NR4a transcription factor protein degraders and methods of use thereof for immunotherapy

Abstract

Targeted protein degrader compounds, and methods of use thereof for targeted protein degradation, are provided. Thus provided are compounds including a first ligand that binds to an NR4A transcription factor(s) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof that induces or increases T cell exhaustion linked to a second ligand that binds a protein that induces or increases protein degradation. In exemplary forms, the NR4A transcription factor(s) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof is an NR4A transcription factor(s) such as NR4A1, NR4A2, and / or NR4A3 and protein that induces or increases protein degradation is an E3 ligase, preferably expressed selectively in immune cells, such as KLHL6 or KBTB8. Pharmaceutical compositions including an effective amount of a provided compounds, and methods of using the provided compounds and compositions for increasing and / or prolonging immune responses, such as in subjects with cancer or an infection are also provided.

Description

[0001] NR4A TRANSCRIPTION FACTOR PROTEIN DEGRADERS AND METHODS OF USE

[0002] THEREOF FOR IMMUNOTHERAPY

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of and priority to U.S.S.N. 63 / 617,384 filed January 3, 2024, and U.S.S.N. 63 / 571,937 filed March 29, 2024, each of which is specifically incorporated by reference in its entirety.

[0005] REFERENCE TO SEQUENCE LISTING

[0006] The Sequence Listing XLM submitted as a file named “LJI2023-110-02PCT-1” created on January 3, 2025, and having a size of 15,076 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1).

[0007] FIELD OF THE INVENTION

[0008] The present invention is generally in the field of immunology and increasing immune responses.

[0009] BACKGROUND OF THE INVENTION

[0010] Tumor-infiltrating CD8+T cells (CDS TILs) differentiate to become cytotoxic effector cells after recognizing ‘foreign’ tumor-expressed antigens that are often associated with tumorspecific mutations. Over time, however, especially under conditions of persistent (chronic) antigen exposure and poor co-stimulation, their effector function is diminished by a physiological mechanism that prevents overactivation and aberrant immunopathology. Specifically, the formation of memory CD8+T cells is disrupted and cells are instead led down a divergait signaling pathway towards a state of hypo-responsiveness that has been termed ‘exhaustion’. Exhausted CD8+T cells show a progressive decrease in cytokine production (i.e. IL-2, TNF, IFNg), upregulation of diverse inhibitory surface receptors (i.e. PD-1, CTLA-4, TIM3), and an altered transcription factor (TF) landscape that directly influences downstream gene expression.

[0011] Current “checkpoint blockade” approaches to cancer immunotherapy involve treatment with antibodies targeting the coinhibitory receptors (e.g. PD-1) that are upregulated in exhausted CD8+T cells. While these therapies have shown considerable promise, they only achieved a complete cure in a small fraction of patients. The likely reason is that blocking individual inhibitory receptors, or even combinations of inhibitory receptors, does not suffice to overcome exhaustion. Similarly, although CD8+T cells expressing chimeric antigen receptors (CAR T cells) have been remarkably effective against blood cancers, they too become exhausted in solid tumors. There is a need for means of reducing T cell exhaustion.

[0012] Thus, it is an object of the invention to provide compositions and methods of use thereof for reducing T cell exhaustion.

[0013] SUMMARY OF THE INVENTION

[0014] Targeted protein degrader compounds, and methods of use thereof for targeted protein degradation, are provided. The compositions and methods can be used as a next generation immunotherapy to, for example, counter T cell exhaustion in solid tumors and / or to facilitate precise immunotherapy delivery by targeting proteins for degradation by directing them to degradation proteins such as E3 ligases, preferably expressed selectively in immune cells.

[0015] Thus provided are compounds including a first ligand that binds an NR4A transcription factors) such as NR4A1 , NR4A2, and / or NR4A3 or variant thereof linked to a second ligand that binds a protein that induces or increases protein degradation. Exemplary NR4A transcription factors) such as NR4A1, NR4A2, NR4A3 and variants include those with an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NOS: 1-8.

[0016] The ligand that binds to NR4A transcription factors) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof, for example, selected from Amodiaquine, Chloroquine, benzimidazole, SR10098 (compound 12), isoxazolo-pyridinone SR10658 (compound 7a), IP7e (analog of SR10658), imidazopyridine SR24237 (compound 3), C-DIM12 or l,l-bis(3'-indolyl)- l-(p-chlorophenyl)methane, Camptothecin, Celastrol, Isoalantolactone, Cytosporone B, ethyl 2- [2,3,4-trimethoxy-6-(l-octanoyl)phenyl]acetate (TMPA), pentyl 2-(3,5-dihydroxy-2- nonanoylphenyl)acetate (PDNPA), 5,6-dihydroxyindole (DHI), prostaglandin Al (PGA1), DHI, analogs 5-chloroindole and 5-bromoindole, 6-mercaptopurine, benzimidazole scaffolds, and Prostaglandin El and Al.

[0017] In some forms, the protein that induces or increases protein degradation is an E3 ligase, bi exemplary forms, the E3 ligase is specifically expressed in an immune cell(s). Exemplary, non-limiting E3 ligases include KLHL6, KB TBS (also referred to herein as TAKRP / KIAA1842), CBLL1, CCM2, DAZAP2, LAPTM5, OSTM1, RNF113A, RNF166, RNF4, TNFRSF1B, TRIM22, TRIM34, and TRIM4.

[0018] In some forms, the ligand that binds a protein that induces or increases protein degradation is an E3 ubiquitin ligase binding group.

[0019] In some forms, the first and second ligands are linked by a linker.

[0020] Pharmaceutical compositions including an effective amount of the compound are also provided as are methods of use thereof. For example, a method of maintaining or prolonging an immune response by an immune cell can include contacting the immune cell with an effective amount of a provided compound to increase degradation of the NR4A transcription factors) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof in the cell or a pharmaceutical composition thereof. The contacting can occur in vitro, ex vivo, or in vivo in a subject in need thereof.

[0021] A method of treating cancer can include administering a subject with cancer an effective amount of a disclosed compound or a pharmaceutical composition thereof. The cancer can include solid and / or liquid tumors.

[0022] Methods of treating an infection are also provided and can include administering a subject with an infection an effective amount of a disclosed compound or a pharmaceutical composition thereof.

[0023] Any of the in vivo methods can include oral administration of the compound or pharmaceutical composition thereof to the subject. Additionally or alternatively, the compound or pharmaceutical composition thereof can be administered locally to a site in need of a prolonged immune response in the subject

[0024] The immune cell can be, for example, a T cell, optionally a Chimeric Antigen Receptor (CAR) T cell.

[0025] Any of the methods can further include administering the subject an effective amount of T cells, optionally CAR T cells, before, during, and / or after administration of the compound or pharmaceutical composition thereof.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figures 1A-1B are plots showing the frequency of PD-1+TIM3+ in unstimulated cells among live, retrovirus reporter-positive cells (Fig. 1A) and TNF expression after 4-hour restimulation of T cells with PMA / ionomycin (Fig. IB).

[0028] Figure 2 is a bar graph showing NR4A3 expression by anti-V5 tag antibody in T cells from harvested spleens of mice treated according to Example 2.

[0029] Figure 3 is a line graph showing tumor volume measurements by digital caliper in MC38 tumor-bearing mice that were WT, heterozygous, or deficient for Nr4a3.

