TRANS-VACCENIC ACID (TVA) AND DERIVATIVES THEREOF IN T CELL-BASED CANCER THERAPIES
20250221953 ยท 2025-07-10
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
A61K31/201
HUMAN NECESSITIES
A61K31/198
HUMAN NECESSITIES
A61K40/11
HUMAN NECESSITIES
C07K16/2809
CHEMISTRY; METALLURGY
C12N15/1138
CHEMISTRY; METALLURGY
A61K31/713
HUMAN NECESSITIES
A61K31/165
HUMAN NECESSITIES
A61K31/202
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61K31/7105
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
International classification
A61K31/201
HUMAN NECESSITIES
A61K31/202
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K40/11
HUMAN NECESSITIES
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Provided herein are compositions and methods for stimulating T-cell-based anti-tumor immunity by administration of trans-vaccenic acid (TVA), an active derivative thereof, or an inhibitor of GPR43 expression or activity. In particular embodiments, TVA, a TVA derivative, or an inhibitor of GPR43 expression or activity is administered to boost an endogenous T-cell response and/or is co-administered with a T-cell-based therapy, such as immune checkpoint blockade therapies, CAR-T therapies, monoclonal antibody therapies, bispecific T-cell engagers therapies, etc.
Claims
1. A method of treating, preventing, reducing the likelihood, or reducing the severity of cancer in a subject in need thereof comprising administering an effective amount of a trans-vaccenic acid (TVA) or an active TVA derivative to the subject.
2. The method of claim 1, wherein the active TVA derivative can enhance CD8+ T cell activity and/or can enhance antitumor immunity.
3. The method of claim 1 or 2, wherein the TVA or the active TVA derivative comprises a compound of formula (I): ##STR00061## wherein: R.sup.1 is selected from hydrogen and C.sub.1-C.sub.4 alkyl; X is selected from C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, and C.sub.2-C.sub.24 alkynylene; R.sup.2 is selected from hydrogen, optionally substituted aryl, optionally substituted heteroaryl, C.sub.1-C.sub.4 alkoxy, hydroxy, and halo.
4. The method of claim 3, wherein R.sup.1 is hydrogen.
5. The method of claim 3, wherein X is selected from C.sub.8-C.sub.24 alkylene, C.sub.8-C.sub.24 alkenylene, C.sub.8-C.sub.24 alkynylene.
6. The method of claim 3, wherein R.sup.2 is selected from hydrogen and optionally substituted aryl.
7. The method of claim 3, wherein the TVA or the active TVA derivative is selected from: ##STR00062##
8. The method of claim 1, wherein the TVA or the active TVA derivative is: ##STR00063##
9. The methods of any of claims 1-8, wherein administration of the TVA or the active TVA derivative comprises oral, intravenous, transdermal, or subcutaneous administration.
10. The methods of any of claims 1-9, wherein administration of the TVA or the active TVA derivative occurs once or more weekly.
11. The methods of any of claims 1-9, wherein administration of the TVA or the active TVA derivative occurs once or more daily.
12. The method of claim 1, wherein the cancer is melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer, pancreatic cancer, Merkel cell carcinoma.
13. The method of claim 1, wherein the subject is a human.
14. The methods of any of claims 1-13, wherein the TVA or the active TVA derivative is co-administered with one or more additional therapeutic or prophylactic agents.
15. The method of claim 14, wherein the one or more additional therapeutic agents are selected from chemotherapeutics and immunotherapeutics.
16. The method of any of claims 1-15, wherein the TVA or the active TVA derivative is co-administered with a T-cell-based immunotherapeutic.
17. The method of claim 16, wherein the T-cell-based immunotherapeutic is selected from a therapy comprising the administration of immune checkpoint inhibitor, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T-cell engager therapy.
18. The method of any of claims 1-17, wherein the TVA or active TVA derivative is co-administered with: an immune checkpoint inhibitor that binds to and inhibits the activity of an immune checkpoint protein; and wherein the immune checkpoint protein is selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3, VISTA.
19. The method of any of claims 1-18, wherein the TVA or active TVA derivative is co-administered with an immune checkpoint inhibitor selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012, STI-A1010.
20. Use of an effective dose of trans-vaccenic acid (TVA) or active TVA derivative for treating a subject suffering from cancer.
21. The use of claim 20, wherein the TVA or active TVA derivative is co-administered with an immunotherapeutic selected from a therapy comprising the administration of immune checkpoint inhibitor, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T-cell engager therapy.
22. Use of an effective dose of trans-vaccenic acid (TVA) or active TVA derivative in the manufacture of a medicament for use in a method of treating a subject suffering from cancer.
21. The use of claim 22, wherein the method further comprises an immune checkpoint inhibitor, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T-cell engager therapy.
24. A composition or kit comprising a trans-vaccenic acid (TVA) or an active TVA derivative and an additional therapeutic or prophylactic agent for the treatment of cancer.
25. The composition or kit of claim 24, wherein the active TVA derivative can enhance CD8+ T cell activity and/or can enhance antitumor immunity.
26. The composition or kit of claim 24 or 25, wherein the TVA or an active TVA derivative is a compound of formula (I): ##STR00064## wherein: R.sup.1 is selected from hydrogen and C.sub.1-C.sub.4 alkyl; X is selected from C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, and C.sub.2-C.sub.24 alkynylene; and R.sup.2 is selected from hydrogen, optionally substituted aryl, optionally substituted heteroaryl, C.sub.1-C.sub.4 alkoxy, hydroxy, and halo.
27. The composition or kit of claim 26, wherein R.sup.1 is hydrogen.
28. The composition or kit of claim 26, wherein X is selected from C.sub.8-C.sub.24 alkylene, C.sub.8-C.sub.24 alkenylene, and C.sub.8-C.sub.24 alkynylene.
29. The composition or kit of claim 26, wherein R.sup.2 is selected from hydrogen and optionally substituted aryl.
30. The composition or kit of claim 26, wherein the TVA or an active TVA derivative is selected from: ##STR00065##
31. The composition or kit of claim 24, wherein the TVA or an active TVA derivative is: ##STR00066##
32. The composition or kit of claim 24, wherein the additional therapeutic agent is selected from a chemotherapeutic or immunotherapeutic.
33. The composition of claim 32, wherein the additional therapeutic agent is a T-cell-based immunotherapeutic.
34. The composition of claim 33, wherein the T-cell-based immunotherapeutic is selected from a therapy comprising the administration of immune checkpoint inhibitor, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T-cell engager therapy.
35. A TVA-containing or active-TVA-derivative-containing composition for use in a method of treating cancer in a human subject.
36. The composition of claim 35, wherein the method comprises co-administering the TVA or active TVA derivative to the subject with an immunotherapeutic selected from a therapy comprising the administration of immune checkpoint inhibitor, CAR-T cell therapy, monoclonal antibody therapy, and bispecific T-cell engager therapy.
37. A method of preventing, reducing the likelihood, or reducing the severity of cancer in a subject comprising administering an effective amount of a trans-vaccenic acid (TVA) or an active TVA derivative to the subject.
38. A method of treating or preventing, reducing the likelihood, or reducing the severity of cancer in a subject comprising administering a composition that results in inhibition of GPR43 activity or expression.
39. The method of claim 38, wherein the composition comprises a trans-vaccenic acid (TVA) or an active TVA derivative.
40. The method of claim 38, wherein the composition comprises a nucleic acid inhibitor of GPR43 expression or activity.
41. The method of claim 40, wherein the composition comprises a siRNA, shRNA, antisense RNA, or CRIPSR system to inhibit expression of GPR43.
42. The method of claim 38, wherein the composition comprises a small molecule inhibitor of GPR43 activity.
43. The method of claim 42, wherein the small molecule inhibitor of GPR43 activity is a GPR43 antagonist.
44. The method of claim 43, wherein the GPR43 antagonist has the structure: ##STR00067##
45. The method of claim 42, wherein the small molecule inhibitor of GPR43 activity is an 5 inverse agonist of GPR43.
46. The method of claim 43, wherein the inverse agonist of GPR43 has the structure: ##STR00068##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DEFINITIONS
[0042] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein.
[0043] Unless otherwise defined, 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 invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.
[0044] As used herein and in the appended claims, the singular forms a, an and the include plural reference unless the context clearly dictates otherwise.
[0045] As used herein, the term comprise and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term consisting of and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase consisting essentially of denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open comprising language. Such embodiments encompass multiple closed consisting of and/or consisting essentially of embodiments, which may alternatively be claimed or described using such language.
[0046] As used herein, the term subject broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term patient typically refers to a subject that is being treated for a disease or condition.
[0047] As used herein, the terms trans-vaccenic acid or TVA refer to a compound of the formula:
##STR00007##
[0048] As used herein, the term active TVA derivative refers to a TVA derivative is a compound of formula (I):
##STR00008## [0049] wherein: R.sup.1 is selected from hydrogen and C.sub.1-C.sub.4 alkyl; X is selected from C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, and C.sub.2-C.sub.24 alkynylene; and R.sup.2 is selected from hydrogen, optionally substituted aryl, optionally substituted heteroaryl, C.sub.1-C.sub.4 alkoxy, hydroxy, and halo; wherein the compound is capable of enhancing CD8+ T cell activity and/or enhancing antitumor immunity via the mechanisms and/or interactions described herein for TVA. In some embodiments, R.sup.1 is selected from hydrogen and methyl. In some embodiments, R.sup.1 is hydrogen. In some embodiments, X is selected from C.sub.8-C.sub.24 alkylene, C.sub.8-C.sub.24 alkenylene, and C.sub.8-C.sub.24 alkynylene. In some embodiments, X is selected from C.sub.12-C.sub.24 alkylene, C.sub.12-C.sub.24 alkenylene, and C.sub.12-C.sub.24 alkynylene. In some embodiments, X is C.sub.12-C.sub.24 alkylene. In some embodiments, X is C.sub.12-C.sub.24 alkenylene. In some embodiments, X is C.sub.12-C.sub.24 alkynylene. In some embodiments, R.sup.2 is selected from hydrogen and optionally substituted aryl. In some embodiments, R.sup.2 is selected from hydrogen and phenyl, wherein the phenyl is unsubstituted or substituted with one substituent selected from C.sub.1-C.sub.4 alkyl (e.g., methyl). In some embodiments, R.sup.2 is hydrogen. In some embodiments, the compound is selected from:
##STR00009##
In some embodiments, a TVA derivative is not TVA.
[0050] As used herein, the term alkyl refers to a radical of a straight or branched saturated hydrocarbon chain. The alkyl chain can include, e.g., from 1 to 24 carbon atoms (C.sub.1-C.sub.24 alkyl), 1 to 16 carbon atoms (C.sub.1-C.sub.16 alkyl), 1 to 14 carbon atoms (C.sub.1-C.sub.14 alkyl), 1 to 12 carbon atoms (C.sub.1-C.sub.12 alkyl), 1 to 10 carbon atoms (C.sub.1-C.sub.10 alkyl), 1 to 8 carbon atoms (C.sub.1-C.sub.8 alkyl), 1 to 6 carbon atoms (C.sub.1-C.sub.6 alkyl), 1 to 4 carbon atoms (C.sub.1-C.sub.4 alkyl), 1 to 3 carbon atoms (C.sub.1-C.sub.3 alkyl), or 1 to 2 carbon atoms (C.sub.1-C.sub.2 alkyl). Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.
[0051] As used herein, the term alkylene refers to a divalent alkyl group.
[0052] As used herein, the term alkenyl refers to a radical of a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond and no triple bonds. The double bond(s) may be located at any position(s) with the hydrocarbon chain. The alkenyl chain can include, e.g., from 2 to 24 carbon atoms (C.sub.2-C.sub.24 alkenyl), 2 to 16 carbon atoms (C.sub.2-C.sub.16 alkenyl), 2 to 14 carbon atoms (C.sub.2-C.sub.14 alkenyl), 2 to 12 carbon atoms (C.sub.2-C.sub.12 alkenyl), 2 to 10 carbon atoms (C.sub.2-C.sub.10 alkenyl), 2 to 8 carbon atoms (C.sub.2-C.sub.8 alkenyl), 2 to 6 carbon atoms (C.sub.2-C.sub.6 alkenyl), 2 to 4 carbon atoms (C.sub.2-C.sub.4 alkenyl), 2 to 3 carbon atoms (C.sub.2-C.sub.3 alkenyl), or 2 carbon atoms (C.sub.2 alkenyl). Representative examples of alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, 2-methyl-2-propenyl, 3-butenyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, octatrienyl, and the like.
[0053] As used herein, the term alkenylene refers to a divalent alkenyl group.
[0054] As used herein, the term alkynyl means a radical of a straight or branched hydrocarbon chain containing at least one carbon-carbon triple bond. The alkynyl chain can include, e.g., from 2 to 24 carbon atoms (C.sub.2-C.sub.24 alkynyl), 2 to 16 carbon atoms (C.sub.2-C.sub.16 alkynyl), 2 to 14 carbon atoms (C.sub.2-C.sub.14 alkynyl), 2 to 12 carbon atoms (C.sub.2-C.sub.12 alkynyl), 2 to 10 carbon atoms (C.sub.2-C.sub.10 alkynyl), 2 to 8 carbon atoms (C.sub.2-C.sub.8 alkynyl), 2 to 6 carbon atoms (C.sub.2-C.sub.6 alkynyl), 2 to 4 carbon atoms (C.sub.2-C.sub.4 alkynyl), 2 to 3 carbon atoms (C.sub.2-C.sub.3 alkynyl), or 2 carbon atoms (C.sub.2 alkynyl). The triple bond(s) may be located at any position(s) with the hydrocarbon chain. Representative examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and the like.
[0055] As used herein, the term alkynylene refers to a divalent alkynyl group.
[0056] As used herein, the term alkoxy refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.
[0057] As used herein, aryl refers to a radical of a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system (e.g., having 6, 10, or 14 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms (C.sub.6-C.sub.14 aryl). In some embodiments, an aryl group has six ring carbon atoms (C.sub.6 aryl; i.e., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (C.sub.10 aryl; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (C.sub.14 aryl; e.g., anthracenyl and phenanthrenyl).
[0058] As used herein, the term halogen or halo refers to F, Cl, Br, or I.
[0059] As used herein, heteroaryl refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (5-10 membered heteroaryl). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.
[0060] As used herein, the term hydroxy or hydroxyl refers to an OH group.
[0061] When a group or moiety can be substituted, the term substituted indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens on the group indicated in the expression using substituted can be replaced with a selection of recited indicated groups or with a suitable substituent group known to those of skill in the art (e.g., one or more of the groups recited below), provided that the designated atom's normal valence is not exceeded. Substituent groups include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.
[0062] The term supplement as used herein refers to a nutritional product that provides nutrients (e.g. vitamins, minerals, fatty acids (e.g., TVA)) to a subject that may otherwise not be consumed in sufficient quantities (e.g., to enhance cancer treatment) by the subject. Supplements may be, for example, provided in the form of a pill, a tablet, a lozenge, a chewy capsule or tablet, a capsule, or a powder supplement that can be, for example, dissolved in water or a beverage (e.g., milk), or sprinkled on food. Supplements typically provide one or more selected compounds (e.g., TVA) without providing a significant portion of the overall nutritional needs of a subject.
[0063] The term pharmaceutical formulation as used herein refers to a composition comprising at least one pharmaceutically-active agent, chemical substance or drug. The pharmaceutical formulation may be in solid or liquid form and can comprise at least one additional active agent, carrier, vehicle, excipient or auxiliary agent identifiable by the skilled person. The pharmaceutical formulation may be in the form of a tablet, capsule, granules, powder, liquid or syrup.
[0064] The term effective dose or effective amount refers to an amount of an agent, e.g., a neutralizing antibody, that results in the reduction of symptoms in a patient, treatment of prevention of a disease or condition, or results in a desired biological outcome.
[0065] As used herein, the terms administration and administering refer to the act of giving a drug, prodrug, or other agent, or therapeutic to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.
[0066] As used herein, the terms co-administration and co-administering refer to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.
[0067] As used herein, an immune response refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (e.g., antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous cells or other abnormal/diseased-associated cells.
[0068] As used herein, the term immunotherapy refers to the treatment or prevention of a disease or condition by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
[0069] As used herein, the term immunotherapeutic refers to any agent (e.g., small molecule, peptide, antibody, engineered cell, etc.) capable of stimulating a host immune system to generate an immune response to a tumor or cancer in the subject.
[0070] As used herein, the term T-cell-based therapy refers to any immunotherapy that acts through T cells. T-cell-based therapies include the administration of exogenous T cells (e.g., CAR-T cell therapies) and therapies that act upon or through a subjects endogenous T cells (e.g., checkpoint inhibitors.
[0071] As used herein, the term antibody refers to a whole antibody molecule or a fragment thereof (e.g., fragments such as Fab, Fab, and F(ab).sub.2), unless specified otherwise; an antibody may be polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, etc.
[0072] As used herein, the term antibody fragment refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab, F(ab).sub.2, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody) produced by recombinant DNA techniques, or chemical polypeptide synthesis.
[0073] As used herein, the term single-chain bispecific antibody construct refers to a polypeptide construct comprising two antibody-derived binding domains. In some embodiments herein the two antibody-derived binding domains are an antigen-recognition domain and an activation domain. The binding domains may comprise variable regions (or parts thereof) of an antibody, antibody fragment or derivative thereof, capable of specifically binding to/interacting with a target antigen and/or an activation molecule. In certain embodiments, a part of a variable region comprises at least one CDR (Complementary determining region), such as at least a CDR1, CDR2, or CDR3 region. The two domains/regions in the single chain antibody construct are preferably covalently connected to one another as a single chain. Illustrative examples of bispecific single chain molecules are known in the art and are described in WO 99/54440; Mack, J. Immunol. (1997), 158, 3965-3970; Mack, PNAS, (1995), 92, 7021-7025; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197; Loffler, Blood, (2000), 95, 6, 2098-2103; and Bruhl, J. Immunol., (2001), 166, 2420-2426; incorporated by reference in their entireties. As used herein, the term engager refers to a molecule that is secreted from a cell and activates immune cells with which it interacts. The engager activates specific immune cells according to the domains present in the engager. Illustrative examples of cells that secrete engagers, but are not limited to, include T-cells, NK cells, NKT cells, CAR T-cells, mesenchymal stem cells (MSCs), neuronal stem cells, hematopoietic stem cells, or a mixture thereof, in some cases.
[0074] As used herein, the term bispecific refers to any molecule or molecular complex that has two different binding specificities. The molecule or molecular complex may comprise two separate binding domains, each with the same specificity (homobispecific) or with specificity for different molecular entities (e.g., antigens) (heterobispecific). For example, a bispecific engager is an engager molecule capable of binding a target antigen (e.g., an antigen on a cancer cell) and a immunostimulatory element.
[0075] As used herein, the term antigen-recognition domain refers to a molecular moiety (e.g. part of an engager molecule or antibody) that recognizes an antigen. In particular embodiments, antigens can be of any nature including, but not limited to, proteins, carbohydrates, and/or synthetic molecules.
[0076] As used herein, the term activation domain refers to a molecular moiety (e.g. part of an engager molecule or antibody) that interacts with immune cells (e.g., T cell receptor (TCR)) and induces a positive or negative immunomodulatory signal. Illustrative examples of positive immunomodulatory signals include signals that induce cell proliferation, cytokine secretion, or cytolytic activity. Illustrative examples of negative immunomodulatory signals include signals that inhibit cell proliferation, inhibit the secretion of immunosuppressive factors, or induce cell death.
[0077] As used herein, the term co-stimulatory domain or co-stimulatory signaling domain refers to an intracellular signaling domain of a co-stimulatory molecule. In particular aspects, it refers to a domain that provides additional signals to the immune cell in conjunction with an activation domain. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Illustrative examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, CD70, CD80, CD86, and CD83.
[0078] As used herein, the term intracellular signaling domain, when used in reference to a cell surface receptor or a CAR, is a moiety responsible for activation of at least one function of the cell upon which the receptor or CAR is displayed. The term effector function refers to a specialized function of a cell. For example, effector function of a T cell includes cytolytic activity or helper activity including the secretion of cytokines. Thus the term intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. To the extent that a truncated portion or variant of a native intracellular signaling domain is active, such a polypeptide may be used in place of the full native chain, as long as it transduces the effector function signal. The term intracellular signaling domain includes any truncated or variant portion of a polypeptide sequence sufficient to transduce the effector function signal. Examples of intracellular signaling domains include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. Cytoplasmic signaling sequences that act in a stimulatory manner comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAM containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and CD66d.
[0079] As used herein, the term transmembrane domain, when used in reference to a cell surface receptor or a CAR, is a moiety that spans the plasma membrane of the cell and is connected to both the intracellular signaling domain and the extracellular antigen-recognition domain. A transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, for example, the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, etc. Alternatively the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signaling domain. A glycine-serine doublet provides a particularly suitable linker.
[0080] As used herein, the term monoclonal antibody refers to an antibody which is a member of a substantially homogeneous population of antibodies that specifically bind to the same epitope. In certain embodiments, a monoclonal antibody is secreted by a hybridoma. In certain such embodiments, a hybridoma is produced according to certain methods known to those skilled in the art. See, e.g., Kohler and Milstein (1975) Nature 256:495-499; herein incorporated by reference in its entirety. In certain embodiments, a monoclonal antibody is produced using recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). In certain embodiments, a monoclonal antibody refers to an antibody fragment isolated from a phage display library. See, e.g., Clackson et al. (1991) Nature 352:624-628; and Marks et al. (1991) J. Mol. Biol. 222:581-597; herein incorporated by reference in their entireties. The modifying word monoclonal indicates properties of antibodies obtained from a substantially-homogeneous population of antibodies, and does not limit a method of producing antibodies to a specific method. For various other monoclonal antibody production techniques, see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); herein incorporated by reference in its entirety.
[0081] As used herein, the term antibody fragment refers to a portion of a full-length antibody, including at least a portion antigen binding region or a variable region. Antibody fragments include, but are not limited to, Fab, Fab, F(ab).sub.2, Fv, scFv, Fd, diabodies, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al. (2003) Nat. Med. 9:129-134; herein incorporated by reference in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies (e.g., papain digestion and pepsin digestion of antibody). produced by recombinant DNA techniques, or chemical polypeptide synthesis.
[0082] As used herein, the term native immune cell refers to an immune cell that naturally occurs in the immune system of a subject. Illustrative examples include, but are not limited to, T-cells, NK cells, NKT cells, B cells, and dendritic cells.
[0083] As used herein, the term engineered immune cell refers to an immune cell (e.g., T-cell, NK cell, NKT cell, B cell, dendritic cell, etc.) that is genetically modified.
[0084] The term chimeric antigen receptor (CAR) refers to a recombinant polypeptide construct comprising at least an extracellular antigen-recognition domain, a transmembrane domain and an intracellular signaling domain. Upon binding to their target (e.g., displayed on a cancer cell), CARs typically modify the immune response of the immune cells they are displayed upon.
[0085] As used herein, the term CAR-T cell refers to a T cell that has been engineered to express a chimeric antigen receptor. In particular embodiments, T cells (e.g., from a subject) are engineered to express a CAR that binds to a cancer-specific antigen of cancer cells, thereby allowing CAR-T cells to effectively recognize and kill cancer cells,
[0086] As used herein, the term adoptive cell transfer (ACT) is the transfer of cells into a patient. The cells may have originated from the patient or from another individual or cell line.
[0087] The cells are most commonly derived from the immune system, with the goal of improving immune functionality or eliciting a desired immune response. In some embodiments, cells are extracted from a subject, genetically modified (e.g., to express a desired construct (e.g., CAR or endanger molecule)), cultured in vitro, and returned to the subject.
DETAILED DESCRIPTION
[0088] Provided herein are compositions and methods for stimulating T-cell-based anti-tumor immunity by administration of trans-vaccenic acid (TVA), an active derivative thereof, or an inhibitor of GPR43 expression or activity. In particular embodiments, TVA, a TVA derivative, or an inhibitor of GPR43 expression or activity is administered to boost an endogenous T-cell response and/or is co-administered with a T-cell-based therapy, such as immune checkpoint blockade therapies, CAR-T therapies, monoclonal antibody therapies, bispecific T-cell engagers therapies, etc.
[0089] Provided herein arc compositions (e.g., pharmaceutical compositions, supplements, etc.) comprising TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, TVA-containing or active-TVA-derivative-containing compositions are provided for the treatment of cancer and/or as a supplement to enhance a cancer treatment. In some embodiments, TVA-containing or active-TVA-derivative-containing compositions are provided for the prevention of cancer. In some embodiments, TVA-containing or active-TVA-derivative-containing compositions are provided for reducing the likelihood or reducing the severity of cancer.
[0090] In some embodiments, provided herein is a compound of formula (I):
##STR00010## [0091] wherein: [0092] R.sup.1 is selected from hydrogen and C.sub.1-C.sub.4 alkyl; [0093] X is selected from C.sub.1-C.sub.24 alkylene, C.sub.2-C.sub.24 alkenylene, and C.sub.2-C.sub.24 alkynylene; and [0094] R.sup.2 is selected from hydrogen, optionally substituted aryl, optionally substituted heteroaryl, C.sub.1-C.sub.4 alkoxy, hydroxy, and halo.
[0095] In some embodiments, R.sup.1 is selected from hydrogen and methyl. In some embodiments, R.sup.1 is hydrogen.
[0096] In some embodiments, X is selected from C.sub.8-C.sub.24 alkylene, C.sub.8-C.sub.24 alkenylene, and C.sub.8-C.sub.24 alkynylene. In some embodiments, X is selected from C.sub.12-C.sub.24 alkylene, C.sub.12-C.sub.24 alkenylene, and C.sub.12-C.sub.24 alkynylene. In some embodiments, X is C.sub.12-C.sub.24 alkylene. In some embodiments, X is C.sub.12-C.sub.24 alkenylene. In some embodiments, X is C.sub.12-C.sub.24 alkynylene.
[0097] In some embodiments, R.sup.2 is selected from hydrogen and optionally substituted aryl. In some embodiments, R.sup.2 is selected from hydrogen and phenyl, wherein the phenyl is unsubstituted or substituted with one substituent selected from C.sub.1-C.sub.4 alkyl (e.g., methyl). In some embodiments, R.sup.2 is hydrogen.
[0098] In some embodiments, the compound is selected from:
##STR00011##
[0099] Experiments conducted during development of embodiments herein demonstrate that TVA exerts a therapeutic activity via attenuation of GPR43 activity. In some embodiments, provided herein are inhibitors of GPR43 activity or expression and methods of their use to boost an endogenous T-cell response and/or for use with a T-cell-based therapy as described herein.
[0100] In some embodiments, the composition comprises a nucleic acid inhibitor of GPR43 expression or activity. In some embodiments, the composition comprises a siRNA, shRNA, antisense RNA, or CRIPSR system to inhibit expression of GPR43.
[0101] In some embodiments, the composition comprises a small molecule or peptide inhibitor of GPR43 activity. In some embodiments, the small molecule inhibitor of GPR43 activity is a GPR43 antagonist. In some embodiments, the GPR43 antagonist has the structure:
##STR00012##
In some embodiments, the small molecule inhibitor of GPR43 activity is an inverse agonist of GPR43. In some embodiments, the inverse agonist of GPR43 has the structure:
##STR00013##
[0102] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is formulated for administration to a subject (e.g., human subject). In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is formulated as a supplement. In some embodiments, provided herein is a supplement consisting of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity and suitable carriers. In some embodiments, the supplement comprises TVA (or an active TVA derivative or an inhibitor of GPR43 expression or activity) and other nutrients, vitamins, and/or minerals. In some embodiments, the other components of a supplement are present to assist or enable delivery of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, other components of a supplement are present to enhance the health of a subject or to otherwise effect the treatment of cancer. Nutrients that may be provided with TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity include, but are not limited to minerals (e.g., iron, manganese, magnesium, copper, calcium, phosphorous, etc.), vitamins (e.g., biotin, choline, folate, niacin, pantothenic acid, riboflavin, thiamin, and vitamins A, B6, B12, C, D, E, K, etc.), and other fatty acids (e.g., omega-3 fatty acids (e.g., a-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc.), omega-6 fatty acids (e.g., linoleic acid (LA), etc.), trans fatty acids, saturated fatty acids, unsaturated fatty acids, polyunsaturated fatty acids (PUFA), etc.). In some embodiments, a supplement comprises TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity or activity and one or more active bacterial cultures, wherein the bacterial cultures comprise species of bacteria that are beneficial to human health or the treatment/prevention of cancer. In some embodiments, a TVA-containing, an active-TVA-derivative-containing, or GPR43-inhibitor-containing supplement is formulated according to standard supplement formulations that are understood in the art. In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is provided as a nutrient supplement to T cell activity when administered to, for example, an infant, an elderly subject, an immunocompromised patient, etc.
