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Definitions

• APCs antigen-presenting cells
• DCs dendritic cells
• cytotoxic T lymphocytes CTLs
• CTLs capable of killing infected or tumor cells expressing their target antigen.
• signals activate CD4 T cells, inducing their expansion and differentiation into Thl or Th2 T helper cells (3) .
• cancer immunotherapy One of the most important targets of improved adjuvant technology lies in the field of cancer immunotherapy, where the adaptive immune system is exploited to kill cancer cells based on their expression of cancer associated antigens or neo-antigens (4, 5) .
• the effectiveness of cancer immunotherapy depends on the generation and
• immune checkpoint inhibitors such as anti-PD-1, anti-PD-Ll and anti-CTLA-4 have achieved remarkable clinical success in the treatment of melanoma and other cancers through their action in blocking pathways that inhibit CTL activation (7, 8).
• T cell support is thought to be at least in part responsible for the lack of vigor exhibited by many synthetic vaccines against various pathogens as well as cancer-related cell surface antigens .
• TLR ligands have long been known to act as adjuvants in adaptive immune responses ⁇ 10, 11) signaling via adapter proteins (MyD88, TRIF, TRAM, MAL) , kinases, and ubiquitin ligases to activate NF- KB and IRFs (12-14) . These and other transcription factors induce the expression of thousands of genes that carry out the innate immune response (15) .
• nucleotide-based adjuvants such as TLR3 agonist poly I:C (9), TLR9 agonist CpG (16), and STING agonist cGAMP (6) have been reported to improve the efficacy of immune checkpoint inhibitors in pre- clinical models for cancer treatment. These approaches aim to increase the number of tumor-specific CTLs upon which checkpoint
• TLR ligands which are difficult to synthesize, and in some instances quite toxic, presumably because they become 'widely disseminated in vivo, and activate myeloid cells indiscriminately, producing cytokine storm (17) .
• Diprovocim illustratively elicits strong adjuvant activity in mice, successfully inhibiting tumor growth and prolonging survival when combined with a cancer antigen and immune checkpoint blockade in the B16-OVA melanoma model.
• Adjuvants enhance adaptive immune responses, sometimes through unknown mechanisms, and can be used to augment both humoral and cellular responses to cancer antigens.
• the present invention thus contemplates a method of inhibiting the growth of diseased cells in a mammal.
• Those diseased cells express a marker molecule that is absent from cells of the same type that are free of the disease or is present in the disease-free cells in significantly reduced numbers compared to said diseased cells.
• a contemplated method comprises the steps of immunizing a diseased mammal in need by: a) administering to the diseased mammal (i) an adjuvant-sufficient amount of a
• Diprovocim compound (ii) a T cell-stimulating amount of an immune checkpoint inhibitor, and (iii) an immunizing amount of a disease marker molecule.
• the immunized mammal is b) maintained for a time period sufficient for the mammal to mount an immune response to the immunization and inhibit growth of diseased cells .
• a contemplated Diprovocim compound corresponds in structure to structural Formula V,
• -A is -H (hydrido) or -C ⁇ 0)NH-R ⁇ ;
• R ⁇ , R ⁇ , R3 are the same or different and are a 2- ( 4-fluorophenyl) ethyl, a trans-2- phenylcyclopropyl, a trans-2- (4-fluorophenyl) - cyclopropyl or a C 2 -C ] _g hydrocarbyl group with the provisos that:
• pyrrolidinyldicarboxamido group has the (S,S) configuration, and each depicted R substituent other than a Cg-Cpg hydrocarbyl group is a trans-2- phenylcyclopropyl , a trans-2- (4-fluorophenyl) - cyclopropyl group or a mixture thereof 'when each of P.l- j_g other than 2- (4-fluorophenyl) ethyl,
• R ⁇ _ 3 when A is hydrido, one of R ⁇ _ 3 can p e a Cf-Cig hydrocarbyl group and the depicted R ⁇ - containing pyrrolidinylcarboxamido group can have either the R or S configurations, or a mixture of both configurations;
• -Z is one or more of halogen -H, -NH 2 , -OH, -OCH3, -N0 2 , -0CH 2 C0 2 H, -0 (CH 2 CH 2 0) n CH 2 CH 2 C0 2 H, -0CH 2 C0NH (CH 2 CH 2 0) n CH 2 CH 2 C0 2 H, -NHC0CH 2 0- (CH 2 CH 2 0) n CH 2 C0 2 H, -OCHgCONHCHgCONHCH (CHOH) C0 2 H, -0CH 2 C0NHCH 2 C0NHCHC0 2 H (CH 2 C0 2 H) ,
• W is nitrogen (N) or CH;
• n is a number whose average value is one to about eight;
• m is a number whose value is 1 to about 6;
• p is a number whose value is 1 to about
• Diprovocim family As disclosed in WO 2018/005812 and Morin et al . , J Am Chem Soc, In Press (2016), several members of the Diprovocim family have been prepared, their activety 3ssaved, and have been given numbers such as Diprovocim-1 through Diprovocim-6. Several other compounds of the Diprovocim family having similar activity profiles, similar to somewhat lesser activity values in the assays used, have also been prepared and assayed.
• Diprovocim-1 has been used herein as an exemplary member of the whole family, and is to be understood hereinafter to mean a Diprovocim family member is used when the word Diprovocim is used without an added hyphenated numeral.
• Diprovocim when used in connection with the words "family" or "a” as in “Diprovocim family” or "a Diprovocim”, the word “Diprovocim” is used to mean a member of the
• Diprovocim family as defined by Formula V.
• the word "Diprovocim” used with the word “preferred” refers to a compound of Formula I
• the phrase “more preferred” or “more preferably” refers to a
• Diprovocim family compound of Formula la A "most preferred” Diprovocim is one of the compounds lettered A-I noted below.
• a preferred member of the Diprovocim family of compounds is a compound that corresponds in structure to structural Formula I , below, in which -A -C(0)NH-R ⁇ .
• the depicted W and Z moieties are as described above.
• Diprovocim family of compounds is a compound that corresponds in structure to structural Formula la, below,
• a Diprovocim is utilized in an adjuvant- sufficient amount to contact host mammal cells that express an antigenic disease-related marker molecule.
• the host mammal cells that express an antigenic disease-related marker compound such as a peptide sequence are also contacted with an immune response-stimulating amount of a checkpoint
• inhibitor preferably an antibody or paratope- containing antibody portion.
• Fig. 1A through Fig. IF provide a series of graphs that illustrate that Diprovocim induces cytokine secretion by mouse and human cells.
• Fig. 1A-1D illustrates amounts of TNF in the supernatants of human THP-1 cells ⁇ Fig. 1A) , human PBMC (Fig. IB), mouse peritoneal macrophages (Fig. 1C), or mouse BMDC (Fig. ID) after treatment with Diprovocim-1 for 4 hours (Fig. 1A, Figs. 1C-1D) or 24 hours (Fig. IB) .
• Fig. IE illustrates the amounts of IL-6 in the supernatants of mouse BMDC after treatment with Diprovocim for 4 hours.
• the means of three independent samples are plotted; P values were determined by one-way ANOVA to compare the responses to different doses; in all studies P ⁇ 0.0001. Results in Fig. 1A - Fig. IE are
• Fig. 2A through Fig. 2D illustrate that a Diprovocim activates mouse and human TLR1/TLR2.