[0030] Figures 4A-4G show the protein abundance of FKBP12F36V-NR4A fusion proteins decreases with increasing doses of dTAG-13, and this diminishes several features of T cell exhaustion. Figure 4A is a plot showing FKBP12F36V-V5-NR4A3 protein abundance in wildtype CD8+T cells treated with increasing concentrations of dTAG-13 for 4 hours. Figure 4B is an experimental scheme: CD8+T cells were isolated from wildtype (C57BL / 6) or Nr4alflynNr4a2fl / nNr4a3"Amice and retrovirally transduced with Cre and an empty vector or an expression plasmid for FKBP12 -NR4A fusion proteins. Figure 4C is a plot showing the frequencies of TNF* IFNg"1" CD8+T cells transduced to express NR4A fusion proteins and treated or untreated with dTAG-13, then stimulated with PMA and ionomycin for 4 hr. Figure 4D is a series of flow cytometry plots. For each expression plasmid, reporter expression was gated on quartile bins (-25%) and then MFI was calculated for each marker within each bin for TNF and IFNg; representative histograms are shown. Figure 4E is a plot showing the frequencies of PD- lhiTIM3"1" CDS* cells after transduction to express NR4A fusion proteins and left untreated or treated with dTAG-13. Figure 4F is a series of representative histograms showing TIM3 expression after treatment with or without dTAG-13. Figure 4G is bar graph showing quantification of the TIM3+T cells. Data are representative of at least two experiments. Statistical significance determined by One-way ANOVA with Tukey’s correction for multiple comparisons, or Two-way ANOVA with Sidak’s correction for multiple comparisons; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0031] Figures 5A-5E show PD-1 and TIM3 expression are temporally diminished by degrading FKBP12F36V-NR4A fusion proteins. Figure 5 A is an experimental scheme for timecourse analysis of TIM3 and PD-1 expression in CD8"1" T cells were isolated from wildtype (C57BL / 6) or Nr4alfl / flNr4a2fl / flNr4a3v" mice and retrovirally transduced with Cre and an empty vector or an expression plasmid for FKBP12F36V-NR4A fusion proteins and treated (or not) with dTAG-13. Figure SB is a series of plots of TIM3+cell median fluorescence intensity (top) and TIM3"1" T cell frequency (bottom) with and without treatment with dTAG-13. Figure SC is a series of plots showing PD-1 median fluorescence intensity (top) and PD-1MT cell frequency (bottom). Figure 5D is an experimental scheme for time-course analysis of TIM3 where treatment of FKBP12F36V-NR4A3 expressing cells began on day 5. Figure 5E is a plot of TIM3+T cell frequency. Data are representative of at least two experiments. Statistical significance determined by One-way ANOVA with Tukey’s correction for multiple comparisons, or Two- way ANOVA with Sidak’s correction for multiple comparisons; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0032] Figures 6A-6I show PD-lhiTIM3+CDS* T cells display distinct tumor killing ability after degrading FKBP12F36V-NR4A fusion proteins. Figure 6A is an experimental scheme for purification and analysis of PD-lhiTIM3+CD8* T cells; CD8* T cells were isolated from OT-I mice and electroporated with gRNA / Cas9 RNP complexes targeting all three Nr4a loci and then retrovirally transduced with an expression plasmid for FKBP12F36V-NR4A fusion proteins. Figure 6B is a sorting scheme for PD-lhiTIM3+cells; Figures 6C-6G are series of plots showing PD-lhiTIM3+cells were then incubated with B16FO-OVA-mKate2 tumor cells at a 1:1 effector- to-target ratio for 96 hours, treated with DMSO or dTAG-13 (500nM) (Fig. 6C); separately, PD- lhiTIM3+cells were treated with DMSO or dTAG-13 (500nM) in vitro and analyzed by flow cytometry for PD-1 and TIM3 expression (Fig. 6D); TIM3+frequency (Fig. 6E), representative histogram of TIM3+cells (Fig. 6F), and median fluorescence intensity of TIM3 expression within TIM3+(Fig. 6G). Figure 6H is an experimental scheme: CD8+T cells were isolated from OT-I mice and electroporated with gRNA / Cas9 RNP complexes targeting individual Nr4a loci to create single-knockout OT-I T cells and used to culture with tumor cells. Figure 61 is a smes of plots showing single-Nr4aKOOT-I CD8+T cells were incubated with B16FO-OVA-mKate2 tumor cells at a 1:2 effector-to-target ratio for 96 hours. Data are representative of at least two experiments. Statistical significance determined by Two-way ANOVA with Sidak’s correction for multiple comparisons; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0033] Figures 7A-7D show generation of endogenous NR4A fusion proteins in primary CD8"1" T cells susceptible to dT AG-mediated degradation. Figure 7 A is scheme showing gRNAs were designed such that the cut site would be within a 10 bp proximity to the transcription start site of each NR4A locus. CD8+ T cells were isolated from healthy donor blood and gRNAs screened for indel frequency determined by ICE analysis. Figure 7B is a series of plots showing ICE analysis of each gRNA. Figure 7C is a series of schematics showing each HDR template used to achieve unique fluorescent reporters and in-frame knock-in of V5-FKBP12ra6Vto each NR4A locus. Figure 7D is a series of histograms. T cells that received AAV-delivered HDRTs were activated for 4 hours with CD3 / CD28 Immunocult or Dynabeads and fluorescent protein expression was measured by flow cytometry.

[0034] Figures 8A-8D show human 19-28z CD8+CAR T cells improve tumor killing by degrading endogenous NR4A proteins. Figure 8A is an illustration of CD8+human T cells electroporated with gRNAs targeting intron preceding the TRAC locus and subsequently delivered HDRT to insert a CAR redirected against human CD 19; successfill integration of the CAR leads to loss of endogenous TCR and expression of the CAR. The associated plot shows CAR expression determined using an antibody against the extracellular linker domain. Figure 8B is a graph showing CAR T cells or normal CD8+T cells incubated at a 1:1 effector-to-target ratio with AsPC-1 tumor cells engineered to express nuclear mKate2 fluorescent protein. Tumor cell growth (red counts) was monitored using Incucyte at an interval of 3 hours for a total of 39 hours; data normalized to timepoint = 0. Figure 8C is a plot showing cancer cell growth in control (DMSO) and dTAG-13 conditions. CD8+human T cells were electroporated with gRNAs targeting the TRAC locus and NR4A1 and subsequently HDRTs were delivered to express 19-28z CAR and knock-in a GFP and FKBP12F36Vinto the NR4A1 locus. Cells wore then incubated with A375-CD19-RFP tumor cells at a 1:8 effector-to-target ratio for 96 hours, treated with DMSO or dTAG-13 (500nM). Similarly, Figure 8D is a plot showing CD8+human T cells electroporated with gRNAs targeting the TRAC locus and NR4A3 and subsequently HDRTs were delivered to express 19-28z CAR and knock-in a BFP and FKBP12F36Vinto the NR4A3 locus; statistical analysis by Two-way ANOVA with Sidak correction for multiple comparison; n=4, one donor, **** p<0.0001.

[0035] Figure 9 is a heat map illustrating a clustering of E3 ligases highly expressed in immune cells.

[0036] Figure 10 is a heat map illustrating a clustering of highly expressed CBLL1 , CCM2, DAZAP2, LAPTM5, OSTM1, RNF113A, RNF166, RNF4, TNFRSF1B, TRIM22, TRIM34, and TRIM4 in immune cells.

[0037] Figures 11A-11F illustrate the characterization of E3 ligase expression across cells and tissues uncovered immune-restricted substrate-recognition domains. Figure 11B is a pair of dot plots showing the Tau score for each tissue type (left plot) and cell type (right plot). Figure 11C is a plot showing the correlation of E3 ligase expression specificity between tissue types and cell types. Figure HD is an illustration of hierarchical clustering on the z-score of normalized E3 ligase expression values and manual curation of immune cell types compared to non-immune cells. Figure HE is a tau plot re-displaying immune-specific genes selected from Fig. 6D. Figure 11F is a heatmap displaying the expression of genes of Figs. 6D and 6E across various tissues, and corresponding dot plots for KLHL6 and KBTBD8.

[0038] Figures 12A-12B illustrate KBTBD8 and KLHL6 expression in purified immune cell subsets. Figure 12A is a pair of graphs showing expression of KBTBD8 (top) and KLHL6 (bottom) in various immune cells of healthy donors from the DICE Database. Figure 12B is a pair of graphs showing protein expression of KBTBD8 and KLHL6 (measured by mass spectrometry) in various activation contexts (top) and timepoints before / after activation (bottom).

[0039] DETAILED DESCRIPTION OF THE INVENTION

[0040] Definitions

[0041] As used herein, the term “immune cell” refers to cells of the innate and acquired immune system including neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells, lymphocytes including B cells, T cells, and natural killer cells. As used herein, the term “immunogenic agent” or “immunogen” or “antigen” is capable of inducing an immunological response against itself on administration to a mammal, optionally in conjunction with an adjuvant.

[0042] As used herein, the term “carrier” refers to an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.

[0043] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0044] As used herein, the term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, dilutants or encapsulating substances which are suitable for administration to a human or other vertebrate animal.

[0045] As used herein, the term “treating” includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with a disease or disorder.

[0046] As used herein, the term “reduce”, “inhibit”, “alleviate” or “decrease” are used relative to a control. One of skill in the art would readily identify the appropriate control to use for each experiment.

[0047] As used herein, the terms “subject,” “individual,” and “patient” refer to any individual who is the target of treatment using the disclosed compositions. Hie subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. A subject can include a control subject or a test subject.

[0048] The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0049] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0050] Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. + / - 10%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 5%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - 2%; in other forms the values may range in value either above or below the stated value in a range of approx. + / - !%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0051] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0052] These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

[0053] All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. n. Compositions

[0054] A. Targeted protein degraders (TPDs)

[0055] Targeted protein degraders (TPDs) that can increase an immune response are provided. Proteolysis-targeting chimeras (PROTACs), and other systems for inducible protein degradation have countered the original view of transcription factors as “undruggable” protein targets. See also B6k6s, et al., “PROTAC targeted protein degraders: the past is prologue,” Nat Rev Drug Discov 21, 181-200 (2022). doi.org / 10.1038 / s41573-021-00371-6, and U.S. Published Applications Nos. 2019 / 0175612, 2019 / 0142961, and 2018 / 0050021 each of which are specifically incorporated by reference herein in their entireties.

[0056] TPDs are typically bivalent molecules including a ligand binding domain linked to an element that recruits degradation-inducing proteins or other machinery. Most TPDs under development take aim at overexpressed proteins (such as activated kinases or oncogenes) in cancer or other diseases; however, much like the use of small-molecule drugs, this approach is vulnerable to tumor escape by acquired mutation and resistance.