[0103] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is formulated as a pharmaceutical composition. In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity and any co-formulated agents (when present) are provided in pharmaceutical formulations for administration to a subject by a suitable route. The pharmaceutical formulations described herein can be administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. Moreover, TVA-containing or active-TVA-derivative-containing pharmaceutical compositions are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, aerosols, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, and capsules.
[0104] Pharmaceutical preparations comprising TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity provided for oral use can be obtained by mixing one or more solid excipients with TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity (and other therapeutic agents desired in the formulation) with any suitable substituents and functional groups disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets, pills, or capsules. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[0105] In some embodiments, routes of administration, formation of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity, etc. are selected to provide efficient and effective delivery. In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is provided with a suitable carrier. In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is encapsulated of embedded into a carrier. In some embodiments, a carrier may comprise a liposome, nanoparticle, or other suitable system for delivery of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is conjugated to a carrier molecule. Suitable carrier molecules for conjugation of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity may include small molecules, peptides, proteins, polymers, etc. In some embodiments, the carrier and/or delivery system is selected to optimize the solubility, stability, bioavailability, targeting, etc. of the TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity.
[0106] The supplement or pharmaceutical compositions described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multiple-dose reclosable containers can be used, in which case it is typical to include a preservative in the composition.
[0107] Dosing and administration regimes are tailored by the clinician, or others skilled in supplements or the pharmacological arts, based upon well-known pharmacological and therapeutic considerations including, but not limited to, the desired level of therapeutic effect, and the practical level of therapeutic effect obtainable. Generally, it is advisable to follow well-known pharmacological principles for administrating chemotherapeutic agents (e.g., it is generally advisable to not change dosages by more than 50% at time and no more than every 3-4 agent half-lives). For compositions that have relatively little or no dose-related toxicity considerations, and where maximum efficacy is desired, doses in excess of the average required dose are not uncommon. This approach to dosing is commonly referred to as the maximal dose strategy. In certain embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is administered to a subject at a dose of about 0.01 mg/kg to about 200 mg/kg (e.g., 0.01 mg/kg, 0.02 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, 200 mg/kg, or ranges therebetween). Dosing may be once per day, multiple times per day (e.g., 2, 3, 4, etc.), once per week, or according to any suitable protocol. In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is administered a single time, each time a co-administered agent is delivered, or for a time period of days, weeks, months, indefinitely, etc.
[0108] In some embodiments, compositions comprising TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity are administered alone or in combination with any other cancer treatments (e.g., immunotherapies, chemotherapies, etc.) using standard delivery systems and methods, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient.
[0109] In some embodiments, methods are provided relating to the prevention, treatment or amelioration of a cancer comprising the step of administering to a subject in the need thereof an effective amount of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, compositions comprising TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity are provided for the prevention, treatment or amelioration of a cancer. In some embodiments, the methods of administering TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity and compositions comprising TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity are provided in combination with other therapies and therapeutics for the treatment/prevention of cancer.
[0110] In some embodiments, indications for administration of the composition(s) herein are cancerous diseases. Examples of hematological (or hematogenous) cancers that are treated/prevented in embodiments herein include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myeiodysplastic syndrome, hairy cell leukemia and myelodysplasia. Examples of solid tumors that are treated/prevented in embodiments herein include, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medu!loblastoma, Schwannoma craniopharyogioma, ependymoma, pincaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).
[0111] The disclosure further encompasses protocols for the co-administration of TVA-containing or active-TVA-derivative-containing compositions (e.g., alone, with a pharmaceutically-acceptable carrier, with other natural products, etc.) with other therapeutics, such as, bispecific antibody constructs, targeted toxins or other blocking or functional antibodies or compounds, immune cells (e.g., CAR-T cells), immune checkpoint blockade therapies, monoclonal antibody therapies, and other treatments described herein. The clinical regimen for co-administration may encompass co-administration at the same time, before or after the administration of the other component. Particular combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy.
[0112] Many chemotherapeutics are presently known in the art and can be used in combination with the TVA or active-TVA-derivative therapy. In some embodiments, the chemotherapeutic is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.
[0113] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is co-administered with one or more chemotherapeutics. Chemotherapies for use with TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity include all classes of chemotherapeutic agents, such as, alkylating agents, antimetabolites, plant alkaloids, antibiotics, hormonal agents, and miscellaneous anticancer drugs. Specific agents include, for example, abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, fuldarabine, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, and vinblastin, or any analog or derivative variant of the foregoing and also combinations thereof. In some embodiments, chemotherapy is employed before, during and/or after administration of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity.
[0114] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is co-administered with radiotherapy, methods of which are understood in the field. In some embodiments, radiotherapy is employed before, during and/or after administration of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity.
[0115] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity co-administered with non-immune based targeted therapies, such as, agents that inhibit signaling pathways such WNT, p53, and/or RB-signaling pathways. Other examples include agents that inhibit tyrosine kinases, BRAF, STAT3, c-met, regulate gene expression, induce cell death or block blood vessel formation. Examples of specific agents include imatinib mesylate, dasatinib, nilotinib, bosutinib, lapatinib, gefinitib, erlotinib, tensirolimus, everolimus, vemurafenib, crizotinib, vorinostat, romidepsin, bexarotene, alitrionin, tretionin, bortezomib, carfilzomib, pralatrexate, sorafenib, sunitinib, pazopanib, regorafenib, or cabozantinib. In some embodiments, non-immune based targeted therapy is employed before, during and/or after administration of TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity.
[0116] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is co-administered with a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the composition comprising TVA. A variety of expression products are encompassed, including inducers of cellular proliferation, inhibitors of cellular proliferation, or regulators of programmed cell death.
[0117] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is administered before, during, and/or after surgery. Surgeries include resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that embodiments herein may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
[0118] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is co-administered with other agents to improve the therapeutic efficacy of treatment.
[0119] In some embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is provided as part of a kit or system along with one or more additional components, such as instructions, devices for administration, additional therapeutic agents, diagnostic agents, research agents, etc.
[0120] In particular embodiments, TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity is co-administered with an immunotherapy. Immunotherapeutics generally rely on the use of immune effector cells (e.g., NK cells, T cells (e.g., CAR-T cells), etc.) and/or molecules (e.g., checkpoint inhibitors, bispecific engager molecules, monoclonal antibodies, etc.) to target and destroy cancer cells.
[0121] A immune effector for co-administration with TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity may be, for example, an antibody, antibody fragment, bispecific engager, etc. that is specific (e.g., binds to) a marker on the surface of a cancer cell, tumor cell, cancer stem cell, etc. The immune effector alone may serve as a therapy or it may recruit cells to effect cell killing. The immune effector may also prevent cancer immunoevasion or immunosuppression. The immune effector also may be conjugated to a drug or toxin (e.g., chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T-cells, NKT cells, and NK cells. In some embodiments, immunotherapy is employed before, during and/or after administration of TVA. In some embodiments, TVA is co-administered with an immune checkpoint inhibitor (e.g., anti-PD1, anti-PDL1, anti-CTLA-4, etc.).
[0122] In some embodiments, methods comprise co-administering TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity to a subject with an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a small molecule, protein, or peptide that specifically binds to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is selected from the group consisting of CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3 or VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof. In some embodiments, the immune checkpoint inhibitor is an interfering nucleic acid molecule. In some embodiments, the interfering nucleic acid molecule is an siRNA molecule, an shRNA molecule or an antisense RNA molecule. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, BMS-936558, MK-3475, CT O1 1, MPDL3280A, MEDI-4736, MSB-0020718C, AUR-012 and STI-A1010. In some embodiments, the immune checkpoint inhibitor is administered before the TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, the immune checkpoint inhibitor is administered at least one day before the TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, the immune checkpoint is administered at about the same time as the TVA, an active TVA derivative, or an inhibitor of
[0123] GPR43 expression or activity. In some embodiments, the immune checkpoint inhibitor is administered on the same day as the TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, the immune checkpoint inhibitor is administered after the TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, the immune checkpoint inhibitor is administered at least one day after the TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity. In some embodiments, the immune checkpoint inhibitor is administered by injection. In some embodiments, the injection is an intravenous, intramuscular, intratumoral or subcutaneous injection.
[0124] In some embodiments, methods comprise co-administering TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity to a subject with a bispecific agent (e.g., bispecific antibody, bispecific engager, etc.). In some embodiments, TVA is co-administered with bispecific antibodies that bind with one arm to a surface antigen on a target cells (e.g., a tumor antigen), and with the second arm to an activating, invariant component of the T cell receptor (TCR) complex. The simultaneous binding of such a bispecific agent to both of its targets forces an interaction between target cell (e.g., cancer cell) and T cell, causing activation of any cytotoxic T cell and subsequent lysis of the target cell. Bispecific T cell engager (BiTE) molecules are tandem scFv molecules wherein two scFv molecules are fused by a flexible linker. BiTEs have been very well characterized and already shown some promise in the clinic (reviewed in Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); incorporated by reference in its entirety). Further bispecific engager formats for T cell engagement and targeting to cancer cells that find use with TVA co-administration include diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); incorporated by reference in its entirety); derivatives thereof, such as tandem diabodies (Kipriyanov et al, J Mol Biol 293, 41-66 (1999); incorporated by reference in its entirety); dual affinity retargeting (DART) molecules, which are based on the diabody format but feature a C-terminal disulfide bridge for additional stabilization (Moore et al, Blood 117, 4542-51 (2011); incorporated by reference in its entirety); triomabs, which are whole hybrid mouse/rat IgG molecules (reviewed in Seimetz et al, Cancer Treat Rev 36, 458-467 (2010); incorporated by reference in its entirety). In some embodiments, the bispecific engager comprises an activation domain that binds to a T cell activating antigen, such as CD3. In some embodiments, the bispecific engager comprises an antigen-binding domain capable of binding is a tumor or cancer cell antigen. In some embodiments, antigen-binding domain binds a tumor associated antigen (TAA). In some embodiments, antigen-binding domain binds a tumor specific antigen (TSA). Non-limiting examples of TSAs or TAAs include the following: Differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.
[0125] In some embodiments, methods comprise co-administering TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity to a subject with a CAR or CAR-T cell. CARs are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif, (2) linking/transmembrane motifs, and (3) intracellular T-cell signaling motifs (Long A H, Haso W M, Orentas R J. Oncoimmunology. 2013; 2 (4): e23621; incorporated by reference in its entirety). The antigen-binding motif of a CAR is commonly fashioned after an single chain Fragment variable (ScFv), the minimal binding domain of an immunoglobulin (Ig) molecule. The linking motifs of a CAR can be a relatively stable structural domain, such as the constant domain of IgG, or designed to be an extended flexible linker. Structural motifs, such as those derived from IgG constant domains, can be used to extend the ScFv binding domain away from the T-cell plasma membrane surface. Signaling motifs used in CARs typically comprise the CD3-zeta chain because this core motif is the key signal for T cell activation. CARs and CAR-T cells capable of interacting (binding) any antigens described herein are provided for co-administration with TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity.
[0126] In some embodiments, methods comprise co-administering TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity to a subject with a monoclonal antibody (mAb). Examples of mAb for co-administration with TVA, an active TVA derivative, or an inhibitor of GPR43 expression or activity include known antibodies such as brentuximab, trastuzumab, inotuzumab, gemtuzumab, glembatumumab, labetuzumab, sacituzumab, lifastuzumab, indusatumab, polatuzumab, pinatuzumab, coltuximab, indatuximab, milatuzumab, rovalpituzumab, anetumab, tisotumab, mirvetuximab, lorvotuzumab, rituximab, depatuxizumab, denintuzumab, enfortumab, telisotuzumab, vandortuzumab, sofituzumab, vorsetuzumab, mirvetuximab, naratuximab, cantuzumab, laprituximab, bivatuzumab, vadastuximab, lupartumab, aprutumab, abagovomab, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atorolimumab, avelumab, azintuxizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, begelomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camrelizumab, caplacizumab, capromab, carlumab, carotuximab, catumaxomab, cedelizumab, certolizumab, cetuximab, citatuzumab, cixutumumab, clenoliximab, clivatuzumab, codrituzumab, conatumumab, concizumab, cosfroviximab, crenczumab, crizanlizumab, crotedumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab, daratumumab, dectrekumab, demcizumab, denosumab, detumomab, dezamizumab, dinutuximab, diridavumab, domagrozumab, dorlimomab, drozitumab, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enavatuzumab, enlimomab, enoblituzumab, enokizumab, enoticumab, ensituximab, epitumomab, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evinacumab, evolocumab, exbivirumab, faralimomab, farletuzumab, fasinumab, felvizumab, fezakinumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, firunevetmab, fulranumab, futuximab, galcanezumab, galiximab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gevokizumab, gilvetmab, girentuximab, golimumab, guselkumab, ibalizumab, ibritumomab, icrucumab, idarucizumab, ifabotuzumab, igovomab, imalumab, imciromab, imgatuzumab, inclacumab, inebilizumab, infliximab, inolimomab, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, keliximab, lacnotuzumab, lampalizumab, lanadelumab, landogrozumab, larcaviximab, lebrikizumab, lemalesomab, lenzilumab, lerdelimumab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifatuzumab, ligelizumab, lilotomab, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab, losatuximab, lucatumumab, lulizumab, lumretuzumab, lutikizumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, minretumomab, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, motavizumab, moxetumomab, muromonab, nacolomab, namilumab, naptumomab, narnatumab, natalizumab, navicixizumab, navivumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, opicinumab, oportuzumab, oregovomab, oreticumab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, pembrolizumab, perakizumab, pertuzumab, pexelizumab, pidilizumab, placulumab, plozalizumab, ponczumab, porgaviximab, prczalumab, priliximab, pritoxaximab, pritumumab, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, refanezumab, regavirumab, remtolumab, reslizumab, rilotumumab, rinucumab, risankizumab, rivabazumab, robatumumab, roledumab, romosozumab, rontalizumab, rosmantuzumab, rovelizumab, rozanolixizumab, ruplizumab, samalizumab, sarilumab, satralizumab, satumomab, secukinumab, selicrelumab, seribantumab, setoxaximab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sontuzumab, stamulumab, sulesomab, suptavumab, suvizumab, suvratoxumab, tabalumab, tadocizumab, talizumab, tamtuvetmab, tanezumab, taplitumomab, tarextumab, tavolixizumab, fanolesomab, nofetumomab, pintumomab, tefibazumab, telimomab, telisotuzumab, tenatumomab, teneliximab, teplizumab, teprotumumab, tesidolumab, tezepelumab, tigatuzumab, tildrakizumab, timigutuzumab, timolumab, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, tregalizumab, tremelimumab, trevogrumab, tucotuzumab, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vantictumab, vanucizumab, vapaliximab, varisakumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, votumumab, vunakizumab, tacatuzumab, zalutumumab, zanolimumab, ziralimumab, zolimomab or anti-embigin antibody; examples of another aspect thereof include brentuximab, trastuzumab, inotuzumab, gemtuzumab, labetuzumab, polatuzumab, coltuximab, indatuximab, anetumab, rituximab, denintuzumab, laprituximab, vadastuximab, glembatumumab, cetuximab, alemtuzumab or depatuxizumab; examples of another aspect thereof include brentuximab, trastuzumab, rituximab or anti-embigin antibody; and examples of another aspect thereof include brentuximab or trastuzumab, and preferably, examples of another aspect include brentuximab.
[0127] Some embodiments described herein find use with TVA. Some embodiments herein find use with the full genus of TVA derivatives described herein. Some embodiments herein find use with specific TVA derivatives described herein.
EXPERIMENTAL
Example 1
Dietary Trans-Vaccenic Acid Promotes Anti-Tumor Immunity
Results
Dietary TVA Enhances Anti-Tumor Immunity
[0128] A blood chemical compound library was constructed for the screening purpose. There are roughly 633 circulating blood chemicals (Table 1), including inorganics (e.g., minerals), organic metabolites (e.g., carbohydrates and amino acids), lipids (e.g., unsaturated and saturated fatty acids), dietary supplements (e.g., vitamins and antioxidants), and proteins (e.g., hormones and cytokines). This summary combines the United States Department of Agriculture (USDA) list of defined diet-derived nutrients (www.nal.usda.gov/legacy/fnic/nutrient-lists-standard-reference-legacy-2018), GNC list of dietary supplements (www.gnc.com/vitamins-supplements), and Wikipedia list of human blood components (www.wikipedia.org/wiki/List_of_human_blood_components). The library used for cell-based screens contains 255 compounds (Table 2) that are commercially available. Proteins that have been extensively studied and some supplements that only function at the organismal level such as fish oils and herbs were excluded. The reported physiological range of serum levels and working levels (2 of highest reported serum level) in the screens of individual blood chemicals are included in Table 2.
[0129] Two preliminary screens were designed and performed. The 1.sup.st screen 1a (Table 3) was to identify blood chemicals that enhance activation of Jurkat T cells stimulated by anti-CD3/CD28 antibodies (
[0130] To determine whether TVA enhances cytotoxic T cell function, TVA's effects on co-cultured mouse melanoma B16F10 cells and pmel-1-specific mouse T cells was tested. It was found that TVA treatment enhances cytotoxicity of B16F10 cells induced by co-cultured pmel-1-specific T cells (
Dietary TVA Enhances Tumor-Infiltrating and Cytotoxic Function of Effector CD8.SUP.+ T Cells
[0131] To understand how dietary TVA influences tumor bearing mice, gut microbiota was first examined. The results suggest that TVA-enriched diet does not significantly alter the diversity and patterns of gut microbial distribution in mice (
[0132] Dietary TVA effects on different T cell populations were next examined. It was found that TVA diet results in a larger percentage of CD8.sup.+ T cells as a fraction of the CD45.sup.+ leukocytes infiltrated in B16F10 tumors and dLNs but not spleens (
[0133] Consistent with these findings, anti-CD3/CD28-stimulated primary CD8.sup.+ T cells with TVA treatment in vitro (
TVA Exhibits a Nutrient-Independent, Signaling Function Through a GPCR-CREB Axis
[0134] To determine the underlying mechanisms by which TVA activates CD8.sup.+ T cells, it was first determined whether TVA functions in an extracellular or intracellular manner. Quantitative mass spectrometry detected isotope-labeled .sup.13C-TVA imported from media to primary murine CD8.sup.+ T cells in a dose-dependent manner, which is CD36-dependent because treatment with CD36 inhibitor sulfosuccinimidyl oleate (SSO) drastically reduced import of .sup.13C-TVA (
[0135] To elucidate the underlying mechanisms, integrated temporal mechanistic investigations on TVA's effects using primary human or murine CD8.sup.+ T cells (
[0136] Consistent with these findings, Gene Ontology (GO) enrichment analysis (Ashburner et al., 2000; Carbon et al., 2021) and global Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005) identified a set of increased signal pathways which are responsible for the effects of TVA treatment on CD8.sup.+ T cells at 24 h including T cell proliferation and activation, and a set of decreased signal pathways including apoptosis (
TVA Signals Through the GPCR-cAMP-PKA-CREB Axis to Enhance CD8.SUP.+ T Cell Function
[0137] Next, which downstream pathway(s) of GPCR is required for TVA function (results summarized in
TVA-Enhanced CD8.SUP.+ T Cell Function is Primarily Mediated Through CREB and its Target Gene Sets
[0138] Next, the importance of CREB and its gene targets was explored. Transcriptome-wide RNA-seq was performed using CD8.sup.+ T cells with siRNA-mediated knockdown of CREB compared to control CD8.sup.+ T cells treated with non-targeting control siRNA (siNTC) in the presence and absence of TVA treatment (
[0139] To further identify the TVA-CREB downstream targets, genes were characterized that were upregulated or downregulated only in the siNTC+TVA group, compared to other 3 groups (marked by yellow boxes in
[0140] As shown in
TVA Antagonizes Immunosuppressive SCFA-Binding GPR43
[0141] To identify the GPCR target of TVA, six of the known fatty acid-binding GPCRs were screened including GPR40, GPR41, GPR43, GPR84, GPR119, GPR120 (Swaminath, 2008) (
[0142] To explore the underlying mechanism by which TVA antagonizes GPR43, structure-activity relationship (SAR) studies were performed on TVA (
[0143] Next, whether TVA may function like a ligand-like antagonist of GPR43 by competing off its SCFA agonists was examined. Indeed, it was found that GPR43 SCFA agonists (20 mM) significantly decrease mouse CD8.sup.+ T cell activation assessed by TNF- level, while TVA at a 1,000 lower level (20 M) is able to reverse SCFA-dependent suppression on CD8.sup.+ T cells (
[0144] Lastly, it was found that stimulation by anti-CD3/CD28 antibodies results in a much higher increased GPR43 expression level in CD8.sup.+ T cells than that in CD4.sup.+ T cells (
TVA Augments Effectiveness of Multiple T Cell-Based Anti-Cancer Therapies
[0145] To explore translational potential of these findings, TVA-dependent effects on multiple T cell-based immunotherapies were examined. Immune checkpoint inhibitors (ICIs) including antibodies targeting PD-1 and PD-L1 have changed the treatment landscape of many tumors (DallOlio et al., 2021). However, immunological treatment of cold tumors due to lack of tumor-infiltrating T cells or not eliciting an immune response remains a great challenge (Jenkins et al., 2018; Vukadin et al., 2021). It was found that TVA treatment significantly reverses exhaustion of primary human bulk T cells (
[0146] Next, the effects of TVA on efficacy of Blinatumomab were tested. Blinatumomab is a bi-specific T-cell engager (BiTE) designed as a constructed monoclonal antibody targeting CD19 antigen on B cells and CD3 on T cells for treatment of human B cell acute lymphoblastic leukemia (B-ALL). Clinically, Blinatumomab is effective for minimal residual disease, but the response rate in relapsed disease is <50% due to lack of T-cell activation (Kantarjian et al., 2017). It was found that TVA treatment significantly enhances in vitro killing efficiency of human peripheral blood mononuclear cells (PBMCs) on human B-ALL RS4;11 cells in the presence of Blinatumomab in a dose-dependent manner (
[0147] Furthermore, TVA treatment improves in vitro expansion of chimeric antigen receptor (CAR) T cells derived from primary T cells from 3 lymphoma patients with age between 42 and 47 (
[0148] This study demonstrates that TVA as a diet-derived nutrient exhibits a nutrient-independent, extracellular signaling function to enhance CD8.sup.+ T cell function and consequent anti-tumor immunity. Thus, as a natural food component, TVA has translational potential to be used as a dietary element in therapeutic approaches to ameliorate clinical outcomes. For example, since TVA signals through the GPR43-CREB pathway that is independent of the PD-L1/PD-1 axis, the finding experimentally provides a mechanism-based rationale supporting a combination of TVA and immune checkpoint inhibitors for improved immunotherapies to treat cancer patients with immunologically cold tumors. In addition, TVA can be combined with specific T-cell engagers such as Blinatumomab to treat B-ALL patients. TVA can also be used to help expand and prime CAR T cells for improved efficacy of treating cancer patients. Lastly, serum level of TVA may be a useful biomarker for prediction of responsiveness to T cell-based immunotherapies such as CAR T-cell therapy and T cell receptor (TCR)-T cell therapy. Epidemiological studies suggest that the circulating levels of TVA in humans are associated with lower adiposity, diabetes risk and systemic inflammation, but the effects of dietary TVA on risk of cancer and cardiovascular diseases are unclear, despite that, in rodent models of dyslipidemia, TVA-enriched diet has hypolipidemic effects by lowering circulating triglyceride (Gebauer et al., 2011; Pranger et al., 2019; Wang et al., 2008). The reported normal ranges of blood TVA levels in human adults vary widely from 10.7+/5.0 M (Psychogios et al., 2011) to 186.5+/45.8 M (Dercksen et al., 2016), which are lower than these measurements using serum samples from lymphoma patients. This may be due to variations of sample collections and measurement approaches. Regardless, these SAR studies reveal that the bioactivity of TVA could be improved through chemical modifications including, for example, shifting the double bond to a more optimized position or changing the chain length. Thus, the next generation of TVA-derivatives could be designed and developed with improved effectiveness to target GPR43 and consequently enhance CD8.sup.+ T cell function and anti-tumor immunity.
[0149] This study also validates a new dimension through a comprehensive evaluation of blood chemicals in order to uncover and understand mechanistic links between diet and cancer or other human diseases. Circulating diet-derived blood chemicals have been inextricably linked to human physiology during evolution, which not only provide energy and precursors for biosynthesis for growth but also function as signaling molecules (Rodriguez et al., 2017). Accordingly, despite the vast diversity of food and diet origins, studies with a focus on blood chemicals get to the heart of the molecular mechanisms underlying nutritional influences on cancer and effectiveness of anti-cancer therapies. This evaluation of blood chemicals, as a representative, also indicates that the approach may have broad implications for future elucidation of previously unknown physiological and pathological roles of the circulating blood chemicals in human health and disease, respectively.
[0150] The integrated mechanistic studies identified cell-surface immunosuppressive GPR43, a SCFA-binding GPCR, as the target of TVA. It was shown that GPR43 contributes to immunosuppression through regulation of CD8.sup.+ T cell activation and exhaustion by signaling through the cAMP-PKA-CREB axis. This signaling mechanism may be cell type-specific for CD8.sup.+ T cells because GPR43 agonism was reported to promote colonic expansion and function of Group 3 innate lymphoid cells (ILC3s) and IL-22 production through AKT and STAT3 signaling pathways (Chun et al., 2019). However, TVA-dependent antagonism of GPR43 results in an increased phosphorylation level of STAT3 (p-STAT3) with a reduced phosphorylation level of AKT (p-AKT) in CD8.sup.+ T cells at 2 h post-TVA treatment (
[0151] Also observed was a much higher increase in GPR43 expression upon activation of CD8.sup.+ T cells than that in CD4.sup.+ T cells (
Materials and Methods
Data and Code Availability
[0152] 16S amplicon sequencing data have been deposited in the GEO repository with the accession number GSE202266 and are publicly available as of the date of publication. The KAS-seq data have been deposited in the GEO repository with the accession number GSE202730. The RNA-sequencing data have been deposited in the GEO repository with the accession number GSE202276 and GSE202274. Project number and accession links are listed in the Resources Table. Original western blot images have been deposited on Mendeley and are publicly available as of the date of publication. The DOI is also listed in the Resources Table. This paper does not report original code
Animal Study
[0153] 5-8 weeks old C57BL/6 mice, C57BL/6 nude mice, TCR-a KO, and Pmel-1 mice were purchased from the Jackson Laboratory. Please also refer to Resources Table for more details
Primary Cells
[0154] Cas9; OT-I cells were isolated from the spleen and peripheral lymph nodes (PLN) (provided by Dr. Hongbo Chi's lab) of Cas9; OT-I mice (Wei et al., 2019) by magnetic bead purification using Easy Sep Mouse nave CD8.sup.+ T Cell Isolation Kit according to the manufacturer's instructions (Stem Cell Technologies). Cas9; OT-I cells were activated in vitro for 18 hours with plate-bound anti-CD3 (10 g/mL; Biolegend) and anti-CD28 (5 g/mL; Biolegend) antibodies in Click's media at 37 C. and 5% CO.sub.2 incubator for further experiments
Cell Line
[0155] All cell lines were authenticated by genomic short tandem repeat (STR) profiling in University of Chicago Integrated Genomics Core (EIGC) upon purchase and at least annually as appropriate. Human T lymphocyte cell line Jurkat T was purchased from American Type Culture Collection (ATCC). Human Plat-E cells were provided by Dr. Hongbo Chi's lab. Human RS4;11 cells were provided by Dr. Wendy Stock lab. Mouse melanoma cancer cell line B16F10, breast cancer cell line E0771, and Lewis lung carcinoma cell line LLC1 were purchased from ATCC. Mouse colorectal adenocarcinoma cell line MC38 was purchased from Kerafast. Plat-E, B16F10, E0771, and LLC1 cells were cultured in Dulbecco Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) (Sigma, F2442) and penicillin/streptomycin. Jurkat T cells were cultured in RPMI 1640 medium with 10% FBS and penicillin/streptomycin. All the cells were cultured at 37 C. and 5% CO.sub.2. Please also refer to Resources Table for detailed information of each cell line.
Human Samples Collection
[0156] Serums of patients that have undergone commercial CAR T cell therapy were from University of Chicago cell therapy biobank. Please also refer to Resources Table and Table 8 for more details.