• Fig. 2B is a graph showing the amount of TNF in the supernatants of human THP-1 cells pretreated with control antibody, anti-TLRl (20 g/rrtl) , or anti-TLR2 (20 Lig/rrtl) for 1 hour, followed by treatment with vehicle or Diprovocim (250 pM) for another 4 hours. P values were determined by
• Fig. 2A and Fig. 2B the means of three independent samples are plotted.
• Fig. 2C and Fig. 2D are immunoblot show the results of analysis of lysates of human THP-1 cells (Fig. 2C) and mouse peritoneal macrophages (Fig. 2D) treated with Diprovocim (5 nM in THP-1 and 500 nM in mouse peritoneal macrophages) for the indicated times. All results are representative of two independent studies .
• Fig. 3A through Fig. 3G illustrate through graphs and schemes that Diprovocim enhances antigen- specific antibody and CTL responses.
• Fig. 3A - Fig. 3C show results WT or Tlr2 / C57BL/6J mice (4 mice per group) were immunized i.m. 'with 100 ,ug OVA mixed with vehicle, Diprovocim (10 mg/kg) , or alum (2 mg/kg) . After 14 days, serum titers of OVA-specific IgG (Fig. 3A) , OVA-specific IgGl (Fig. 3B) and OVA- specific IgG2b (Fig. 3C) were measured by ELISA.
• Fig. 3D shows a schematic of the experimental setup (left) and results for detection (center) and quantification (right) of CD69 expression on OT-I CD8 T cells by flow cytometery after 24 hours co-culture with DC collected from mice 24 hours after they were immunized i.m. with OVA mixed with vehicle or
• Fig. 3E and Fig. 3F show results from mice that were unimmunized or immunized i.m. with 100 gg OVA mixed with vehicle or Diprovocim (10 mg/kg) (4 mice per group) .
• mice Seven days after immunization, mice were injected i.v. with Celltrace Violet-labeled mouse splenocytes that were unpulsed (control cells) or pulsed with OVA peptide (a. a. 257-263) ⁇ target cells) . Two days later, blood 'was collected to measure remaining live dye-labeled cells.
• FIG. 3E illustrates representative flow cytometry plots that count remaining target cells (right peak) and control cells (left peak) in wild type mice.
• Fig. 3F shows a quantitative comparison of the percentage of target cells killed in WT, Tlrl ' or Tlr2 ⁇ mice. P values were determined by Student's t test. All results are representative of two independent studies.
• Fig. 4A through Fig. 4J illustrate inhibition of B16-OVA tumor growth by pre- or post tumor treatment with Diprovocim-adj uvanted tumor vaccination and checkpoint blockade.
• Fig. 4A provides a schematic of pre- and post-tumor treatment protocols.
• mice were injected s.c. with 2x10° B16-OVA melanoma cells on day 0.
• mice were immunized i.m. with OVA ⁇ 100 gg) mixed with vehicle or Diprovocim (10 mg/kg) or alum (2 mg/kg) on the same day prior to tumor injection.
• mice received a booster immunization seven days later in which anti- PD-L1 (200 gg) was administered on days 3, 6 and 9 after tumor inoculation by i.p. injection.
• anti-PD-L1 200 gg
• initial immunization was on day 3 after tumor inoculation, with a booster seven days later.
• Anti-PD-Ll 200 gg was administered on days 3, 6, 9, 12, and 15 after tumor inoculation by i.p. injection.
• Fig.4B through Fig. 4F illustrate pre tumor treatment results. Tumor volume (Fig. 4B and Fig. 4D) and percent mouse survival ⁇ survivors/total mice) (Fig. 4C and Fig. 4E) .
• Fig. 4G through Fig. 4J illustrate a comparison of pre-tumor (Fig. 4G and Fig. 4H) vs. post-tumor treatment (Fig. 41 and Fig. 4J) .
• Tumor volume (Fig. 4G and Fig. 41) and percent mouse survival ( survivors /total mice) (Fig. 4H and Fig. 4J) are shown.
• P values for tumor volume analysis apply to the final time point as indicated in graphs and were calculated by Student's t test.
• P values for survival analysis were calculated by Kaplan-Meier analysis. All results are
• Fig. 5A through Fig. 5M illustrate that a Diprovocim enhances TILs and anti-tumor CTL
• FIG. 5A is a schematic of treatment protocol for Fig. 5A through Fig. 5J in which
• Mice received a booster immunization on day 10 after tumor inoculation in which anti-PD-Ll (200 pg) was administered on days 3, 6, 5, and 12 after tumor inoculation by i.p. injection.
• Tumors were harvested on day 14 to isolate and analyze tumor-infiltrating leukocytes (TILs) .
• Fig. 5A through Fig. 5J illustrate the frequency of each cell type out of total tumor cells is shown.
• Fig. 5B shows TILs (CD45 + ) .
• Fig. 5C shows CD4 T cells
• FIG. 5D shows activated CD4 T cells (CD44 high CD4 ⁇ CD3 ⁇ CD45 + ) .
• FIG. 5E shows CDS T cells (CD8 + CD3 + CD45 ) .
• Fig. 5F shows activated CDS T cells ⁇ CD44 high CD8 ⁇ CD3 ⁇ CD45 + ) .
• Fig. 5G shows OVA-specific CDS T cells bearing a T-cell receptor specific for OVA (2 57- 264) _ H2Kb tetramer .
• Fig. 5H shows NK cells (NK1.1 CD3 ⁇ CD45 + ) .
• Fig. 51 shows DCs (CD1 lc + CD3 CD45 ⁇ ) .
• Fig. 5J shows macrophages (F4/8CrCDllb " CD45 + ) .
• anti-CD4 300 pg
• anti-CD8 300 pg
• anti-NKl .1 300 pg
• a mixture of these three antibodies was administered to C57BL/6J mice by i.p. injection.
• Tumor volume Fig. 5L
• percent mouse survival survivors/total mice
• P values for tumor volume analysis apply to the final time point as indicated in graphs and 'were calculated by Student's t test.
• P values for survival analysis were calculated by Kaplan-Meier analysis. All results are representative of two independent studies.
• Fig. 6 shows a schematic model of key cellular events mediating the antitumor effect of Diprovocim-adjuvant immunization plus checkpoint inhibition .
• Fig. 7 illustrates that a Diprovocim does not stimulate IFN-b secretion by mouse peritoneal macrophages. IFN-b in the supernatants of mouse peritoneal macrophages after treatment with
• Fig. 8A and Fig. 8B illustrate that anti- PD-L1 antibodies do not inhibit B16 tumor growth in mice.
• Anti-PD-Ll 200 pg
• mouse IgG2a isotype control antibody was administered on days 3, 6 and 9 after tumor
• Fig. 8A is a graph of tumor volume versus time and Fig. 8B is a graph showing percent mouse survival (survivors/total mice) versus time .
• the control values in both plots are shown above those for the anti-PD-Ll values where the two values diverge.
• the P value for tumor volume analysis applies to the final time point and was calculated by Student's t test; no significant difference was found between treatments.
• P values for survival analysis were calculated by Kaplan-Meier analysis; no significant difference was found between treatments . Results are representative of two independent studies.