[0057] The disclosed compositions and methods address this problem by targeting protein degradation 1) as a next generation immunotherapy to NR4A transcription factors) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof e.g., in tumors and 2) to permit precise immunotherapy delivery by targeting TPDs to degradation-inducing proteins, preferably expressed selectively in immune cells.

[0058] As used herein, the term “proteolysis targeting chimera” (“PROTAC’) refers to a compound including two functional moieties, a binding moiety for a target of degradation of NR4A transcription factors) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof and a degradation moiety, tethered together by a suitable linker. PROTACs bind to the target molecule (e.g., an NR4A) and signal for degradation of the target molecule (e.g., by recruitment of the E3 ligase, resulting in ubiquitination and subsequent degradation of the target protein by the proteasome). PROTACs may inhibit the activity of the target through their binding to the target active site (e.g., as with a conventional enzyme inhibitor) or may bind to the target without significant inhibition of activity.

[0059] In some forms, the compounds described herein are PROTACs. In exemplary forms, the composition is a PROTACs where the degradation-inducing protein is an E3 ligase. However, the success of PROTACs in hijacking the ubiquitin-proteasome system (UPS) for targeted protein degradation (TPD) has motivated research into other classes of heterobifunctional molecules that depend on non-UPS pathways to degrade a protein of interest. These classes include autophagy-targeting chimeras (AUTACs) and autophagosome-tethering compounds (ATTECs). Collectively, AUTACs and ATTECs have also been categorized as macroautophagy degradation-targeting chimeras (MADTACs). Thus, although the disclosed compositions are preferably PROTACs that target an E3 ligase, alternative forms such as AUTACs and AlT'ECs are also contemplated and expressly provided.

[0060] Degradation Targets and Binders Thereof

[0061] The disclosed compositions include a ligand or other compound that binds to a degradation target protein (also referred to herein as “DP”). The DP binding group of the disclosed compositions can be e.g., a peptide, nucleic acid, or small molecule. a. Targets

[0062] The disclosed compositions typically target NR4A transcription factors) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof . The target is typically expressed by immune cells that when reduced by the disclosed compositions leads to an increased immune response mediated by the immune cell. For example, in some forms, the target NR4A transcription factors) such as NR4A1, NR4A2, and / or NR4A3 or variant thereof induces, causes, or otherwise enhances T cell exhaustion, and thus targeting the target for degradation can reduce, prevent, and / or reverse this / these process(es).

[0063] The immune cells can be, for example, T lymphocytes. In exemplary forms, the T lymphocytes are CD8+ T cells.

[0064] The immune cells can be T lymphocytes that express an o0 T cell receptor (TCR) as well as a co-receptor CD4 or CDS. Such conventional T cells are present in the peripheral blood, lymph nodes, and tissues. See, Roberts and Girardi, “Conventional and Unconventional T Cells”, Clinical and Basic Immunodermatology, pp. 85-104, (Gaspari and Tyring (ed.)), Springer London (2008).

[0065] The immune cells can be T lymphocytes that express a y5 TCR and may commonly reside in an epithelial environment such as the skin, gastrointestinal tract, or genitourinary tract. Another subset of unconventional T cells is the invariant natural killer T (NKT) cell, which has phenotypic and functional capacities of a conventional T cell, as well as features of natural killer cells (e.g., cytolytic activity). See, Roberts and Girardi, “Conventional and Unconventional T Cells”, Clinical and Basic Immunodermatology, pp. 85-104, (Gaspari and Tyring (ed.)), Springer London (2008).

[0066] 5 NR4A TF family (NR4A1, NR4A2, and NR4A3) drives the transcriptional program that leads to CDS T cell exhaustion in TILs. Triple deletion of Nr4al, 2 and 3 markedly enhanced anti-tumor responses in mouse models. In addition, NR4A TFs are needed to maintain Foxp3 expression in Tregs, and deletion of Nr4a TFs abrogates Treg suppressive functions. Deleting Nr4al and Nr4a2 specifically in Tregs of tumor-bearing mice resulted in slowed tumor growth,

[0067] 10 with increased CD8+TIL infiltration and reduced tumor burden.

[0068] NR4A deletion in other immune cells may also have potential benefits for anti-tumor responses. In CD4+T cells, NR4A deletion led to increased cytokine expression and proliferation. Nr4al deletion in macrophages yielded more proinflammatory Ml type macrophages with higher expression of TNF and iNOS. Moreover, TLR signaling induced

[0069] 15 NR4A2, which binds the Arginase 1 promoter and upregulates anti-inflammatory (M2-related) genes, indicating that both NR4A1 and NR4A2 deletion would skew macrophage polarization towards proinflammatory Ml type macrophages. Patients with Ml -skewed macrophages in tumors typically show more favorable outcomes.

[0070] Sequences for NR4A transcription factors are known in the art, and can serve as the

[0071] 20 reference sequence(s) for targeted degradation. Non-limiting examples include the human NR4A1, NR4A2, and NR4A3 proteins. Thus, in some forms, the compositions target for degradation of one or more isoforms of one or more of human NR4A1 , NR4A2, and NR4A3.

[0072] See, e.g., UniProt Accession No. P22736 • NR4A1_HUMAN, three isoforms:

[0073] 25

[0074] 30

[0075] 11 5

[0076] 10

[0077] 15

[0078] 20

[0079] 25

[0080] 30

[0081] Consensus sequences are SEQ ID NO:1 for NR4A1, SEQ ID NO:4 for NR4A2, and SEQ

[0082] ID NO:6 for NR4A3, and the remaining proteins (i.e., SEQ ID NOS:2, 3, 5, 7, and 8, are the result of alternatively splicing. In some forms, the disclosed compositions target for degradation 13 is one or more proteins including the amino acid sequence any one of SEQ ID NOS:l-8, or a variant thereof with at least 70%, 75%, 80%, 85%, 90%, or 95% sequence identity thereto. b. Binders

[0083] Exemplary ligands and other compounds that bind to DPs are also provided for use in the

[0084] 5 disclosed protein degrader compositions.

[0085] In exemplary forms, the ligand or other molecules binds to a NR4A transcription factor or variant thereof. Exemplary ligands are provided in Table 1.

[0086] Table 1 - Exemplary NR4A Binders

[0087] 14

[0088]

[0089] Any of the compounds of Table 1 can be used as a DP binder in the disclosed compositions.

[0090] Select structures for the compounds of Table 1 are:

[0091] 5

[0092] 16

[0093]

[0094] 2. Degradation-inducing Proteins and Binders Thereof

[0095] The disclosed protein degrader compositions include a ligand or other compound that binds to a degradation-inducing protein. The degradation-inducing protein binding group of the disclosed compositions can be e.g., a peptide, nucleic acid, or small molecule. In some forms, the protein that induces or increases protein degradation is specifically expressed in immune cell(s) and / or lymphoid tissue. Specifically expressed means the protein that induces or increases protein degradation has increased expression relative to one or more or all non-immune cells and / or lymphoid tissue, or is uniquely expressed in the immune cell(s) and / or lymphoid tissue. See e.g., the experiments below and the associate Figures and their descriptions for nonlimiting examples. a. Degradation Protein Targets

[0096] The degradation-inducing protein is most typically an E3 ubiquitin ligase. E3 ubiquitin ligases (of which over 600 are known in humans) confer substrate specificity for ubiquitination. There are known ligands which bind to these ligases. Thus, the degradation-inducing protein binding group can be an E3 ubiquitin ligase binding group (as referred to as “E3LB”).

[0097] Any E3 ligase can serve as a target for the disclosed composition.

[0098] In exemplary forms, the composition target is an E3 ligase that is expressed, and preferrable has increased or specific expression, in immune cells. Examples include, but are not limited to, KLHL6, KBTB8, CBLL1, CCM2, DAZAP2, LAPTM5, OSTM1, RNF113A, RNF166, RNF4, TNFRSF1B, TRIM22, TRIM34, and TRIM4.

[0099] Other specific E3 ubiquitin ligases include: von Hippel-Lindau (VHL); cereblon, XIAP, E3A; MDM2; Anaphase-promoting complex (APC); UBR5 (EDD1); SOCS / BC- box / eloBC / CUL5 / RING; LNXpSO; CBX4; CBLL1; HACE1; HECTD1; HECTD2; HECTD3; HECW1; HECW2; HERC1; HERC2; HERC3; HERC4; HUWE1; ITCH; NEDD4; NEDD4L; PPIL; PRPF19; PIAS1; PIAS2; PIAS3; PIAS4; RANBP2; RNF4; RBX1; SMURF1; SMURF2; STUB1; TOPOR5; TRIP12; UBE3A; UBE3B; UBE3C; UBE4A; UBE4B; UBOX5; UBR5; WWP1; WWP2; Parkin; A20 / TNFAIP3; AMFR / gp78; ARA54; beta-TrCPl / BTRC; BRCA1; CBL; CHIP / STUB1; E6; E6AP / UBE3A; F-box protein 15 / FBXO15; FBXW7 / Cdc4; GRAII / RNF128; HOIP / RNF31; cIAP-l / HIAP-2; cIAP-2 / HIAP-l; cIAP (pan); ITCH / AIP4; KAP1; MARCH8; Mind Bomb 1 / MIB1; Mind Bomb 2 / MIB2; MuRFl / TRIM63; NDFEPl; NEDD4; NleL; Parkin; RNF2; RNF4; RNF8; RNF168; RNF43; SART1; Skp2; SMURF2; TRAF-1; TRAF-2; TRAF-3; TRAF-4; TRAF-5; TRAF-6; TRIMS; TRIM21; TRIM32; UBR5; andZNRF3.