Construction of PD-1.SUP.+ Jurkat T Cell Line
[0157] Jurkat T cells were infected with pre-made lentivirus expresses human PD-1 (Gen Target Inc, Cat #LVP1076-PBS) according to the manufacturer's instructions. After infection for 24 hours, cells were selected with 2 g/mL puromycin to obtain PD-1.sup.+ Jurkat T cells. The PD-1 expression level was checked using western blot
Blood Chemicals Library Screens
[0158] To construct blood chemical compound library for cell-based screening purposes, components such as antibodies that are difficult to distinguish due to a wide variety, and some supplements that only function at a whole organism level including fish oils and herbs were excluded. Physiological ranges of serum levels of individual blood chemicals are available in the human metabolome database (https://hmdb.ca/) and applied in the experimental design. For first screen 1a, Jurkat T cells were treated with blood chemical library for 48 hours and then activated with 2.5 g/mL anti-CD3 and 0.5 g/mL anti-CD28 antibodies for another 12 hours, followed by measurement of IL-2 level in medium supernatant using ELISA kit (Biolegend). For first screen 1b, 110.sup.5 PD-1.sup.+ Jurkat T cells were co-cultured with 210.sup.4 H596 (PD-L1.sup.+) cells in a well of 96-well plate for 60 hours, and then activated with 2.5 g/mL anti-CD3 and 0.5 g/mL anti-CD28 antibodies for another 12 hours, followed by measurement of IL-2 level in medium supernatant using ELISA kit. Please also refer to Resources Table and Table 1-2 for more details.
Mouse Tumor Models
[0159] For C57BL/6 mice and TCR-a KO mice tumor model, mice were anesthetized with isoflurane, shaved the injection site, and then injected subcutaneously in the abdominal flank with 110.sup.5 B16F10, MC38, or LLC1 cells, or in the mammary gland with 210.sup.5 E0771 cells. C57BL/6 nude mice were injected subcutaneously in the abdominal flank with 110.sup.5B16F10 cells. The tumor bearing mice were assigned to TVA enriched diet (1% TVA, special order from Research Diets Inc), CVA enriched diet (1% CVA, special order from Research Diets Inc) or control diet (Research Diets Inc) as of the day of tumor inoculation, with mice body weight monitored. Tumors were measured the tumors using a caliper every 2-3 days. Tumor volumes were calculated using the following formula: lengthwidth[(lengthwidth).sup.0.5]/6. Mice were euthanized at humane endpoints or day 11-15 for tissue collection.
[0160] For CREB inhibitor 666-15 treatment mouse model, 6-8 weeks old C57BL/6 mice were anesthetized with isoflurane, shaved the injection site, and then injected subcutaneously in the abdominal flank with 110.sup.5 B16F10 for tumor development. The tumor bearing mice were assigned to TVA enriched diet or control diet as of the day of tumor inoculation, when the tumor volume reached approximately 100 mm.sup.3, the mice were treated with either vehicle or 666-15 at 20 mg/kg. 666-15 was dissolved in 1% N-methylpyrrolidone (NMP), 5% Tween-80 in H.sub.2O. The mice were treated once a day for 5 consecutive days per week for 2 weeks. Tumors were measured the tumors using a caliper every 2-3 days. Tumor volumes were calculated using the following formula: lengthwidth[(lengthwidth).sup.0.5]/6. Mice were euthanized at humane endpoints.
[0161] For anti-PD-1 treatment mouse model, 6-8 weeks old C57BL/6 mice were anesthetized with isoflurane, shaved the injection site, and then injected subcutaneously in the abdominal flank with 110.sup.5 B16F10 for tumor development. The tumor bearing mice were assigned to TVA enriched diet or control diet as of the day of tumor inoculation. In day 3, 6, 9, 12, and 15, 250 g anti-PD-1 (BioXCell) or IgG control (BioXCell) was injected intraperitoneally per mouse. Tumors were measured the tumors using a caliper every 2-3 days. Tumor volumes were calculated using the following formula: lengthwidth[(lengthwidth).sup.0.5]/6. Mice were euthanized at humane endpoints. Please also refer to Resources Table for more details.
Antibody-Mediated T Cell Depletions
[0162] 5-8 weeks old C57BL/6 mice were injected subcutaneously in the abdominal flank with 110.sup.5 B16F10 cells, and then injected intraperitoneally with six doses of depleting antibodies (anti-CD8a, BioXCell, Clone #2.43) or isotype control (rat IgG2b isotype control, BioXCell, Clone #LTF-2) on days 1 (200 g), 2 (200 g), 4 (200 g), 8 (200 g), 12 (200 g), and 16 (200 g) relative to tumor injection (day 0). Cheek bleeds were collected and subjected to flow cytometry to check CD8.sup.+ T cell depletion efficiency on days 3, 12, and 18 using antibodies targeting non-competing CD8 epitopes (BV711 anti-mouse CD8a).
Secreted Cytokines Level
[0163] Human or mouse T cell secreted IL-2, TNF-, and IFN- levels were detected by ELISA MAX Standard Set (Biolegend) as the manufacturer's instructions. Please also refer to Resources Table for more details.
Pmel-1 Killing Assay
[0164] Pmel-1 cells were isolated from Pmel mouse and seeded at density of 110.sup.6 cells/well in 6-well plate (pre-coated with anti-CD3 and anti-CD28) for 18 hours. 110.sup.6 activated Pmel-1 cells were then co-cultured with 210.sup.5 B16F10 cells for 24 hours with or without 20 M TVA. All cells in suspension were collected, stained with anti-mouse CD45 and PI, and analyzed by flow cytometry. Please also refer to Resources Table for more details.
Cell Proliferation Assay
[0165] Cell proliferation assays were conducted by seeding 510.sup.4 cells in a 6-well plate. Cell numbers were recorded daily within 5 days using TC20 Automated Cell Counter (BioRad).
Extraction and Quantification of TVA Levels by NMR
[0166] Tumor interstitial fluid (TIF) and serum from tumor-bearing animals were isolated as described (Sullivan et al., 2019). In brief, tumors were dissected away from the euthanized animal, rinsed in the saline containing Petri dish, and then placed on top of the 20 m mesh nylon filter (Spectrum Labs, Cat #148134) that was affixed to the conical tube (Falcon, Cat #1495949A). After the conical tube lid was placed on top without screwing and taped using laboratory tape in place, the tumor containing conical tube was subject to centrifugation at 4 C. for 10 minutes at 106g using a refrigerated clinical centrifuge. 10-50 L of fluid were obtained in the bottom of the conical tube. To prepare the mouse serum, once the tumors were dissected, blood was isolated from the mouse heart by cardiac puncture using 1 mL 25G TB syringe, allowed to clot by leaving it undisturbed at room temperature, and then centrifuged at 1,500g for 10 min in a refrigerated centrifuge. The resulting supernatant was designated serum. The human serum samples were obtained from University of Chicago cell therapy biobank. The TIL and serum samples are lyophilized and subjected to quantification of TVA levels by .sup.1H nuclear magnetic resonance (NMR) spectroscopy using a Bruker Ascend 600 spectrometer equipped with CryoProbeProdigy. In a typical procedure, 350 L deuterated methanol (Methanol-d.sup.4) with 0.03% tetramethylsilane (TMS) purchased from Oakwood Chemical (Estill, SC, USA) was added to a lyophilized sample to dissolve TVA. After being vortexed, the sample was then centrifuged at 10000g for 5 min to collect the supernatant. .sup.1H-NMR spectrum of the supernatant (250 L) in a 335-pp Precision 3 mm NMR tube (Wilmad-Lab Glass, Vineland, NJ, USA) was acquired with delay time (d.sup.1) set to 2 s for 3072 scans. TVA concentration was calculated based on the integral of peak at 1.97 ppm and the TMS internal standard
CD45.SUP.+ Tumor-Infiltrating Leukocytes Isolation
[0167] Tumor tissue were dissected from euthanized tumor-bearing mice, minced into small pieces (2 mm) using a scalpel in a dish, and then transferred to a 14 mL round-bottom tube containing 5 mL tumor digestion medium (500 L Collagenase/Hyaluronidase Solution, 750 L 1 mg/mL DNase I Solution, and 3.75 mL RPMI 1640 Medium). After incubation at 37 C. for 25 min on a shaking platform, the digested tumor tissues were transferred into a 70 m mesh nylon strainer on a 50 mL conical tube, pushed through the strainer using the rubber end of a syringe plunger, and rinsed with the recommended medium. After centrifugation at 300g for 10 min at room temperature with the brake on low, the resulting cell pellets were added 10 mL of ammonium chloride solution for incubation at room temperature for 5 minutes, followed by centrifugation at 300g for 10 minutes at room temperature with the brake on low. The resulting cell pellets were re-suspended at 1-1010.sup.6 cells/mL in PBS and then subjected to CD45.sup.+ tumor-infiltrating leukocytes isolation by magnetic bead purification using EasySep Mouse TIL (CD45) Positive Selection Kit according to the manufacturer's instructions (Stemcell Technologies). Please also refer to Resources Table for more details.
Mouse Tumor-Infiltrating Lymphocytes Isolation
[0168] Freshly excised mouse tumor tissues were minced into small pieces (volume smaller than 1 mm.sup.3) by scissors in PBS, digested by Collagenase IV (1 mg/mL) and DNase I (200 U/mL) at 37 C. for 20 minutes in PBS with gentle rocking. The digested tumor tissues were added 5 times volume 0.01M EDTA, filtered into new tube through 200 mesh screen (100 m strainer), and then centrifuged at 300g for 5 minutes at room temperature. The resulting cell pellets were re-suspended with 3 mL PBS, laid gently on 3 mL lymphocytes isolation liquid (pre-warmed to room temperature), and subjected to density gradient centrifugation (300g, 30 minutes, room temperature, accelerate 6, decelerate 2). The middle layer of cells was moved carefully to a new tube, added PBS to 15 mL, and centrifuged (300g, 10 minutes, room temperature). The cell pellets (lyse red cells if necessary) were designated tumor-infiltrating lymphocytes and used for following experiments. Please also refer to Resources Table for more details.
Mouse Spleen Lymphocytes Isolation
[0169] Mouse spleens were disrupted with syringe plunger in 1 mL PBS in a 40 m strainer filtered to a 15 mL tube, washed with PBS and centrifuged at 300g for 5 minutes. The resulting cell pellets were re-suspended the with 2 mL red cell lysis buffer (Invitrogen), incubated at room temperature for 10 minutes, and centrifuged at 300g for 5 minutes after adding 13 mL PBS. The resulting cell pellets was designated splenocytes and used for following experiments.
Mouse Draining Lymph Nodes Lymphocytes Isolation
[0170] Draining lymph nodes were disrupted with syringe plunger in 1 mL PBS in a 40 m strainer filtered to a 15 mL tube, washed with PBS, and centrifuged at 300g for 5 minutes. The resulting cell pellets was designated LN Lymphocytes and used for following experiments.
Primary CD8.sup.+ or CD4.sup.+ T Cell Isolation and Activation
[0171] Mouse primary CD8.sup.+ or CD4.sup.+ T cells were isolated from the spleen and peripheral lymph nodes (PLN) of C57BL/6 mice by magnetic bead purification using EasySep Mouse CD8.sup.+ or CD4.sup.+ T Cell Isolation Kit according to the manufacturer's instructions (Stemcell Technologies). Human primary CD8.sup.+ T cells were isolated from PBMC (Zen-Bio) by human CD8.sup.+ T Cell Isolation Kit according to the manufacturer's instructions (Stemcell Technologies). Isolated primary CD8.sup.+ or CD4.sup.+ T cells were activated in vitro for 18 hours with plate-bound anti-CD3 (10 g/mL; Biolegend) and anti-CD28 (5 g/mL; Biolegend) antibodies in click's media at 37 C. and 5% CO.sub.2 incubator. Activated CD8.sup.+ or CD4.sup.+ T cells were ready for further experiments. A naive CD8.sup.+ T cells control was maintained in Click's media containing 10 ng/ml IL-7 (BioLegend). Please also refer to Resources Table for more details.
Flow Cytometry and Staining
[0172] Mouse primary cells isolated from tumor, spleen, draining lymph node were stained with fluorescent antibodies and analyzed by flow cytometry.
[0173] For experiments with live/dead criteria, cells were first stained with Fixable Viability Dyes (FVD) (Thermo Fisher Scientific) according to the manufacturer's instructions. Subsequent surface marker staining was performed in Flow Cytometry Staining Buffer (Thermo Fisher Scientific). Intracellular staining for flow panels containing nuclear proteins was performed using the eBioscience FoxP3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's instructions. For intracellular staining of cytoplasmic proteins, the Fixation/Permeabilization Solution Kit (BD Biosciences) was used according to the manufacturer's instructions.
[0174] For phospho antibody staining, cells were incubated with FVD (cell viability dye) for 15 minutes at room temperature in a tube, re-suspended with 200 L pre-warmed 1BD Phosflow Lyse/Fix buffer directly into the tube, and incubated at 37 C. for 10-15 minutes, followed by centrifugation at 300g for 5 minutes. The resulting cell pellets were washed once with FACS buffer, permeabilized with 200 L of BD Phosflow Perm Buffer III for 45 minutes on ice, and centrifuged at 300g for 5 minutes. The cell pellets were washed again with FACS buffer, centrifuged at 300g for 5 minutes, and incubated with antibodies in FACS buffer for 45 minutes-1 hour at room temperature.
[0175] Mouse anti-CD11b antibody was used for myeloid cell (CD11b.sup.+) marker. After gated with CD11b.sup.+ cells, anti-F4/80 and Gr1 antibodies were used for macrophage (Gr1.sup. F4/80.sup.+) markers, anti-CD11c antibody was used for dendritic cells (CD11c.sup.+) marker, anti-CD16 and CD63 antibodies were used for neutrophils (CD16.sup.CD63.sup.+) markers, anti-CD14 antibody was used for monocytes (CD14.sup.+) marker.
[0176] Data was collected on LSR-Fortessa 4-15 flow cytometer or Attune NxT 4-14 and analyzed using FlowJo v10.4. Please also refer to Resources Table for more details.
Microbiome 16S Sequencing
[0177] Gut feces of CON and TVA group (7 samples per group) B16F10 tumor bearing mice were collected at day 16, and then subjected to microbiome 16S sequencing by Zymo Research (Irvine, CA). In brief, The ZymoBIOMICS-96 MagBead DNA Kit (Zymo Research) was used to extract DNA using an automated platform. Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S NGS Library Prep Kit (Zymo Research). The bacterial 16S primers amplified the V3-V4 region of the 16S rRNA gene. The sequencing library was prepared using an innovative library preparation process in which PCR reactions were performed in real-time PCR machines to control cycles and therefore limit PCR chimera formation. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator (Zymo Research), then quantified with TapeStation (Agilent Technologies) and Qubit (Thermo Fisher Scientific). The ZymoBIOMICS Microbial Community Standard (Zymo Research) was used as a positive control for each DNA extraction, if performed. The final library was sequenced on Illumina MiSeq with a v3 reagent kit (600 cycles). The sequencing was performed with 10% PhiX spike-in. For Bioinformatics Analysis, unique amplicon sequences variants were inferred from raw reads using the DADA2 pipeline (Callahan et al., 2016). Potential sequencing errors and chimeric sequences were also removed with the Dada2 pipeline. Chimeric sequences were also removed with the DADA2 pipeline. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 with the Zymo Research Database, a 16S database that is internally designed and curated, as reference. Composition visualization, alpha-diversity, and beta-diversity analyses were performed with Qiime v.1.9.1.
.SUP.13.C1-TVA Metabolic Flux Analysis by GC-MS
[0178] 110.sup.6 activated mouse primary CD8.sup.+ T cells were cultured for 24 hours in RPMI-1640 medium containing 0, 20, 50 UM .sup.13C1-TVA (Sigma), rinsed with 0.9% saline solution, and lysed with lysis buffer (400 L of cold MeOH, 300 L of 0.88% (w/v) KCl). Cell lysis were scraped off the plate into a glass vial, added 800 L of cold dichloromethane, and vortexed for 15 minutes at 4 C., followed by centrifugation at max speed for 10 minutes at 4 C. Samples were kept at room temperature after centrifugation to form two distinct phases. 650 L of the bottom dichloromethane fraction was transferred into a new glass tube and dried with nitrogen gas to obtain a lipid fraction pellet. FAME Derivatization was performed as previously described (Lien et al., 2020). In brief, the dried lipid pellet was dissolved in 100 L of toluene, added 200 L of 2% sulfuric acid in MeOH for incubation at 50 C. overnight and then added 500 L of 5% NaCl to extract twice with 500 L hexane. FAME cleanup was applied if analyzing animal tissues (FAME Cleanup: a Florisil column was pre-conditioned with 3 mL of hexane, added methyl ester, and eluted twice with 1 mL isohexane-diethyl ether (95:5 v/v), with drying down in between elutions). Otherwise, samples were dried down under nitrogen and re-suspended in 50 L of hexane to load onto gas chromatography (GC) mass spectrometry (MS). Derivatized samples were injected into a GC-MS using DB-FastFAME column (Agilent Technologies, Cat #G3903-63011) installed in an Agilent GC system. TVA-FAME has retention time of 9.6 minutes and m/z of 264 and 292, .sup.13C1-TVA-FAME has retention time of 9.6 minutes and m/z 265. Metabolite levels and mass isotopomer distributions of derivatized fragments were analyzed with an in-house MATLAB script, which integrated the metabolite fragment ions and corrected for natural isotope abundances.
Cell Culture Treatment
[0179] Mouse primary CD8.sup.+ T cells were isolated, activated, and subjected to further treatment. Treatment with sulfosuccinimidyl oleate (SSO) was performed by incubating cells with 100 M SSO for 24 hours. For inhibitors and modulators treatments, SCH-202676 (200 nM), 666-15 (100 nM), H-89 dihydrochloride (200 nM), Rhosin HCl (10 M), NFAT inhibitor (1 M), U0126 (100 nM), Ruxolitinib (100 nM), short chain fatty acid mix (10 mM), acetate (0.02, 0.2, 2, 20 mM), butyrate (0.02, 0.2, 2, 20 mM), propionate (0.02, 0.2, 2, 20 mM) were added to medium synchronized with 20 M TVA for 24 hours. For TVA washing experiment, mouse CD8.sup.+ T cells were treated with TVA for 24 hours, washed off, and then cultured for another 24 hours in media with or without TVA. For 8-Bromo-cAMP and TVA different doses treatment experiment, activated mouse CD8.sup.+ T cells were treated with 8-Bromo-cAMP (0.01, 0.1, 1, 10, 100 M) or TVA (10, 20, 100, 500, 1000 M) for 24 hours, cells were collected for p-CREB level detection. For human T cell and CD8.sup.+ T cell rhPD-L1 assay, activated cells were treated with 0.5 g/mL rhPD-L1 (R&D) for 72 hours in the presence or absence of 20 M TVA, followed by ELISA detection of IL-2 and TNF- level. Please also refer to Resources Table for more details.
Kethoxal-Assisted Single-Stranded DNA Sequencing (KAS-Seq) and Data Analysis
[0180] Mouse and human CD8.sup.+ T cells were isolated, activated, and treated with or without 20 M TVA for 20 minutes, 40 minutes, and 2 hours. 500 mM N3-kethoxal was then supplemented into the media followed by brief, vigorous shaking to fully homogenize the solution. The 6-well plates were then moved into the cell incubator (37 C., 5% CO2) for 10 minutes to promote labeling of genomic single-stranded DNA (ssDNA). Cell suspensions were then transferred to 15 mL conical tubes and centrifuged at 300g for 5 minutes at 4 C. The supernatant media was discarded, and genomic DNA was then extracted. The ssDNA with N3-kethoxal label was biotinylated, enriched, and fragmented following the established KAS-seq protocol (Lyu et al., 2022; Wu et al., 2020a). Dual index libraries were constructed for high throughput sequencing using an Accel-NGS Methyl-seq DNA library kit and then sequenced at the Genomics Facility (University of Chicago) via Illumina NovaSeq 6000 (SP flowcell, 100 bp cassette) in two separate runs. Raw sequencing data under each condition was then catenated from the two runs, and adapter trimming; read deduplication and mapping; and differential KAS-seq analysis (comparing TVA-treated to untreated cells) was performed following the KAS-pipe analysis pipeline (Lyu et al., 2022; Wu et al., 2020a) Volcano plots were subsequently generated in RStudio. For Gene Ontology (GO) enrichment analysis, a list of gene bodies exhibiting differential ssDNA levels following TVA treatment (p-values as indicated in
Phospho Antibody Array
[0181] To analyze signaling pathways, mouse primary CD8.sup.+ T cells were isolated, activated, and treated with TVA for indicated time, followed by Phospho Antibody Array (R&D Systems) according to the manufacturer's instructions. The quantification for pixel density in each spot of the array was carried out by subtracting background signals from the spot intensity using software ImageJ (ImageJ, RRID: SCR_003070). Please also refer to Resources Table and Table 6 for more details.
Quantitative Real-Time PCR (RT-PCR)
[0182] Total RNA was extracted with TRIzol Reagent (Invitrogen) and then used for synthesizing the first strand cDNA with PrimeScript 1st strand cDNA Synthesis Kit (Takara) according to the manufacturer's instructions. Quantitative RT-PCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad). Please also refer to Resources Table for more details of reagents and RT-PCR primers.
RNA Interference (RNAi) with Accell siRNA
[0183] Mouse primary CD8.sup.+ T cells were isolated and cultured in replete media (RPMI 1640 medium or Click's medium containing 15% FBS, 55 M 2-mercaptoethanol, 2 mM glutamine, penicillin/streptomycin, and either PHA, CD3 or IL-2) for 24 hours, followed by incubation with Accell delivery mix (Accell siRNA Delivery Media (Horizon Discovery) with 1 M siRNA, 20 IU/mL IL-2 and 1% FBS) for 72 hours. Cells were collected for subsequent function analysis as well as depletion efficiency validation using RT-PCR. Please also refer to Resources Table for more details.
RNA-Sequencing
[0184] To check effect of TVA treatment on gene expression of mouse CD8.sup.+ T cells, primary mouse CD8.sup.+ T cells were isolated, activated, and then treated with or without 20 M TVA (TVA group vs. CON group) for 24 hours, followed by RNA extraction using the PureLink RNA Mini Kit as the manufacturer's instructions. RNA samples in triplicate were used for Illumina Next Generation Sequencing. To check effect of Creb1 knockdown on TVA dependent CD8.sup.+ T cell gene expression changes, mouse primary CD8.sup.+ T cells were isolated, activated, and then transfected with siNTC or siCreb1 using Accell siRNA method, followed by treatment with or without 20 M TVA for 24 hours. RNA samples from four groups (siNTC, siNTC+TVA, siCreb1, siCreb1+TVA) were extracted and used for Illumina Next Generation Sequencing. Data processing and analysis were performed as previous described (Su et al., 2018). Please also refer to Resources Table and Table 7 for more details.
cAMP Level
[0185] Mouse primary CD8.sup.+ T cells were isolated, activated, and then treated with 20 M TVA for 0, 20 minutes, 40 minutes, and 2 hours. cAMP-Screen Cyclic AMP Immunoassay System (Fisher Scientific Company) was used to check cAMP level according to the manufacturer's instructions. Please also refer to Resources Table for more details.
CRISPR Editing of Mouse OT-I Cells
[0186] CRISPR editing of mouse primary CD8.sup.+ T cells was performed as previously described. In brief, LMPd-sgNTC-mPGK-Ametrine, LMPd-sgGPR43 #1-mPGK-Ametrine, LMPd-sgGPR43 #2-mPGK-Ametrine, and LMPd-sgGPR43 #3-mPGK-Ametrine were generated and co-transfected with pCL-Eco (Addgene, Cat #12371) into Plat-E cells using TransIT-LT1 Transfection Reagent to produce retrovirus. Virus harvest media was changed 18 hours after transfection and then collected 48 hours later. Primary Cas9-expressing OT-I (Cas9; OT-I) cells were derived from the spleen and peripheral lymph nodes (PLN) of Cas9; OT-I mice (Wei et al., 2019) by magnetic bead purification using EasySep Mouse T Cell Isolation Kit according to the manufacturer's instructions (Stemcell Technologies). 2-5 million Cas9; OT-I cells were activated, placed into one well of 24-well plate, and supplemented with retrovirus supernatant containing 10 g/mL polybrene, followed by spin infection (800g, brake 3) for 3 hours. After infection, cells were moved to 37 C. cell culture incubator for another 2 hours and then changed with new complete Click's medium (containing 20 IU/mL human IL-2, 2.5 ng/ml mIL-7, 25 ng/ml mIL-15) for 72 hours. Cells were harvested, sorted with flow cytometry to enrich virus-transduced cells (Ametrine positive), and subjected to subsequent function analysis as well as knockout efficiency validation using RT-PCR. Please also refer to Resources Table for more details.
Pull Down Assay to Identify the Crosslinked Protein-TVA Complexes
[0187] Mouse primary CD8.sup.+ T cells were isolated and activated. 10 million cells were collected, pelleted, and re-suspended in 0.3 mL ice-cold PBS containing EDTA-free protease and phosphatase inhibitor Cocktail (Thermo Fisher Scientific, Cat #A32961), followed by sonication on ice. TVA Probe 3 was added and incubated at 37 C. for 2 hours under dark conditions. After probe labeling, the sample was irradiated under 365 nm UV light for 6 minutes on ice, diluted with 1% SDS and sonicated on ice. The proteome concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) on a microplate reader (Bio-Rad) and normalized to 1.5 mg/mL. 700 L protein sample was conjugated with a biotin tag by click chemistry (100 M biotin-azide, 100 M TBTA, 1 mM CuSO4 and 1 mM TCEP) in dark at room temperature for 1 hour. The sample was added 4 volumes of acetone, chilled to 20 C., vortexed, and then incubated for 3 hours at 20 C. to completely precipitate the proteins and remove unreacted biotin-azide. The sample was centrifuged at 17,000g for 15 minutes at 4 C. to pellet the precipitated proteins. The resulting protein pellets were resolved in 1% SDS by sonication, added PBS to dilute the concentration of SDS to 0.2% and then added 60 l pre-washed high-capacity streptavidin C.sub.1 beads, followed by incubation overnight at 4 C. with rotation. The beads were washed 3 times with 0.1% SDS in PBS and once with PBS, added 2SDS sample buffer, and boiled for western blot analysis of TVA binding proteins. Please also refer to Resources Table for more details.
Co-Culture Assay with Blinatumomab
[0188] The RS4;11 target cells were stained using the CellTrace Far Red Cell Proliferation Kit (Invitrogen, Cat #C.sub.34564), and then co-cultured for 24 hours with PBMC in flat bottom 24 well-plate at a 1:5 ratio (210.sup.5 target cells to 10.sup.6 PBMC) at indicated concentrations of Blinatumomab (0, 10, 100, 1000 g/mL). derived from the leftover of infusions (Provided by Dr. Wendy Stock's lab). The cell mixture was resuspended in a flow cytometry staining buffer, stained using a Fixable Viability Dye780 (R&D), and then analyzed by flow cytometry. Please also refer to Resources Table for more details.
CAR-T Cell Expansion Assay
[0189] 63,000 anti-human CD19-CD28z-GFP CAR T cells (Provided by Justin Kline lab) were cultured in T cell expansion medium (StemCell) with IL-7 (5 ng/mL) and IL-15 (5 ng/ml) in the presence or absence of 20 M TVA. Medium was changed to fresh medium (with IL-7 and IL-15 in the presence or absence of 20 M TVA) in Day 2, 5, 7, and 9. Cell number was counted in day 7, 9, and 10.
Quantification and Statistical Analysis
[0190] A two-tailed Student's t test was subjected to data statistical analysis, except a two-way ANOVA test was performed for cell proliferation assay and tumor growth analysis. p values less than 0.05 were considered significant. Data with error bars represent meanSD, except for cell proliferation and tumor growth curves which represent meanSEM. Statistical analysis and graphical presentation was performed using Prism 5.0 (GraphPad).
