a peptide disclosed herein, and combinations thereof. The cells can be isolated by negative or positive selection using a binding agent attached to a solid support, such as a bead, a magnetic bead, or a particle of a fluorescent binding agent.

[0141] In certain embodiments, the anti-CD3 antibody and the anti-CD28 antibody are immobilized on a bead, a magnetic bead, or a solid surface. In certain embodiments, more than 5.0%, or 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%, or 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.

[0142] In certain embodiments, the purified T cells are obtained by centrifuging blood under conditions such that the plasma and red blood cells are separated to provide the purified T cells in a mixture of white blood cells between the plasma and the red blood cells. In certain embodiments, the purified T cells are obtained by a bone marrow aspiration or a bone marrow biopsy.

[0143] In certain embodiments, the purified T cells are obtained by mixing the cells with a fluorescent marker that binds CD3 and purifying the cells by fluorescence activated cell sorting. In certain embodiments, the purified T cells are obtained by mixing the cells with a magnetized marker that binds CD3 and purifying the cells by magnetic sorting. In certain embodiments, the purified T cells are obtained by mixing the 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, the purified T cells are obtained by mixing the cells with a magnetized marker that binds CD3 and / or CD4 and / or CD8 and purifying the cells by magnetic sorting.

[0144] In certain embodiments, the present disclosure contemplates a solid substrate, such as a bead, that has anti-CD3 antibodies and anti-CD28 antibodies and has a VIP-degrading enzyme coupled to the surface. In certain embodiments, it is contemplated that the beads are arranged in a culture medium and T cells are expanded on top of the culture medium such that the beads are subcellular.

[0145] In certain embodiments, the VIP-degrading enzyme comprises human CMA1 Accession Number GenBank: AAI03975.1:

[0146]

[0147] In certain embodiments, the VIP-degrading enzyme is human recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) having (SEQ ID NO: 14):

[0148]

[0149] 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 or nanoparticles thereof disclosed herein on the proliferation of T cells stimulated in vitro with antibodies against CD3 and CD28.

[0150] In certain embodiments, the present invention relates to expanding T cells or expanding or reversing senescence in T cells, where a naturally occurring reactivity to cancer can be found infiltrating a tumor in a subject. The tumor can be harvested and these tumor infiltrating lymphocytes (TILs) can be expanded using the methods disclosed herein.

[0151] Some cancers are caused by viruses and traditional vaccines against these viruses, such as HPV vaccines and hepatitis B vaccines, will prevent these cancers. It is contemplated that the peptides disclosed herein can be administered in combination with these vaccines to improve the efficacy of the treatment.

[0152] It is believed that cancer cells produce and are destroyed by the immune system, and that cancer forms when the immune system is unable to destroy the cancer cells. One method of cancer vaccination is to isolate proteins from cancer cells and immunize cancer patients against these proteins, thereby stimulating an immune response that kills cancer cells. Cancer vaccines are contemplated for the treatment of breast cancer, lung cancer, colon cancer, skin cancer, kidney cancer, prostate cancer, and other cancers. In certain embodiments, the present disclosure relates to the treatment of cancer by administering a peptide disclosed herein in combination with a cancer antigen.

[0153] In certain embodiments, the present disclosure relates to a method of treating or preventing a viral infection, comprising administering a peptide disclosed herein to a subject at risk of a viral infection, exhibiting symptoms of a viral infection, or diagnosed with a viral infection. 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 undergoing hemodialysis, diagnosed with cancer, receiving an immunosuppressive drug, and / or diagnosed with an HIV infection. In certain embodiments, the present disclosure relates to preventing a viral infection in an immunocompromised subject at risk of infection by administering a peptide disclosed herein and optionally one or more antiviral agents.

[0154] In some embodiments, the present disclosure relates to the use of a peptide disclosed herein for the manufacture of an antiviral medicament for the treatment of a viral infection. In some embodiments, the subject is diagnosed with a chronic viral infection. In certain embodiments, the subject is undergoing serological monitoring. In some embodiments, the administration is performed under conditions in which viral infection is no longer detected. In some embodiments, the subject is diagnosed with an RNA virus, a DNA virus, or a retrovirus. In some embodiments, the subject is diagnosed with a virus that is a double-stranded DNA virus, a positive-sense single-stranded DNA virus, a double-stranded RNA virus, a positive-sense single-stranded RNA virus, a negative-sense single-stranded RNA virus, a positive-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 viral titer of the subject is reduced after treatment compared to prior to treatment.