[0100] In some forms, the E3 ligase is selected from Tables 2-16, which lists certain E3 ligases.

[0101] Table 2

[0102] 21

[0103]

[0104]

[0105]

[0106]

[0107] 35

[0108]

[0109]

[0110] Table 16

[0111] The Examples below illustrate that KLHL6 (listed above in Table 14) and KBTB8 (also

[0112] 5 listed above in Table 14, using the synonym TAKRP / KIAA1842) are selectively expressed in immune tissues and / or cells. Thus, in some exemplary forms, the E3 ligase is KLHL6 or

[0113] KBTB8.

[0114] Sequences for E3 ligases, including those provided herein such as KLHL6 and KBTB8, are known in the art, and in many cases accession numbers are provided in the Tables above.

[0115] 10 In a particular form, the E3 ligase has the amino acid sequence

[0116] 15

[0117] 20

[0118] Accession No. Q8WZ60 • KLHL6_HUMAN), or a variant thereof with at least 70, 75, 80, 85,

[0119] 90, 95, or more percent sequence identity thereto.

[0120] 41 In another particular form, the E3 ligase has the amino acid sequence

[0121] 5

[0122] 10 sequence identity thereto. b. E3 Ligase Binders

[0123] 15 Exemplary E3LBs are also provided.

[0124] In some forms, the Ligase ligand is a Von HippeLLindau E3 ubiquitin ligase binding moiety (e.g., hydroxyproline, hydroxyproline derivatives, or binding moieties described in U.S. Patent Application Pub. No. 2014 / 03022523 (herein incorporated by reference in its entirety)), a cereblon E3 ubiquitin ligase binding moiety (e.g., thalidomide, lenalidomide, pomalidomide,

[0125] 20 analogs thereof, isosteres thereof, derivatives thereof, or binding moieties described in U.S. Patent Application Publication US 2015 / 0291562 (herein incorporated by reference in its entirety)), a mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase binding moiety (e.g., binding moieties described in U.S. patent application Ser. No. 15 / 206,497 (herein incorporated by reference in its entirety)), or an LAP E3 ubiquitin ligase binding moiety. Suitable ligands for

[0126] 25 binding the aforementioned E3 ubiquitin ligases, as well as other known E3 ubiquitin ligases, are understood in the field and described in, for example, U.S. Pub. Nos. 2015 / 0291562, 2014 / 0356322, 2018 / 0256586, 2018 / 0228907, 2018 / 0193470, 2018 / 0179183, 2018 / 0134684; 2017 / 0327469; and US Patent No. 11,485,743 herein incorporated by reference in their entireties. The compounds and formulas within the scope of forms herein are not limited to

[0127] 30 specific ligase ligand structures described herein, or incorporated by reference, but include ligase ligands understood in the field.

[0128] A particular E3 ubiquitin ligase is von Hippel-Lindau (VHL) tumor suppressor, the substrate recognition subunit of the E3 ligase complex VCB, which also consists of elongins B and C, CuL and Rbxl. The primary substrate of VHL is Hypoxia Inducible Factor la (HIF-la), 4Z a transcription factor that upregulates genes such as the pro-angiogenic growth factor VEGF and the red blood cell inducing cytokine erythropoietin in response to low oxygen levels. Compounds that bind VHL may be hydroxyproline compounds such as those disclosed in WO2013 / 106643, and other compounds described in US2016 / 0045607, WO2014187777, US20140356322, and U.S. Pat No. 9,249,153.

[0129] Another particular E3 ubiquitin ligase is MDM2. Examples of small molecular binding compounds for MDM2 include the “nutlin” compounds, e.g., nutlin 3a and nutlin 3, having the structure:

[0130] Also included as MDM2 binding compounds are those described in WO2012 / 121361 ;

[0131] W02014 / 038606; W02010 / 082612; W02014 / 044401; W02009 / 151069; W02008 / 072655;

[0132] W02014 / 100065; W02014 / 100071; WO2014 / 123882; WO2014 / 120748; WO2013 / 096150;

[0133] W02015 / 161032; WO2012 / 155066; WO2012 / 065022; WO2011 / 060049; W02008 / 036168;

[0134] W02006 / 091646; WO2012 / 155066; W02012 / 065022; WO2011 / 153509; WO2013 / 049250;

[0135] WO2014 / 151863; W02014 / 130470; WO2014 / 134207; WO2014 / 200937; WO2015 / 070224;

[0136] WO2015 / 158648; WO2014 / 082889; WO2013 / 178570; WO2013 / 135648; WO2012 / 116989;

[0137] WO2012 / 076513; WO2012 / 038307; WO2012 / 034954; W02012 / 022707; WO2012 / 007409;

[0138] WO2011 / 134925; WO2011 / 098398; WO2011 / 101297; WO2011 / 067185; WO2011 / 061139;

[0139] WO2011 / 045257; W02010 / 121995; W02010 / 091979; W02010 / 094622; W02010 / 084097;

[0140] W02009 / 115425; W02009 / 080488; W02009 / 077357; W02009 / 047161A1;

[0141] WO2008 / 141975A1; W02008 / 141917A1; WO2008 / 125487A1; W02008 / 034736A2;

[0142] W02008 / 055812A1; W02007 / 104714A1; W02007 / 104664A1; W02007 / 082805A1;

[0143] W02007 / 063013A1; W02006 / 136606A2; W02006 / 097261A1; WO2005 / 123691A1;

[0144] W02005 / 110996A1 ; W02005 / 003097A1 ; W02005 / 002575A1; W02004 / 080460A1 ;

[0145] W02003 / 051360A1; W02003 / 051359A1; WO 1998 / 001467; WO2011 / 023677;

[0146] WO2011 / 076786; WO2012 / 066095; WO2012 / 175487; WO2012 / 175520; WO2012 / 176123;

[0147] W02013 / 080141; W02013 / 111105; WO2013 / 175417; WO2014 / 115080; WO2014 / 115077; WO2014 / 191896; WO2014 / 198266; WO2016 / 028391A9; WO2016 / 028391A2;

[0148] WO2016 / 026937; W02016 / 001376; WO2015 / 189799; WO2015 / 155332A1;

[0149] W02015 / 004610A8; W02013 / 105037A1; WO2012 / 155066A3; WO2012 / 155066A2;

[0150] WO2012 / 033525A3; WO2012 / 047587A2; WO2012 / 033525A2; W02011 / 106650A3;

[0151] W02011 / 106650A2; W02011 / 005219A1; W02010 / 058819A1; W02010 / 028862A1;

[0152] W02009 / 037343A1; W02009 / 037308A1; W02008 / 130614A3; W02009 / 019274A1;

[0153] W02008 / 130614A2; W02008 / 106507A3; W02008 / 106507A2; W02007 / 107545A1;

[0154] W02007 / 107543A1; W02006032631A1; W02000 / 015657A1; WO 1998 / 001467A2;

[0155] WO1997 / 009343A3; WO1997 / 009343A2; WO1996 / 002642A1; US2007 / 0129416; Med. Chem.

[0156] Utt, 2013, 4, 466-469; J. Med. Chem., 2015, 58, 1038-1052; Bioorg. Med. Chem. Utt. 25 (2015) 3621-3625; Bioorg. Med. Chem. Utt. 16 (2006) 3310-3314. Further specific examples of small molecular binding compounds for MDM2 contemplated for use with a PAC include RG7112, RG7388, MI 773 / SAR 405838, AMG 232, DS-3032b, RO6839921, RO5045337, RO5503781, Idasanudin, CGM-097, and MK-8242.

[0157] Another particular E3 ubiquitin ligase is X-linked inhibitor of apoptosis (XIAP). XIAP is a protein that stops apoptotic cell death. Deregulation of XIAP has been associated with cancer, neurodegenerative disorders and autoimmunity. In the development of lung cancer, the overexpression of XIAP inhibits caspases. In developing prostate cancer, XIAP is one of four IAPS overexpressed in the prostatic epithelium. Mutations in the XIAP gene can result in a severe and rare type of inflammatory bowel disease. Defects in the XIAP gene can also result in an extremely rare condition called X-linked lymphoproliferative disease. Degradation of XIAP can enhance apoptosis by preventing XIAP from binding to caspases. This allows normal caspase activity to proceed.