TABLE-US-00001 KeyResourcesTable REAGENTorRESOURCE SOURCE IDENTIFIER Antibodies Ratanti-IgG2bisotype BioXCell Cat#BE0090; Clone#LTF-2; RRID:AB_1107780. Mouseanti-CD8 BioXCell Cat#BE0061; Clone#2.43; RRID:AB_1125541. Mouseanti-PD-1(CD279) BioXCell Cat#BE0146; Clone#RMP1-14; RRID:AB_10949053. MousePerCP/Cyanine5.5anti-Ki-67 Biolegend Cat#652423; Antibody Clone#16A8; RRID:AB_2629530. BrilliantViolet605anti-T-bet Biolegend Cat#644817; Antibody Clone#4B10; RRID:AB_11219388. MouseAPCanti-CD223(LAG-3)Antibody Biolegend Cat#125209; Clone#C9B7W; RRID:AB_10639935. MouseBrilliantViolet650anti-CD223 Biolegend Cat#125227; (LAG-3)Antibody Clone#C9B7W; RRID:AB_2687209. MouseAPCanti-Ly108Antibody Biolegend Cat#134609; Clone#330-AJ; RRID:AB_2728154. MousePerCP/Cyanine5.5anti-CD366(Tim-3) Biolegend Cat#134012; Antibody Clone#B8.2C12; RRID:AB_2632736. PEanti-TCF1(TCF7)Antibody Biolegend Cat#655207; Clone#7F11A10; RRID:AB_2728491. Human/mouse/ratFITCanti-CD278(ICOS) Biolegend Cat#313505; Antibody Clone#C398.4A; RRID:AB_416329. Human/mouseFITCanti-GranzymeB Biolegend Cat#372205; RecombinantAntibody Clone#QA16A02; RRID:AB_2687029. MousePE/Cyanine5anti-CD69Antibody Biolegend Cat#104509; Clone#H1.2F3; RRID:AB_313112. MouseAPCanti-CD152Antibody Biolegend Cat#106309; Clone#UC10-4B9; RRID:AB_2230158. MouseAPCanti-CD279(PD-1)Antibody Biolegend Cat#135209; Clone#29F.1A12; RRID:AB_2251944. MousePE/Cyanine5anti-CD4Antibody Biolegend Cat#100409; Clone#GK1.5; RRID:AB_312694. MouseBrilliantViolet421anti-IL-2 Biolegend Cat#503825; Antibody Clone#JES6-5H4; RRID:AB_10895901. MouseAPCanti-CD45.2Antibody Biolegend Cat#109813; Clone#104; RRID:AB_389210. MouseAPCanti-IFN-Antibody Biolegend Cat#505810; Clone#XMG1.2; RRID:AB_315404. Human/mousePE/Cyanine7anti-GranzymeB Biolegend Cat#372213; RecombinantAntibody Clone#QA16A02; RRID:AB_2728380. MousePerCP/Cyanine5.5anti-TNF- Biolegend Cat#506321; Antibody Clone#MP6-XT22; RRID:AB_961435. MouseBrilliantViolet711anti-CD8a Biolegend Cat#100747; Antibody Clone#53-6.7; RRID:AB_11219594. MouseBrilliantViolet421anti-FOXP3 Biolegend Cat#126419; Antibody Clone#MF-14; RRID:AB_2565933. MouseAPCanti-CD3Antibody Biolegend Cat#100235; Clone#17A2; RRID:AB_2561455. FITCanti-Bcl-2 Biolegend Cat#633503; Clone#BCL/10C4; RRID:AB_2028392. MousePEanti-CD71 Biolegend Cat#113807; Clone#RI7217; RRID:AB_313568. MouseAPCanti-CD98(4F2) Biolegend Cat#128211; Clone#RL388; RRID:AB_2750544. PEanti-RPS6Phospho(Ser235/Ser236) Biolegend Cat#608603; Clone#A17020B; RRID:AB_2750251. PEanti-NFATc1 Biolegend Cat#649605; Clone#7A6; RRID:AB_2562546. MouseFITCanti-F4/80Recombinant Biolegend Cat#157309; Antibody Clone#QA17A29; RRID:AB_2876535. MouseAPCanti-Ly-6G(Gr1)Antibody Biolegend Cat#127613; Clone#1A8; RRID:AB_1877163. Mouse/humanAPCanti-CD11bAntibody Biolegend Cat#101211; Clone#M1/70; RRID:AB_312794. MousePerCPanti-CD11cAntibody Biolegend Cat#117325; Clone#N418; RRID:AB_893236. AlexaFluor647anti-mouseCD16Antibody Biolegend Cat#158021; Clone#S17014E; RRID:AB_2904300. PE/Cyanine5anti-mouseCD28Antibody Biolegend Cat#102108; Clone#37.51; RRID:AB_312873. MousePE/Cyanine7anti-CD14Antibody Biolegend Cat#123315; Clone#Sa14-2; RRID:AB_10641133. Mouse/humanPEanti-Ki-67Antibody Biolegend Cat#151210; Clone#11F6; RRID:AB_2716008. PEanti-LckPhospho(Tyr394) Biolegend Cat#933103; Clone#A18002D; RRID:AB_2820203. PETOXMonoclonalAntibody(TXRX10) ThermoFisherScientific Cat#12-6502-82; Clone#TXRX10; RRID:AB_10855034. APCPhospho-CREB(Ser133)Recombinant ThermoFisherScientific Cat#MA5-36992; RabbitMonoclonalAntibody Clone#CREBS133- 4D11; RRID:AB_2896927. RabbitPEActiveCaspase-3 ThermoFisherScientific Cat#BDB561011; Clone#C92-605; RRID:AB_2033931. Phospho-NFATC1(Ser172)Polyclonal ThermoFisherScientific Cat#PA564696; Antibody Clone#N/A; RRID:AB_2662248. RabbitPEPhospho-Stat1(Tyr701) ThermoFisherScientific Cat#MA5-37039; RecombinantMonoclonalAntibody Clone#Stat1Y701- 3E6; RRID:AB_2896974. GPR43PolyclonalAntibody ThermoFisherScientific Cat#PA5-111780; Clone#N/A; RRID:AB_2857189. BiotinMonoclonalAntibody(Z021) ThermoFisherScientific Cat#03-3700; Clone#Z021; RRID:AB_2532265. PKAC-Antibody CellSignaling Cat#4782S; Technology Clone#N/A; RRID:AB_2170170. Phospho-PKAC(Thr197)Antibody CellSignaling Cat#4781S; Technology Clone#N/A; RRID:AB_2300165. RabbitStat1(D1K9Y)mAb CellSignaling Cat#14994S; Technology Clone#D1K9Y; RRID:AB_2737027. Mousemonoclonalanti--actinantibody Sigma-Aldrich Cat#A1978; Clone#AC-15; RRID:AB_476692. Goatanti-MouseIgG(H+L)Secondary ThermoFisherScientific Cat#31430; Antibody,HRP Clone#N/A; RRID:AB_228307. Goatanti-RabbitIgG(H+L)Secondary ThermoFisherScientific Cat#31460; Antibody,HRP Clone#N/A; RRID:AB_228341. GoatPolyclonalIFN-alpha/betaR1Antibody NovusbiologicalsInc. Cat#AF3039-SP; Clone#N/A; RRID:AB_664107. HamsterMonoclonalTNFRI/TNFRSF1A NovusbiologicalsInc. Cat#MAB430-SP; Antibody Clone#55R170; RRID:AB_2208782. Bacterialandvirusstrains MAXEfficiencyStbl2CompetentCells ThermoFisherScientific Cat#10268019 hPDCD1(CD279)Expression,Concentrated GenTargetInc Cat#:LVP1076-PBS Lentivirus Biologicalsamples Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-035, commercialCARTcelltherapy therapybiobank RF Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-033, commercialCARTcelltherapy therapybiobank PB Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-031, commercialCARTcelltherapy therapybiobank EG Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-049, commercialCARTcelltherapy therapybiobank MC Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-086, commercialCARTcelltherapy therapybiobank LT Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-032, commercialCARTcelltherapy therapybiobank LB Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-051, commercialCARTcelltherapy therapybiobank MH Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-075, commercialCARTcelltherapy therapybiobank KY Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-112, commercialCARTcelltherapy therapybiobank ND Serumofpatientthathaveundergone UniversityofChicagocell Cat#18-0025-122, commercialCARTcelltherapy therapybiobank TM PatientPeripheralBloodMononuclearCells JustinKlinelab N/A (PBMCs) HumanPeripheralBloodMononuclearCells Zen-BioInc Cat#SER-PBMC- (PBMCs) 200-F CAS9;OT-Imousespleenandlymphnode HongboChilab N/A Chemicals,peptides,andrecombinantproteins HumanIL-2IS MiltenyiBiotec Cat#130-097-743 MouseIL-2IS MiltenyiBiotec Cat#130-120-662 MouseIL-7 FisherScientificCompany Cat#50813299 MouseIL-15 FisherScientificCompany Cat#50813273 RecombinantHumanPD-L1/B7-H1Fc R&D Cat#156-B7-100 ChimeraProtein Fixableviabilitydye780 R&D Cat#65-0865-14 DNaseI Millipore Cat#EN0521 N-methylpyrrolidone Sigma-Aldrich Cat#PHR1352 CAS:872-50-4 TWEEN-80 Sigma-Aldrich Cat#P1754 H89Dihydrochloride FisherScientificCompany Cat#29101 CAS:130964-39-5 666-15 FisherScientificCompany 501871791 CAS:1433286-70-4 Rhosinhydrochloride FisherScientificCompany 500310R CAS:1281870-42-5 RhoinhibitorI FisherScientificCompany Cat#NC9588154 XL019 FisherScientificCompany Cat#501364339 8-Bromo-cAMP FisherScientificCompany Cat#11-401-0 AcetateSolution Sigma-Aldrich Cat#3863 NFATInhibitor CaymanChemical Cat#13855-1 SCH202676 CaymanChemical Cat#16945-10 cis-VaccenicAcid CaymanChemical Cat#20023-500 Trans-Vaccenicacid Sigma-Aldrich Cat#V1131 CAS:693-72-1 Trans-Vaccenicacid-1-13C Sigma-Aldrich Cat#645516 Sulfo-N-succinimidylOleatesodium Sigma-Aldrich Cat#SML2148 CAS:135661-44-8 GLPG0974 Sigma-Aldrich Cat#SML2443 CAS:1391076-61-1 AH-7614 Sigma-Aldrich Cat#SML2025 CAS:6326-06-3 DL--Hydroxybutyricacidsodiumsalt Sigma-Aldrich Cat#H6501 CAS:150-83-4 Collagenase/Hyaluronidase StemcellTechnologiesInc Cat#07912 AmmoniumChlorideSolution StemcellTechnologiesInc Cat#07800 T4DNALigaseReactionBuffer NewEnglandBiolabsInc Cat#B0202S NEBufferSet(r1.1,r2.1,r3.1&rCutSmart) NewEnglandBiolabsInc Cat#B7030S Criticalcommercialassays ELISAMAXDeluxeSetHumanIL-2 Biolegend Cat#431804 ELISAMAXDeluxeSetHumanTNF- Biolegend Cat#430204 ELISAMAXDeluxeSetHumanIFN- Biolegend Cat#430104 ELISAMAXDeluxeSetMouseIL-2 Biolegend Cat#431004 ELISAMAXDeluxeSetMouseTNF- Biolegend Cat#430904 ELISAMAXDeluxeSetMouseIFN- Biolegend Cat#430804 FitcannexinVapoptosisdetectionkitI BDBiosciences Cat#556547 CellTraceVioletCellProliferationKit FisherScientificCompany Cat#C34571_372 EasySepMouseTCellIsolationKit StemcellTechnologiesInc Cat#19851 EasySepMouseTIL(CD45)Positive StemcellTechnologiesInc Cat#100-0350 SelectionKit EasySepMouseCD8+TCellIsolationKit StemcellTechnologiesInc Cat#19853 EasySepMouseCD4+TCellIsolationKit StemcellTechnologiesInc Cat#19852 EasySepHumanTCellIsolationKit StemcellTechnologiesInc Cat#17951 EasySepHumanCD8+TCellIsolationKit StemcellTechnologiesInc Cat#17953 EasySepHumanCD4+TCellIsolationKit StemcellTechnologiesInc Cat#17952 CaspaseGlo3/7 Promega Cat#G8090 CellActivationCocktail Biolegend Cat#423304 (withBrefeldinA) eBioscience7-AADViabilityStaining FisherScientificCompany Cat#00-6993-50 Solution eBioscienceICFixationBuffer FisherScientificCompany Cat#00-8222-49 eBioscienceIntracellularFixation& FisherScientificCompany Cat#G762684 PermeabilizationBufferSet FoxP3/TranscriptionFactorStaining ThermoFisherScientific Cat#00-5523-00 BufferSet PhosflowPermBufferIII BDBiocience Cat#558050 PhosflowLyse/Fixbuffer BDBiocience Cat#558049 CAMP-ScreenCyclicAMPImmunoassay FisherScientificCompany Cat#4412182 System PurelinkRNAMiniKit FisherScientificCompany Cat#12183018A PierceRapidGoldBCAProteinAssay ThermoFisherScientific Cat#A53225 Kit QIAprepSpinMiniprepKit Qiagen Cat#27106 RBClysisbuffer Invitrogrn Cat#00-4300-54 TRIzolReagent Invitrogen Cat#15596026 AccellsiRNADeliveryMedia HorizonDiscovery Cat#B-005000-500 QIAquickGelExtractionKit QiagenInc Cat#28706 Lymphocytesisolationliquid GE Cat#17144002 ProteomeProfilerHumanPhospho-Kinase R&DSystems Cat#ARY003C ArrayKit Depositeddata RNA-seqdata:effectofTVAtreatmenton Thispaper GEO:GSE202276 geneexpressionofmouseCD8+Tcells RNA-seqdata:effectofCreb1KDonTVA Thispaper GEO:GSE202274 dependentCD8+Tcellgeneexpression changes KAS-seqdata Thispaper GEO:GSE202730 Microbiome16SrRNAsequencingdata Thispaper GEO:GSE202266 OriginalimagesweredepositedtoMendeley Thispaper https://data. data mendeley.com/ datasets/ 89j7ytsgn5/ draft?a=b922 746d-9ce5-4775- 8e9c-55dfda2e6f90 Experimentalmodels:Celllines Human:JurkatTcells ATCC Cat#TIB-152; RRID:CVCL_0367 Human:JurkatTPD-1+cells Thispaper N/A Human:Plat-Ecells (Weietal.,2019) N/A Human:RS4;11cells Dr.WendyStocklab N/A Mouse:B16F10cells ATCC Cat#CRL-6475; RRID:CVCL_0159 Mouse:MC38cells Kerafast Cat#ENH204-FP; RRID:CVCL_B288 Mouse:E0771cells ATCC Cat#CRL-3461; RRID:CVCL_GR23 Mouse:LLC1cells ATCC Cat#CRL-1642; RRID:CVCL_4358 Experimentalmodels:Organisms/strains Mouse:C57BL/6J TheJacksonLaboratory JAX:000664: RRID:IMSR_JAX: 000664 Mouse:C57BL/6nude(B6.Cg-Foxn1nu/J) TheJacksonLaboratory JAX:000819; RRID:IMSR_JAX: 000819 Mouse:TCRKnock-out(B6.129S2- TheJacksonLaboratory JAX:002116; Tcratm1Mom/J) RRID:IMSR_JAX:00 2116 Mouse:PmeI-1(B6.Cg- TheJacksonLaboratory JAX:005023; Thy1a/CyTg(TcraTcrb)8Rest/J) RRID:IMSR_JAX: 005023 Mouse:OT-I(C57BL/6- TheJacksonLaboratory JAX:003831; Tg(TcraTcrb)1100Mjb/J) RRID:IMSR_JAX: 003831 Oligonucleotides AccellMouseFfar1(233081)siRNA- HorizonDiscoveryLtd. E-051167-00-0005 SMARTpool AccellMouseFfar3(233080)siRNA- HorizonDiscoveryLtd. E-058296-00-0005 SMARTpool AccellMouseGpr84(80910)siRNA- HorizonDiscoveryLtd. E-047398-00-0005 SMARTpool AccellMouseGpr119(236781)siRNA- HorizonDiscoveryLtd. E-042791-00-0005 SMARTpool AccellNon-targetingPool HorizonDiscoveryLtd. D-001910-10-05 AccellMouseFfar2(233079)siRNA- HorizonDiscoveryLtd. E-061251-00-0020 SMARTpool AccellMouseFfar4(107221)siRNA- HorizonDiscoveryLtd. E-042801-00-0005 SMARTpool AccellMouseCreb1(12912)siRNA- HorizonDiscoveryLtd. E-040959-00-0020 SMARTpool, AccellNon-targetingsiRNA#1 HorizonDiscoveryLtd. D-001910-01-20 AccellGAPDsiRNA-Mouse HorizonDiscoveryLtd. D-001930-02-05 AccellMouseII18(16173)siRNA- HorizonDiscoveryLtd. E-046634-00-0005 SMARTpool AccellMouseTbx21(57765)siRNA- HorizonDiscoveryLtd. E-040669-00-0005 SMARTpool AccellMouseIlf2(67781)siRNA-SMARTpool HorizonDiscoveryLtd. E-060911-00-0005 AccellMouseFoxo4(54601)siRNA- HorizonDiscoveryLtd. E-045377-00-0005 SMARTpool AccellMouseBcl6(12053)siRNA- HorizonDiscoveryLtd. E-057495-00-0005 SMARTpool AccellMouseDffb(13368)siRNA- HorizonDiscoveryLtd. E-058666-00-0005 SMARTpool AccellMouseEbi3(50498)siRNA- HorizonDiscoveryLtd. E-047002-00-0005 SMARTpool sgRNAtargetingGPR43sequence#1, Thispaper N/A Forward:5- CACCGGCGCGCACACGATCTTTGGT-3 (SEQIDNO:1) sgRNAtargetingGpr43sequence#1, Thispaper N/A Reverse:5- AAACACCAAAGATCGTGTGCGCGCC-3 (SEQIDNO:2) sgRNAtargetingGpr43sequence#2, Thispaper N/A Forward:5- CACCGGCAGCGATCACTCCATACAG-3 (SEQIDNO:3) sgRNAtargetingGpr43sequence#2, Thispaper N/A Reverse:5- AAACCTGTATGGAGTGATCGCTGCC-3 (SEQIDNO:4) sgRNAtargetingGpr43sequence#3, Thispaper N/A Forward:5- CACCGCTGAACTCAACTGAGCAGGT-3 (SEQIDNO:5) sgRNAtargetingGpr43sequence#3, Thispaper N/A Reverse:5- AAACACCTGCTCAGTTGAGTTCAGC-3 (SEQIDNO:6) RT-PCRPrimer:Dffb,Forward: Thispaper N/A 5-GGATGGGAGGGAGGTGAACTGG-3 (SEQIDNO:7) RT-PCRPrimer:Dffb,Reverse: Thispaper N/A 5-GGTCTTCTTGTGGCAGGCGATG-3 (SEQIDNO:8) RT-PCRPrimer:Bcl6,Forward: Thispaper N/A 5-AGGTCGTGAGGTCGTGGAGAAC-3 (SEQIDNO:9) RT-PCRPrimer:Bcl6,Reverse: Thispaper N/A 5-GGATAAGAGGCTGGTGGTGTTGAC-3 (SEQIDNO:10) RT-PCRPrimer:Tnf,Forward: Thispaper N/A 5-GCCTCTTCTCATTCCTGCTTGTGG-3 (SEQIDNO:11) RT-PCRPrimer:Tnf,Reverse: Thispaper N/A 5-GTGGTTTGTGAGTGTGAGGGTCTG-3 (SEQIDNO:12) RT-PCRPrimer:Foxo4,Forward: Thispaper N/A 5-GATGGGTCTTTGTCAGCAGGAGAAG-3 (SEQIDNO:13) RT-PCRPrimer:Foxo4,Reverse: Thispaper N/A 5-AGCAGGAGGTGGTGGTGTATCAG-3 (SEQIDNO:14) RT-PCRPrimer:Ilf2,Forward: Thispaper N/A 5-AGCCAGCACCTGATGAGACCTC-3 (SEQIDNO:15) RT-PCRPrimer:Ilf2,Reverse: Thispaper N/A 5-TGTCACCAAAGAAAGGATGGAAGCC-3 (SEQIDNO:16) RT-PCRPrimer:Tbx21,Forward: Thispaper N/A 5-ATCACTAAGCAAGGACGGCGAATG-3 (SEQIDNO:17) RT-PCRPrimer:Tbx21,Reverse: Thispaper N/A 5-ACCAAGACCACATCCACAAACATCC-3 (SEQIDNO:18) RT-PCRPrimer:Il18,Forward: Thispaper N/A 5-CAAAGTGCCAGTGAACCCCAGAC-3 (SEQIDNO:19) RT-PCRPrimer:Il18,Reverse: Thispaper N/A 5-ACAGAGAGGGTCACAGCCAGTC-3 (SEQIDNO:20) RT-PCRPrimer:Gpr41,Forward: Thispaper N/A 5-CCACACTGCTCATCTTCTTCGTCTG-3 (SEQIDNO:21) RT-PCRPrimer:Gpr41,Reverse: Thispaper N/A 5-ACGGACTCTCACGGCTGACATAG-3 (SEQIDNO:22) RT-PCRPrimer:Gpr43,Forward: Thispaper N/A 5-CTGTATGGAGTGATCGCTGCTCTG-3 (SEQIDNO:23) RT-PCRPrimer:Gpr43,Reverse: Thispaper N/A 5-CTGCTCTTGGGTGAAGTTCTCGTAG-3 (SEQIDNO:24) RT-PCRPrimer:Il2,Forward: Thispaper N/A 5-GCAGCTCGCATCCTGTGTCAC-3 (SEQIDNO:25) RT-PCRPrimer:Il2,Reverse: Thispaper N/A 5-CTGCTGTGCTTCCGCTGTAGAG-3 (SEQIDNO:26) RT-PCRPrimer:Creb1,Forward: Thispaper N/A 5-GCTGGCTAACAATGGTACGGATGG-3 (SEQIDNO:27) RT-PCRPrimer:Creb1,Reverse: Thispaper N/A 5-AACTTGGTTGCTGGGCACTAGAATC-3 (SEQIDNO:28) RT-PCRPrimer:Prkaca,Forward: Thispaper N/A 5-GGCTCTCGGAGTCCTCATCTACG-3 (SEQIDNO:29) RT-PCRPrimer:Prkaca,Reverse: Thispaper N/A 5-CGCAGCAGGTCCTTCAAGTCAG-3 (SEQIDNO:30) RT-PCRPrimer:Lag3,Forward: Thispaper N/A 5-GCCATCTCGTTCTCGTTCTCATCC-3 (SEQIDNO:31) RT-PCRPrimer:Lag3,Reverse: Thispaper N/A 5-TTCTCCACCAGTGAAAGCCAAAGG-3 (SEQIDNO:32) RT-PCRPrimer:Batf,Forward: Thispaper N/A 5-AGAGCCGACAGAGACAGACACAG-3 (SEQIDNO:33) RT-PCRPrimer:Batf,Reverse: Thispaper N/A 5-TCGGTGAGCTGTTTGATCTCTTTGC-3 (SEQIDNO:34) RT-PCRPrimer:Ebi3,Forward: Thispaper N/A 5-TCAAGTACCGACTCCGCTACCG-3 (SEQIDNO:35) RT-PCRPrimer:Ebi3,Reverse: Thispaper N/A 5-CTGAGCTGACACCTGGATGCAATAC-3 (SEQIDNO:36) RT-PCRPrimer:Gpr40,Forward: Thispaper N/A 5-GAAGCTGGCTGGACAACAGTACC-3 (SEQIDNO:37) RT-PCRPrimer:Gpr40,Reverse: Thispaper N/A 5-AAGGGCAGAAAGAAGAGCAGAATGG-3 (SEQIDNO:38) RT-PCRPrimer:Gpr120,Forward: Thispaper N/A 5-AATCGCACCCACTTCCCTTTCTTC-3 (SEQIDNO:39) RT-PCRPrimer:Gpr120,Reverse: Thispaper N/A 5-GCCCAGCAGTGAGACGACAAAG-3 (SEQIDNO:40) RT-PCRPrimer:Gpr84,Forward: Thispaper N/A 5-CAGCCTTTCTCCGTGGACACATAC-3 (SEQIDNO:41) RT-PCRPrimer:Gpr84,Reverse: Thispaper N/A 5-TAGAGCAATGAGACAGAGGGTGAGG-3 (SEQIDNO:42) RT-PCRPrimer:Gpr119,Forward: Thispaper N/A 5-TCCATATTCCAGCAGACCACCTACC-3 (SEQIDNO:43) RT-PCRPrimer:Gpr119,Reverse: Thispaper N/A 5-AGAGGCAATCTTGAGCATGTCACAG-3 (SEQIDNO:44) RecombinantDNA LMPd-gRNA-mPGK-Ametrine (Weietal.,2019) N/A LMPd-sgNTC-mPGK-Ametrine (Weietal.,2019) N/A LMPd-sgGPR43#1-mPGK-Ametrine Thispaper N/A LMPd-sgGPR43#2-mPGK-Ametrine Thispaper N/A LMPd-sgGPR43#3-mPGK-Ametrine Thispaper N/A anti-humanCD19-CD28z-GFP Thispaper N/A Softwareandalgorithms GraphPadPrism9software GraphPadSoftware https://www. graphpad.com/ ImageJ https://imagej.nih.gov/ij/ RRID:SCR_003070 FlowJo10.4 FlowJoSoftware https://www. flowjo.com/ solutions/flowjo/ Other ControlRodentDiets ResearchDiets,Inc. D12450J RodentDietswith1%Trans-VaccenicAcid ResearchDiets,Inc. D19110701 RodentDietswith1%Cis-VaccenicAcid ResearchDiets,Inc. D19110582
Example 2
TVA Suppresses Tumorigenesis and Tumor Progression
[0191] Apc.sup.+/fl and Cdx-2P-Cre-NLS mice were interbred to generate Apc.sup.+/fl; Cdx-2P-Cre-NLS 5 mice. Genotypes were verified using PCR protocols recommended by Jackson Laboratories. At 12 wks age, mice were started on lipid rich diet (20% mixed lipids) that mimics Western diet. After 2 wks, mice were divided into two cohorts balanced for males and females and gavaged with TVA 35 mg/kg bw or vehicle (ETOH/DMSO 2:1 v/v) daily5 days for 4 wks. After 4 weeks, mice were killed, and samples collected. Left panel. Representative colons showing 10 tumors in situ (see
Example 3
Enhanced Anti-Tumor Immunity Function of TVA
[0192] TVA treatment enhances anti-tumor immunity by coupling tumor infiltrating CD8+ T cells with increased pro-immune dendritic cells and by reducing anti-immune Treg and tumor associated macrophages (TAMs) (see
Example 4
Oral Gavage of TVA to Mice Ameliorates Immune Response to Infection by Influenza Virus (by Zhong Zheng and Hao Fan)
[0193] Zhong Zheng in Chuan's lab helped Hao study the effects of TVA exposure during fetal and infant stages on mouse immune response to viral challenge using influenza virus. The TVA mice were born by mothers on TVA enriched diet and fed on TVA-containing milk from mothers, followed with TVA diet since weaning. Control mice were born by mothers on normal diet, followed by normal milk feeding and normal diet since weaning. It was found that TVA mice showed improved immune responses to influenza virus with significantly increased survival (see
Example 5
TVA Enhances Immune Response to Infection by Influenza Virus with Sex Preference to Male (by Zhong Zheng and Hao Fan)
[0194] Intriguingly, it appears that male TVA mice showed better immune responses to viral exposure compared to female TVA mice, suggesting that TVA might enhance immune response in mice with sex preference to male (see
Example 6
TVA Derivative #203 Enhances CD8+ T Cell Function In Vitro
[0195] Also tested was a spectrum of TVA derivatives. #203 (top left) also enhances CD8+ T cells mediated killing of mouse melanoma B16 cells (top right) through enhanced CD8 T cell function (lower 3 panels) better than cells treated with TVA in vitro (see
Example 7
Oral Gavage of TVA Derivative #203 Enhances Anti-Tumor Immunity in Syngeneic Mice
[0196] Oral gavage of #203 resulted in increased anti-tumor immunity in a dose dependent manner in syngeneic mice inoculated with murine melanoma B16 cells (see
Example 8
TVA Derivatives and Probe Synthetic Procedures
Synthesis of Compound 9
##STR00014##
##STR00015##
[0197] The bromide S1 (1.0 g, 3.6 mmol) was added to a stirred solution of 1-phenyl-1H-tetrazol-5-thiol S2 (1.14 g, 6.4 mmol) and potassium carbonate (1.0 g, 7.2 mmol) in acetone (20 mL) at room temperature. The mixture was heated to 65 C. and stirred for 2.5 h, then cooled to room temperature, the mixture was filtered, then the solvent was evaporated and the residue was diluted with a mixture of DCM (100 mL) and water (20 mL). The organic layer was separated and the aqueous layer was extracted with the DCM (250 mL). The combined organic layers were dried over NaSO.sub.4 and evaporated. Silica gel column chromatography (EtOAc:hexane=1:6) gave a white solid (1.4 g, 96%). Obtained characterization data were in agreement with those published in the literature.