• Fig. 9A and Fig. 9B illustrate that a Diprovocim is more potent than PanruCSKi in activation of TNF production in human cells.
• FIG. 9B show TNF amounts assayed from the supernatants of human THP-1 cells (Fig. 9A) and human PBMC (Fig. 9B) after treatment with Diprovocim or Pam 3 CSK 4 for 4 hours (Fig. 9A) or 24 hours (Fig. 9B) .
• Data points for Diprovocim are shown generally ⁇ to the left of data points for Pam 3 CSK 4 in Fig. 9A, above data points for Pam 3 CSK4 where the lines diverge in Fig. 9B .
• the means of three independent samples are plotted.
• Antibody a polypeptide that
• Antibodies as used herein, are immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules . Such portions known in the art as Fab, Fab'; F(ab')2 and Fy are included. Typically, antibodies bind ligands that range in size from about 6 through about 34 Angstroms (A) with association constants in the range of about 10 ⁇ to about 10-*-*-*
• Antibodies can bind a wide range of ligands, including small molecules such as steroids and prostaglandins, biopolymers such as nucleic acids, proteins and polysaccharides, and synthetic polymers such as polypropylene.
• an “antibody combining site” or “paratope” is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds to (immunoreacts with) an "antigen” or “epitope".
• monoclonal antibodies that are suitable for injection ⁇ pharmaceutically acceptable) into a diseased mammal in need of treatment without undo adverse effects due to contaminants .
• Such monoclonal antibodies can be obtained from the animal species that is immunized as discussed herein, such as a human. Or, the antibodies can be induced in one animal and the antibody-producing cells modified to produce antibody protein sequences of the mammal to be immunized. Although other species of mammal are contemplated for immunization, a human is a
• a contemplated monoclonal antibody that was originally induced in a mouse can be more useful to a human recipient as a so- called “humanized” antibody, or as a “chimeric” antibody.
• INN International Nonproprietary Names
• antigen has been used historically to designate an entity that is bound by an antibody or receptor, and also to designate the entity that induces the production of the antibody. More current usage limits the meaning of antigen to that entity bound by an antibody or receptor, whereas the word “immunogen” is used for the entity that induces antibody production or binds to the receptor. Where an entity discussed herein is both immunogenic and antigenic, reference to it as either an immunogen or antigen is typically made according to its intended utility.
• immunoreact m its various forms is used herein to refer to specific binding between an antigenic determinant-containing molecule
• an antibody combining site such as a whole antibody molecule or a paratope-containing portion thereof.
• antigenic determinant is the structural portion of the antigen that is
• Antibodies can bind a single epitope of an antigen (monoclonal) or multiple epitopes (poly / clonal) .
• the length of a linear epitope is usually 7 recited as being about 5 to about 7 amino acid residues .
• an element means one element or more than one element.
• hydrocarbyl is used herein as a short hand term for a non-aromatic group that includes straight and branched chain aliphatic as well as alicyclic groups or radicals that contain only carbon and hydrogen.
• alkyl, alkenyl and alkynyl groups are contemplated, whereas aromatic hydrocarbons such as phenyl and naphthyl groups, which strictly speaking are also hydrocarbyl groups, are referred to herein as aryl groups or radicals, as discussed hereinafter.
• a specific aliphatic hydrocarbyl substituent group is intended, that group is recited; i.e., methyl, ethyl, butyl, tert-butyl, hexyl, hexenyl, 2-ethylhexyl, dodecyl (Cyg) r octadecyl (C_3) .
• a particularly 7 preferred hy 7 drocarby 7 l group is an alkyl group.
• a generalized, but more preferred substituent can be recited by replacing the descriptor "hydrocarby1" with "alkyl" in any of the substituent groups enumerated herein,
• alkyl radicals include ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and cyclopropyl .
• suitable alkenyl radicals include ethenyl (vinyl) , 2-propenyl, 3- propenyl, 1, 4-butadienyl, 1-butenyl, 2-butenyl, and 3-butenyl.
• alkynyl radicals examples include ethynyl, 2-propynyl, 1-propynyl, 1-butynyl, 2- butynyl, 3-butynyl, and l-rnethyl-2-propynyl .
• hydrocarbyl ether is referred to as a
• hydrocarbyloxy rather than a “hydrocarboxy” group as may possibly be more proper when following the usual rules of chemical nomenclature.
• Illustrative hydrocarbyloxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, allyloxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy groups.
• Ligand a molecule having a structural region that binds specifically to a particular receptor molecule, usually via electrostatic forces and/or hydrogen bonds .
• An exemplary ligand is the epidermal growth factor molecule .
• the "marker molecule” (antigen or immunogen) can be but need not be expressed on the cell surface, and rather can be expressed anywhere in the diseased cell. The reason for that is that substantially all of the native cellular proteins of mammals are processed into shorter peptides by the cell and bound extracellularly by class I MHC molecules. Such native proteins are typically so processed during the organism's immaturity, and T cells or other immune cells that may be induced by those native protein portions are eliminated by the organism prior to maturity, resulting in "self protein” tolerance. As a result, except in cases of certain immunological diseases, only foreign peptides or neoantigenic peptides caused by disease such as cancer whose cells result from mutation are
• the marker molecule can also be a "tumor antigen;” that is, a protein that can be expressed by other cells during embryonic development, for example, but is
• the marker molecule can be an oncogene product, for example, an abnormal fusion protein created by a recombination event within tumor cells.
• the marker molecule can also be the product of an infectious agent such as a virus or bacterium as we11.
• Peptide/Polypeptide an oligomer or polymer comprising at least two amino acid residues in which adjacent residues are linked by a peptide bond between the alpha-amino group of one residue and the alpha-carboxyl group of an adjacent residue.
• the primary structure of a polypeptide has a primary amine group at one terminus and a carboxylic acid group at the other terminus of the polymer.
• a peptide or polypeptide is depicted herein and usually in the art from left to right and in the direction from amino-terminus to carboxy-terminus . Also, a polypeptide in aqueous solution is usually in one or more zwitterionic forms depending on the pH of the solution.
• the words "peptide” and “polypeptide” are used interchangeably herein.
• Protein a single polypeptide or set of cross-linked polypeptides comprising more than about 100 amino acid residues. Proteins can have chemical crosslinking, e.g., via disulfide bridges, within the same polypeptide chain or between adjacent
• polypeptides When a protein is glycosylated it can be called a glycoprotein.
• a protein When a protein comprises one or more discrete polypeptide /protein subunits linked together, as by a peptide linkage, amino acid residue sequence, disulfide bridge, and the like, the protein is frequently termed a fusion protein, fusion polypeptide, chimeric fusion, and the like.
• Receptor a biologically active substance
• An exemplary receptor molecule is an antibody combining site or a transmembrane cellular protein molecule involved in intra- or intercellular signaling such as the endothelial growth hormone receptor referred to as EGFR, ERBB and also as HER2, and the like.
• amino acid residue is used interchangeably with the phrase amino acid residue . All amino acid residues identified herein are in the natural or L- configuration . In keeping with standard polypeptide nomenclature, [J. Biol. Chera. , 243:3557-59 (1969)], abbreviations for amino acid residues are as shown in the following Table of Correspondence.