[0155] In some embodiments, the subject is diagnosed with a virus: Influenza A virus (including subtypes H1N1), Influenza B virus, Influenza C virus, Rotavirus A, Rotavirus B, Rotavirus C, Rotavirus D, Rotavirus E, SARS coronavirus, Human Adenovirus (HAdV-1 through 55) types, Human Papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, Parvovirus B19, Molluscum contagiosum virus, JC virus (JCV), BK virus, Merkel cell polyomavirus, Coxsackievirus A1, Norovirus, Rubella virus, Lymphocytic choriomeningitis virus (LCMV), Yellow fever virus, Measles virus, Mumps virus, Respiratory syncytial virus, Rinderpest virus, California encephalitis virus, Hantavirus, Rabies virus, Ebola virus, marburg virus, Herpes simplex virus-1 (HSV-1), Herpes simplex virus-2 (HSV-2), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Herpes lymphotropic virus, Roseolovirus, Kaposi's sarcoma-associated herpesvirus, Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis D virus (HDV), Hepatitis E virus (HEV), Human immunodeficiency virus (HIV), Human T-lymphotropic leukemia virus type I (HTLV-1), Friend spleen focus-forming virus (SFFV), or XMRV.

[0156] In some embodiments, the present disclosure relates to treating or preventing a viral infection by administering a combination of a peptide disclosed herein and a second antiviral agent. In further embodiments, the subject is co-administered abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, ampligen, arbidol, atazanavir, atripla, boceprevir, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, ganciclovir, idoxuridine, imiquimod, indinavir, inosine, interferon type III, interferon type II, interferon type I, lamivudine, lopinavir, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, oseltamivir (Tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, tenofovir, tenofovir disoproxil, tipranavir, triglycerides, tromantadine, tyrozanamide, valacyclovir, valganciclovir, vicriviroc, zanamivir, or zidovudine.disoproxil), tipranavir, trifluridine, trizivir, tromantadine, Truvada™ (emtricitabine / tenofovir disoproxil fumarate), valaciclovir, valaciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and / or zidovudine. In certain embodiments, a pharmaceutical composition comprising a peptide disclosed herein and a second antiviral agent is administered to a subject.

[0157] In certain embodiments, the present disclosure relates to treating a subject having a viral infection after being infected by administering a peptide disclosed herein and an immunoglobulin.

[0158] In certain embodiments, the present disclosure relates to treating or preventing a viral infection by administering a peptide disclosed herein and a viral vaccine or in the absence of a viral vaccine.

[0159] In certain embodiments, the disclosure relates to enhancing an immune response to a vaccine comprising administering to a subject in need thereof a peptide disclosed herein. Typically, the vaccine is selected from the group of vaccines consisting of: shingles vaccine, smallpox vaccine, polio vaccine, pertussis vaccine, influenza vaccine, diphtheria vaccine, tetanus vaccine, meningococcal vaccine, influenza A vaccine comprising H1N1 subtype 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 (HAdV-1 to 55) type vaccine, human papillomavirus (HPV) vaccine, parvovirus B19 vaccine, infectious soft-bodied animal 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 lymph vaccine, roseola virus 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-lymphotrophic virus type I (HTLV-1) vaccine, Friend's spleen focus-forming virus (SFFV) vaccine, and xenotropic MuLV-related virus (XMRV) vaccine. In certain embodiments, the vaccine is for a subject diagnosed with a chronic viral infection.

[0160] 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 a live or killed or inactivated prokaryotic or eukaryotic cell.

[0161] In certain embodiments, the human T cells are activated in vitro by co-incubation with a viral antigen. In certain embodiments, the viral antigen is presented on a microvesicle. In certain embodiments, the viral antigen is presented on a dendritic cell.

[0162] Nucleic acid vaccines, usually DNA plasmids, are genetically engineered to encode one or more antigens and / or produce one or more antigens from a pathogen. The nucleic acid transfects or infects host cells, where the cells’ internal machinery expresses the proteins. Because these proteins are recognized as foreign, when the proteins are processed by the host cells and displayed on their surface, an immune response is triggered. 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 peptides disclosed herein can be administered in combination with nucleic acid vaccines or other costimulatory molecules.

[0163] In certain embodiments, the present disclosure relates to vaccine compositions comprising the peptides disclosed herein and methods of administering the peptides disclosed herein in combination with vaccines. In certain embodiments, vaccines contain antigens from a pathogen and are presented to the immune system from a weakened or killed microorganism or its toxin form. The antigens stimulate the immune system. Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of any future infection by a pathogen) or therapeutic, administered after infection or diagnosis of disease.