##STR00016##
[0198] To a solution of sulfide S3 (500 mg, 1.2 mmol) in EtOH/THF=5:1 (12 mL) was added a solution of (NH.sub.4).sub.6Mo7O.sub.24.Math.4H.sub.2O (293.8 mg, 0.23 mmol) in H.sub.2O.sub.2 (2.6 mL, 30%) at 0 C. The reaction was stirred at room temperature overnight and quenched with H.sub.2O (5.0 mL). The mixture was extracted with DCM (320 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:5) gave a white solid (448 mg, 90%). Obtained characterization data were in agreement with those published in the literature.
[0199] To a stirred solution of the S4 (100 mg, 0.25 mmol) in DME (1.6 mL) at 78 C. under
##STR00017##
nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.54 mL, 0.27 mmol) The mixture was then stirred for 30 min before addition of the aldehyde S6 (prepared as reported, 45 mg, 0.25 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (1.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=100:1) gave a colourless oil (65 mg, 73%). .sup.1H NMR (400 MHz, CDCl.sub.3) 5.38 (q, J=5.1 Hz, 2H), 3.66 (s, 3H), 2.29 (t, J=7.5 Hz, 2H), 2.21 (t, J=6.9 Hz, 2H), 1.97 (h, J=5.7 Hz, 4H), 1.60 (m, 2H), 1.56-1.37 (m, 4H), 1.28 (m, 12H), 0.14 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) 174.5, 130.9, 129.9, 107.8, 84.4, 51.6, 34.3, 32.7, 32.1, 29.7, 29.6, 29.5, 29.4, 29.3, 29.3, 28.9, 28.2, 25.1, 19.9, 0.3.
##STR00018##
[0200] To a solution of sulfide S7 (21 mg, 0.057 mmol) in THF (2.0 mL) was added a solution of TBAF (1.0 M in THF, 86 L, 0.086 mmol) at room temperature. The reaction was stirred at room temperature for 30 min then quenched with H.sub.2O (2.0 mL). The mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to yield the product which was used directly in the follow reaction. The above product was dissolved in THF:H.sub.2O=3:1 (0.8 mL), then LiOH (14 mg, 0.34 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (35.0 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:5) gave a white solid (15 mg, 93% for 2 steps). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.48-5.27 (m, 2H), 2.35 (t, J=7.5 Hz, 2H), 2.18 (td, J=6.9, 2.7 Hz, 2H), 2.08-1.91 (m, 5H), 1.69-1.58 (m, 2H), 1.57-1.40 (m, 4H), 1.39-1.27 (m, 12H). .sup.13C NMR (101 MHz, CDCl.sub.3) 180.2, 131.0, 129.8, 84.8, 68.3, 34.2, 32.7, 32.1, 29.8, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 28.8, 28.1, 24.8, 18.4. HRMS ESI (m/z): calcd. for C.sub.18H.sub.30O.sub.2H.sup.+ [M+H].sup.+: 279.2319, found 279.2317.
Synthesis of Compound 10
##STR00019##
##STR00020##
[0201] To a stirred solution of the S4 (50 mg, 0.12 mmol) in DME (1.6 mL) at 78 C. under nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.27 mL, 0.14 mmol) The mixture was then stirred for 30 min before addition of the p-tolualdehyde (16 mg, 0.27 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (1.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=60:1) gave a colorless oil (22 mg, 60%).
##STR00021##
[0202] The above product S5 (12 mg, 0.039 mmol) was dissolved in THF:H.sub.2O=3:1 (0.8 mL), then LiOH (7.0 mg, 0.16 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (35.0 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:6) gave a white solid (10 mg, 90%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.23 (d, J=7.9 Hz, 2H), 7.09 (d, J=7.8 Hz, 2H), 6.34 (d, J=15.8 Hz, 1H), 6.16 (dt, J=15.8, 6.9 Hz, 1H), 2.36 (d, J=7.6 Hz, 2H), 2.32 (d, J=2.9 Hz, 3H), 2.18 (q, J=6.5 Hz, 2H), 1.63 (p, J=7.4 Hz, 2H), 1.45 (p, J=7.0 Hz, 2H), 1.30 (s, 10H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 179.6, 136.6, 135.3, 130.3, 129.7, 129.3, 125.9, 34.1, 33.2, 29.9, 29.6, 29.6, 29.4, 29.3, 29.2, 24.8, 21.3. HRMS-ESI (m/z): calcd. for C.sub.19H.sub.28O.sub.2H.sup.+ [M+H].sup.+: 289.2162, found 289.2149.
Synthesis of Compound 11
##STR00022##
[0203] To a stirred solution of the S4 (100 mg, 0.25 mmol) in DME (1.6 mL) at 78 C. under
##STR00023##
nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.54 mL, 0.28 mmol) The mixture was then stirred for 30 min before addition of the propionaldehyde (16 mg, 0.27 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (1.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:60) gave a colorless oil (43 mg, 73%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.49-5.30 (m, 2H), 3.66 (s, 3H), 2.29 (t, J=7.5 Hz, 2H), 1.98 (dqd, J=14.8, 6.9, 5.1 Hz, 4H), 1.61 (h, J=7.5 Hz, 2H), 1.38-1.20 (m, 12H), 1.02-0.85 (m, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 174.5, 132.0, 129.5, 51.6, 34.3, 32.7, 29.8, 29.6, 29.5, 29.4, 29.3, 29.3, 25.7, 25.1, 14.1. HRMS-ESI (m/z): calcd. for C.sub.15H.sub.28O.sub.2Na.sup.+ [M+Na].sup.+: 263.1982, found 263.1978.
##STR00024##
[0204] The above product S6 (28 mg, 0.12 mmol) was dissolved in THF:H.sub.2O=3:1 (1.2 mL), then LiOH (20 mg, 0.46 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (35.0 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:10-1:5-1:1) gave a white solid (20 mg, 72%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.50-5.31 (m, 2H), 2.35 (t, J=7.5 Hz, 2H), 1.98 (dq, J=13.9, 7.2 Hz, 4H), 1.63 (p, J=7.3 Hz, 2H), 1.40-1.26 (m, 12H), 0.96 (t, J=7.4 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 180.4, 132.0, 129.5, 34.2, 32.7, 29.9, 29.8, 29.6, 29.5, 29.4, 29.3, 29.2, 25.7, 24.8, 14.1. HRMS-ESI (m/z): calcd. for C.sub.14H.sub.26O.sub.2H.sup.+ [M+H].sup.+: 249.1825, found 249.1821.
Synthesis of Compound 13
##STR00025##
##STR00026##
[0205] A solution of PT-SH (568 mg, 3.2 mmol), PPh.sub.3 (836 mg, 3.2 mmol) and alcohol S7 (500 mg, 2.7 mmol) in THF (13 mL) and DEAD (644 mg, 3.2 mmol) was added. The resulting solution was stirred for 3 h at room temperature. The resulting solution was diluted with EtOH (20 mL), cooled to 0 C. and (NH.sub.4).sub.6Mo7O.sub.24.Math.4H.sub.2O (641 mg, 0.51 mmol) in H.sub.2O.sub.2 (5.8 mL, 30%) were added. The resulting yellowish solution was allowed to warm to room temperature and stirred for 10 h. Water (20 mL) was added and the whole mixture was extracted with EtOAc (3100 mL). The combined organic layers were washed with brine (20 mL), dried over Na.sub.2SO.sub.4, filtered and the solvents were evaporated under reduced pressure. Purification by silica gel column chromatography (EtOAc:hexane=1:5) gave a white solid (900 mg, 83% for 2 steps). .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.70 (ddt, J=7.2, 3.4, 2.5 Hz, 2H), 7.65-7.56 (m, 3H), 3.81-3.70 (m, 2H), 3.66 (s, 3H), 2.30 (t, J=7.5 Hz, 2H), 2.01-1.89 (m, 2H), 1.68-1.44 (m, 4H), 1.33 (qd, J=8.8, 4.5 Hz, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 174.3, 153.6, 133.2, 131.6, 129.8, 125.2, 56.1, 51.6, 34.1, 29.0, 28.9, 28.8, 28.2, 24.9, 22.1. HRMS-ESI (m/z): calcd. for C.sub.17H.sub.24N.sub.4O.sub.4SH.sup.+ [M+H].sup.+: 381.1591, found 381.1600.
##STR00027##
[0206] Compounds S10 was synthesized following a similar procedure described for S6. Without purified for the next step.
[0207] Compounds 13 was synthesized following a similar procedure described for 11. White
##STR00028##
solid, yield: 70%. .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.41-5.34 (m, 2H), 2.34 (t, J=7.5 Hz, 2H), 1.96 (q, J=6.2 Hz, 4H), 1.63 (p, J=7.2 Hz, 2H), 1.44-1.15 (m, 22H), 0.88 (t, J=6.7 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 180.0, 130.5, 130.2, 34.0, 32.6, 32.6, 31.9, 29.7, 29.6, 29.6, 29.6, 29.4, 29.2, 29.1, 29.0, 28.9, 24.7, 22.7, 14.1. HRMS-ESI (m/z): calcd. for C.sub.19H.sub.36O.sub.2H.sup.+
[0208] [M+H].sup.+: 297.2788, found 297.2777.
Synthesis of Compound 14
##STR00029##
[0209] Compounds S12 was synthesized following a similar procedure described for S6. Without purified for the next step.
[0210] Compounds 14 was synthesized following a similar procedure described for 11. White
##STR00030##
solid, yield: 65%, .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.44-5.29 (m, 2H), 5.10 (ddt, J=8.6, 7.1, 1.4 Hz, 1H), 2.35 (t, J=7.5 Hz, 2H), 1.97 (dh, J=10.2, 4.4 Hz, 5H), 1.87-1.77 (m, 1H), 1.68 (s, 3H), 1.65-1.62 (m, 1H), 1.60 (s, 3H), 1.49-1.40 (m, 1H), 1.37-1.24 (m, 16H), 0.86 (d, J=6.6 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 180.0, 131.7, 131.0, 128.7, 125.0, 40.0, 36.6, 34.0, 32.8, 32.6, 29.7, 29.6, 29.4, 29.4, 29.2, 29.1, 29.1, 25.7, 25.6, 24.7, 19.4, 17.6. HRMS-ESI (m/z): calcd. for C.sub.21H.sub.38O.sub.2H.sup.+ [M+H].sup.+: 323.2945, found 323.2947.
Synthesis of Compound 15
##STR00031##
##STR00032##
[0211] Compounds S14 was synthesized following a similar procedure described for S6. Without purified for the next step.
##STR00033##
[0212] Compounds 15 was synthesized following a similar procedure described for 11. White solid, yield: 73%. .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.42-5.34 (m, 2H), 2.40-2.30 (m, 2H), 2.01-1.92 (m, 4H), 1.63 (p, J=7.4 Hz, 2H), 1.38-1.21 (m, 24H), 0.88 (t, J=1.9 Hz, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 180.0, 130.5, 130.2, 34.0, 32.6, 32.6, 31.9, 29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 29.0, 28.9, 24.7, 22.7, 14.1. HRMS-ESI (m/z): calcd. for C.sub.20H.sub.38O.sub.2H.sup.+ [M+H].sup.+: 311.2945, found 311.2938.
Synthesis of Compound 16
##STR00034##
[0213] Compounds S16 was synthesized following a similar procedure described for S6. Without purified for the next step.
##STR00035##
[0214] Compounds 16 was synthesized following a similar procedure described for 11. White, solid yield: 78%. .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.42-5.34 (m, 2H), 2.35 (ddd, J=9.2, 6.7, 1.5 Hz, 2H), 2.01-1.92 (m, 4H), 1.63 (p, J=7.4 Hz, 2H), 1.37-1.25 (m, 26H), 0.92-0.84 (m, 3H). .sup.13C NMR (101 MHz, CDCl.sub.3) 179.8, 130.5, 130.2, 34.0, 32.6, 32.6, 31.9, 29.7, 29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 29.0, 28.9, 24.7, 22.7, 14.1. HRMS-ESI (m/z): calcd. for C.sub.21H.sub.40O.sub.2H.sup.+ [M+H].sup.+: 325.3101, found 325.3097.
Synthesis of Compound 17
##STR00036##
[0215] To a stirred solution of the S4 (80 mg, 0.20 mmol) in DME (1.3 mL) at 78 C. under nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.43 mL, 0.22 mmol) The mixture was then stirred for 30 min before addition of the aldehyde S17 (prepared as reported, 53 mg, 0.22 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (1.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Without purified for the next step.
##STR00037##
[0216] To a solution of sulfide S18 (60 mg, 0.15 mmol) in THF (1.5 mL) was added a solution of TBAF (1.0 M in THF, 181 L, 0.18 mmol) at room temperature. The reaction was stirred at room temperature for 30 min then quenched with H.sub.2O (2.0 mL). The mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to yield the product which was used directly in the follow reaction. The above product was dissolved in THF:H.sub.2O=3:1 (2.4 mL), then LiOH (25 mg, 0.60 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (35.0 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:4-1:1) gave a white solid (31 mg, 70% for 2 steps). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.43-5.32 (m, 2H), 3.64 (t, J=6.6 Hz, 2H), 2.34 (t, J=7.5 Hz, 2H), 2.04-1.92 (m, 4H), 1.69-1.51 (m, 5H), 1.41-1.20 (m, 19H). .sup.13C NMR (101 MHz, CDCl.sub.3) & 179.3, 130.5, 130.2, 63.0, 34.0, 32.7, 32.5, 32.5, 29.6, 29.5, 29.4, 29.2, 29.0, 29.02, 29.0, 25.6, 24.7. HRMS-ESI (m/z): calcd. for C.sub.18H.sub.34O.sub.3H.sup.+ [M+H].sup.+: 299.2581, found 299.2582.
Synthesis of Probe-1
##STR00038##
[0217] Oxalyl chloride (46 L, 0.54 mmol) was dissolved in 2.0 mL DCM and brought to 78
##STR00039##
C. A solution of DMSO (78 L, 1.08 mmol) in DCM (1.0 mL) was added dropwise, and the reaction was allowed to stir for 15 min. A solution of the alcohol S19 (50 mg, 0.36 mmol) in DCM (1.0 mL) was added dropwise, and the reaction was allowed to stir an additional 15 min. Then Et.sub.3N (300 L, 2.2 mmol) was added dropwise. After 15 min, the reaction was allowed to warm to room temperature. The reaction mixture was transferred to a separatory funnel and washed with H.sub.2O (2 mL). The phases were separated, and the aqueous phase was extracted with DCM (35.0 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4, and concentrated. Purification by silica gel column chromatography (EtOAc:hexane=1:6) gave a brown oil (20 mg, 40%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 9.74 (s, 1H), 2.46 (d, J=1.6 Hz, 2H), 2.06 (td, J=7.3, 2.7 Hz, 2H), 2.01 (d, J=2.6 Hz, 1H), 1.73 (t, J=7.2 Hz, 2H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 197.5, 82.5, 69.8, 48.4, 32.4, 24.5, 13.3.
##STR00040##
[0218] To a stirred solution of the S4 (40 mg, 0.10 mmol) in DME (0.50 mL) at 78 C. under nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.19 mL, 0.10 mmol) The mixture was then stirred for 30 min before addition of the aldehyde S20 (12 mg, 0.09 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (1.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:30) gave a colorless oil (10 mg, 37%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.56-5.42 (m, 1H), 5.25-5.12 (m, 1H), 3.66 (s, 3H), 2.30 (t, J=7.6 Hz, 2H), 2.08-1.89 (m, 6H), 1.68-1.54 (m, 4H), 1.44-1.12 (m, 13H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 174.5, 135.5, 122.1, 83.0, 69.1, 51.6, 37.0, 34.3, 32.7, 31.9, 29.5, 29.4, 29.3, 29.2, 28.3, 25.1, 13.4. HRMS-ESI (m/z): calcd. for C.sub.19H.sub.30N.sub.2O.sub.2H.sup.+ [M+H].sup.+: 319.2380, found 319.2381.
##STR00041##
[0219] The above product S21 (10 mg, 0.031 mmol) was dissolved in THF:H.sub.2O=3:1 (0.40 mL), then LiOH (5.4 mg, 0.13 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (35.0 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:3) gave a white solid (7.6 mg, 76%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.49 (dt, J=15.3, 6.6 Hz, 1H), 5.25-5.13 (m, 1H), 2.35 (t, J=7.5 Hz, 2H), 2.09-1.89 (m, 6H), 1.63 (td, J=7.5, 3.9 Hz, 4H), 1.42-1.25 (m, 13H). .sup.13C NMR (101 MHz, CDCl.sub.3) 179.7, 135.5, 122.1, 83.0, 69.1, 37.0, 32.7, 31.9, 29.8, 29.5, 29.5, 29.4, 29.3, 29.20, 28.3, 24.8, 13.4. HRMS-ESI (m/z): calcd. for C.sub.18H.sub.28N.sub.2O.sub.2H.sup.+ [M+H].sup.+: 305.2224, found 305.2220.
Synthesis of Probe-2
##STR00042##
[0220] Trimethylaluminium (66 mL, 132 mmol, 2.0 M in toluene) was added over 1 h to a
##STR00043##
solution of N,O-dimethylhydroxylamine hydrochloride (13.6 g, 139.4 mmol) in DCM (50 mL) at 78 C. The solution was warmed to room temperature and stirred for 4 h. The solution was then cooled to 5 C., y-butyrolactone S22 (4.4 ml, 57.2 mmol) was added and the resulting mixture stirred for a further 1.5 h. After this time, the solution was carefully quenched at 0 C. by addition of a solution of potassium sodium L-tartrate tetrahydrate (16 g) in water (20 mL) and stirred overnight. The resulting precipitate was filtered through a plug of Celite and washed with DCM. The organic phase was dried over Na.sub.2SO.sub.4, filtered, and the solvent removed in vacuo to give a light yellow oil (8.31 g, 99%). Obtained characterization data were in agreement with those published in the literature.
##STR00044##
[0221] To a solution of S23 (3.4 g, 23.2 mmol) and imidazole (2.4 g, 34.8 mmol) in DCM (70 mL) was added TBSCl (3.8 g, 25.5 mmol) at 0 C. The mixture was stirred at rt for 3 h. The mixture was then quenched by addition of H.sub.2O (15 mL), and extracted with DCM (3 30 mL). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by silica gel chromatography (EtOAc:hexane=1:3) gave a colorless oil (5.6 g, 92%). Obtained characterization data were in agreement with those published in the literature.
##STR00045##
Synthesis of (5-(trimethylsilyl) pent-4-yn-1-yl) magnesium chloride
[0222] This compound was prepared by following the reported procedure. Magnesium turnings (206 mg, 8.6 mmol) were etched with the back of a glass pipette and added to a flame-dried, two-neck RBF containing a stir bar and fitted with an oven-dried reflux condenser. After purging the reaction vessel with argon, a small bead of 12 was added to the magnesium turnings followed by anhydrous THF (3.0 mL) and the resulting mixture was stirred for 15 min at room temperature. A few drops of (5-chloropent-1-yn-1yl)trimethylsilane (1.0 g, 5.7 mmol) dissolved in anhydrous THF (7.0 mL) was then added to the mixture and the mixture was heated to reflux. The remaining (5-chloropent-1yn-1-yl)trimethylsilane solution was then slowly added to the refluxing reaction mixture over 30 min. When the addition was complete, the reaction was refluxed for an additional 3 h before cooling to room temperature.
[0223] Compound S24 (1.5 g, 5.7 mmol) was dissolved in anhydrous THF (30 mL) and cooled to 0 C. under N.sub.2. The above fresh prepared (5-(Trimethylsilyl) pent-4-yn-1-yl) magnesium chloride S25 was then added dropwise and stirring for an additional 1 h at room temperature. After stirring for 1 h, the reaction was quenched with the addition of sat. NH.sub.4Cl (aq.) (10 mL) and the product was extracted with EtOAc (330 mL). The combined organic layers were washed with brine, then dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc:hexane=1:20) gave a yellow oil (1.2 g, 63%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 3.60 (td, J=5.7, 2.3 Hz, 2H), 2.59-2.45 (m, 4H), 2.25 (tt, J=6.9, 1.2 Hz, 2H), 1.84-1.71 (m, 4H), 0.88 (s, 9H), 0.14 (s, 9H), 0.03 (s, 6H). .sup.13C NMR (101 MHz, CDCl.sub.3) 210.2, 106.2, 85.2, 62.0, 41.1, 39.1, 26.7, 25.8, 22.3, 19.1, 18.2, 0.1, 5.5. HRMS-ESI (m/z): calcd. for C.sub.18H.sub.36O.sub.2Si.sub.2H.sup.+ [M+H].sup.+: 341.2327, found 341.2331.
##STR00046##
[0224] To a solution of S26 (300 mg, 0.88 mmol) in THF (5.0 mL) was added a solution of TBAF (1.0 M in THF, 2.2 mL, 2.2 mmol) at room temperature. The reaction was stirred at room temperature for 30 min then quenched with H.sub.2O (5.0 mL). The mixture was extracted with EtOAc (320 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:hexane=1:1) gave a yellow oil (130 mg, 96%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 3.64 (td, J=6.1, 1.8 Hz, 2H), 2.59 (dtd, J=8.9, 7.1, 1.7 Hz, 4H), 2.24 (ddd, J=7.0, 4.7, 2.2 Hz, 2H), 2.16 (s, 1H), 1.97 (q, J=2.1 Hz, 1H), 1.91-1.74 (m, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 210.9, 83.6, 69.1, 62.2, 41.2, 39.6, 26.5, 22.2, 17.7. HRMS-ESI (m/z): calcd. for C.sub.9H.sub.14O.sub.2H.sup.+ [M+H].sup.+: 155.1067, found 155.1067.
##STR00047##
[0225] Dess-Martin reagent (99 mg, 0.23 mmol) was added to the solution of S27 (30 g, 0.2 mmol) in DCM (2.0 mL) at 0 C., and the mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated Na.sub.2S.sub.2O.sub.3:NaHCO.sub.3=1:1 (5 mL), and the product was extracted with DCM (310 mL). The organic layer was washed with brine and dried over Na.sub.2SO.sub.4, concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:hexane=1:4) gave a yellow oil (20 mg, 70%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 9.80 (s, 1H), 2.76 (p, J=2.8 Hz, 4H), 2.64 (td, J=7.2, 2.8 Hz, 2H), 2.24 (tt, J=6.9, 2.3 Hz, 2H), 2.01-1.93 (m, 1H), 1.88-1.75 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) 208.0, 200.4, 83.5, 69.1, 41.0, 37.5, 34.8, 22.3, 17.7. HRMS-ESI (m/z): calcd. for C.sub.9H.sub.12O.sub.2Na.sup.+ [M+Na].sup.+: 175.0730, found 175.0735.
##STR00048##
[0226] To a stirred solution of the S4 (54 mg, 0.13 mmol) in DME (2.0 mL) at 78 C. under nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.26 mL, 0.13 mmol) The mixture was then stirred for 30 min before addition of the aldehyde S28 (20 mg, 0.13 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (2.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:10) gave a yellow oil (16 mg, 37%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.49-5.29 (m, 2H), 3.66 (s, 3H), 2.63-2.52 (m, 2H), 2.47 (t, J=7.4 Hz, 2H), 2.34-2.16 (m, 6H), 2.06-1.90 (m, 3H), 1.79 (pd, J=7.0, 1.0 Hz, 2H), 1.62 (p, J=6.5 Hz, 2H), 1.37-1.23 (m, 12H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 209.9, 174.3, 131.6, 128.2, 83.6, 69.0, 51.4, 42.8, 41.1, 34.1, 32.5, 29.5, 29.4, 29.4, 29.3, 29.1, 29.1, 26.9, 25.0, 22.2, 17.8. HRMS-ESI (m/z): calcd. for C.sub.21H.sub.34O.sub.3H.sup.+ [M+H].sup.+: 335.2581, found 335.2584.
##STR00049##
[0227] The above product S29 (16 mg, 0.048 mmol) was dissolved in THF:H.sub.2O=3:1 (0.40 mL), then LiOH (8.0 mg, 0.19 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (35.0 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:10) gave a white solid (15 mg, 96%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.49-5.31 (m, 2H), 2.55 (t, J=7.3 Hz, 2H), 2.48 (t, J=7.5 Hz, 2H), 2.35 (t, J=5.8 Hz, 2H), 2.30-2.16 (m, 4H), 2.06-1.90 (m, 3H), 1.85-1.73 (m, 2H), 1.63 (p, J=7.6 Hz, 2H), 1.41-1.24 (m, 12H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 210.0, 179.9, 131.6, 128.2, 83.6, 69.0, 42.8, 41.1, 34.0, 32.5, 29.4, 29.4, 29.4, 29.2, 29.1, 29.1, 26.9, 24.7, 22.2, 17.8. HRMS-ESI (m/z): calcd. for C.sub.18H.sub.32O.sub.3H.sup.+ [M+H].sup.+: 321.2424, found 321.2424.
##STR00050##
[0228] Ketone S30 (16 mg, 0.050 mmol) was dissolved in a solution of NH.sub.3 (7.0 N in MeOH, 0.19 mL, 1.3 mmol) at 0 C. under N.sub.2. After stirring for 3 h at 0 C., a solution of hydroxylamine-O-sulfonic acid (6.5 mg, 0.058 mmol) in MeOH (0.1 mL) was added dropwise. The reaction mixture was allowed to slowly warm to room temperature while stirring overnight. The reaction was then concentrated and the remaining residue was redissolved in anhydrous DCM (1.0 mL) and pyridine (0.1 mL) under the protection of N2. PCC (11 mg, 0.05 mmol) was then added in small portions while the reaction mixture was cooled to 0 C. The reaction was then allowed to warm to room temperature and stirred for an additional 1 h, then 2 M HCl (1.0 mL) was added into above solution. The resulting solution was extracted with DCM (310 mL).
[0229] The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:3) gave a oil (4.0 mg, 30%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.50-5.24 (m, 2H), 2.35 (t, J=7.5 Hz, 2H), 2.16 (td, J=7.0, 2.7 Hz, 2H), 2.02-1.91 (m, 3H), 1.79 (p, J=6.6 Hz, 2H), 1.63 (p, J=7.3 Hz, 2H), 1.56-1.34 (m, 2H), 1.34-1.17 (m, 16H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 179.7, 131.7, 128.3, 83.4, 68.9, 34.0, 33.1, 32.6, 32.5, 31.8, 29.7, 29.6, 29.5, 29.5, 29.4, 29.4, 29.2, 29.1, 29.1, 28.3, 26.8, 24.7, 22.7, 18.0. HRMS-mixed (m/z): calcd. for C.sub.20H.sub.32N.sub.2O.sub.2H.sup.+ [M+H].sup.+: 333.2537, found 333.2535.
Synthesis of Probe-3
##STR00051## ##STR00052##
##STR00053##
[0230] Trimethylaluminium (2.0 M in toluene, 7.5 mL, 35 mmol) was added dropwise to a solution of N,O-dimethylhydroxylamine hydrochloride (3.4 g, 35 mmol) and S31 (2.0 g, 17.5 mmol) in DCM (60 mL) at 0 C. The solution stirred for 24 h at 0 C. After this time, the solution was carefully quenched at 0 C. by addition of a solution of potassium sodium L-tartrae tetrahydrate (3.9 g) in H.sub.2O (5.9 mL). The resulting precipitate was filtered through a plug of Celite and washed with DCM. The organic phase was dried over Na.sub.2SO.sub.4, filtered, and the solvent removed in vacuo to give a light yellow oil (2.9 g, 99%). Obtained characterization data were in agreement with those published in the literature.
##STR00054##
[0231] To a solution of S32 (2.9 g, 16.6 mmol) and imidazole (1.8 g, 26.4 mmol) in DCM (50 mL) was added TBSCl (2.8 g, 18.6 mmol) at 0 C. The mixture was stirred at room temperature for 3 h. The mixture was then quenched by addition of H.sub.2O (15 mL), and extracted with DCM (330 mL). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue was purified by silica gel chromatography (EtOAc:hexane=1:3) gave a colorless oil (4.7 g, 98%). Obtained characterization data were in agreement with those published in the literature.