[0158] Examples of small molecular binding compounds for XIAP include compounds disclosed in U.S. Pat No. 9,096,544; WO 2015187998; WO 2015071393; U.S. Pat. Nos. 9,278,978; 9,249,151 ; US 20160024055; US 20150307499; US 20140135270; US 20150284427; US

[0159] 20150259359; US 20150266879; US 20150246882; US 20150252072; US 20150225449; U.S.

[0160] Pat. No. 8,883,771, J. Med. Chem., 2015, 58(16) 6574-6588 and Small-molecule Pan-IAP Antagonists: A Patent Review (2010) Expert Opin Ther Pat; 20: 251-67 (Flygare & Fairbrother).

[0161] Specific compounds include all the tetrahydro-benzodiazinone compounds of the following formula:

[0162] as disclosed in WO 2015 / 071393. Other small molecular binding compounds for XIAP include AEG35156, Embelin, TWX006 and TWX024. When an XIAP bindig moeity is used as part of a PROTAC, the XIAP binding moiety can bind to the BIR2 or BIR3 domain of XIAP or both.

[0163] Another particular E3 ubiquitin ligase is cereblon. Cereblon is a protein that forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), Cullin-4A (CUL4A), and regulator of cullins 1 (ROC 1). Uris complex ubiquitinates a number of other proteins. Cereblon ubquitination of target proteins results in increased levels of fibroblast growth factor 8 (FGF8) and fibroblast growth factor 10 (FGF10). FGF8 in turn regulates a number of developmental processes, such as limb and auditory vesicle formation. In the absence of cereblon, DDB1 forms a complex with DDB2 that functions as a DNA damage-binding protein.

[0164] Thalidomide, lenalidomide, pomalidomide and analogs thereof are known to bind to cereblon. The crystal structure of cereblon with thalidomide and derivative compounds are described in US2015 / 0374678. Other small molecule compounds that bind to cereblon are also known, e.g., the compounds disclosed as an in US2016 / 0058872 and US2015 / 0291562.

[0165] 3. Linker

[0166] The E3LB and DP groups of PROTACs as described herein are connected and can optionally be connected with a linker (also referred to herein as “L”). The linker group of the disclosed compositions can be e.g., a peptide, nucleic acid, or small molecule.

[0167] In certain forms, the linker group L is a group including one or more covalently connected structural units of A (e.g., -Ai . . . Aq-), wherein Ai is a group coupled to at least one of a E3LB, a DP, or a combination thereof. In certain forms, Ai finks a E3LB, a DP, or a combination thereof directly to another E3LB, DP, or combination thereof. In other forms, Ai links a EL3B, a DP, or a combination thereof indirectly to another E3LB, DP, or combination thereof through Aq.

[0168] In certain forms, Ai to Aqare, each independently, a bond, CRLbR"’, O, S, SO, SO2, NR1*, SChNRLb, SONR1*, CONR1*, NRUCONRU, NRLcSO?NRLd, CO, CR1*— CR"1, C=C, SiRLaRLb, P(O)RLa, P(O)ORLa, NRLcC(=NCN)NRLd, NRUC(=NCN), NRLcC(=CNO2)NRLd, C3- ii cycloalkyl optionally substituted with 0-6 R^* and / or RLbgroups, C3-11 heterocyclyl optionally substituted with 0-6 RLband / or R1^ groups, aryl optionally substituted with 0-6 R1^ and / or R1^ groups, heteroaryl optionally substituted with 0-6 Ruand / or R1-6groups, where Ruor R1^, each independently, can be linked to other A groups to form cycloalkyl and / or heterocyclyl moiety which can be further substituted with 0-4 R1-6groups; wherein RLa, RLb, RLb, RLdand RLeare, each independently, H, halo, C1-8alkyl, OC1-8 alkyl, SCi-g alkyl, NHC1-8alkyl, N(ci-s alkylh, Call cycloalkyl, aryl, heteroaryl, C3-11 heterocyclyl, OC1-8cycloalkyl, SC1-8cycloalkyl, NHC1-8cycloalkyl, N(C1-8cycloalkyl)2, N(C1-8cycloalkylXC1-8alkyl), OH, NHz, SH, SO2C1-8 alkyl, P(O)(OC1-8alkyl)(C1-8alkyl), P(OXOCi-8 alkylfc, CC— C1-8alkyl, CCH, CH=CH(C1-8alkyl), C(C1-8alkyl)=CH(Ci-8 alkyl), C(C1-8alkyl)=C(Ci-8 alkyl)2, Si(OH)3, Si(C1-8alkyl)3, Si(OH)(C1-8alkyl)2, COC1-8alkyl, CO2H, halogen, CN, CF3, CHF2, CH2F, NO2, SFs, SO2NHC1-8 alkyl, SO22N(C1-8alkyl)2, SONHC1-8alkyl, SON(C1-8alkyl)2, CONHC1-8alkyl, CON(C1-8aJkyl)2, N(Ci- 8 alkyl)CONH(Ci-8 alkyl), N(C1-8alkyl)C0N(Ci4 alkyl)2, NHCONH(C1-8alkyl), NHCON(CI-8

[0169] In certain forms, q is an integer greater than or equal to 0. In certain forms, q is an integer greater than or equal to 1.

[0170] In certain forms, e.g., where q is greater than 2, Aq is a group which is connected to an E3LB moiety, and Ai and Aq are connected via structural units of A (number of such structural units of A: q-2).

[0171] In certain forms, e.g., where q is 2, Aqis a group which is connected to Ai and to an E3LB moiety.

[0172] Ai is a group which is connected to an E3LB moiety and a DP moiety.

[0173] In additional forms, q is an integer from 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10.

[0174] In certain forms, the linker (L) is selected from the group consisting of:

[0175]

[0176] In additional forms, the linker group is an optionally substituted (poly)ethyleneglycol having between 1 and about 100 ethylene glycol units, between about 1 and about 50 ethylene glycol units, between 1 and about 25 ethylene glycol units, between about 1 and 10 ethylene glycol units, between 1 and about 8 ethylene glycol units and 1 and 6 ethylene glycol units, between 2 and 4 ethylene glycol units, or optionally substituted alkyl groups interdispersed with optionally substituted, O, N, S, P or Si atoms. In certain forms, the linker is substituted with an aryl, phenyl, benzyl, alkyl, alkylene, or heterocycle group. In certain forms, the tinker may be asymmetric or symmetrical.

[0177] In any of the forms of the compounds described herein, the linker group may be any suitable moiety as described herein. In one form, the linker is a substituted or unsubstituted polyethylene glycol group ranging in size from about 1 to about 12 ethylene glycol units, between 1 and about 10 ethylene glycol units, about 2 about 6 ethylene glycol units, between about 2 and 5 ethylene glycol units, between about 2 and 4 ethylene glycol units.

[0178] Although the E3LB group and DP group may be covalently linked to the tinker group through any group which is appropriate and stable to the chemistry of the tinker. The tinker is independently covalently bonded to the E3LB group and the DP group preferably through an amide, ester, thioester, keto group, carbamate (urethane), carbon or ether, each of which groups may be inserted anywhere on the E3LB group and DP group to provide maximum binding of the E3LB group on the ubiquitin ligase and the DP group on the target protein to be degraded. In certain aspects where the DP group is an E3LB group, the target protein for degradation may be the ubiquitin ligase itself. In certain aspects, the tinker may be linked to an optionally substituted alkyl, alkylene, alkene or alkyne group, an aryl group or a heterocyclic group on the E3LB and / or DP groups. It is noted that an E3LB group or a DP group may need to be derivatized to make a chemical functional group that is reactive with a chemical functional group on the linker. Alternatively, the linker may need to be derivatized to include a chemical functional group that can react with a functional group found on E3LB and / or DP.

[0179] L can also be represented by the formula:

[0180] Where Z is a group which links E3LB to X; and X is a group linking Z to group DP.

[0181] In forms, Z is absent (a bond), — (CH2)i-0, — (CH2)i-S, — (CH2)i-N— R, a (CH2)i-

[0182] X1Y1 group wherein X1Y1 forms an amide group, or a urethane group, ester or thioester group, or a where, each R is H, or a C1-C3 alkyl, an alkanol group or a heterocycle (including a water soluble heterocycle, preferably, a morpholino, piperidine or piperazine group to promote water solubility of the linker group); each Y is independently a bond, O, S or N — R; and each i is independently 0 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5;

[0183] In forms, X is a j is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, I to 8, I to 6, 1, 2, 3, 4 or 5; k is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1, 2, 3, 4 or 5; preferably k is 1, 2, 3, 4, or 5; m' is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, 1 to 8, 1 to 6, l, 2, 3, 4 or 5; n is 1 to 100, 1 to 75, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 2 to 35, 3 to 30, 1 to 15, 1 to 10, to 8, 1 to 6, 1, 2, 3, 4 or 5;

[0184] X1is O, S or N — R, preferably O;

[0185] Y is the same as above; and CON is a connector group (which may be a bond) which connects Z to X, when present in the tinker group.