• the present invention has several benefits and advantages .
• a salient benefit of the invention is that the combination immunization provides synergistic results in inhibiting diseased cell growth.
• An advantage of the invention is that the combination immunization provides T cell help that virus- and bacteria-free vaccines have often lacked.
• Another benefit that the invention provides is that those skilled in the art have been finding, studying and publishing formulas of disease-related immunogens that have been ultimately unsuccessful since the early 1980' " s but can now be successfully put to use.
• the present invention contemplates a method of inhibiting the growth of diseased cells in a mammal .
• Those diseased cells express one or more marker molecules that are absent on cells of the same type that are free of the disease or are present in the disease-free cells in significantly reduced numbers compared to the diseased cells.
• Workers skilled in the art have published numerous articles and reviews discussing marker molecules that are present in diseased cells in amounts significantly greater than the amount present in disease-free cells and methods for determining those differences. See, for example, Kim et al . , BMB Rep 50 ( 6) : 285-298 (2017) and the citations therein.
• techniques such as quantitative western blots performed with optical density scans or radioactivity detection means and other means are well known in the art.
• Cells "of the same type" are disease-free cells from the same organ and tissue as the diseased cells.
• a contemplated method comprises the steps of immunizing a diseased mammal in need by: a) administering to the diseased mammal (i) an adjuvant- sufficient amount of a Diprovocim compound, (ii) a T cell-stimulating amount of an immune checkpoint inhibitor, and (iii) an immunizing amount of that marker molecule.
• the immunized mammal is b)
• a contemplated Diprovocim compound corresponds in structure to structural Formula V,
• -A is -H (hydrido) or -C ⁇ 0)NH-R ⁇ ;
• R-*-, R ⁇ , R3 are the same or different and are a 2- (4-fluorophenyl) ethyl, a trans-2- phenylcyclopropyl, a trans-2- ( 4-fluorophenyl ) - cyclopropyl or a Cg-Cyg hydrocarbyl group with the provisos that:
• R*-, R ⁇ , R- and R ⁇ (R ⁇ -- ⁇ ) or at least two of R ⁇ , R ⁇ , and R ⁇ ⁇ R ⁇ _ 3) are a trans- 2-phenylcyclopropyl, a trans-2- (4-fluorophenyl) - cyclopropyl group or a mixture thereof, or each of R-'-- ⁇ is a 2- (4-fluorophenyl) ethyl group,
• pyrrolidinyldicarboxamido group has the (5, S) configuration, and each depicted R substituent other than a Cy-Cyg hydrocarbyl group is a trans-2- phenylcyclopropyl, a trans-2- (4-fluorophenyl) - cyclopropyl group or a mixture thereof when each of R! j_ s other than 2- (4-fluorophenyl) ethyl,
• R ⁇ - _ 3 when A is hydrido, one of R ⁇ - _ 3 can k e a Cf-C ⁇ g hydrocarbyl group and the depicted R J - containing pyrrolidinylcarboxamido group can have either the R or S configurations, or a mixture of both configurations;
• -Z is one or more of halogen -H, -NH 2 , -OH, -OCH 3 , -N0 2 , -0CH 2 C0 2 H, -0 (CH 2 CH 2 0) n CH 2 CH 2 C02H, -OCHgCONH (CH 2 CH 2 0) n CH 2 CH 2 C0 2 H, -NHCOCHgO- (CH 2 CH 2 0) n CH 2 C0 2 H, -OCH 2 CONHCH 2 CONHCH (CHOH) C0 2 H, -OCH 2 CONHCH 2 CONHCHCO 2 H (CH 2 C0 2 H) ,
• W is nitrogen (N) or CH;
• n is a number whose average value is one to about eight;
• m is a number whose value is 1 to about
• a preferred member of the Diprovocim family of compounds of Formula V is a compound that
• At least one member of substituent pair R ⁇ - and (either or R-) or pair R (either R ⁇ or R ⁇ ) is a trans-2-phenyIcyclopropyl or trans-2- (4- fluorophenyl) cyclopropyl group. It is also preferred that at least one member of substituent pair R ⁇ and R ⁇ or pair R ⁇ and R ⁇ has the (1S,2R) configuration of a trans-2-phenylcyclopropyl or trans-2- (4-fluoro- phenyl ) cyclopropyl group .
• substituents of R- 1 , R , R ⁇ and R ⁇ have the (IS, 2_R) configuration of a trans-2-phenylcyclopropyl or trans-2- ( 4 -fluorophenyl ) cyclopropyl group. More preferably still, each of R ⁇ , R ⁇ , R ⁇ and has the (1S,2R) configuration of a trans-2-phenylcyclopropyl or a trans-2- ( 4-fluorophenyl) cyclopropyl group.
• each depicted pyrrolidinyldicarboxamido group has the (S, S)
• R- 3 ⁇ 4 substituent is a trans-2-phenylcyclopropyl , a trans-2- (4-fluoro ⁇ phenyl ) cyclopropyl group or a mixture thereof, and bonds to the cyclopropyl moiety have a (IS, 2R) configuration .
• up to two of R ⁇ - ⁇ in a compound of Formula I or one of its sub-generic formulas can be a Cg-Cgg hydrocarbyl group.
• the hydrocarbyl group be an alkyl group and have a length of 4 to about 16 carbon atoms, and more preferably still, about 6 to about 10 carbon atoms .
• Straight chained hydrocarbyl groups are also preferred, although up to two methyl and ethyl group substituents or both can be present as can an carbocyclic ring, and also one or two double or triple bonds. Specific Cg-Cgg hydrocarbyl groups are discussed previously in the discussion of the use of the word hydrocarbyl .
• each molecule contains at least one, and preferably two, 3,4- pyrrolidinyldicarboxyl groups.
• the carboxyl groups are bonded to amine-terminated R-*-, R ⁇ , R3 and R ⁇ substituents, forming four (or three) amido linkages.
• the two pyrrolidinyldicarboxyl groups can also therefore also be referred to as two
• Substituents bonded to the carboxyl groups of a pyrrolidinyldicarboxyl group can be in a cis or trans conformation, that is the two substituents can both project above or below the plane of the depicted ring ⁇ cis), or one can project above that plane and the other substituent project below (trans) .
• a cis- disubstituted pyrrolidinyldicarboxyl group with two identical substituents has a symmetric configuration and does not have enantiomeric forms.
• a trans- disubstituted pyrrolidinyldicarboxyl group with those same two identical substituents has an asymmetric (chiral) configuration and has enantiomeric forms.
• At least one, and more preferably both 3, 4-pyrrolidinyldicarboxyl groups have the (S,S) configuration .
• -A of Formula V is hydrido and one of R-'--- 3 is a Cy-Cyg hydrocarbyl group, and more preferably, a C ] _o-Ci_g hydrocarbyl group.
• a preferred compound corresponds in structure to Formula Va , below, in vihich the depicted R- 1 W
• the number of carbon atoms of a group here is preferably 2 to 18, and more preferably 10 to 16 carbon atoms.
• This hydrocarbyl group is also more preferably an alkyl group that is a straight chained substituent although methyl and ethyl branches can be tolerated as can double and/or triple bonds in the chain.
• Cyclic hydrocarbyl substituent compounds and carbocyclic ring-containing substituents can also be utilized.