##STR00055##
[0232] Compound S33 (1.6 g, 5.7 mmol) was dissolved in anhydrous THF (30 mL) and cooled to 0 C. under N.sub.2. The fresh prepared (5-(Trimethylsilyl) pent-4-yn-1-yl) magnesium chloride S25 was then added dropwise and stirring for an additional 1 h at room temperature. After stirring for 1 h, the reaction was quenched with the addition of sat. NH.sub.4Cl (aq.) (10 mL) and the product was extracted with EtOAc (330 mL). The combined organic layers were washed with brine, then dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by silica gel chromatography (EtOAc:hexane=1:30) gave a yellow oil (0.7 g, 34%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 3.45 (td, J=6.5, 1.7 Hz, 2H), 2.39 (td, J=7.3, 1.7 Hz, 2H), 2.28 (td, J=7.5, 1.7 Hz, 2H), 2.11 (td, J=6.9, 1.7 Hz, 2H), 1.63 (pd, J=7.1, 1.7 Hz, 2H), 1.52-1.32 (m, 4H), 1.19 (ttd, J=8.7, 6.3, 3.3 Hz, 2H), 0.74 (d, J=1.7 Hz, 9H), 0.00 (d, J=1.7 Hz, 9H), 0.10 (d, J=1.6 Hz, 6H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 210.4, 106.2, 85.2, 62.8, 42.8, 41.0, 32.5, 25.8, 25.4, 23.6, 22.3, 19.1, 18.2, 5.4. HRMS-ESI (m/z): calcd. for C.sub.20H.sub.40O.sub.2Si.sub.2H.sup.+ [M+H].sup.+: 369.2640, found 369.2640.
##STR00056##
[0233] To a solution of S34 (710 mg, 1.9 mmol) in THF (8.0 mL) was added a solution of TBAF (1.0 M in THF, 4.8 mL, 4.8 mmol) at room temperature. The reaction was stirred at room temperature for 30 min then quenched with H.sub.2O (5.0 mL). The mixture was extracted with EtOAc (330 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:hexane=1:1) gave a yellow oil (350 mg, 100%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 3.64 (td, J=6.5, 4.4 Hz, 2H), 2.56 (td, J=7.2, 3.1 Hz, 2H), 2.45 (td, J=7.2, 3.2 Hz, 2H), 2.23 (tdd, J=6.8, 4.2, 2.5 Hz, 2H), 1.97 (q, J=2.6 Hz, 1H), 1.86-1.71 (m, 2H), 1.68-1.51 (m, 4H), 1.44-1.30 (m, 2H). .sup.13C NMR (101 MHz, CDCl.sub.3) 210.6, 83.6, 69.1, 62.6, 42.8, 41.1, 32.4, 25.4, 23.5, 22.2, 17.8. HRMS-ESI (m/z): calcd. for C.sub.11H.sub.18O.sub.2Na.sup.+ [M+Na].sup.+: 205.1199, found 205.1202.
##STR00057##
[0234] Dess-Martin reagent (413 mg, 0.97 mmol) was added to the solution of S35 (100 mg, 0.65 mmol) in DCM (6.5 mL) at 0 C., and the mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with saturated Na.sub.2S.sub.2O.sub.3:NaHCO.sub.3=1:1 (10 mL), and the product was extracted with DCM (320 mL). The organic layer was washed with brine and dried over Na.sub.2SO.sub.4, concentrated in vacuo. The residue was purified by silica gel chromatography (EtOAc:hexane=1:3) gave a yellow oil (70 mg, 70%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 9.77 (s, 1H), 2.56 (t, 2H), 2.48-2.41 (m, 4H), 2.23 (tdd, J=6.9, 2.7, 1.2 Hz, 2H), 1.97 (dq, J=2.7, 1.3 Hz, 1H), 1.79 (d, 2H), 1.70-1.55 (m, 4H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 209.8, 202.2, 83.5, 77.4, 77.1, 76.8, 69.1, 43.7, 42.5, 41.1, 23.2, 22.2, 21.6, 17.7. HRMS-ESI (m/z): calcd. for C.sub.11H.sub.16O.sub.2H.sup.+ [M+H].sup.+: 181.1213, found 181.1218
##STR00058##
[0235] To a stirred solution of the S4 (174 mg, 0.43 mmol) in DME (2.0 mL) at 78 C. under nitrogen was added dropwise the KHMDS (0.5 M in toluene, 0.85 mL, 0.43 mmol) The mixture was then stirred for 30 min before addition of the aldehyde S36 (70 mg, 0.39 mmol). After stirring for a further 1 h at 78 C. the reaction mixture was quenched with sat. NH.sub.4Cl (3.0 mL), then the mixture was extracted with EtOAc (310 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:10) gave a yellow oil (65 mg, 46%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.46-5.30 (m, 2H), 3.66 (s, 3H), 2.55 (t, J=7.2 Hz, 2H), 2.41 (t, J=7.4 Hz, 2H), 2.30 (t, J=7.5 Hz, 2H), 2.23 (td, J=6.9, 2.6 Hz, 2H), 2.08-1.91 (m, 5H), 1.79 (d, J=6.4 Hz, 2H), 1.69-1.52 (m, 4H), 1.40-1.24 (m, 15H). .sup.13C NMR (101 MHZ, CDCl.sub.3) 210.5, 174.3, 130.9, 129.6, 83.6, 69.0, 51.4, 42.8, 41.0, 34.1, 32.6, 32.3, 29.6, 29.4, 29.4, 29.3, 29.2, 29.2, 25.0, 23.4, 22.3, 17.8. HRMS-ESI (m/z): calcd. for C.sub.23H.sub.38O.sub.3H.sup.+ [M+H].sup.+: 363.2894, found 363.2890.
##STR00059##
[0236] To a solution of S37 (65 mg, 0.18 mmol) in MeOH (0.10 mL) was added an ammonia solution in MeOH (7 N, 2.0 mL) at 0 C. under Ar. The solution was stirred at that temperature for 1 h, then warmed to room temperature stirred for 2 h. Cooled to 0 C. again, to this solution was then added hydroxylamine-O-sulfonic acid (24.3 mg, 0.22 mmol) slowly at 0 C. The resulting mixture was warmed to rt and stirred for 16 h. The white precipitate was removed by filtration and the remaining solution was concentrated by vacuo. The residue was re-dissolved in DCM (1.0 mL). To this solution was added Et.sub.3N (0.043 mL, 0.3 mmol) and a solution of 12 (82 mg, 0.32 mmol) in DCM (1.0 mL) dropwise until the solution stayed red-brown. The reaction mixture was quenched by saturated Na.sub.2S.sub.2O.sub.3, and extracted with DCM (310 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:30) gave a yellow oil (29 mg, 43%). .sup.1H NMR (400 MHz, CDCl.sub.3) 5.46-5.27 (m, 2H), 3.66 (s, 3H), 2.30 (t, J=7.7 Hz, 3H), 2.16 (tdd, J=6.8, 2.6, 1.0 Hz, 2H), 2.02-1.89 (m, 5H), 1.67-1.56 (m, 2H), 1.56-1.44 (m, 2H), 1.41-1.20 (m, 18H), 1.15-1.03 (m, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) 174.3, 130.9, 129.6, 83.4, 68.9, 51.4, 34.1, 32.7, 32.6, 32.3, 31.8, 29.6, 29.4, 29.3, 29.2, 29.1, 29.1, 28.4, 25.0, 23.3, 22.8, 18.0. HRMS-mixed (m/z): calcd. for C.sub.23H.sub.38N.sub.2O.sub.2H.sup.+ [M+H].sup.+: 375.3006, found 375.3018.
##STR00060##
[0237] The above product S38 (28 mg, 0.076 mmol) was dissolved in THF:H.sub.2O=3:1 (2.4 mL), then LiOH (13 mg, 0.30 mmol) was added into the above solution, heated the mixture to 66 C. and stirred for 3 h, cooled to room temperature, 2M HCl was added to the mixture to adjust the pH to 2, then the mixture was extracted with EtOAc (310 mL). The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and evaporated. Purification by silica gel column chromatography (EtOAc:hexane=1:6) gave a colorless oil (24 mg, 96%). .sup.1H NMR (400 MHZ, CDCl.sub.3) 5.47-5.23 (m, 2H), 2.35 (t, J=7.5 Hz, 3H), 2.20-2.06 (m, 2H), 1.98-1.87 (m, 5H), 1.69-1.57 (m, 2H), 1.57-1.43 (m, 2H), 1.41-1.23 (m, 18H), 1.15-1.05 (m, 1H). .sup.13C NMR (101 MHz, CDCl.sub.3) 180.1, 130.9, 129.6, 83.5, 68.9, 34.1, 32.7, 32.6, 32.3, 31.8, 29.6, 29.4, 29.4, 29.2, 29.1, 29.1, 29.1, 28.4, 24.7, 23.3, 22.8, 18.0. HRMS-ESI (m/z): calcd. for C.sub.22H.sub.36N.sub.2O.sub.2H.sup.+ [M+H].sup.+: 361.2850, found 361.2847.
TABLE-US-00002 TABLE 1 Human blood chemicals (633 in total) Organic metabolites & inorganics Lipids Supplements Proteins Inorganics 26 FA 14 Vitamins 16 Antibodies 5 (monounsaturated) Carbohydrates 8 FA 11 Fish oil & Omegas 25 Hormones 49 (polyunsaturated) Amino acids 26 FA 37 Minerals 31 Enzymes 11 (saturated) Glycosaminoglycans 19 FA 3 Antioxidants 31 Coagulation 15 (trans) Factors Nucleotides 4 FA 18 Herbs 44 Complement 14 (trans-monoenoic) system Organic chemicals 45 Other lipids 5 Peptides 5 Growth Factors 4 Cytokines/Chemokines 96 Other proteins 29
TABLE-US-00003 TABLE 2 Stock Physiological Working concentration Number Name concentration concentration 2000X 1 Alpha-linolenic 18.5 +/ 7.9 uM 37 uM 20.6 ul in 2 ml acid (ALA) 2 Arachidonic acid 55.4 +/ 4.48 uM 110.8 uM 0.073 ul in 2 ml 3 Astaxanthin 0.092 +/ 0.025 uM 0.184 uM 109.8186 ug in 1 ml DMSO (1000X) 4 cis -Vaccenic acid 10.7 +/ 5 uM 21.4 uM 0.013 ul in 2 ml 5 Coconut oil 800 mg/L 1.77 ul in 2 ml 6 Docosahexaenoic 94.2 +/ 31.3 uM 188.4 uM 0.131 ul in 2 ml Acid (DHA) 7 Docosapentaenoic 9.0 (7.3-11.5) uM 18 uM 23.7954 ul in 2 ml acid (DPA) 8 Eicosatrienoic Acid 3.61 +/ 2.1 uM 7.22 uM 9 Eicosapentaenoic 11.0 +/ 8.3 uM 22 uM 0.014 ul in 2 ml Acid (EPA) 10 Erucic acid 1.264 +/ 1.590 uM 2.58 uM 873.5106 ug in 1 ml DMSO (1000X) 11 Evening 200 mg/L 0.43 ul in 2 ml primrose oil 12 Flax seed oil 260 mg/L 0.56 ul in 2 ml 13 Gamma linolenic 1.08 +/ 1.5 uM 2.16 uM 1.3 ul in 2 ml acid (GLA) 14 linoleic acid (CLA) 264 +/ 17.0 uM 528 uM 0.328 ul in 2 ml 15 Myristic Acid 25.0 (8.0-70.0) uM 50 uM 11.4185 mg in 1 ml DMSO (1000X) 16 Myristoleic acid 2.016 +/ 0.733 uM 4.032 uM 0.002 ul in 2 ml 17 Nervonic acid 0.900 +/ 0.974 uM 1.8 uM 659.916 ug mg in 1 ml DMSO (1000X) 18 Oleic acid 122 +/ 56 uM 244 uM 0.154 ul in 2 ml 19 Palmitic Acid 122 +/ 48 uM 244 uM 0.146 ul in 2 ml 20 Palmitoleic acid 31.0 (5.0-85.0) uM 62 uM 0.035 ul in 2 ml 21 Pumpkin seed oil 400 mg/L 0.87 ul in 2 ml 22 Stearic Acid 85 (31-470) uM 170 uM 48.3616 mg in 1 ml DMSO (1000X) 23 Stearidonic acid 0.408 +/ 0.4 uM 0.816 uM 24 trans - 10.7 +/ 5 uM 21.4 uM 6.0446 mg in 1 ml Vaccenic acid DMSO (1000X) 25 alpha-lipoic acid 0.077 +/ 0.017 uM 0.154 uM 31.7748 ug in 1 ml DMSO (1000X) 26 CoQ-10 4.31 +/ 0.42 uM 8.62 uM 7.442 mg in 1 ml DMSO (1000X) 27 Inositol N/A 120 mg/L 120 mg in 1 ml DMSO (1000X) 28 L-glutathione 800.50 +/ 201.73 uM 1601 uM 492.0193 mg 1 ml DMSO (1000X) 29 Lutein 0.52 (0.24-0.79) uM 1.04 uM 591.624 ug 1 ml DMSO (1000X) 30 Lycopene 0.743 +/ 0.168 uM 1.486 uM 797.7888 ug 1 ml DMSO (1000X) 31 L-- N/A 84 mg/L 25 mg in 298 ul Phosphatidylcholine (1000X) 32 Pterostibene N/A 10 mg/L 10 mg in 1 ml (1000X) 33 Quercetin 0.06 +/ 0.03 uM 0.12 uM 37 ug in 1 ml (1000X) 34 Thymoquinone N/A 200 mg/L 200 mg in 1 ml (1000X) 35 Trimethylglycine N/A 130 mg/L 130 mg in 1 ml (1000X) 36 Ubiquinol N/A 20 mg/L 50 mg in 2.5 ml (1000X) 37 Zeaxanthin 0.04 +/ 0.04 uM 0.08 uM 46 ug in 1 ml (1000X) 38 6-gingerol N/A 5.5 mg/L 5.5 mg in 1 ml (1000X) 39 Boswellia Serrata 90 mg/L 90 mg in 1 ml (1000X) Extract 40 Bromelain from 100 mg/L 100 mg in 1 ml (1000X) pineapple stem 41 Caffeic acid 0.012 mg/L 0.012 mg in 1 ml (1000X) phenethyl ester 42 capsaicin N/A 100 mg/L 100 mg in 1 ml (1000X) 43 Chondroitin 9.58 +/ 2.71 uM 300 mg/L 300 mg in 1 ml (1000X) sulfate sodium 44 Collagen, type II, 0.2 g/L 200 mg in 1 ml (1000X) chicken-sternal cartilage 45 Crucuminoids 95 mg/L 95 mg in 1 ml (1000X) 46 Curcumin 0.17 +/ 0.013 uM 0.34 uM 133 mg in 1 ml (1000X) 133 mg/L 47 D-glucosamine 3-sulfate N/A 100 mg/L 100 mg in 1 ml (1000X) 48 Gelatin solution 115 mg/L 49 Glucosamine 0.29 (0.0-0.6) uM 0.58 uM 125 ug in 1 ml (1000X) Hydrochloride 50 Hyaluronic acid 0.24 (0.12-0.36) uM 0.48 uM 20 mg/ml in 1 ml (1000X) sodium salt 20 mg/L 51 Methylsulfonylmethane 8.8 +/ 7.3 uM 17.6 uM 1.65 mg in 1 ml (1000X) 52 N-acetyl D-glucosamine N/A 140 mg/L 140 mg in 1 ml (1000X) 53 Phenolics, total 20 mg/L 20 mg in 1 ml (1000X) 54 Resveratrol N/A 4 mg/L 4 mg in 1 ml (1000X) 55 Rutoside trihydrate 20 mg/L 20 mg in 1 ml (1000X) 56 Taurine 162.0 +/ 60.0 uM 324 uM 40.54 mg in 1 ml (1000X) 57 Calcium citrate Ca2+: 2420.0 200 mg/L 200 mg in 1 ml (1000X) (2250.0-2590.0) uM Citrate: 190.0 (30.0-400.0) uM 58 calcium d-glucarate Ca2+: 2420.0 200 mg/L 200 mg in 1 ml (1000X) (2250.0-2590.0) uM Glucarate: N/A in blood 59 chromium Chloride Cr3+: 100 ug/L 100 ug in 1 ml (1000X) 0.0075 +/ 0.0015 uM Cl: 103700.0 +/ 1900.0 uM 60 Chromium picolinate Cr3+: 100 ug/L 100 ug in 1 ml (1000X) 0.0075 +/ 0.0015 uM Picolinate: 0.299 +/ 0.034 uM 61 Copper Gluconate Cu2+: 37.6 400 ug/L 400 ug in 1 ml (1000X) (23.6-49.9) uM Gluconate: 3.295 +/ 0.534 uM 62 copper sulfate Cu2+: 37.6 600 ug/L (23.6-49.9) uM Sulfate: 490.0 (310.0-580.0) uM 63 Dicalcium Phosphate Ca2+: 2420.0 50 mg/L 50 mg in 1 ml (1000X) (2250.0-2590.0) uM PO43: 379.1 +/ 31.6 uM 64 Ferrous Fumarate Fe2+: 9766 +/ 1246 uM 6 mg/L 6 mg in 1 ml (1000X) Fumarate: 1.5 (0.0-4.0) uM 65 Ferrous gluconate Fe2+: 9766 +/ 1246 uM 3.6 mg/L 3.6 mg in 1 ml (1000X) Gluconate: 3.295 +/ 0.534 uM 66 Ferrous Sulfate Fe2+: 9766 +/ 1246 uM 13 mg/L 13 mg in 1 ml (1000X) Sulfate: 490.0 (310.0-580.0) uM 67 Iron Gluconate Fe3+: N/A 3.6 mg/L 3.6 mg in 1 ml (1000X) Gluconate: 3.295 +/ 0.534 uM 68 L-Aspartic acid L-Aspartic acid: 50 mg/L 50 mg in 1 ml (1000X) potassium salt 21.0 +/ 5.0 uM K+: 4200.0 (3600.0-4800.0) uM 69 Magnesium Aspartate Mg2+: 50 mg/L 50 mg in 1 ml (1000X) 833.0 +/ 208.0 uM L-Aspartic acid: 21.0 +/ 5.0 uM 70 Magnesium citrate Mg2+: 50 mg/L 50 mg in 1 ml (1000X) 833.0 +/ 208.0 uM Citrate: 190.0 (30.0-400.0) uM 71 Manganese sulfate Mn2+: 0.17292 400 ug/L 400 ug in 1 ml (1000X) (0.16382-0.18202) uM Sulfate: 490.0 (310.0-580.0) uM 72 Malic acid N/A 120 mg/L 120 mg in 1 ml (1000X) 73 Monopotassium K+: 4200.0 40 mg/L 40 mg in 1 ml (1000X) Phosphate (3600.0-4800.0) uM PO43: 379.1 +/ 31.6 uM 74 Potassium Citrate K+: 4200.0 20 mg/L 20 mg in 1 mk (1000X) (3600.0-4800.0) uM Citrate: 190.0 (30.0-400.0) uM 75 Potassium Gluconate K+: 4200.0 20 mg/L 20 mg in 1 ml (1000X) (3600.0-4800.0) uM Gluconate: 3.295 +/ 0.534 uM 76 Potassium phosphate K+: 4200.0 10 mg/L 10 mg in 1 ml (1000X) dibasic (3600.0-4800.0) uM PO43: 379.1 +/ 31.6 uM 77 Potassium sorbate N/A 2.4 mg/L 2.4 mg in 1 ml (1000X) 78 Silicic acid N/A 1 mg/L 1 mg in 1 ml (1000X) 79 Sodium citrate Na+: 142600.0 5 mg/L 5 mg in 1 ml (1000X) (138000.0-146000.0) uM Citrate: 190.0 (30.0-400.0) uM 80 Sodium Molybdate Na+: 142600.0 2.5 ug/L 2.5 ug in 1 ml (1000X) (138000.0-146000.0) uM Molybdate: N/A 81 sodium selenite Na+: 142600.0 60 ug/L 60 ug in 1 ml (1000X) (138000.0-146000.0) uM Selenite: 0.0013 (0.00-0.0063) uM 82 strontium chloride Sr2+: 0.44 +/ 0.17 uM 20 mg/L 20 mg in 1 ml (1000X) Cl: 103700.0 +/ 1900.0 uM 83 Tricalcium phosphate Ca2+: 2420.0 100 mg/L 100 mg in 1 ml (1000X) hydrate (2250.0-2590.0) uM PO43: 379.1 +/ 31.6 uM 84 Vanadylsulfate Sulfate: 490.0 2 mg/L 2 mg in 1 ml (1000X) (310.0-580.0) uM 85 Zinc Citrate Zn2+: 16.8 +/ 6.3 uM 6 mg/L 6 mg in 1 ml (1000X) Citrate: 190.0 (30.0-400.0) uM 86 Zinc Oxide 4 mg/L 4 mg in 1 ml (1000X) 87 Zinc Picolinate Zn2+: 16.8 +/ 6.3 uM 10 mg/L 10 mg in 1 ml (1000X) Picolinate: 0.299 +/ 0.034 uM 88 p-Aminobenzoic Acid 15.0 (5.01-32.0) uM 30 uM 4.1142 ug in 1 ml (1000X) 89 d-Biotin 0.11-0.13 uM 0.26 uM 63.5206 ng in 1 ml (1000X) 90 Folic Acid 0.021 (0.017-0.025) uM 0.042 uM 18.5388 ng in 1 ml (1000X) 91 Niacinamide 0.44 +/ 0.0054 uM 0.88 uM 107.4656 ng in 1 ml (1000X) 92 D-Pantothenic Acid, 4.91 +/ 0.38 uM 9.82 uM 2.3398 ug in 1 ml (1000X) hemicalcium salt 93 Pyridoxal Hydrochloride 0.251 +/ 0.051 uM 0.502 uM 102.2172 ng in 1 ml (1000X) 94 Pyridoxamine 0.164 +/ 0.038 uM 0.328 uM 79.0841 ng in 1 ml (1000X) Dihydrochloride 95 Pyridoxine 0.025 (0.007-0.060) uM 0.05 uM 10.282 ng in 1 ml (1000X) Hydrochloride 96 Riboflavin 0.530 (0.265-1.30) uM 1.06 uM 398.9416 ng in 1 ml (1000X) 97 Thiamine Hydrochloride 0.34 (0.14-0.79) uM 0.68 uM 229.3436 ng in 1 ml (1000X) 98 DL-6,8-Thioctic Acid 0.077 +/ 0.017 uM 0.154 uM 31.7748 ng in 1 ml (1000X) 99 Tryptophan 100 Threonine 101 Isoleucine 102 Leucine 103 Lysine Blood Chemicals 2000* physilogical concentration stock: Number Name mass (mg) in 1 ml ALDOSTERONE 0.04992094 105 ALUMINUM 0.2080104 CHLORIDE, ANHYDROUS 106 ANDROSTERONE 0.1277936 VETRANAL,/androsterone 107 TIN(II) CHLORIDE/Tin 0.3432122 108 PHOSPHORUS 300FIX PENTACHLORIDE 109 FUROSEMIDE/LASIX 800 110 CALCIUM CHLORIDE/calcium 77.1311 111 CYTOSINE 3.31078 112 L-THYROXINE 9.94394E05 113 (+)-DELTA-TOCOPHEROL 22.22628 114 THEOBROMINE 2.34208 115 O-METHYLGUANINE 300FIX 116 17A-HYDROXYPROGESTERONE 0.17811794 117 THYMIDINE 0.823582 118 119 CAFFEINE 50.10102 120 PREGNENOLONE 2.5310806 ACETATE/pregnenolone 121 FOLIC ACID, 0.366362 122 GUANINE 29.31922 123 ESTRIOL 0.230704 124 D-LACTOSE MONOHYDRATE 307FIX 125 CADMIUM CHLORIDE/cadmium 0.1246576 126 SULFUR/sulfide 0.009621 127 PURINE 305FIX 128 ESTRONE 0.0324444 129 LEAD(II) SULFIDE 3.325853 130 TETRAETHYLAMMONIUM 0.4 HYDROGEN SULFATE, FOR IPC 131 D-GLUCURONIC ACID 76.49116 132 CHOLINE CHLORIDE 3.4905 133 CREATININE, ANHYDROUS, 24.614912 134 L-CYSTEINE BIOULTRA, 0.460408 135 ACETYLCHOLINE 0.0781138 CHLORIDE 136 L-CITRULLINE 16.11748 137 GLYCEROL MOLECULAR 8.84064 BIOLOGY REAGENT 138 COPPER(II) SULFATE, 4.859600979 ANHYDROUS,/copper 139 140 CORTISONE 0.25735416 141 MERCURY(II) CHLORIDE 0.192765 142 URIC ACID 121.71164 143 POTASSIUM 242.2875 CHLORIDE/potassium 144 UREA 270.27 145 CHITIN 302FIX 146 CELLULOSE FIBROUS 304FIX 147 D(+)-GALACTOSE 44.31936 ANHYDROUS 148 SUCROSE 2.0538 149 SODIUM BICARBONATE/ 1121.11345 hydrogen carbonate 150 SODIUM SULFATE 164.7664 151 NICOTINIC ACID 14.330004 152 POTASSIUM 297.5 BROMIDE/potassium 153 0% deoxycholic acid 8.064 sodium salt + 50% cholic acid sodium sa 154 TAURINE 42.551 155 GUANIDINE 0.19106 HYDROCHLORIDE, 156 L-ALANINE 72.51926 157 THIAMINE 2.664433 HYDROCHLORIDE/thiamine 158 MANGANESE SULFATE 3.09002364 MONOHYDRATE, 159 MYO-INOSITOL 6.161472 160 L-ASCORBIC ACID 40.15536 BIOXTRA, 161 ZINC CHLORIDE 6.29706 162 L-ORNITHINE 47.55084 MONOHYDROCHLORIDE 163 DL-MALIC ACID 5.63178 164 ZINC SULFATE 13.285272 HEPTAHYDRATE 165 BETAINE 66.7755 166 MAGNESIUM 1.73301242 CHLORIDE ANHYDROUS 167 D-(+)-GLUCOSAMINE 0.258756 HYDROCHLORIDE 168 SUCCINIC ACID 7.55776 169 URACIL 3.497208 170 OXALIC ACID SODIUM SALT 5.9496 171 D-(+)-MALTOSE 305.362725 MONOHYDRATE/D-Maltose 172 SODIUM BROMIDE 19.178696 BIOXTRA/Bromide 173 D()FRUCTOSE 12.25088 174 PECTIN 303FIX 175 SODIUM FLUORIDE/fluoride 0.5450302 176 SODIUM CHLORIDE 4266.12 BIOXTRA/sodium 177 LIPASE FROM 1 PORCINE PANCREAS, 178 D-PANTOTHENIC 2.5208966 ACID HEMICALCIUM 179 2,3-DPG, D-Glycerate 250.00675 2,3-diphosphate sodium salt 180 TRANS-B-CAROTENE, 0.7140371 TYPE I,/all trans B carotene 181 L-GLUTATHIONE REDUCED 154.0026618 182 VITAMIN K1 0.76619 183 (+/)-A-TOCOPHEROL/ 49.96236 Alpha-Tocopherol 184 3-HYDROXYBUTYRIC ACID 16.656 185 L(+)LACTIC ACID SODIUM 104.249418 186 ADENOSINE FREE BASE 1.7290428 187 PYRUVIC ACID 45.43896 188 3-HYDROXYTYRAMINE 0.0123266 HCL/dopamine 189 PHENOL 1.6017522 190 4-AMINOBENZOIC ACID 8.77696 191 LYCOPENE 4.8908857 192 (+)-GAMMA-TOCOPHEROL 55.3017696 193 ALBUMIN HUMAN 25000 194 ()-EPINEPHRINE 0.161216 195 ERGOCALCIFEROL 0.277655 196 BIOTIN, POWDER, 0.2369807 197 2-DEOXY-L-RIBOSE/ 0.67065 Deoxycytidine 198 CHONDROITIN SULFATE, 307.25 A SODIUM SALT 199 CHOLECALCIFEROL 300FIX CRYSTALLINE 200 5-HYDROXYTRYPTAMINE 4.2536 HYDROCHLORIDE/Serotonin 201 OXYTOCIN 179.27982 202 VASOACTIVE 124.7175 INTESTINAL PEPTIDE 203 C-PEPTIDE FRAGMENT 0.13289144 3-33, HUMAN/C-peptide 204 OLECYSTOKININ 1.6798718 FRAGMENT 26-33 AMIDE NON SULFAT 205 GLYCOGEN 20FIX 206 ALPHA 1 ANTITRYPSIN, 0.6 HUMAN PLASMA 207 ,3,5-TRIIODO-L-THYRONINE 3.76858E05 SODIUM SALT/Liothyronin 208 HYALURONIC ACID 100FIX SODIUM SALT 209 ARG8-VASOPRESSIN 0.000138781 ACETATE SALT/Vasopressin 210 L-A- 25FIX PHOSPHATIDYLETHANOLAMINE TYPE I 211 CHORIONIC 50FIX GONADOTROPIN HUMAN, 212 THYROCALCITONIN 0.000002 SALMON/calcitonin 213 BETA-CRYPTOXANTHIN 1.1167974 214 L-(+)- 0.697072 ERGOTHIONEINE INNER SALT 215 FERRITIN TYPE IV 0.6 FROM HUMAN LIVER 216 HISTAMINE FREE 0.122265 BASE CRYSTALLINE 217 ANGIOTENSIN II HUMAN 0.031594636 218 L-A- 27.81696 PHOSPHATIDYLCHOLINE TYPE XVI-E 219 MELATONIN 0.00015795 CRYSTALLINE/melatonin 220 INDOXYL beta-D- 3.395835 GLUCOSIDE/Inodxylglucuronide 221 SPHINGOMYELIN/ 50FIX SM(d18:1/16:0) 222 GUANOSINE 3:5-CYCLIC 0.2129702 MONOPHOSPHATE SODIUM 223 ARSENIC(III) CHLORIDE 0.056613744 224 SODIUM CYANIDE, exclude 225 INIZING HORMONE all prepared (fix) RELEASING HORMONE HUMAN ACE 226 5alpha- 300FIX Tetrahydrocorticosterone 227 CHOLESTEROL 2513.225 228 ADENOSINE 5- 429.8892 TRIPHOSPHATE DISODIUM SALT 229 CITRIC ACID 153.696 230 CALCIUM 77.1311 CHLORIDE/calcium 231 BILIRUBIN MIXED ISOMERS 24.55572 232 ACETONE 19.7472 233 LITHIUM ACETOACETATE/ 18.57944 Acetoacetic acid 234 Carcinoembryonic 10 Antigen (CEA) 235 PHYTANIC ACID 8.000768 236 ETHYL ALCOHOL/Ethanol 7.3712 237 ALCOHOL 7.3712 238 A-CAROTENE 6.012944 239 PYRIMIDINE 4.8054 240 PROTOPORPHYRIN IX 4.726344 241 A-CRYPTOXANTHIN 4.5114192 242 LEPTIN 4 243 PROINSULIN C-PEPTIDE 3.1829512 (55-89)/c-peptide 244 SELENIUM TETRACHLORIDE/ 1.175723881 Selenium ion (se2+) 245 PYRIDOXINE 1.01508 246 XANTHOPHYLL FROM 1.0012112 MARIGOLD/xanthophyll (Lutein) 247 TESTOSTERONE--DEA 1.0008174 SCHEDULE III 248 IODINE 0.925452 MONOCHLORIDE/IODINE 249 IN ADENINE DINUCLEOTIDE 0.8046247 DISODIUM SALT HYDRATE/ 250 5ALPHA- 0.493748 DIHYDROTESTOSTERONE (DHT)/DHT 251 THIAMINE PYROPHOSPHATE 0.3041082 252 ADENOSINE 3:5- 0.296289 CYCLIC MONOPHOSPHATE 253 ANDROSTENEDIONE 0.2823666 254 NOREPINEPHRINE 0.186098 255 11-DEOXYCORTISOL/Cortexolone 0.1489778