[0186] In forms, CON is a bond (absent), a heterocycle including a water soluble heterocycle such as a piperazinyl or other group or a group, where X2is O, S, NR4, S(O), S(O)2, — S(O)2O, — OS(O)2, or OS(O)2O;

[0187] X3is O, S, CHR4, NR4; and

[0188] R is H or a C1-C3 alkyl group optionally substituted with one or two hydroxyl groups, or a pharmaceutically acceptable salt, enantiomer or stereoisomer thereof.

[0189] In alternative exemplary aspects, the tinker group is a (poly)ethyleneglycol having between 1 and about 100 ethylene glycol units, between about 1 and about 50 ethylene glycol units, between 1 and about 25 ethylene glycol units, between about 1 and 10 ethylene glycol units, between 1 and about 8 ethylene glycol units and 1 and 6 ethylene glycol units, between 2 and 4 ethylene glycol units.

[0190] In forms, CON is

[0191] Although the E3LB group and DP group may be covalently linked to the linker group through any group which is appropriate and stable to the chemistry of the linker, in exemplary aspects, the linker is independently covalently bonded to the E3LB group and the DP group through an amide, ester, thioester, keto group, carbamate (urethane) or ether, each of which groups may be inserted anywhere on the E3LB group and DP group to allow binding of the E3LB group to the ubiquitin ligase and the DP group to the target protein to be degraded. In other words, as shown herein, the linker can be designed and connected to E3LB and DP to minimize, eliminate, or neutralize any impact its presence might have on the binding of E3LB and DP to their respective binding partners. In certain aspects, the targeted protein for degradation may be an ubiquitin ligase.

[0192] Additional linkers L are disclosed in US Application Publication Nos. 2016 / 0058872; 2016 / 0045607; 2014 / 0356322; and 2015 / 0291562, and WO2014 / 063061.

[0193] B. Formulations

[0194] Formulations including the disclosed compositions and for use in the disclosed methods are provided.

[0195] 1. Pharmaceutical Compositions

[0196] Pharmaceutical compositions including the disclosed composition are provided.

[0197] Pharmaceutical compositions can be for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV), intradermal, or subcutaneous injection), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.

[0198] In some forms, the compositions are administered systemically, for example, by oral or intravenous or intraperitoneal administration, in an amount effective for delivery of the compositions to targeted cells.

[0199] The compositions can also be administered by intramuscular, intradermal, subcutaneous injection or infusions, or intravenous injection or infusion, or by intranasal delivery. In certain forms, the compositions are administered locally, for example by injection directly into a site to be treated. In some forms, the compositions are injected or otherwise administered directly to one or more tumors. Typically, local injection causes an increased localized concentration of the compositions which is greater than that which can be achieved by systemic administration.

[0200] In some forms, the compositions are delivered by using a catheter or syringe. Other means of delivering such compositions include using infusion pumps (for example, from Alza Corporation, Palo Alto, Calif.) or incorporating the compositions into polymeric implants (see, for example, P. Johnson and J. G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England: Ellis Horwood Ltd., 1987), which can effect a sustained release of the composition to the immediate area of the implant.

[0201] As further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired.

[0202] The compositions can be formulated for immediate release, extended release, or modified release. A delayed release dosage form is one that releases a drug (or drugs) at a time other than promptly after administration. An extended release dosage form is one that allows at least a twofold reduction in dosing frequency as compared to that drug presented as a conventional dosage form (e.g. as a solution or prompt drug-releasing, conventional solid dosage form). A modified release dosage form is one for which the drug release characteristics of time course and / or location are chosen to accomplish therapeutic or convenience objectives not offered by conventional dosage forms such as solutions, ointments, or promptly dissolving dosage forms. Delayed release and extended release dosage forms and their combinations are types of modified release dosage forms.

[0203] Formulations are prepared using a pharmaceutically acceptable “carrier” composed of materials that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The “carrier” is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. The term “carrier” includes but is not limited to diluents, binders, lubricants, desintegrators, fillers, and coating compositions.

[0204] “Carrier” also includes all components of the coating composition which may include plasticizers, pigments, colorants, stabilizing agents, and glidants. The delayed release dosage formulations may be prepared as described in references such as “Pharmaceutical dosage form tablets”, eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, and “Pharmaceutical dosage forms and drag delivery systems”, 6thEdition, Ansel et.al., (Media, PA: Williams and Wilkins, 1995) which provides information on carriers, materials, equipment and process for preparing tablets and capsules and delayed release dosage forms of tablets, capsules, and granules.

[0205] The compositions can be administered to a subject with or without the aid of a delivery vehicle. Appropriate delivery vehicles for the compounds are known in the art and can be selected to suit the particular active agent. For example, in some forms, the active agent(s) is incorporated into or encapsulated by a nanoparticle, microparticle, micelle, synthetic lipoprotein particle, or carbon nanotube. For example, the compositions can be incorporated into a vehicle such as polymeric microparticles which provide controlled release of the active agent(s). In some forms, release of the composition is controlled by diffusion of the active agent(s) out of the microparticles and / or degradation of the polymeric particles by hydrolysis and / or enzymatic degradation.

[0206] Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives. Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials for drag containing microparticles or particles. Other polymers include, but are not limited to, polyanhydrides, poly (ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybut rate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof. In some forms, both agents are incorporated into the same particles and are formulated for release at different times and / or over different time periods. For example, in some forms, one of the agents is released entirely from the particles before release of the second agent begins. In other forms, release of the first agent begins followed by release of the second agent before the all of the first agent is released. In still other forms, both agents are released at the same time over the same period of time or over different periods of time. a. Formulations for Parenteral Administration

[0207] Compounds and pharmaceutical compositions thereof can be administered in an aqueous solution, by parenteral injection. The formulation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of the active agent(s) and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. b. Oral Immediate Release Formulations

[0208] Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the art. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.

[0209] Examples of suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name Eudragit® (Roth Pharma, Westerstadt, Germany), Zein, shellac, and polysaccharides.

[0210] Additionally, the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.

[0211] Optional pharmaceutically acceptable excipients present in the drug-containing tablets, beads, granules or particles include, but are not limited to, diluents, binders, lubricants, disintegrants, colorants, stabilizers, and surfactants. Diluents, also termed "fillers," are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules. Suitable diluents include, but are not limited to, , dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powder sugar. Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms. Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydorxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, polyacrylic acid / polymethacrylic acid and polyvinylpyrrolidone.

[0212] Lubricants are used to facilitate tablet manufacture. Examples of suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.

[0213] Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone XL from GAP Chemical Corp).

[0214] Stabilizers are used to inhibit or retard drug decomposition reactions which include, by way of example, oxidative reactions.

[0215] Surfactants may be anionic, cationic, amphoteric or nonionic surface active agents. Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions. Examples of anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate. Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene and coconut amine. Examples of nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, soibitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide. Examples of amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-.beta.- iminodipropionate, myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

[0216] If desired, the tablets, beads granules or particles may also contain minor amount of nontoxic auxiliary substances such as wetting or emulsifying agents, dyes, pH buffering agents, and preservatives. c. Extended release dosage forms

[0217] The extended release formulations are generally prepared as diffusion or osmotic systems, for example, as described in “Remington - The science and practice of pharmacy” (20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000). A diffusion system typically consists of two types of devices, reservoir and matrix, and is well known and described in the art The matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form. The three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds. Plastic matrices include, but not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene. Hydrophilic polymers include, but are not limited to, methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and carbopol 934, polyethylene oxides. Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate.

[0218] Alternatively, extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form. In the latter case, the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.

[0219] The devices with different drug release mechanisms described above could be combined in a final dosage form comprising single or multiple units. Examples of multiple units include multilayer tablets, capsules containing tablets, beads, granules, etc.

[0220] An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.

[0221] Extended release tablets containing hydrophilic polymers are prepared by techniques commonly known in the art such as direct compression, wet granulation, or dry granulation processes. Their formulations usually incorporate polymers, diluents, binders, and lubricants as well as the active pharmaceutical ingredient. The usual diluents include inert powdered substances such as any of many different kinds of starch, powdered cellulose, especially crystalline and microcrystalline cellulose, sugars such as fructose, mannitol and sucrose, grain flours and similar edible powders. Typical diluents include, for example, various types of starch, lactose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar. Powdered cellulose derivatives are also useful. Typical tablet binders include substances such as starch, gelatin and sugars such as lactose, fructose, and glucose. Natural and synthetic gums, including acacia, alginates, methylcellulose, and polyvinylpyrrolidine can also be used. Polyethylene glycol, hydrophilic polymers, ethylcellulose and waxes can also serve as binders. A lubricant is necessary in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant is chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and hydrogenated vegetable oils.