[0164] Some vaccines contain killed but previously virulent microorganisms that have been destroyed by chemicals or heat. Influenza vaccine, cholera vaccine, plague vaccine, polio vaccine, hepatitis A vaccine, and rabies vaccine are examples of inactivated vaccines contemplated by the present disclosure.

[0165] Some vaccines contain live attenuated microorganisms. Typically, these are live viruses that have been cultured under conditions that disable certain virulent properties or use closely related but less dangerous organisms to produce a broad immune response; however, some are bacterial in nature.

[0166] In certain embodiments, vaccines are protein subunits. Fragments of these can be used to generate an immune response rather than introducing an inactivated or attenuated microorganism to the immune system. Examples include subunit vaccines against hepatitis B virus composed of only viral surface proteins, virus-like particle (VLP) vaccines against human papillomavirus (HPV) composed of the viral major coat protein, and hemagglutinin and neuraminidase subunits of influenza virus.

[0167] In certain embodiments, vaccines include polysaccharides. Certain bacteria have a polysaccharide coat that is typically immunogenic. By linking these polysaccharides to proteins (e.g., toxins), the immune system can recognize the polysaccharides as if they were protein antigens.

[0168] Toxoid vaccines are made from inactivated toxic compounds. Examples of toxoid-based vaccines include diphtheria and tetanus toxoids. In certain embodiments, a peptide disclosed herein is administered in combination with DPT. DPT (also known as DTP and DTwP) refers to a combination vaccine against three infectious diseases in humans: diphtheria, pertussis (whooping cough), and tetanus. The vaccine components include diphtheria and tetanus toxoids, and killed whole cells of the organism that causes pertussis (wP). DTaP (also known as Tdap, DTPa, and TDaP) refers to a similar combination vaccine that is acellular for the pertussis component. DT or TD vaccines that lack the pertussis component are also contemplated.

[0169] Other specific vaccines contemplated by the present disclosure include anthrax vaccines, such as unencapsulated culture filtrate of an avirulent strain known as V770-NP1-R; Bacillus Calmette-Guerin (BCG), such as a live attenuated Mycobacterium bovis strain; Haemophilus influenzae type B vaccine, such as a Hib polysaccharide-protein conjugate vaccine; Hepatitis A vaccine, such as inactivated Hepatitis A virus; Hepatitis B vaccine, such as Hepatitis B surface antigen; Human Papillomavirus (HPV) vaccine, such as non-infectious virus-like particles assembled from HPV types 6, 11, 16, and 18 LI proteins; Meningococcal vaccine, such as capsular polysaccharide antigens of Neisseria meningitidis serogroups A, C, Y, and W-135 individually conjugated to diphtheria toxoid protein.

[0170] In certain embodiments, the present disclosure relates to methods of treating an active cytomegalovirus infection, comprising administering to a subject diagnosed with an active cytomegalovirus infection and exhibiting signs or symptoms thereof, 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 group, and is optionally modified with a hydrocarbyl or polyethylene glycol group.

[0171] In certain embodiments, the immune system of the subject is compromised. In certain embodiments, the subject is a transplant recipient.

[0172] In certain embodiments, the present disclosure relates to methods of reducing an active cytomegalovirus infection, comprising administering to a subject having 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 group, and is optionally modified with a hydrocarbyl or polyethylene glycol group. In certain embodiments, the titer of cytomegalovirus in the subject is reduced after administration of the vasoactive intestinal peptide antagonist as compared to pretreatment.

[0173] Examples

[0174] Improved VIP antagonists

[0175] To assess whether tumor-specific expression of vasoactive intestinal polypeptide represents a tumor-mediated immune escape mechanism. There is a spectrum of VIP expression across tumors, with the highest expression seen in pancreatic exocrine carcinomas and the lowest expression seen in melanomas. Generally, the VIP expression level of a tumor is inversely proportional to the expression of other co-inhibitory pathway molecules, such as PDL1. Tumors that express and secrete VIP can have mutations in the VIP coding sequence that result in a peptide molecule with improved pharmacokinetics or pharmacodynamics in the tumor microenvironment. The pharmacokinetic advantage can be the result of mutations that reduce VIP susceptibility to proteases, improving its half-life. The pharmacodynamic advantage can be the result of mutations that enhance binding affinity to the receptor, enhancing signaling.