TABLE-US-00004 TABLE 3 Rank Blood chemicals Relative changed percentage (%) 1 Nicotinic Acid 205.602475 2 Potassium Phosphate Dibasic 177.4846 3 Caffeine 170.0384 4 Trans-Vaccenic Acid 133.920675 5 Stearidonic Acid 109.251075 6 Folic Acid 105.595875 7 Potassium Gluconate 97.7133 8 Sphingomyelin 95.238125 9 Niacinamide 95.233175 10 Pregnenolone Acetate 82.906525 11 POTASSIUM BROMIDE 78.357625 12 Oleic acid 77.53305 13 CYTOSINE 74.6228 14 SUCROSE 70.452825 15 PURINE 63.4263 16 Gelatin 59.80065 17 Potassium Citrate 58.7511 18 MELATONIN CRYSTALLINE/melatonin 56.15455 19 Palmitoleic acid 54.625525 20 alpha-lipoic acid 54.6153775 21 THIAMINE HYDROCHLORIDE 53.320075 22 ()-EPINEPHRINE 53.119875 23 COPPER(II) SULFATE, ANHYDROUS,/copper 51.653075 24 Tricalcium phosphate hydrate 51.487425 25 SODIUM BICARBONATE 50.3453675 26 L-GLUTATHIONE REDUCED 48.2820975 27 p-Aminobenzoic Acid 47.979275 28 D-Glycerate 2,3-diphosphate sodium salt 47.739595 29 L-ALANINE 44.49305 30 Caffeic acid phenethyl ester 43.8461725 31 6-gingerol 42.62105 32 GUANIDINE IIYDROCIILORIDE, 41.8290325 33 (+)-DELTA-TOCOPHEROL 41.421275 34 17A-HYDROXYPROGESTERONE 41.357225 35 L-Alanine 41.06955 36 MYO-INOSITOL 40.7157 37 (+/)-A-TOCOPHEROL/Alpha-Tocopherol 40.654625 38 NOREPINEPHRINE 40.2469 39 DL-MALIC ACID 40.0104375 40 THYMIDINE 39.62865 41 GLYCEROL MOLECULAR BIOLOGY REAGENT 39.3621 42 SODIUM SULFATE 38.986945 43 POTASSIUM CHLORIDE/potassium 38.973175 44 Stearic Acid 38.766525 45 Malic acid 38.522425 46 ALBUMIN HUMAN 38.341525 47 L-A-PHOSPHATIDYLCHOLINE TYPE XVI-E 38.095225 48 Nervonic acid 37.681155 49 2-DEOXY-L-RIBOSE/Deoxycytidine 36.6995175 50 SODIUM CHLORIDE 36.528025 51 BIOTIN, POWDER, 36.4532125 52 D(+)-GALACTOSE ANHYDROUS 35.840375 53 TRANS-B-CAROTENE, TYPE I,/all trans B carotene 35.08137 54 L-Cysteine 32.819475 55 L-A-PHOSPHATIDYLETHANOLAMINE TYPE I 32.242225 56 CADMIUM CIILORIDE/cadmium 32.11155 57 Lutein 31.92307 58 D-Pantothenic Acid, hemicalcium salt 31.9171 59 MAGNESIUM CHLORIDE ANHYDROUS 31.903775 60 strontium chloride 30.89245 61 INDOXYL beta-D-GLUCOSIDE/Inodxyl glucuronide 30.458225 62 Lycopene 30.0000125 63 d-Biotin 29.844575 64 Manganese sulfate 29.5514375 65 A-CRYPTOXANTHIN 29.100525 66 FUROSEMIDE/LASIX 29.012345 67 Monopotassium Phosphate 28.67195 68 L-Asparagine 28.4787 69 LEPTIN 27.08995 70 PHENOL 26.78725 71 CHORIONIC GONADOTROPIN HUMAN, 26.677575 72 CoQ-10 26.5384625 73 ANDROSTENEDIONE 25.925925 74 L-Glutamine 25.51725 75 CHOLECALCIFEROL CRYSTALLINE 25.451545 76 THEOBROMINE 25.41615 77 ESTRONE 25.0199 78 BETA-CRYPTOXANTHIN 24.87725 79 SULFUR/sulfide 24.143425 80 Riboflavin 23.14715 81 D-LACTOSE MONOHYDRATE 22.470125 82 Sodium Molybdate 22.1968 83 11-DEOXYCORTISOL/Cortexolone 21.85185 84 BoswelliaSerrata Extract 20.45585 85 TIN(II) CHLORIDE/Tin 18.86145 86 3-HYDROXYTYRAMINE HCL/dopamine 18.776925 87 L-THYROXINE 18.7243 88 PROINSULIN C-PEPTIDE (55-89)/c-peptide 17.8836 89 4-AMINOBENZOIC ACID 17.743325 90 ANGIOTENSIN II HUMAN 17.2506625 91 Bromelain from pineapple stem 16.0114 92 ERGOCALCIFEROL 16.00985 93 ANDROSTERONE VETRANAL,/androsterone 15.912225 94 Inositol 15.3846175 95 Eicosatrienoic Acid 15.3571425 96 GUANINE 15.13945 97 Rutoside trihydrate 14.9881 98 L-ASCORBIC ACID 14.4874425 99 Sodium citrate 13.8444075 100 GLYCOGEN 13.7470725 101 L-glutathione 13.4615325 102 ZINC CHLORIDE 13.3368125 103 sodium selenite 13.272315 104 Resveratrol 13.164175 105 Evening primrose oil 12.31885 106 Astaxanthin 12.261905 107 THYROCALCITONIN SALMON/calcitonin 11.374785 108 PYRIDOXINE 11.1111 109 L-Arginine 10.74767 110 Erucic acid 10.71428 111 PYRUVIC ACID 10.4220675 112 VITAMIN K1 10.1265725 113 ALCOHOL 9.841275 114 D-glucosamine 3-sulfate 9.8006575 115 TIIIAMINE PYROPIIOSPIIATE 9.753075 116 Trimethylglycine 9.458665 117 CHONDROITIN SULFATE, A SODIUM SALT 9.1953975 118 FERRITIN TYPE IV FROM HUMAN LIVER 9.074575 119 Gamma linolenic acid 9.0579725 120 Thiamine Hydrochloride 8.9151325 121 Collagen type II 8.55482 122 GUANOSINE 3:5-CYCLIC MONOPIIOSPIIATE 8.265975 SODIUM 123 L-ORNITHINE MONOHYDROCHLORIDE 8.0543775 124 Pumpkin seed oil 7.9295 125 Pyridoxal Hydrochloride 7.787315 126 XANTHOPHYLL FROM MARIGOLD/xanthophyll (Lutein 7.53085 127 Vanadylsulfate 7.0938175 128 Pterostibene 6.87431 129 FOLIC ACID, 6.8525775 130 A-CAROTENE 6.56085 131 Ferrous Fumarate 6.27762 132 O-METHYLGUANINE 6.27401 133 Curcumin 5.9800925 134 CHITIN 5.909425 135 Zinc Picolinate 5.492225 136 L-Valine 5.29597 137 trans-4-Hydroxy-L-proline 5.2812025 138 cis-Vaccenic acid 5.238115 139 L(+)LACTIC ACID SODIUM 5.0818225 140 3-HYDROXYBUTYRIC ACID 5.0818225 141 Arachidonic acid 4.583345 142 Thymoquinone 4.55838 143 LECYSTOKININ FRAGMENT 26-33 AMIDE NON SULFA 4.556175 144 L-Aspartic acid potassium salt 4.420875 145 SUCCINIC ACID 4.1401275 146 PYRIMIDINE 4.126995 147 Copper Gluconate 4.0672 148 Magnesium citrate 3.781875 149 ADENOSINE FREE BASE 3.703725 150 Zinc Oxide 3.6269575 151 L-Histidine hydrochloride 3.5306375 152 Iron Gluconate 3.27146 153 Hyaluronic acid sodium salt 3.09278 154 ESTRIOL 2.7888525 155 ADENOSINE 3:5-CYCLIC MONOPHOSPHATE 2.71604 156 URACIL 2.45679 157 TAURINE 2.3857 158 L-Glutamic Acid 2.2718 159 Ubiquinol 2.16524 160 Dicalcium Phosphate 1.94519 161 Capsaicin 1.661115 162 (+)-GAMMA-TOCOPHEROL 1.559935 163 L-Tryptophan 1.3580175 164 ZINC SULFATE HEPTAHYDRATE 1.046025 165 VASOACTIVE INTESTINAL PEPTIDE 1.0212 166 UREA 0.31116 167 ADENINE DINUCLEOTIDE DISODIUM SALT HYDRAT 0.3703625 168 Silicic acid 1.029765 169 L-Lysine hydrochloride 1.6049375 170 Zcaxanthin 1.6524075 171 Zinc Citrate 2.0725275 172 Potassium sorbate 2.28832 173 Palmitic Acid 2.643185 174 MERCURY(II) CHLORIDE 2.99494 175 phenolics 3.0134675 176 L-CITRULLINE 3.3839075 177 5ALPHA-DIHYDROTESTOSTERONE (DIIT)/DIIT 3.4567975 178 HISTAMINE FREE BASE CRYSTALLINE 3.5938975 179 L-Proline 3.935085 180 N-acetyl D-glucosamine 4.044425 181 Chromium picolinate 4.0671975 182 DL-6,8-Thioctic Acid 4.135335 183 Magnesium Aspartate 4.155615 184 % deoxycholic acid sodium salt + 50% cholic acid 4.29424 sodium s 185 Crucuminoids 4.4019975 186 Quercetin 4.501425 187 PECTIN 4.9135575 188 D-(+)-MALTOSE MONOHYDRATE 4.9135625 189 Dipotassium Phosphate 5.3934625 190 Pyridoxamine Dihydrochloride 6.1761475 191 L-Threonine 6.543205 192 URIC ACID 6.65112 193 Glucosamine Hydrochloride 6.9785875 194 OXYTOCIN 6.99136 195 L-Leucine 7.16049 196 ARG8-VASOPRESSIN ACETATE SALT/Vasopressin 7.610475 197 L-Isoleucine 8.64197 198 Calcium Carbonate 8.7499975 199 ACETYLCHOLINE CHLORIDE 8.9394725 200 Glycine 8.9655275 201 Flax seed oil 9.0579825 202 LIPASE FROM PORCINE PANCREAS 9.4032675 203 linoleic acid 9.4202875 204 L-Aspartic Acid 9.6146025 205 HYALURONIC ACID SODIUM SALT 9.7381275 206 L-(+)-ERGOTHIONEINE INNER SALT 10.3927975 207 ALPHA 1 ANTITRYPSIN, HUMAN PLASMA 10.6048725 208 Chondroitin sulfate sodium 11.4617925 209 C-PEPTIDE FRAGMENT 3-33, HUMAN/C-peptide 11.7046425 210 L-Methionine 11.83801 211 Calcium d-glucarate 12.1428575 212 CORTISONE 12.2909425 213 CELLULOSE FIBROUS 13.200305 214 ,5-TRIIODO-L-THYRONINE SODIUM SALT/Liothyroni 13.4206225 215 L-Tyrosine 13.4475575 216 LEAD(II) SULFIDE 13.6320975 217 MANGANESE SULFATE MONOHYDRATE, 14.2345875 218 Calcium citrate 14.5833325 219 OXALIC ACID SODIUM SALT 16.878975 220 L-Phenylalanine 17.2378 221 Myristic Acid 17.3913125 222 Pyridoxine Hydrochloride 17.45435 223 Methylsulfonylmethane 17.52577 224 Taurine 17.6843775 225 PHYTANIC ACID 18.606555 226 BETAINE 18.828455 227 D-PANTOTHENIC ACID HEMICALCIUM 18.9873425 228 Docosapentaenoic acid 19.39394 229 IZING HORMONE RELEASING HORMONE HUMAN AC 19.590155 230 L-Cystine 19.73001 231 Myristoleic acid 21.37681 232 LITHIUM ACETOACETATE/Acetoacetic acid 21.47541 233 L-Serine 22.1095325 234 -HYDROXYTRYPTAMINE HYDROCHLORIDE/Serotoni 22.38806 235 L-CYSTEINE BIOULTRA, 22.735805 236 Alpha-linolenic acid 24.529505 237 D()FRUCTOSE 25.5232025 238 Copper sulfate 26.31579 239 SODIUM BROMIDE 26.9790725 240 Docosahexaenoic Acid 29.8883575 241 ACETONE 31.065575 242 CHOLESTEROL 31.885245 243 Chromium Chloride 35.279105 244 TETRAETHYLAMMONIUM HYDROGEN SULFATE 35.945565 245 Citric Acid 36.80328 246 L-a-Phosphatidylcholine 37.161085 247 Bilirubin Mixed Isomers 37.6229525 248 D-(+)-Glucosamine Hydrochloride 38.232215 249 Adenosine 5-Triphosphate Disodium 38.2786875 250 Coconut Oil 38.8516725 251 Creatine 40.990145 252 Choline Chloride 41.1074625 253 Sodium Fluoride 43.8125575 254 D-Glucuronic Acid 45.8000925
TABLE-US-00005 TABLE 4 Rank Blood chemicals Relative changed percentage (%) 1 GUANINE 236.028968 2 THEOBROMINE 211.643836 3 THYMIDINE 162.160531 4 GUANOSINE 3:5-CYCLIC MONOPHOSPHATE SODIUM 161.1621 5 FERRITIN TYPE IV FROM HUMAN LIVER 146.7316 6 L-CITRULLINE 137.20195 7 CREATININE, ANHYDROUS, 125.820975 8 COPPER(II) SULFATE, ANHYDROUS,/copper 108.412075 9 BILIRUBIN MIXED ISOMERS 98.6544475 10 Pyridoxine Hydrochloride 96.1931291 11 FUROSEMIDE/LASIX 93.8356164 12 D-Glycerate 2,3-diphosphate sodium salt 92.245325 13 Riboflavin 88.0222841 14 D(+)-GALACTOSE ANHYDROUS 87.926375 15 CITRIC ACID 82.446475 16 L-Tryptophan 76.3431151 17 CELLULOSE FIBROUS 72.1710725 18 Trans-Vaccenic Acid 69.862825 19 p-Aminobenzoic Acid 68.3070257 20 D-Pantothenic Acid, hemicalcium salt 67.4094708 21 TIN(II) CHLORIDE/Tin 66.3013699 22 D-(+)-MALTOSE MONOHYDRATE 63.1480275 23 GUANIDINE HYDROCHLORIDE 62.929575 24 L-Methionine 62.8893905 25 ACETONE 54.128425 26 ANGIOTENSIN II HUMAN 52.665725 27 Alpha-linolenic acid (ALA) 52.430375 28 L-Threonine 49.4808126 29 ACETYLCHOLINE CHLORIDE 46.018875 30 Glycine 43.9869281 31 ADENOSINE 5-TRIPHOSPHATE DISODIUM SALT 42.629985 32 Pyridoxal Hydrochloride 42.3398329 33 calcium d-glucarate 41.6058394 34 Pyridoxamine Dihydrochloride 40.9470752 35 THYROCALCITONIN SALMON/calcitonin 39.4258125 36 SODIUM CHLORIDE 38.7733375 37 BETA-CRYPTOXANTIIIN 36.94945 38 L-Aspartic acid potassium salt 36.7029549 39 L-A-PHOSPHATIDYLCHOLINE TYPE XVI-E 36.39315 40 INDOXYL beta-D-GLUCOSIDE/Inodxyl glucuronide 34.0751 41 Iron Gluconate 33.437014 42 Nervonic acid 32.9066 43 L-Glutamine 32.6143791 44 Evening primrose oil 32.4109 45 trans-4-Hydroxy-L-proline 31.8954248 46 L-Isoleucine 31.5575621 47 POTASSIUM BROMIDE 30.326475 48 Docosapentaenoic acid (DPA) 29.35347 49 D()FRUCTOSE 28.93901 50 Magnesium Aspartate 27.8382582 51 sodium selenite 26.8647478 52 CoQ-10 26.1655 53 Monopotassium Phosphate 26.1446164 54 Malic acid 25.6609642 55 Inositol 25.39915 56 chromium Chloride 25.3041363 57 Calcium citrate 24.6553122 58 Chromium picolinate 24.0064882 59 GLYCEROL MOLECULAR BIOLOGY REAGENT 23.6167425 60 Glucosamine Hydrochloride 23.4064125 61 Bromelain from pineapple stem 23.00595 62 Stearidonic acid 22.77165 63 MERCURY(II) CHLORIDE 22.68544 64 CHITIN 21.06117 65 DL-6,8-Thioctic Acid 20.3160271 66 Pumpkin seed oil 20.0438 67 alpha-lipoic acid 20.016425 68 Erucic acid 19.77587 69 L-Serine 18.7581699 70 Dipotassium Phosphate 17.7293935 71 Tricalcium phosphate hydrate 17.3630455 72 L-a-Phosphatidylcholine 17.214025 73 CHOLINE CHLORIDE 15.6995125 74 Flax seed oil 14.847725 75 L-Cystine 14.1327623 76 Zcaxanthin 14.04087 77 PECTIN 13.74691 78 MELATONIN CRYSTALLINE/melatonin 13.6300325 79 Folic Acid 13.5871247 80 HISTAMINE FREE BASE CRYSTALLINE 13.58368 81 PYRUVIC ACID 13.110525 82 SODIUM SULFATE 12.88661 83 ZINC SULFATE HEPTAHYDRATE 12.3672725 84 NICOTINIC ACID 12.242275 85 L-Lysine hydrochloride 11.2866817 86 Myristoleic acid 11.07895 87 Astaxanthin 10.193495 88 SODIUM FLUORIDE 9.5875875 89 SODIUM BICARBONATE 9.45016 90 Zinc Citrate 9.37790158 91 OXALIC ACID SODIUM SALT 7.9325475 92 D-(+)-GLUCOSAMINE HYDROCHLORIDE 7.3703925 93 Copper Gluconate 7.21816707 94 ALBUMIN HUMAN 6.7961275 95 3,3,5-TRIIODO-L-THYRONINE SODIUM SALT/Liothyronin 5.58025 96 d-Biotin 5.04487775 97 D-GLUCURONIC ACID 3.553765 98 Ferrous Fumarate 2.95489891 99 Caffeic acid phenethyl ester 2.439025 100 Lutein 2.386235 101 Magnesium citrate 2.33281493 102 Gelatin 1.98878123 103 L-(+)-ERGOTHIONEINE INNER SALT 1.8080625 104 D-glucosamine 3-sulfate 1.38734739 105 Thiamine Hydrochloride 0.72234763 106 A-CRYPTOXANTHIN 0.706805 107 Hyaluronic acid sodium salt 0.61193269 108 TAURINE 0.6013675 109 L-Proline 0.32679739 110 Carcinoembryonic Antigen (CEA) 0.3876075 111 Niacinamide 0.5261529 112 Curcumin 1.8868075 113 L-Leucine 2.6636569 114 VITAMIN K1 3.1362375 115 TRANS-B-CAROTENE, TYPE I,/all trans B carotene 3.187675 116 Capsaicin 3.4406215 117 L-glutathione 4.0510575 118 Potassium phosphate dibasic 4.4995264 119 Calcium Carbonate 5.3527981 120 ARG8-VASOPRESSIN ACETATE SALT/Vasopressin 5.4185025 121 ALPHA 1 ANTITRYPSIN, HUMAN PLASMA 5.4185175 122 PROINSULIN C-PEPTIDE (55-89)/c-peptide 5.533555 123 ALCOHOL 5.9393525 124 CAFFEINE 6.2160531 125 Sphingomyelin 6.3420275 126 D-LACTOSE MONOHYDRATE 6.5769806 127 LEPTIN 6.6005475 128 A-CAROTENE 7.3985325 129 PYRIMIDINE 7.9799475 130 LITHIUM ACETOACETATE/Acetoacetic acid 8.413135 131 ANDROSTENEDIONE 8.442685 132 LUTEINIZING HORMONE RELEASING HORMONE HUMAN 8.4632925 133 L-Phenylalanine 8.7437545 134 THIAMINE PYROPHOSPHATE 9.50731 135 ESTRIOL 10.313901 136 L-ALANINE 10.442568 137 L-Asparagine 12.810458 138 PHYTANIC ACID 12.916098 139 SUCROSE 13.144325 140 BoswelliaSerrata Extract 13.71128 141 TETRAETHYLAMMONIUM HYDROGEN SULFATE, FOR IPC 14.050822 142 Zinc Picolinate 14.856082 143 O-METHYLGUANINE 16.535908 144 UREA 16.838108 145 Zinc Oxide 16.867843 146 L-Glutamic Acid 17.581699 147 FLAVIN ADENINE DINUCLEOTIDE DISODIUM SALT HYDR 17.863335 148 Potassium Citrate 17.950742 149 ADENOSINE 3:5-CYCLIC MONOPHOSPHATE 17.999503 150 cis-Vaccenic acid 18.40491 151 copper sulfate 19.545823 152 Lycopene 19.700335 153 50% deoxycholic acid sodium salt + 50% cholic acid sodium 19.888333 154 URIC ACID 20.628048 155 L-Valine 20.770878 156 HYALURONIC ACID SODIUM SALT 21.593205 157 strontium chloride 23.150727 158 CADMIUM CHLORIDE/cadmium 23.318386 159 L-CYSTEINE 23.886633 160 CHOLESTEROL 24.097855 161 BIOTIN, POWDER, 24.341195 162 SODIUM BROMIDE 24.920693 163 XANTHOPHYLL FROM MARIGOLD/xanthophyll (Lutein) 24.981428 164 Collagen type II 25.471698 165 phenolics 25.522693 166 Sodium Molybdate 25.528892 167 URACIL 26.983125 168 N-acetyl D-glucosamine 27.205507 169 Docosahexaenoic Acid (DHA) 27.371385 170 L-Tyrosine 27.694504 171 Nicotinic Acid 27.82182 172 5ALPHA-DIHYDROTESTOSTERONE (DHT)/DHT 28.076258 173 Potassium sorbate 28.702242 174 17A-HYDROXYPROGESTERONE 28.907664 175 L(+)LACTIC ACID SODIUM 29.30591 176 Coconut oil 29.68382 177 Manganese sulfate 29.860031 178 (+)-DELTA-TOCOPHEROL 31.643836 179 ANDROSTERONE VETRANAL,/androsterone 32.054795 180 Dicalcium Phosphate 32.927818 181 PYRIDOXINE 33.17653 182 Folic Acid 33.308168 183 Eicosatrienoic Acid 33.55307 184 Rutoside trihydrate 33.860275 185 CORTISONE 34.650788 186 ESTRONE 34.828102 187 CYTOSINE 34.931507 188 L-Arginine 34.939329 189 Arachidonic acid 35.913165 190 ERGOCALCIFEROL 35.92233 191 L-GLUTATHIONE REDUCED 36.349615 192 NOREPINEPHRINE 37.505568 193 3-HYDROXYBUTYRIC ACID 38.354755 194 L-THYROXINE 38.356164 195 MAGNESIUM CHLORIDE ANHYDROUS 38.600875 196 VASOACTIVE INTESTINAL PEPTIDE 38.677018 197 LEAD(II) SULFIDE 39.312407 198 SULFUR/sulfide 39.760837 199 Methylsulfonylmethane 40.489546 200 GLYCOGEN 40.51758 201 ADENOSINE FREE BASE 41.491003 202 Potassium Gluconate 41.506157 203 L-Alanine 43.361884 204 POTASSIUM CHLORIDE/potassium 43.638333 205 Taurine 44.212137 206 L-Aspartic Acid 45.503212 207 Resveratrol 46.175421 208 L-Histidine hydrochloride 46.680942 209 5-HYDROXYTRYPTAMINE HYDROCHLORIDE/Serotonin 47.013163 210 PURINE 47.533632 211 L-ORNITHINE MONOHYDROCHLORIDE 48.433615 212 D-PANTOTHENIC ACID HEMICALCIUM 48.46669 213 6-gingerol 49.505603 214 Adenosine 5-Triphosphate Disodium 49.528125 215 SPHINGOMYELIN/SM(d18:1/16:0) 49.663608 216 L-A-PHOSPHATIDYLETHANOLAMINE TYPE I 49.979783 217 Silicic acid 50.078939 218 2-DEOXY-L-RIBOSE/Deoxycytidine 50.556865 219 CHOLECYSTOKININ FRAGMENT 26-33 AMIDE NON SULF 50.691865 220 ZINC CHLORIDE 51.16857 221 ()-EPINEPHRINE 51.595008 222 Ubiquinol 52.052658 223 L-ASCORBIC ACID BIOXTRA, 52.461463 224 THIAMINE HYDROCHLORIDE 52.66037 225 Chondroitin sulfate sodium 52.663708 226 CHORIONIC GONADOTROPIN HUMAN, 53.740398 227 DL-MALIC ACID 54.898063 228 Quercetin 55.667115 229 Trimethylglycine 55.823293 230 Myristic Acid 55.849253 231 (+/)-A-TOCOPHEROL/Alpha-Tocopherol 56.143958 232 11-DEOXYCORTISOL/Cortexolone 56.715753 233 C-PEPTIDE FRAGMENT 3-33, HUMAN/C-peptide 56.901788 234 Niacinamide 56.92714 235 Sodium citrate 60.767288 236 Crucuminoids 61.653718 237 SUCCINIC ACID 65.833855 238 FOLIC ACID, 65.962583 239 LIPASE FROM PORCINE PANCREAS 66.02044 240 Pterostibene 66.845158 241 CHONDROITIN SULFATE, A SODIUM SALT 67.16166 242 L-a-Phosphatidylcholine 67.469877 243 (+)-GAMMA-TOCOPHEROL 68.16921 244 OXYTOCIN 68.747893 245 MYO-INOSITOL 68.771755 246 BETAINE 69.206745 247 PHENOL 71.08183 248 CHOLECALCIFEROL CRYSTALLINE 71.751605 249 linoleic acid 73.48477 250 4-AMINOBENZOIC ACID 73.578365 251 3-HYDROXYTYRAMINE HCL/dopamine 73.994453 252 PREGNENOLONE ACETATE/pregnenolone 76.946288 253 MANGANESE SULFATE MONOHYDRATE, 79.462953 254 Thymoquinone 82.307006 255 Palmitoleic acid 82.70231 256 Stearic Acid 85.813338 257 Palmitic Acid 86.33945 258 Gamma linolenic acid 94.207538