• W be CH .
• Structural Formula la shown below, incorporates several of the above preferences.
• "h” in the depicted amido group is 1 to 17, preferably 7 to 17, and more preferably 9 to 15.
• "j” in the depicted amido group is 1 to 17, preferably 3 to 11, and more preferably 5 to 9.
• a contemplated immune checkpoint inhibitor is typically an intact antibody or the paratope- containing portion of an antibody. Such a
• contemplated antibody or antibody paratope-containing portion is preferably a monoclonal humanized, chimeric or human antibody.
• Illustrative checkpoint US FDA approved checkpoint inhibitors include
• CTLA-4 itself binds to proteins B7-1 and B7-2 to inhibit T cell activity.
• Anti-B7-1 and anti-B7-2 paratope-containing molecules can also be used to block the CTLA-4/B7-1+B7-2 interaction, thereby providing checkpoint inhibitor activity much as do antibodies to either of the binding pair PD-1 and PD-L1.
• proteinaceous and can be the whole protein or an immunogenic portion of the protein.
• the checkpoint inhibitor is administered intravenously (IV) , whereas the
• Diprovocim and antigen are administered together in an immunizing pharmaceutical composition
• IM intramuscularly
• SC subcutaneously
• Those administrations can be made within the span of a few minutes, hours, days or weeks.
• checkpoint inhibitors are antibodies or
• a checkpoint inhibitor can be administered prior to, coincidently with or a few days after administration of the Diprovocim and immunogen.
• Each of the components can be administered a plurality of times during a course of treatment.
• a Diprovocim compound is administered in an adjuvant-sufficient amount.
• Diprovocim-1 (A) used illustratively herein acted as a robust in vivo adjuvant or TLR1/TLR2 agonist that evoked a potent TLR2-dependent adjuvant activity in vivo in mice at about 0.25 to about 5 mg/kg (i.m.) when co-injected with an immunogen in an immunizing pharmaceutical composition by an intramuscular route.
• An adjuvant- sufficient amount can be readily determined for mammals of greater weight by techniques well known in the art.
• Diprovocim-1 did not display the overt toxicity that is characteristic of LPS administration when used as an adjuvant.
• a checkpoint inhibitor is typically utilized in an amount discussed in the product label.
• Illustrative dosage and administrations include the following using melanoma as exemplary diseased cell for use in the recited amounts: Keytruda®—
• melanoma 2 mg/kg every 3 weeks; Yervoy®— adjuvant melanoma: 10 mg/kg administered intravenously over 90 minutes every 3 weeks for 4 doses, followed by 10 mg/ kg every 12 weeks for up to 3 years or until documented disease recurrence or unacceptable toxicity; Tecentriq®--administer 1200 mg as an intravenous infusion over 60 minutes every 3 weeks; and Opdivo®--unresectable or metastatic melanoma 240 mg every 2 weeks .
• telomeres having a length of about 5 to about 20 residues are themselves poorly immunogenic, and are often best utilized as haptens chemically linked to a carrier molecule.
• Illustrative proteinaceous carrier molecules include keyhole limpet hemocyanin (KLH) , hepatitis B surface molecule (HBsAg) , the hepatitis B core (capsid; HBcAg) , ovalbumin, bovine serum albumin, bovine gammaglobulin and human gammaglobulin have been used as a hapten carrier, as have many other molecules have been used in the literature .
• KLH keyhole limpet hemocyanin
• HBsAg hepatitis B surface molecule
• capsid HBcAg
• ovalbumin ovalbumin
• bovine serum albumin bovine gammaglobulin
• human gammaglobulin have been used as a hapten carrier, as have many other molecules have been used in
• a contemplated disease-related marker molecule is present in and/or on diseased cells.
• Diseased cells are typically cancerous or pathogen- infected .
• CSC cancer stem cell
• Exemplary CSC markers that are largely absent in normal (disease-free) cells and present in diseased cells include CDS6, CD20, DLL4, CD55, TIM-3, CXCR1 , CD54, CD114, LGR5 , CD105, CD56, CD13, CD271 , CD34, CXCR4 , CD26, CD117, CD10, CD146, Notch.2 , CD49f, CD24, ABCG2 , PODXL-2, Cripto-1, CD326, CD90 , CD133, SSEA1, TRA-1-81, TRA-1-60, SSEA4 , SSEA3 , CD151 , CD340 and CD44.
• Exemplary disease-related marker molecules are typically present in and/or on solid tumor cells.
• Illustrative solid tumors include osteosarcoma cells, Kaposi's sarcoma cells, melanoma cells, prostate cancer cells, glioblastoma cells, small cell lung carcinoma cells, breast cancer cells, liver cancer cells, colon cancer cells, ovarian cancer cells, renal cancer cells, gastric cancer cells,
• neuroblastoma cells pancreatic cancer cells
• Hodgkin's lymphoma cells Hodgkin's lymphoma cells
• a contemplated disease-related marker molecule or portion thereof can also be present in and/or on pathogen-infected cells.
• Illustrative infecting pathogens include one or more of a virus, bacterium, fungus and unicellular parasite.
• Illustrative viruses include influenza, hepatitis viruses A, B, C and D, herpes viruses such as Varicella zoster (chickenpox) , Herpes simplex 1 and 2 (HSV1 and HSV2 ) , human papilloma virus (HPV) , and the like.
• Illustrative bacterial pathogens include E. coli, E. faecalis, S. aureus, and the like.
• An illustrative unicellular parasite is the malaria sporozoite of P. falciparum, P. vivax, P. bergeii or P. ycelli .
• Illustrative proteinaceous immunogens include the following disease-related marker molecule peptides that are listed below with a citation to their publication source.
• NANPNVDP (NANP) 3NVDP SEQ ID NO:
• NANPNVDP (NANP) 3 SEQ ID NO:
• NPNVDP (NANP) 3NV SEQ ID NO:
• M2 protein is expressed in cells infected by the influenza A strains.
• the N-terminal residues 1-24 of the M2 protein extends through the infected cell's membrane. That extracellular portion of the protein is referred to as M2e.
• M2e that extracellular portion of the protein.
• use of the influenza A extracellular M2e portion of that protein as the immunogenic marker can provide protection from all of the influenza strains.
• the yearly changes in influenza vaccine selection can be avoided .
• MSLLTE ETPIRNEWGARANDSSD MSLLTEVETPIRNEWGCRANDSSD
• MSLLTEVETPIRNEWGARCNDSSD MSLLTEVETPIRNEWGCRSNDSSD
• MSLLTEVETPIRNEWGSRCNDSSD SLLTEVETPIRNEWGSRSNDSSDSLL- TEVETPIRNEWGSRSNDSSD
• Hepatitis B virus surface antigen provides both B cell and T cell polypeptide epitopes.
• a number of each epitope type as disclosed in US Patent No. 4,599,231 are set out below in the table along with their peptide denominations, and parenthesized sequence position from the N-terminus, as recited in that patent based on DNA from an ayw donor (P49) and an adw donor (P72 and P73) .