[0222] Extended release tablets containing wax materials are generally prepared using methods known in the art such as a direct blend method, a congealing method, and an aqueous dispersion method. In a congealing method, the drug is mixed with a wax material and either spray- congealed or congealed and screened and processed. d. Delayed release dosage forms

[0223] Delayed release formulations are created by coating a solid dosage form with a film of a polymer which is insoluble in the acid environment of the stomach, and soluble in the neutral environment of small intestines.

[0224] The delayed release dosage units can be prepared, for example, by coating a drug or a drug-containing composition with a selected coating material. The drug-containing composition may be, e.g., a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coaled core" dosage form, or a plurality of drug-containing beads, particles or granules, for incorporation into either a tablet or capsule. Exemplary coating materials include bioerodible, gradually hydrolyzable, gradually water-soluble, and / or enzymatically degradable polymers, and may be conventional "enteric" polymers. Enteric polymers, as will be appreciated by those skilled in the art, become soluble in the higher pH environment of the lower gastrointestinal tract or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon. Suitable coating materials for effecting delayed release include, but are not limited to, cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropylmethyl cellulose phthalate, methylcellulose, ethyl cellulose, cellulose acetate, cellulose acetate phthalate, cellulose acetate trimellitate and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and / or ethyl methacrylate, and other methacrylic resins that are commercially available under the tradename Eudragit®. (Rohm Pharma; Westerstadt, Germany), including Eudragit®. L30D-55 and LI 00-55 (soluble at pH 5.5 and above), Eudragit®. L-100 (soluble at pH 6.0 and above), Eudragit®. S (soluble at pH 7.0 and above, as a result of a higher degree of esterification), and Eudragits®. NE, RL and RS (water-insoluble polymers having different degrees of permeability and expandability); vinyl polymers and copolymers such as polyvinyl pyrrolidone, vinyl acetate, vinylacetate phthalate, vinylacetate cratonic acid copolymer, and ethylene- vinyl acetate copolymer; enzymatically degradable polymers such as azo polymers, pectin, chitosan, amylose and guar gum; zein and shellac. Combinations of different coating materials may also be used. Multi-layer coatings using different polymers may also be applied.

[0225] The preferred coating weights for particular coating materials may be readily determined by those skilled in the art by evaluating individual release profiles for tablets, beads and granules prepared with different quantities of various coating materials. It is the combination of materials, method and form of application that produce the desired release characteristics, which one can determine only from the clinical studies.

[0226] The coating composition may include conventional additives, such as plasticizers, pigments, colorants, stabilizing agents, glidants, etc. A plasticizer is normally present to reduce the fragility of the coating, and will generally represent about 10 wt % to 50 wt. % relative to the dry weight of the polymer. Examples of typical plasticizers include polyethylene glycol, propylene glycol, triacetin, dimethyl phthalate, diethyl phthalate, dibutyl phthalate, dibutyl sebacate, triethyl citrate, tributyl citrate, triethyl acetyl citrate, castor oil and acetylated monoglycerides. A stabilizing agent is preferably used to stabilize particles in the dispersion. Typical stabilizing agents are nonionic emulsifiers such as sorbitan esters, polysorbates and polyvinylpyrrolidone. Glidants are recommended to reduce sticking effects during film formation and drying, and will generally represent approximately 25 wt. % to 100 wt % of the polymer weight in the coating solution. One effective glidant is talc. Other glidants such as magnesium stearate and glycerol monostearates may also be used. Pigments such as titanium dioxide may also be used. Small quantities of an anti-foaming agent such as a silicone (e.g., simethicone), may also be added to the coating composition.

[0227] Methods of manufacturing

[0228] As will be appreciated by those skilled in the art and as described in the pertinent texts and literature, a number of methods are available for preparing drug-containing tablets, beads, granules or particles that provide a variety of drug release profiles. Such methods include, but are not limited to, the following: coating a drug or drug-containing composition with an appropriate coating material, typically although not necessarily incorporating a polymeric material, increasing drug particle size, placing the drug within a matrix, and forming complexes of the drug with a suitable complexing agent.

[0229] The delayed release dosage units may be coated with the delayed release polymer coating using conventional techniques, e.g., using a conventional coating pan, an airless spray technique, fluidized bed coating equipment (with or without a Wurster insert). For detailed information concerning materials, equipment and processes for preparing tablets and delayed release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6.sup.th Ed. (Media, PA: Williams & Wilkins, 1995).

[0230] An method for preparing extended release tablets is by compressing a drug-containing blend, e.g., blend of granules, prepared using a direct blend, wet-granulation, or dry-granulation process. Extended release tablets may also be molded rather than compressed, starting with a moist material containing a suitable water-soluble lubricant. However, tablets are preferably manufactured using compression rather than molding. An exemplary method for forming extended release drug-containing blend is to mix drug particles directly with one or more excipients such as diluents (or fillers), binders, disintegrants, lubricants, glidants, and colorants. As an alternative to direct blending, a drug-containing blend may be prepared by using wetgranulation or dry-granulation processes. Beads containing the active agent may also be prepared by any one of a number of conventional techniques, typically starting from a fluid dispersion. For example, a typical method for preparing drug-containing beads involves dispersing or dissolving the active agent in a coating suspension or solution containing pharmaceutical excipients such as polyvinylpyrrolidone, methylcellulose, talc, metallic stearates, silicone dioxide, plasticizers or the like. The admixture is used to coat a bead core such as a sugar sphere (or so-called "non-pareil") having a size of approximately 60 to 20 mesh.

[0231] An alternative procedure for preparing drug beads is by blending drug with one or more pharmaceutically acceptable excipients, such as microcrystalline cellulose, lactose, cellulose, polyvinyl pyrrolidone, talc, magnesium stearate, a disintegrant, etc., extruding the blend, spheronizing the extrudate, drying and optionally coating to form the immediate release beads. e. Formulations for Topical and Mucosal Administration

[0232] The compositions can be applied topically. Topical administration can include application to the skin, lungs (pulmonary), nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. Compositions can be delivered to the lungs while inhaling and traverse across the lung epithelial lining to the blood stream when delivered either as an aerosol or spray dried particles having an aerodynamic diameter of less than about 5 microns.

[0233] A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent® nebulizer (Mallinckrodt Inc., St Louis, Mo.); the Acorn® II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin® metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powder inhaler (Pisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

[0234] Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.

[0235] 2. Immunogenic Compositions

[0236] The compositions disclosed herein can be used in combination with immunogenic compositions and as components in vaccines, in the same or different pharmaceutical compositions. Typically, immunogenic compositions disclosed herein include an adjuvant, an antigen, or a combination thereof. When administered to a subject in combination, the adjuvant and antigen can be administered in separate pharmaceutical compositions, or they can be administered together in the same pharmaceutical composition.

[0237] When present in the same pharmaceutical composition, or administered in combination, an adjuvant and an antigen can be referred to as a vaccine. a. Antigen

[0238] Antigens for use in the disclosed compositions and methods are provided. Antigen refers to the molecule to which an immune response is desired. The antigen can be a component of the adjuvant (e.g., nanocage or nanoparticulate structure itself) and / or separate and distinct therefrom (e.g., distinct from the saponin, sterol, lipid, and optional additional adjuvant (e.g., TLR4 agonist) components). Thus, in some forms, the adjuvant, particularly nanocage and other nanoparticulate adjuvants can optionally include, encapsulate, or incorporate one or more antigens. Such adjuvants can thus serve as both adjuvant and antigen in an immunogenic or vaccine formulation. In other forms, the nanocages or particulate adjuvants are absent or free of an antigen. In such forms, the adjuvants typically serve as an adjuvant only. In some such forms, antigen (e.g., free antigen) is present in a pharmaceutical composition in combination with an adjuvant that is free from / of the antigen.

[0239] Antigens can be peptides, proteins, polysaccharides, saccharides, lipids, nucleic acids, or combinations thereof. In exemplary forms, the antigen is polypeptide. The antigen can be derived from a virus, bacterium, parasite, plant, protozoan, fungus, tissue or transformed cell such as a cancer or leukemic cell and can be a whole cell or immunogenic component thereof, e.g., cell wall components or molecular components thereof.

[0240] Suitable antigens are known in the art and are available from commercial, government, and scientific sources. The antigens can be whole inactivated or attenuated organisms, or derived therefrom. These organisms may be infectious organisms, such as viruses, parasites and bacteria. These organisms may also be tumor cells, or derived therefrom. For example, the antigens may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources. The antigens can be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system. The antigens can be DNA or RNA (e.g., mRNA) encoding all or part of an antigenic protein. The DNA may be in the form of vector DNA such as plasmid DNA.

[0241] Antigens may be provided as single antigens or may be provided in combination. Antigens may also be provided as complex mixtures of polypeptides or nucleic acids. Exemplary sources of antigens are provided below. b. Adjuvants

[0242] Suitable adjuvants include immunostimulators including those that include a lipid tail, or can be modified to contain a lipid tail. Examples of molecules that include a lipid tail, or can be modified to include one, can be, for example, pathogen-associated molecular patterns (PAMPs). PAMPS are recognized by pattern recognition receptors (PRRs). Five families of PRRs have been shown to initiate pro-inflammatory signaling pathways: Toll-like receptors (TLRs), NOD- like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs). Also, some NLRs are involved in the formation of pro- inflammatory complexes called inflammasomes.