[0176] Experiments were performed to determine whether mutated VIP produced by tumors would result in more durable suppression of anti-cancer T cells in the tumor microenvironment. Mutations in specific genes picked from deposited tumor sequences were analyzed, and there were mutations in the VIP coding sequence in multiple cancers. Specifically, there were 140 missense mutations and 17 truncating mutations within the VIP gene cluster listed in the Cancer Genome Atlas. Within the coding sequence of the 28 amino acid VIP peptide, mutations were identified in the VIP coding sequence present in breast adenocarcinoma, prostate adenocarcinoma, esophageal adenocarcinoma, cutaneous melanoma, small cell lung cancer, gastric adenocarcinoma, endometrial carcinoma, cutaneous melanoma, esophageal adenocarcinoma, large intestine adenocarcinoma, uterine carcinosarcoma, hepatocellular adenoma, lung adenocarcinoma, and gastric adenocarcinoma. Eight specific mutations were present in the C-terminal amino acids, including the alpha helix of the VIP that binds the receptor.

[0177] Alpha helix sequences are common among immunosuppressive VIPs, i.e., peptide agonists. VIPhyb (SEQ ID NO: 1), an antagonist peptide that differs from six internal amino acids from native VIP (SEQ ID NO: 2) (see Figure 1 ). Eight sequences were made in the presence of the six N-terminal amino acids of VIPhyb, namely ANT-1 through 8 (see SEQ ID NOs: 3-10 in Figure 1 ). Specifically, the ANT-1 sequence has the amino acid T substituted for A at amino acid position seven. The ANT-2 sequence has the amino substitution D substituted for V at amino acid position number 8. ANT-3 has the amino acid substitution Y substituted for C at amino acid position number 10. ANT-4 has the amino acid substitution R substituted for S at amino acid position 12. ANT-5 has the amino acid substitution M substituted for I at amino acid position number 17. ANT-6 has the amino acid substitution K substituted for N at amino acid position 20. ANT-7 has the amino acid substitution L substituted for M at amino acid position number 23. ANT-8 has the amino acid substitution S substituted for L at amino acid position number 25. Each of the alternative antagonist peptide sequences ANT-1 through ANT-8 were synthesized.

[0178] peptides ( Figure 1 The SEQ ID NO: 3-10) was added to a short-term culture of luciferase-positive T cells cultured in a 96-well plate in the presence of anti-CD3 antibody binding to the plate. The concentration of the anti-CD3 antibody was 0.5 mcg / ml or 1 mcg / ml. T cells from luciferase-transgenic mice were added in the presence of a low dose of IL-2, and 0.5 μmol, 1 μmol, or 3 μmol of the original VIPhyb peptide or the alternative ANT-1 to ANT-8 peptide sequence was added. Figure 2 The study showed T cell proliferation at 24 hours in the presence of 1 μmol concentrations of Ant-1 to Ant-8. Figure 3 The study demonstrated enhanced T cell proliferation at 24 hours in the presence of ANT-8 peptide at concentrations of 0.5 μmol, 1 μmol, and 3 μmol, compared to the original VIPhyb peptide at the same corresponding concentrations.

[0179] Compared to control cultures containing anti-CD3 antibody with a binding plate but without the added peptide, the enhanced T cell proliferation using lower concentrations of anti-CD3 with the ANT-8 peptide was more moderate and not significantly different from control cultures containing only a small increase. A unique peptide sequence ANT-8 (SEQ ID NO: 10) with improved antagonistic activity compared to native VIPhyb was identified.

Claims

1. A peptide consisting of KPRRPYTDNYTRLRKQMAVKKYLNLILN (SEQ ID NO: 10).

2. The peptide according to claim 1, wherein the peptide is conjugated to a nanoparticle.

3. A pharmaceutical composition comprising the peptide according to claim 1 or 2 and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition according to claim 3 in the form of a capsule, tablet, pill, powder or granule.

5. The pharmaceutical composition according to claim 3 in the form of a sterilized pH buffered aqueous salt solution.

6. The pharmaceutical composition according to claim 3 in the form of a container configured as a container for spraying a liquid or a sealed container with a propellant.

7. A nucleic acid encoding the peptide according to any one of claims 1 to 2 in operable combination with a promoter.

8. A recombinant vector comprising the nucleic acid according to claim 7.

9. A cell comprising the recombinant vector according to claim 8.

10. Use of the peptide according to any one of claims 1 to 2 for the manufacture of a kit for in vitro or ex vivo boosting T cell activation and expansion, wherein the peptide is for mixing with T cells.

11. The use according to claim 10, wherein T cells are mixed in combination with an anti-CD3 antibody and / or an anti-CD28 antibody.

12. The use according to claim 10, wherein T cells are mixed in combination with a phosphoinositide 3-kinase delta (PI3Kd) inhibitor.

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