• Papillomaviruses induce benign, dysplastic and malignant hyperproliferations of skin or mucosal epithelium. More than 50 types (strains) of human papillomavirus (HPV) have been identified. In humans, different papillomavirus types are known to cause distinct diseases. For example, HPV types 1 and 2 cause common warts, and types 6 and 11 cause condylomas and genital flat warts. In contrast, HPV types 16, 18 and 33 are carried in a majority of cervical cancers and do not cause the usual
• U.S. Patent No. 5,180,806 discloses several peptide sequences that induce the production of antibodies. Illustrative peptide markers of type 16-related HPV sequences disclosed in US Patent No. 5,180,806 are set out below as illustrative. That patent also discloses peptide sequences from type 18 and type 33, as well as sequences encoded by the E2 ORE of HPV types 6, 11, 18 and 33.
• a contemplated immunizing composition also typically contains pharmaceutically acceptable salts, buffers and the like excipients that collectively are referred to as pharmaceutically (or physiologically) acceptable diluents or carriers as compared to those that can be present in a composition that is not intended for pharmaceutical use, as in an in vitro assay. These compositions are discussed in further detail hereinafter.
• a Diprovocim compound useful herein can be provided for use by itself, or as a pharmaceutically acceptable salt.
• Exemplary salts useful for a contemplated compound include but are not limited to the following: sulfate, hydrochloride, hydro
• bromides acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate,
• ethanesulfonate glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy- ethanesulfonate , lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate , oxalate,
• Salts of the carboxylate group include sodium, potassium, magnesium, calcium, aluminum, ammonium, and the many substituted ammonium salts .
• the salts can also be used as an aid in the isolation, purification or
• the acid or base used and the salt prepared need not be pharmaceutically acceptable.
• a contemplated immunizing pharmaceutical composition contains an adjuvant-effective amount of a Compound of Formula V or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically (pharmaceutically) acceptable carrier along with the immunogenic marker.
• composition can be administered to mammalian cells in vitro as in a cell culture to contact those cells, or the cells can be contacted in vivo as in a living, host mammal in need.
• a contemplated Diprovocim compound present at femtomolar to nanomolar amounts provides an adjuvant effect in in vivo and in in vitro assay studies.
• a Compound of Formula V When used as a vaccine adjuvant, a Compound of Formula V is preferably administered together with the selected marker immunogen. Both components are preferably present together in a single immunizing pharmaceutical composition as noted above. However, the two ingredients can be present in separately administered immunizing pharmaceutical compositions, and those separate compositions can be administered up to about one to about two hours apart. It is preferred when two separate compositions are
• the Diprovocim compound utilized can be chemically bonded to the immunizing marker compound. That chemical bond can be formed using the Z substituent shown in Formula V as where a Z substituent that includes a carboxyl group can be bonded to an amino group of an immunogenic peptide marker compound.
• the Diprovocim compound can also be chemically bonded to the same carrier molecule.
• a contemplated immunizing pharmaceutical composition is typically administered in vivo to a subject in need thereof a plurality of times within one month, such as daily or weekly, and can be administered over a period of several months to several years. More usually, a contemplated
• composition is administered a plurality of times over a course of treatment.
• a contemplated immunizing pharmaceutical composition is preferably adapted for parenteral administration.
• an immunizing pharmaceutical composition is preferably in liquid form when administered, and most preferably, the liquid is an aqueous liquid, although other liquids are
• composition contemplated as discussed below, and a presently most preferred composition is an injectable preparation.
• injectable preparations for example, sterile injectable aqueous or oleaginous solutions or suspensions can be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• suitable dispersing or wetting agents and suspending agents for example, sterile injectable aqueous or oleaginous solutions or suspensions.
• preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 , 3-butanediol .
• a nontoxic parenterally acceptable diluent or solvent for example, as a solution in 1 , 3-butanediol .
• acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution, phosphate-buffered saline.
• liquid pharmaceutical compositions include, for example, solutions suitable for
• a contemplated Compound of Formula V is provided as a dry powder that is to be dissolved in an appropriate liquid medium such as sodium chloride for injection prior to use.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil can be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid find use in the preparation of an injectable composition.
• Dimethyl acetamide, surfactants including ionic and non-ionic detergents, polyethylene glycols can be used. Mixtures of solvents and wetting agents such as those discussed above are also useful. Sterile solutions can be prepared by dissolving the active component in the desired solvent system, and then passing the resulting solution through a membrane filter to sterilize it or, alternatively, by dissolving the sterile compound in a previously sterilized solvent under sterile conditions.
• a mammal in having diseased cells in need of treatment (a subject) and to which a
• composition containing at least a Compound of Formula V and a immunogenic marker compound is administered can be a primate such as a human, an ape such as a chimpanzee or gorilla, a monkey such as a cynomolgus monkey or a macaque, a laboratory animal such as a rat, mouse or rabbit, a companion animal such as a dog, cat, horse, or a food animal such as a cow or steer, sheep, lamb, pig, goat, llama or the like.
• the pharmaceutical composition is in unit dosage form.
• the pharmaceutical composition is in unit dosage form.
• the pharmaceutical composition is in unit dosage form.
• composition is divided into unit doses containing appropriate quantities of the Diprovocim and
• the unit dosage form can be a packaged preparation, the package containing discrete
• quantities of the preparation for example, in vials or ampules .
• Diprovocim induces cytokine production in both human and mouse cells
• THP-1 cells EC50 110 pM
• human PBMC EC50 875 pM
• mouse peritoneal macrophages EC50 1.3 nM
• bone marrow-derived dendritic cells EC50 6.7 nM
• Diprovocim-1 induced IL-6 production by mouse BMDC (Fig. 0E)
• Diprovocim-1 failed to stimulate type I IFN production by mouse peritoneal macrophages (Fig. 7) .
• the other numbered Diprovocims studied and those defined by structural Formula I also share these activities.
• Diprovocim targets TLR1/TLR2 and activates downstream MAPK and NF-kB signaling pathway
• Diprovocims effects were analyzed on peritoneal macrophages from wild type C57BL/6J mice and C57BL/6J mice deficient in various TLR signaling components. Induction of TNF by Diprovocim was completely absent in TLR1- or TLR2-deficient macrophages, but not in TLR-6 deficient macrophages (Fig. 1A) . Diprovocim activity was also dramatically reduced in macrophages from MyD88-, TIRAP-, and IRAK4-deficient cells (Fig. 1A) . These data suggested that Diprovocim targets the mouse TLR1/TLR2 heterodimer.
• TLR1 or TLR2 antibody significantly reduced the effect of Diprovocim on THP-1 cells, indicating that human TLR1/TLR2 is also a target of Diprovocim molecules (Fig. IB) .
• OVA ovalbumin
• OVA+alum induced primarily the Th2-related Ig subclass IgGl
• OVA ⁇ Diprovocim induced both IgGl and the Thl-related IgG2b (Fig. 2B and Fig. 3C) .
• DCs Dendritic cells purified from draining lymph nodes and spleens 24 hours after immunization of mice with OVA+Diprovocim activated OT-I CDS T cells co-cultured with them, as evidenced by CD69 upregulation on the OT-I cells (Fig. 2D) .
• DCs from mice immunized with OVA+vehicle failed to induce CD69 expression on OT-I CDS T cells (Fig. 2D) .
• Diprovocims activate antigen cross-presentation by DCs and cross priming of CDS T cells in vivo.