TABLE-US-00006 TABLE 5 Mouse T cell IL-2 Mouse CD4.sup.+ T cell IL-2 Mouse CD8.sup.+ T cell IL-2 level changes after level changes after level changes after Candidate name treatment treatment treatment Trans-Vaccenic Acid 183.5% 155.6% 193.6% Thymidine 145.6% 135.8% 128.9% D-Glycerate2,3- 106.2% 97.9% 121.4% diphosphate sodium salt p-Aminobenzoic Acid 160.3% 140.5% 145.2% D(+)-galactose anhydrous 130.1% 110.4% 130.3% GUANINE 148.6% 110.4% 150.8%
TABLE-US-00007 TABLE 6 Relative Pixel Density (%) Log10(P-value) p-HSP27(S78/S82) 120.689435 0.316964977 p-CREB(S133) 111.6374 1.702018135 p-EGFR(Y1086) 109.55535 0.733795502 p-STATI(Y701) 109.29665 0.533230814 p-GSK-3?/?(S21/S9) 104.3625 0.670840896 p-STAT6(Y641) 104.173575 0.148230402 HSP60 99.96418 0.007185034 ?-Catenin 98.64661 0.288927992 p-STAT5a/b(Y694/Y699) 97.09414 0.591725263 p-Yes(Y426) 92.31604 1.066073081 p-PDGF R?(Y751) 91.23745 1.345515631 p-Lck(Y394) 89.85713 0.814107011 p-MSK1/2(S376/S360) 89.695615 1.637991908 p-STAT3(Y705) 89.61953 1.328125213 p-PLC-?1(Y783) 89.1635 0.89090845 p-STAT2(Y689) 88.72418 0.715027718 p-GSK-3?(S9) 87.430175 1.297616719 p-Fgr(Y412) 87.28216 1.724642219 p-Lyn(Y397) 87.24125 1.079569568 p-PYK2(Y402) 86.97589 1.477158775 p-Src(Y419) 85.88792 0.841504158 p-eNOS(S1177) 84.26072 1.245335838 p-Akt1/2/3(S473) 83.05442 1.193659351 p-Chk-2(T68) 80.693955 1.043199032 p-p70 S6 Kinase(T421/S424) 80.33929 2.061011185 p-p38?(T180/Y182) 79.60737 0.723801794 p-p53(S15) 79.533465 2.040065894 p-STAT3(S727) 79.499035 1.433119205 p-ERK1/2(T202/Y204 T185/Y187) 79.269935 2.250374315 p-JNK1/2/3(T183/Y185T221/Y223) 78.521155 2.28989768 p-p53(S46) 78.5116 1.501913129 p-WNK1(T60) 78.342385 1.379441613 p-Akt1/2/3(T308) 77.687955 0.826150604 p-p70 S6 Kinase(T389) 77.295155 2.001311438 p-p53(S392) 75.85709 1.231509636 p-PRAS40(T246) 72.29019 2.473862729 p-c-Jun(S63) 70.59788 1.84147218 p-RSK1/2(S221/S227) 69.16812 2.45867683 p-RSK1/2/3(S380/S386/S377) 64.02901 1.241087258
TABLE-US-00008 TABLE 7 Table 7: DEGs only in NTC + TVA group gene.sub. gene.sub. gene.sub. gene.sub. gene.sub. gene.sub. gene.sub. gene.sub. tf.sub. gene_id
FC log2FoldCha pvalue name chr start end strand length biotype descrip family ENSMUSG0
0
17
+ 8514 protein_codin
ENSMUSG0
0
5
+ 1807 protein_codin
ENSMUSG0
0
1
777 protein_codin
ENSMUSG0
0
9
+ 1031 protein_codin
ENSMUSG0
0
11
+ 4287 protein_codin
ENSMUSG0
0
1
2759 protein_codin
ENSMUSG0
0
4
13978 protein_codin
ENSMUSG0
0
7
6120 protein_codin
ENSMUSG0
0
1
+ 2012 protein_codin
ENSMUSG0
0
15
2435 protein_codin
ENSMUSG0
9
3026 protein_codin
ENSMUSG0
6
+ 3335 protein_codin
ENSMUSG0
7
+ 2758 protein_codin
ENSMUSG0
X
+
protein_codin
ENSMUSG0
2
+ 4194 protein_codin
ENSMUSG0
5
+ 10815 protein_codin
ENSMUSG0
13
485 protein_codin
ENSMUSG0
1
+ 5313 protein_codin
ENSMUSG0
15
2728 protein_codin
ENSMUSG0
X
4165 protein_codin
ENSMUSG0
9
1592 protein_codin
ENSMUSG0
17
3872 protein_codin
ENSMUSG0
2
+ 1873 protein_codin
ENSMUSG0
17
+ 1154 protein_codin
ENSMUSG0
5
5593 protein_codin
ENSMUSG0
1
8467 protein_codin
ENSMUSG0
12
+ 1832 protein_codin
ENSMUSG0
10
protein_codin
ENSMUSG0
7
+ 3747 protein_codin
ENSMUSG0
14
+ 5506 protein_codin
ENSMUSG0
11
2089 protein_codin
ENSMUSG0
13
2564 protein_codin
ENSMUSG0
15
5170 protein_codin
ENSMUSG0
11
+ 2146 protein_codin
ENSMUSG0
19
864 protein_codin
ENSMUSG0
5
+ 1037 protein_codin
ENSMUSG0
1
+ 16449 protein_codin
ENSMUSG0
1
742 protein_codin
ENSMUSG0
13
+ 4936 protein_codin
ENSMUSG0
18
+ 2837 protein_codin
ENSMUSG0
14
+ 2413 protein_codin
ENSMUSG0
17
+
protein_codin
ENSMUSG0
17
1418 protein_codin
ENSMUSG0
X
+ 1255 protein_codin
ENSMUSG0
7
2204 protein_codin
ENSMUSG0
1
+
422 protein_codin
ENSMUSG0
4
+ 1275 protein_codin
ENSMUSG0
16
+ 9141 protein_codin
ENSMUSG0
7
+ 5579 protein_codin
ENSMUSG0
13
5773 protein_codin
ENSMUSG0
7
4199 protein_codin
zf- C2H2 ENSMUSG0
7
+ 1164 protein_codin
ENSMUSG0
10
+ 3465 protein_codin
ENSMUSG0
8
4141 protein_codin
ENSMUSG0
2
177
protein_codin
ENSMUSG0
8
+ 5528 protein_codin
ENSMUSG0
X
2106 protein_codin
ENSMUSG0
5
5850 protein_codin
ENSMUSG0
19
+ 2778 protein_codin
ENSMUSG0
9
2000 protein_codin
ENSMUSG0
19
6384 protein_codin
ENSMUSG0
5
+ 6433 protein_codin
ENSMUSG0
4
+ 4924 protein_codin
ENSMUSG0
2
6679 protein_codin
ENSMUSG0
5
5216 protein_codin
ENSMUSG0
2
+ 6558 protein_codin
ENSMUSG0
16
+ 3213 protein_codin
ENSMUSG0
7
3455 protein_codin
ENSMUSG0
9
3641 protein_codin
ENSMUSG0
17
386 protein_codin
ENSMUSG0
10
3594 protein_codin
ENSMUSG0
1
4195 protein_codin
ENSMUSG0
10
6016 protein_codin
ENSMUSG0
15
1617 protein_codin
ENSMUSG0
2
+ 1000 protein_codin
ENSMUSG0
2
+ 9128 protein_codin
ENSMUSG0
9
+ 5392 protein_codin
ENSMUSG0
2
5039 protein_codin
ENSMUSG0
11
3460 protein_codin
ENSMUSG0
15
+ 2742 protein_codin
ENSMUSG0
7
7170 protein_codin
ENSMUSG0
2
+ 9832 protein_codin
ENSMUSG0
2
2531 protein_codin
ENSMUSG0
2
2400 protein_codin
ENSMUSG0
11
2762 protein_codin
ENSMUSG0
6
+ 3020 protein_codin
ENSMUSG0
2
546
protein_codin
ENSMUSG0
3
12692 protein_codin
ENSMUSG0
11
5965 protein_codin
ENSMUSG0
4097 protein_codin
ENSMUSG0
3
+ 9831 protein_codin
ENSMUSG0
1
9079 protein_codin
ENSMUSG0
7
676 protein_codin
ENSMUSG0
19
2053 protein_codin
ENSMUSG0
5
3611 protein_codin
ENSMUSG0
9
+ 3512 protein_codin
ENSMUSG0
6
1720 protein_codin
ENSMUSG0
3
1126 protein_codin
ENSMUSG0
11
+ 4257 protein_codin
ENSMUSG0
7
7342 protein_codin
ENSMUSG0
8
2043 protein_codin
ENSMUSG0
2
+ 1893 protein_codin
ENSMUSG0
13
+ 2231 protein_codin
ENSMUSG0
11
+ 6797 protein_codin
ENSMUSG0
3
+ 4200 protein_codin
ENSMUSG0
5
947 protein_codin
ENSMUSG0
18
+ 837 protein_codin
ENSMUSG0
X
+ 7996 protein_codin
ENSMUSG0
2
+ 3991 protein_codin
ENSMUSG0
13
6180 protein_codin
ENSMUSG0
13
11223 protein_codin
ENSMUSG0
17
2283 protein_codin
ENSMUSG0
4
4683 protein_codin
ENSMUSG0
6
5796 protein_codin
ENSMUSG0
19
1402 protein_codin
ENSMUSG0
8
+ 3140 protein_codin
ENSMUSG0
17
+ 4209 protein_codin
zf- C2H2 ENSMUSG0
12
14093 protein_codin
ENSMUSG0
17
+
protein_codin
ENSMUSG0
9
6148 protein_codin
ENSMUSG0
4
1441 protein_codin
ENSMUSG0
7
4601 protein_codin
ENSMUSG0
19
2548 protein_codin
ENSMUSG0
X
1934 protein_codin
ENSMUSG0
1
2878 protein_codin
ENSMUSG0
2
1463 protein_codin
ENSMUSG0
9
+ 6540 protein_codin
ENSMUSG0
14
+
637 protein_codin
ENSMUSG0
14
1623 protein_codin
ENSMUSG0
7
1716 protein_codin
ENSMUSG0
13
3968 protein_codin
zf- C2H2 ENSMUSG0
19
protein_codin
ENSMUSG0
6
5280 protein_codin
MBD ENSMUSG0
13
+ 3350 protein_codin
ENSMUSG0
16
5223 protein_codin
ENSMUSG0
11
4461 protein_codin
ENSMUSG0
7
5141 protein_codin
ENSMUSG0
2
9154 protein_codin
ENSMUSG0
14
2378 protein_codin
ENSMUSG0
1
8287 protein_codin
ENSMUSG0
15
4908 protein_codin
ENSMUSG0
17
3530 protein_codin
ENSMUSG0
2
+ 2820 protein_codin
ENSMUSG0
13
5600 protein_codin
ENSMUSG0
13
+ 692 protein_codin
ENSMUSG0
17
2163 protein_codin
ENSMUSG0
6
1136 protein_codin
ENSMUSG0
14
+ 6243 protein_codin
ENSMUSG0
17
8466 protein_codin
ENSMUSG0
18
+ 3
67 protein_codin
ENSMUSG0
X
+ 1289 protein_codin
ENSMUSG0
9
4645 protein_codin
ENSMUSG0
5
+
protein_codin
ENSMUSG0
17
+ 2076 protein_codin
ENSMUSG0
11
2873 protein_codin
ENSMUSG0
9
+ 5363 protein_codin
ENSMUSG0
7
5210 protein_codin
ENSMUSG0
5
5820 protein_codin
ENSMUSG0
1
+ 2921 protein_codin
ENSMUSG0
X
5437 protein_codin
ENSMUSG0
5
+ 6303 protein_codin
ENSMUSG0
X
+ 2785 protein_codin
ENSMUSG0
3
+ 7286 protein_codin
ENSMUSG0
2
1131 protein_codin
ENSMUSG0
1
5195 protein_codin
ENSMUSG0
16
+ 1503 protein_codin
ENSMUSG0
4
7857 protein_codin
SAND ENSMUSG0
6
6576 protein_codin
ENSMUSG0
9
+ 3731 protein_codin
ENSMUSG0
4
+ 1898 protein_codin
ENSMUSG0
8
+ 2709 protein_codin
ENSMUSG0
4
+ 2993 protein_codin
ENSMUSG0
15
6256 protein_codin
ENSMUSG0
8
+ 5227 protein_codin
HMG ENSMUSG0
5
+ 5860 protein_codin
ENSMUSG0
4
+ 17627 protein_codin
zf- C2H2 ENSMUSG0
14
+
526 protein_codin
ENSMUSG0
16
protein_codin
ENSMUSG0
9
+
protein_codin
ENSMUSG0
3
2344 protein_codin
ENSMUSG0
4
+
protein_codin
ENSMUSG0
11
2464 protein_codin
ENSMUSG0
17
4840 protein_codin
ENSMUSG0
14
2563 protein_codin
ENSMUSG0
2
+ 2330 protein_codin
ENSMUSG0
15
3738 protein_codin
ENSMUSG0
7
+ 4176 protein_codin
ENSMUSG0
14
4298 protein_codin
ENSMUSG0
X
+ 6098 protein_codin
ENSMUSG0
12
3931 protein_codin
ENSMUSG0
3
2366 protein_codin
ENSMUSG0
4
2766 protein_codin
ENSMUSG0
17
5950 protein_codin
ENSMUSG0
14
11570 protein_codin
ENSMUSG0
6
4171 protein_codin
ENSMUSG0
10
4091 protein_codin
ENSMUSG0
19
3798 protein_codin
ENSMUSG0
13
5553 protein_codin
ENSMUSG0
6
+ 2818 protein_codin
ENSMUSG0
12
+ 4599 protein_codin
ENSMUSG0
1
+ 8400 protein_codin
ZBTB ENSMUSG0
11
2488 protein_codin
T-box ENSMUSG0
13
5429 protein_codin
E2F ENSMUSG0
12
20676 protein_codin
ENSMUSG0
14
6015 protein_codin
ENSMUSG0
5
+ 2590 protein_codin
ENSMUSG0
1
2005 protein_codin
ENSMUSG0
2
+ 6195 protein_codin
ENSMUSG0
10
9028 protein_codin
ENSMUSG0
16
3221 protein_codin
ENSMUSG0
15
+ 4198 protein_codin
ENSMUSG0
11
+ 872 protein_codin
ENSMUSG0
X
419 protein_codin
ENSMUSG0
4
+ 5093 protein_codin
ENSMUSG0
11
6248 protein_codin
ENSMUSG0
4
3088 protein_codin
ENSMUSG0
13
21824 protein_codin
ENSMUSG0
4
2040 protein_codin
ENSMUSG0
8
5253 protein_codin
ENSMUSG0
2
1032 protein_codin
ENSMUSG0
5
+ 13135 protein_codin
ENSMUSG0
11
3454 protein_codin
ENSMUSG0
2
+ 2703 protein_codin
ENSMUSG0
7
2684 protein_codin
ENSMUSG0
2
2088 protein_codin
ENSMUSG0
10
3850 protein_codin
ENSMUSG0
17
7315 protein_codin
NF-YA ENSMUSG0
2
+ 3728 protein_codin
ENSMUSG0
2
+ 5707 protein_codin
ENSMUSG0
14
5039 protein_codin
ENSMUSG0
11
3420 protein_codin
ENSMUSG0
10
3210 protein_codin
ENSMUSG0
11
1633 protein_codin
ENSMUSG0
10
+ 1740 protein_codin
ENSMUSG0
5
+ 3709 protein_codin
ENSMUSG0
12
1518 protein_codin
ENSMUSG0
18
+ 3698 protein_codin
ENSMUSG0
12
5080 protein_codin
ENSMUSG0
5
9664 protein_codin
ENSMUSG0
X
5570 protein_codin
ENSMUSG0
17
+ 1799 protein_codin
ENSMUSG0
19
2813 protein_codin
ENSMUSG0
18
+ 4257 protein_codin
ENSMUSG0
13
1131 protein_codin
ENSMUSG0
7
3828 protein_codin
ENSMUSG0
15
+ 2287 protein_codin
ENSMUSG0
15
2731 protein_codin
ENSMUSG0
8
1178 protein_codin
ENSMUSG0
39
+ 1751 protein_codin
ENSMUSG0
4
+ 4500 protein_codin
ENSMUSG0
17
1467 protein_codin
ENSMUSG0
9
+ 836 protein_codin
ENSMUSG0
5
7721 protein_codin
ENSMUSG0
12
7590 protein_codin
ENSMUSG0
10
+ 7267 protein_codin
ENSMUSG0
7
+ 2854 protein_codin
ENSMUSG0
3
+ 8580 protein_codin
ENSMUSG0
16
+ 3970 protein_codin
ENSMUSG0
4
+ 5314 protein_codin
ENSMUSG0
2
+ 5373 protein_codin
ENSMUSG0
6
5347 protein_codin
ENSMUSG0
4
8744 protein_codin
ENSMUSG0
3
+ 6008 protein_codin
ENSMUSG0
14
+ 2377 protein_codin
ENSMUSG0
10
863 protein_codin
ENSMUSG0
9
+ 3077 protein_codin
ENSMUSG0
15
3567 protein_codin
ENSMUSG0
1
3392 protein_codin
ENSMUSG0
X
+ 6851 protein_codin
ENSMUSG0
19
820 protein_codin
ENSMUSG0
X
+ 2517 protein_codin
ENSMUSG0
6
2509 protein_codin
ENSMUSG0
11
+ 3332 protein_codin
ENSMUSG0
4
+ 770 protein_codin
ENSMUSG0
9
+ 3827 protein_codin
ENSMUSG0
19
5030 protein_codin
ENSMUSG0
17
7060 protein_codin
ENSMUSG0
12
+ 3299 protein_codin
ENSMUSG0
2
+ 8490 protein_codin
ENSMUSG0
7
+ 11369 protein_codin
ENSMUSG0
12
1717 protein_codin
ENSMUSG0
9
+ 6378 protein_codin
ENSMUSG0
6
+ 5992 protein_codin
ENSMUSG0
17
+ 8333 protein_codin
ENSMUSG0
6
+ 12640 protein_codin
ENSMUSG0
10
5011 protein_codin
ENSMUSG0
3
+ 8105 protein_codin
ENSMUSG0
10
+ 4435 protein_codin
ENSMUSG0
3
4333 protein_codin
ENSMUSG0
11
+ 1872 protein_codin
ENSMUSG0
14
7734 protein_codin
ENSMUSG0
2
3463 protein_codin
ENSMUSG0
9
+ 7786 protein_codin
ENSMUSG0
16
3130 protein_codin
ENSMUSG0
4
+ 2140 protein_codin
ENSMUSG0
15
1675 protein_codin
ENSMUSG0
14
+ 3498 protein_codin
ENSMUSG0
12
+ 5732 protein_codin
ENSMUSG0
1
+ 3826 protein_codin
ENSMUSG0
4
+ 5324 protein_codin
ENSMUSG0
1
+ 7265 protein_codin
ENSMUSG0
6
+ 4619 protein_codin
ENSMUSG0
13
5771 protein_codin
ENSMUSG0
17
8173 protein_codin
ENSMUSG0
12
+ 3261 protein_codin
ENSMUSG0
13
6393 protein_codin
ENSMUSG0
8
14828 protein_codin
ENSMUSG0
1
+ 4817 protein_codin
ENSMUSG0
17
2147 protein_codin
ENSMUSG0
3
4172 protein_codin
ENSMUSG0
3
7423 protein_codin
ENSMUSG0
6
+ 4663 protein_codin
ENSMUSG0
8
+ 7018 protein_codin
ENSMUSG0
11
7017 protein_codin
ENSMUSG0
4
6447 protein_codin
ENSMUSG0
7
3280 protein_codin
ENSMUSG0
6
3943 protein_codin
ENSMUSG0
11
+ 6868 protein_codin
ENSMUSG0
14
10124 protein_codin
ENSMUSG0
14
+ 6502 protein_codin
ENSMUSG0
15
+ 8106 protein_codin
ENSMUSG0
4
10710 protein_codin
ZBTB ENSMUSG0
11
4698 protein_codin
ENSMUSG0
11
4595 protein_codin
ENSMUSG0
15
+ 2414 protein_codin
ENSMUSG0
11
6463 protein_codin
ENSMUSG0
10
8933 protein_codin
MYB ENSMUSG0
11
+ 13896 protein_codin
ENSMUSG0
1
4515 protein_codin
ENSMUSG0
7
+ 2800 protein_codin
ENSMUSG0
5
+ 5505 protein_codin
ENSMUSG0
1
6460 protein_codin
ENSMUSG0
4
1334 protein_codin
THAP ENSMUSG0
15
23395 protein_codin
ENSMUSG0
2
+ 8561 protein_codin
ENSMUSG0
2
+ 9807 protein_codin
zf- C2H2 ENSMUSG0
13
+ 16704 protein_codin
ENSMUSG0
3
+ 6971 protein_codin
ENSMUSG0
12
10049 protein_codin
ENSMUSG0
11
+ 3056 protein_codin
ENSMUSG0
11
+ 5631 protein_codin
ENSMUSG0
5
protein_codin
zf- C2H2 ENSMUSG0
5
+ 8541 protein_codin
zf- C2H2 ENSMUSG0
9
2578 protein_codin
ENSMUSG0
2
+ 8451 protein_codin
ENSMUSG0
9
+ 2458 protein_codin
ENSMUSG0
8
+ 9698 protein_codin
ENSMUSG0
10
5949 protein_codin
ENSMUSG0
5
+ 4319 protein_codin
ENSMUSG0
8
9131 protein_codin
ENSMUSG0
15
+ 2067 protein_codin
ENSMUSG0
12
2715 protein_codin
ENSMUSG0
19
4640 protein_codin
ENSMUSG0
6
+ 6449 protein_codin
ENSMUSG0
7
+ 2592 protein_codin
ENSMUSG0
17
+ 3903 protein_codin
ENSMUSG0
4
2875 protein_codin
ENSMUSG0
13
12106 protein_codin
ENSMUSG0
9
5619 protein_codin
ENSMUSG0
13
4322 protein_codin
ENSMUSG0
11
+ 5626 protein_codin
ENSMUSG0
2
+ 7496 protein_codin
ENSMUSG0
8
4220 protein_codin
ENSMUSG0
8
3490 protein_codin
ENSMUSG0
31
7038 protein_codin
ENSMUSG0
33
+ 5395 protein_codin
ENSMUSG0
11
5968 protein_codin
ENSMUSG0
11
3329 protein_codin
ENSMUSG0
18
5101 protein_codin
HMG ENSMUSG0
9
4538 protein_codin
ENSMUSG0
17
2075 protein_codin
ENSMUSG0
8
+ 5530 protein_codin
zf- C2H2 ENSMUSG0
33
+ 7308 protein_codin
zf- C2H2 ENSMUSG0
19
+ 1968 protein_codin
ENSMUSG0
2
5594 protein_codin
ENSMUSG0
X
+ 10048 protein_codin
ESR- like ENSMUSG0
13
8017 protein_codin
zf- C2H2 ENSMUSG0
18
15167 protein_codin
zf- C2H2 ENSMUSG0
7
5239 protein_codin
ENSMUSG0
1
+ 16555 protein_codin
ARID ENSMUSG0
9
+ 5845 protein_codin
ENSMUSG0
3967 protein_codin
ENSMUSG0
8
+ 4108 protein_codin
ENSMUSG0
X
+ 3485 protein_codin
Fork ENSMUSG0
5
+ 4937 protein_codin
ENSMUSG0
15
+ 3932 protein_codin
ENSMUSG0
X
3891 protein_codin
ENSMUSG0
6
+ 5691 protein_codin
ENSMUSG0
8
+ 1824 protein_codin
ENSMUSG0
6
6022 protein_codin
ENSMUSG0
18
5473 protein_codin
ENSMUSG0
8
1498 protein_codin
ENSMUSG0
3
+ 5946 protein_codin
ENSMUSG0
2
2066 protein_codin
ENSMUSG0
6
4740 protein_codin
ENSMUSG0
X
+ 3330 protein_codin
ENSMUSG0
7
2734 protein_codin
zf- C2H2 ENSMUSG0
10
+ 7878 protein_codin
ENSMUSG0
2
+ 2604 protein_codin
ENSMUSG0
4
2882 protein_codin
ENSMUSG0
6
+ 5041 protein_codin
ENSMUSG0
15
+ 2055 protein_codin
ENSMUSG0
16
7361 protein_codin
ENSMUSG0
4144 protein_codin
ENSMUSG0
X
2820 protein_codin
ENSMUSG0
1
6458 protein_codin
ENSMUSG0
7
+ 7556 protein_codin
ENSMUSG0
18
3990 protein_codin
ENSMUSG0
4
2768 protein_codin
ENSMUSG0
5
8305 protein_codin
ZBTB ENSMUSG0
4
+ 5999 protein_codin
ENSMUSG0
5
+ 7025 protein_codin
ENSMUSG0
16
12996 protein_codin
ENSMUSG0
9
+ 1543 protein_codin
ENSMUSG0
8
+ 6153 protein_codin
ENSMUSG0
9
11152 protein_codin
ENSMUSG0
18
3520 protein_codin
ENSMUSG0
12
2583 protein_codin
ENSMUSG0
4
+ 6216 protein_codin
ENSMUSG0
7
+ 1304 protein_codin
T-box ENSMUSG0
4
3516 protein_codin
ENSMUSG0
17
1407 protein_codin
ENSMUSG0
16
6700 protein_codin
ZBTB ENSMUSG0
19
+ 4209 protein_codin
ENSMUSG0
15
4662 protein_codin
ENSMUSG0
4
8487 protein_codin
zf- C2H2 ENSMUSG0
10
2789 protein_codin
ENSMUSG0
X
1195 protein_codin
ENSMUSG0
11
4847 protein_codin
ENSMUSG0
6
+ 3419 protein_codin
ENSMUSG0
7
+ 2497 protein_codin
ENSMUSG0
11
1732 protein_codin
ENSMUSG0
3
+ 8815 protein_codin
ENSMUSG0
15
4740 protein_codin
ENSMUSG0
9
1582 protein_codin
ENSMUSG0
12
6895 protein_codin
ENSMUSG0
13
3682 protein_codin
ENSMUSG0
11
+ 3626 protein_codin
T-box ENSMUSG0
17
5505 protein_codin
ENSMUSG0
8
+ 2674 protein_codin
ENSMUSG0
7
+ 6078 protein_codin
zf- C2H2 ENSMUSG0
2
3723 protein_codin
ENSMUSG0 0
4
+ 3609 protein_codin
ENSMUSG0 0
11
5149 protein_codin
ENSMUSG0 0
2
+ 7639 protein_codin
ENSMUSG0 0
6
6014 protein_codin
Homeo- box ENSMUSG0 0
2
+ 2580 protein_codin
ENSMUSG0
5
+ 10604 protein_codin
ENSMUSG0 0
X
468 protein_codin
ENSMUSG0 0
17
5703 protein_codin
ENSMUSG0 0
X
627 protein_codin
indicates data missing or illegible when filed
TABLE-US-00009 TABLE 8 Cellular Therapy Serum Biobank ID Sample collection time point volume(uL) Response Treatment 18-0025-035, RF CR Tisagenlecleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 2 800 d + 14 800 d + 21 800 18-0025-033, PB CR Axicabtagene Ciloleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 2 800 d + 14 800 d + 28 800 18-0025-031, EG CR Axicabtagene Ciloleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 4 800 d + 14 800 d + 30 800 18-0025-049, MC CR Axicabtagene Ciloleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 2 800 d + 12 800 d + 28 800 18-0025-086, LT CR Tisagenlecleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 6 800 d + 14 800 d + 28 800 18-0025-032, LB PD Axicabtagene Ciloleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 4 800 d + 10 800 d + 21 800 18-0025-051, MH PD Tisagenlecleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 4 800 d + 10 800 d + 28 800 18-0025-075, KY PD Tisagenlecleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 2 800 d + 10 800 d + 28 800 18-0025-112, ND PD Axicabtagene Ciloleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 4 800 d + 14 800 d + 28 800 18-0025-122, TM Suspected Lisocaptagene maraleucel Immediately prior to start of LD chemo (i.e. Day 5) 800 d + 4 800 d + 14 800 d + 28 800