• mice with already established B16-OVA tumors C57BL/6J mice were immunized with OVA with or without a Diprovocim on the day of or three days after tumor inoculation and received a booster immunization seven days later ⁇ Fig. 3A) .
• alum was substituted for Diprovocim to permit direct comparison between these two adjuvants.
• anti-PD-Ll treatment was initiated on day 3 after tumor
• Diprovocim+OVA still significantly inhibited tumor growth and prolonged average survival compared to OVA alone (41 days vs.
• P 0.082
• TILs Tumor- infiltrating leukocytes
• Fig. 4A Tumors were collected 14 days after inoculation, and single-cell suspensions were antibody stained and analyzed by flow cytometry to detect total leukocytes, CD4 and CDS T cells, NK cells, DCs, and macrophages.
• the leukocytes were also stained with antibody to the H-2K b MHC-class I tetramer bound to the OVA peptide (residues 257-264), as well as antibody against CDS to identify tumor-specific CDS T cells.
• OVA immunizations containing a Diprovocim significantly increased the frequency of leukocytes in tumors compared to vehicle+OVA (Fig. 4B) .
• Further analysis of these TILs revealed that a Diprovocim increased the frequencies of CD4 and CDS T cells including activated CD4 and CDS T cells (CD44 nig!1 ) and OVA-specific CDS T cells, as well as the frequency of NK cells (Fig. 4C - Fig. 5H) .
• Alum+OVA immunization showed a trend towards increasing TILs (Fig. 4B) , which reached statistical significance for total and CD44 nign CDS T cells (Fig. 4E and Fig. 5F) .
• the magnitude of the increase was reduced compared to that induced by Diprovocim+OVA.
• OVA-specific CDS T cells were not increased by alum+OVA immunization (Fig. 4G) on day 14 after tumor inoculation; neither were total and CD44 high CD4 T cells (Fig. 4C and Fig. 5D) , nor NK cells compared to vehicle+OVA (Fig. 4H) .
• mice were depleted of CDS T cells, CD4 T cells, NK cells, or all three cell populations using cell type-specific antibodies.
• the depletion antibodies were administered i.p. on the day of BIG- OVA tumor inoculation ⁇ day 0) and every three days thereafter for 15 days ⁇ Fig. 4K) .
• the effect of Diprovocim+OVA on both tumor growth and mouse survival was abrogated when mice were depleted of CDS T cells or all three cell types together ⁇ CD4 T, CDS T, NK cells) (Fig. 4L and Fig. 5M) .
• depletion of CD4 T cells or NK cells had little effect on the anti-tumor activity of Diprorocim+OVA (Fig. 4L and Fig. 5M) .
• Diprovocim+OVA plus anti-PD-Ll in which tumor growth was greater in mice depleted of all three cell types. However, this difference did not translate to a difference in either survival rate or time. This finding suggests a minor role of either CD4 T cells, NK cells, or both, in mediating the anti-tumor effects of Diprovocim+OVA plus anti-PD-Ll. These data demonstrate that CDS T cells are necessary for tumor eradication by therapeutic Diprovocim+OVA immunization and checkpoint inhibition in mice.
• cancer vaccines targeted to tumor neoantigens can boost the success of immune checkpoint inhibition for cancer treatment by increasing the number and activation of tumor- specific CTLs capable of responding to checkpoint inhibitors.
• type and magnitude of the T cell response to immunization depends critically on the vaccine adjuvant; currently only few adjuvants are approved for use in humans .
• Diprovocim a novel and potent adjuvant that engages and activates human and mouse TLR1/TLR2 heterodimers.
• a Diprovocim bears no structural similarity to other reported synthetic chemical ligands, nor to the natural ligands that activate TLR1/TLR2 (22-26) .
• a Diprovocim is more potent and efficacious in
• a Diprovocim induces strong TLR1- and TLR2-dependent humoral and CTL responses to a co administered antigen.
• a Diprovocim-adj uvanted immunization causes antigen-specific eradication of a rapidly fatal tumor, and induces memory responses capable of preventing tumor regrowth. Cure of the tumor is observed despite the fact that checkpoint inhibition alone is insufficient to prevent a fatal outcome ⁇ Fig. 8A and Fig. 8B) , supporting the premise of this combination immunotherapy.
• Diprovocim binds to TLR1/TLR2 on APCs, activating them to produce pro-inflammatory cytokines and take up the administered tumor-specific antigens for processing and presentation via MHC I and MHC II.
• Antigen presentation, costimulatory molecule expression, and cytokine secretion by APCs induce proliferation and activation of antigen- specific CD4 T cells and CD8 T cells, which develop cytolytic activity toward tumor cells.
• NK cells are also activated by pro-inflammatory cytokines and infiltrate the tumor site.
• anti-PD- L1 inhibits the major immunosuppressive mechanism active in the tumor microenvironment, permitting uninhibited T cell activation and proliferation in response to TCR/CD28 ligation (27-29) , further promoting tumor cell lysis mediated by CDS T cells.
• TLR2 signaling supports tumor growth through induction of immune suppressive cytokines such as IL-10, and activation of myeloid derived suppressor cells and tumor associated macrophages (30-32) .
• TLR2 signaling also promotes tumor regression by stimulating DC activation and cross-presentation (33) , and
• the therapeutic index of an adjuvant presumably depends upon the efficiency of conjoint targeting of antigen to an APC, and activation of that APC .
• Diprovoci and TLR2 has been studied by X-ray crystallography, and its contacts with this subunit of the receptor will be reported elsewhere.
• the structure of a Diprovocim-TLRl/2 complex points to opportunities for a Diprovocim modification to incorporate immunogenic peptides, which might permit optimization of the therapeutic index by assuring that all active Diprovocim molecules are accompanied by antigen.
• Diprovocim is easy to synthesize and can be rapidly adapted to incorporate tumor-associated antigens and neoantigens. These features make it an attractive candidate for clinical development.
• mice C57BL/6J, mice were purchased from The Jackson Laboratory. (MD-2' ) mice were from RIKEN BRC . Tlr4 Ips>/lps3 , Tlr6 nt/int ,
• Ticaml lps2/lps ‘ /irak4 ot ose/otl s mice ses in generated on a pure C57BL./ 6J background by ENU mutagenesis and are described at http : / /mutagenetix . utsout ’ nwestern . edu .
• Tlrl y mice were created by CRISPR/Cas 9 gene targeting.
• Female C57BL/6J mice were super- ovulated by injection of 6.5 U pregnant mare serum gonadotropin (PMSG; Millipore) , followed by injection of 6.5 U human chorionic gonadotropin ⁇ hCG; Sigma- Aldrich) 48 hours later. The superovulated mice were subsequently mated overnight with C57BL/6J male mice.
• PMSG pregnant mare serum gonadotropin
• ⁇ hCG human chorionic gonadotropin
• fertilized eggs were collected from the oviducts and in vi tro-transcribed Cas9 mRNA ⁇ 50 ng/m ⁇ ) and Tlrl small base-pairing guide RNA (50 ng/m ⁇ ; 5 ' -CAAACCGATCGTAGTGCTGA-3 ' ; SEQ ID NO: XX) were injected into the cytoplasm or pronucleus of the embryos.
• the injected embryos were cultured in Ml 6 medium ( Sigma-AIdrich) at 37 °C in 5% C02.
• mutant mice For the production of mutant mice, two-cell stage embryos were transferred into the ampulla of the oviduct (10— 20 embryos per oviduct) of pseudo-pregnant Hsd:ICR (CD-I) female mice (Harlan Laboratories) .
• mice were maintained at the University of Texas
• Thioglycollate-elicited macrophages concentrated 4 days after i.p. injection of 2 ml BBL thioglycollate medium, brewer modified (4% wt/vol; BD Biosciences) by peritoneal lavage with 5 ml phosphate buffered saline (PBS) .
• the peritoneal macrophages were cultured in DMEM cell culture medium [DMEM containing 10% vol/vol FBS (Gemini Bio Products), 1%
• BMDCs bone morrow cells were cultured in Petri dishes in 10 ml DMEM cell culture medium containing 10 ng/ml of murine GM-CSF (R&D Systems) . On day 3 of culture, this was replaced with fresh GM-CSF medium. Loosely adherent cells were transferred to a fresh Petri dish and cultured for an additional 4 d.
• Human PBMC were purchased from Stemcell Technologies. THP-1 (ATCC) cells v/ere differentiated by treatment with 100 nM PMA (Sigma) in RPMI cell culture medium [RPMI containing 10% vol/vol FBS (Gemini Bio Products) , 1% penicillin and streptomycin (Life Technologies)] for 24 hours.
• mouse cells were for 1 hour. Unless otherwise indicated, mouse cells 'were from wild type C57BL/6J mice .
• Membranes were probed with the following antibodies: phospho-IKKa ( Ser176 ) /IKKb (Serl77), IkB , phospho-p38 (Thrl80/Tyrl82) , phospho- JNK ⁇ Thrl83/Tyrl85) , phospho-ERKl/2 (Thr202/Tyr204 ) (Cell Signaling Technology) and b-Actin (Sigma) .
• OVA ovalbumin
• SDS-PAGE purity minimum
• endotoxin levels ⁇ 1 EU/rng was purchased from Invivogen.
• serum titers of OVA- specific IgG, IgGl, or IgG2b were measured by ELISA.
• C57BL/6J male mice 'were injected i.m. 'with 100 pg OVA plus 10 mg/kg Diprovocim (n 4 mice per group) .
• naive C57BL/6J mice were killed, and splenocytes were collected.
• One-half of the splenocytes were left unpulsed, and half were pulsed with OVA257-263 peptides for 2 hours in complete medium [RPMI containing 10% vol/vol FBS, 1% penicillin and streptomycin] at 37 °C.
• the unpulsed and peptide-pulsed cells were labeled, respectively, with 0.5 mM ("low”) or 5 mM ("high”) CellTrace Violet (Invitrogen) in serum-free medium for 20 minutes.
• Equal numbers (2x10°) of CellTrace Violet high (OVA pulsed) and CellTrace Violet 1 'TM (unpulsed) cells were mixed together and injected intravenously into the immunized mice. After 48 hours, blood from treated mice was collected and subjected to flow cytometry analysis. The numbers of remaining live CellTrace Violet uign and CellTrace Violet 0 " ’ cells were determined and used to calculate the percentage of OVA peptide- pulsed target cells killed. Specific killing was defined as
• target cell lysis [1 - unimmunized ratio/immunized ratio] x 100.
• B16-OVA cells (B16F10 melanoma cells stably expressing chicken ovalbumin) were grown in DMEM containing 10% vol/vol FBS .
• a total of 2x10 J B16-OVA cells in 100 pL PBS were injected s.c. into the right flank of 8-12 week old male C57BL/6J mice to
• mice 10 mg/kg Diprovocim-1 or 2 mg/kg alum with or without OVA (100 pg) was injected i.m. into mice on the sarnie day as tumor inoculation (day 0) .
• checkpoint inhibitor anti-mPD-Ll, BioXcell
• CD4 T cells For depletion of CD4 T cells, CDS T cells, and/or NK cells, 300 gg anti-mCD4 (BioXcell), 300 gg anti-mCDS (BioXcell), 300 gg anti-mNKl .1 (BioXcell), or the three antibodies together in 200 gl saline were injected i.p. into mice on day 0, 3, 6, 9, 12, and 15 after tumor inoculation. 10 mg/ kg Diprovocim with OVA (100 gg) or vehicle was injected i.m. into mice on day 3 after tumor inoculation. Mice received a booster shot seven days after the first
• mice were also injected i.p. with 200 gg anti-mPD-Ll in 100 gl saline.
• tumors were harvested, minced and filtered through a 40-ym strainer to obtain single-cell suspensions.
• Red blood cells were lysed with RBC lysis buffer (Sigma) .
• cells were stained with a mixture of antibodies for 45 minutes, including anti-mouse CD45.2-PE or CD45.2-APC (BioLegend) , anti-mouse CD3- FITC (BD Biosciences), anti-mouse CD4-BY786 (BD Biosciences), anti-mouse CD8-BV510 (BioLegend), anti mouse CD44-PE-CF594 (BioLegend) , APC-conj ugated H- 2Kb/OVA (SIINFEKL; SEQ ID NO: XX) tetramer (Baylor College of Medicine) , anti-mouse F4/80-PE ⁇ Tonbo Bioscience) , anti-mouse CDllb-BV605 (BioLegen
• mice were injected i.rn. with 100 yg OVA mixed with vehicle or with 10 mg/kg
• DCs from draining lymph nodes and spleen were purified by Mouse Pan Dendritic Cell Isolation Kit (Miltenyi Biotech) .
• CDS T cells from OT-I transgenic mice were purified by Mouse CD8+ T Cell Isolation Kit (Miltenyi Biotech) .
• 3x10 J DCs were co-cultured with
• Li0H * H 2 0 500 mg, 11.9 mmol was added. After 2 hours, additional LiOH (470 mg, 11.2 mmol) was added, along 'with H 2 0 (10 mL) and THF (15 mL) .
• the aqueous THF reaction mixture was stirred 3 hours, warming to room temperature.
• the aqueous phase was acidified with the addition of aqueous 1 N HC1 to pH 2 (ca. 75 mL) .
• the aqueous phase ' was extracted with ethyl acetate
• aqueous phase was extracted 'with EtOAc (2 x 75 mL) , and the combined organic phases were washed with aqueous 1 N HC1 (75 mL) , saturated aqueous aHCOs (75 mL) , and saturated aqueous NaCl (50 mL) sequentially.
• the organic phase was dried over Na 2 S0 4 , filtered and concentrated. Flash column chromatography (SiCt, 50% EtOAc/hexanes ) provided 1.02 g (70%) of 5.
• Diprovocim-1 ( 3S, 3 ' S, 4 S, 4 ' S) -1 , 1 ' -
• Diprovocim-1 Diprovocim-1 could be further purified by trituration with cold (0 °C) 1:1 Et 2 0/Et0Ac ⁇ 3 *5 mL) , decanting off the liquid phase to provide 421 mg
• B7-H1 a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion.
• TLR2 ligation protects effector T cells from regulatory T-cell mediated suppression and repolarizes T helper responses following MVA-based cancer immunotherapy. Oncoimmunology 1(8) : 1271-1280.

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