[0243] Thus, in some forms, the adjuvant is a TLR ligand, a NOD ligand, an RLR ligand, a CLR ligand, and inflammasome inducer, a STING ligand, or a combination thereof. Such ligands are known in the art can obtained through commercial vendors such as InvivoGen.

[0244] The ligands and other adjuvants can be modified (e.g., through chemical conjugation, for example, maleimide thiol reaction, amine N-hydroxy succinimide ester reaction, click chemistry, etc.) to include a lipid tail to facilitate incorporation of the adjuvant into the nanocage structure during self-assembly. Exemplary lipids will include a 16:0 dipalmitoyl tail such as 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [4-(p-maleimidophenyl)butyramide] , these, however, are non-limiting examples. For example, lipids of different lengths are also contemplated. In exemplary forms, the lipid or lipids is / are unsaturated. Chemically functionalized lipids that that can be used for conjugation are known in the art and commercially available. See, for example, AV ANTI® Polar Lipids, Inc. (e.g., “Headgroup Modified Lipids” and “Functionalized Lipids”).

[0245] The adjuvant can be an immunostimulatory oligonucleotide, preferable a lipidated immunostimulatory oligonucleotide. Exemplary lapidated immunostimulatory oligonucleotides and methods of making them are described in Liu, et al., Nature Letters, 507:519-22 (+ 11 pages of extended data) (2014)) (lipo-CpG) and U.S. Patent No. 9,107,904, that contents of which are incorporated by reference herein in their entireties. In some forms, the immunostimulatory oligonucleotide portion of the adjuvant can serve as a ligand for PRRs. Therefore, the oligonucleotide can serve as a ligand for a Toll-like family signaling molecule, such as Toll-Like Receptor 9 (TLR9).

[0246] For example, unmethylated CpG sites can be detected by TLR9 on plasmacytoid dendritic cells and B cells in humans (Zaida, et al., Infection and Immunity, 76(5):2123-2129, (2008)). Therefore, the sequence of the oligonucleotide can include one or more unmethylated cytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs. Hie ‘p* refers to the phosphodiester backbone of DNA, as discussed in more detail below, some oligonucleotides including CG can have a modified backbone, for example a phosphorothioate (PS) backbone.

[0247] In some forms, an immunostimulatory oligonucleotide can contain more than one CG dinucleotide, arranged either contiguously or separated by intervening nucleodde(s). The CpG motif(s) can be in the interior of the oligonucleotide sequence. Numerous nucleotide sequences stimulate TLR9 with variations in the number and location of CG dinucleotide(s), as well as the precise base sequences flanking the CG dimers.

[0248] Typically, CG ODNs are classified based on their sequence, secondary structures, and effect on human peripheral blood mononuclear cells (PBMCs). The five classes are Class A (Type D), Class B (Type K), Class C, Class P, and Class S (Vollmer, J & Krieg, AM, Advanced drug delivery reviews 61(3): 195-204 (2009), incorporated herein by reference). CG ODNs can stimulate the production of Type I interferons (e.g., IFNa) and induce the maturation of dendritic cells (DCs). Some classes of ODNs are also strong activators of natural killer (NK) cells through indirect cytokine signaling. Some classes are strong stimulators of human B cell and monocyte maturation (Weiner, GL, PNAS USA 94(20): 10833-7 (1997); Dalpke, AH, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun. 164(3): 1617-2 (2000), each of which is incorporated herein by reference).

[0249] Other PRR Toll-like receptors include TLR3, and TLR7 which may recognize doublestranded RNA, single-stranded and short double-stranded RNAs, respectively, and retinoic acidinducible gene I (RIG-I)-like receptors, namely RIG-I and melanoma differentiation-associated gene 5 (MDA5), which are best known as RNA-sensing receptors in the cytosol. Therefore, in some forms, the oligonucleotide contains a functional ligand for TLR3, TLR7, or RIG-I-like receptors, or combinations thereof.

[0250] Examples of immunostimulatory oligonucleotides, and methods of making them are known in the art, see for example, Bodera, P. Recent Pat Inflamm Allergy Drug Discov. 5(1):87- 93 (2011), incorporated herein by reference.

[0251] In some forms, the oligonucleotide includes two or more immunostimulatory sequences. Microbial cell-wall components such as Pam2CSK4, Pam3CSK4, and flagellin activate TLR2 and TLR5 receptors respectively and can also be used.

[0252] In other forms, the Addavax, Alum, ISCOMATRIX®, and ASO1B. Immunostimulatory complexes called ISCOMs are particulate antigen delivery systems having antigen, cholesterol, phospholipid and saponin (Quil A or other saponin) with potent immunostimulatory activity. ISCOMATRIX® is a particulate adjuvant having cholesterol, phospholipids and saponins (Quil A) but without containing antigen. See, e.g., U.S. Patent No. 9,149,520, Sun, et al., Volume 27, Issue 33, 16 July 2009, Pages 4388-4401, and Morelli, et al., J Med Microbiol. 2012 Jul;61(Pt 7):935-43. doi: 10.1099 / jmm.0.040857-0. Epub 2012 Mar 22. This adjuvant has principally the same structure as ISCOMs, consisting of perforated cage-like particles of approximately 40 nm in diameter. The antigens can be formulated with ISCOMATRIX® to produce vaccines capable of antigen presentation and immunostimulants similar to ISCOMs-type formulations, but with a wider range of applicability, since its use is not limited to hydrophobic membrane proteins. Modifications of ISCOMs formulations and ISCOMATRIX® have also been developed to achieve a better association of some antigens, such as described in WO 98 / 36772.

[0253] Other liposomal systems mainly composed of saponins from Q. saponaria and sterols (primarily cholesterol) have been described, one of which is referred to as ASO1B. See, e.g., WO 96 / 33739, also being formulated as emulsions such as described in US 2005 / 0220814. See, also, U.S. Published Application No. 2011 / 0206758.

[0254] In exemplary forms, the adjuvant does not consist of Alum (e.g., aluminum hydroxide, aluminum phosphate) such as Alhydrogel. Preferable the adjuvant is an ISCOM, ISCOMATRIX®, ASO1B, or most preferably a nanocage adjuvant such as those discussed above containing a TLR4 agonist, a sterol, and a saponin. See also Silva, M. et al. “A particulate saponinTTLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity,” Sci. Immunol. 6(66) :eabfl 152 (2021) doi: 10.1126 / sciimmunol.abfl 152., WO 2020 / 055503, and U.S. Published Application No. 2020 / 0085756 each of which are specifically incorporated by reference in their entireties. m. Methods of Use

[0255] A. Methods of Increasing an Immune Response

[0256] Methods of using the disclosed compositions are provided, and typically include contacting immune cells with a disclosed composition. The disclosed compositions can be administered in an effective amount to induce, increase, enhance, or maintain an immune response. Immune response typically refers to responses that induce, increase, or perpetuate the activation or efficiency of innate or adaptive immunity. In exemplary forms, the compositions can be used to reduce or prevent inactivation and / or prolong activation of immune cells, e.g., T cells (i.e., increase antigen-specific proliferation of T cells, enhance cytokine production by T cells, stimulate differentiation ad effector functions of T cells and / or promote T cell survival) or overcome T cell exhaustion and / or anergy. By reducing reduction, inhibition, or resolution of the immune response, the compositions thus increase the immune response relative to untreated controls.

[0257] In some forms, one or more hallmarks of T cell exhaustion (e.g., PD-1 and / or TIM3) are reduced, and / or expression of one or more effector cytokines (e.g., IFNy and / or TNF) are increased, in treated immune cells such as T cells.

[0258] In vivo, ex vivo, and in vitro uses are provided. In some forms, the disclosed compositions are administered directedly to the subject (i.e., in vivo). In other forms, the disclosed compositions are contacted with immune cells ex vivo or in vitro, and the cells are subsequently administered to the subject. Ex vivo treatment can form part of an adoptive transfer method in which the immune cells are treated to reduce or prevent inactivation and / or prolong activation of the immune cells before they are administered to the subject. The adaptive T cells can be Chimeric Antigen Receptor (CAR) T cells.

[0259] Additionally or alternatively, the disclosed compositions can be administered in combination with adoptive transfer, including adoptive transfer of CAR T cells. Hie disclosed compositions can thus be used as an adjunct to increase or prolong an immune response modulated by the adaptive T cell therapy, including CAR T cell therapy.

[0260] Thus, the disclosed compositions are particularly useful in the context of maintaining or otherwise enhancing or proIongin the immune response of lymphocytes expressing immune

[0261] 66 receptors, particularly chimeric immune receptor (CIR) such as chimeric antigen receptors (CAR). Artificial immune receptors (also known and referred to herein, as chimeric T cell receptors, chimeric immunoreceptors, chimeric antigen receptors (CARs), and chimeric immune receptors (CIR)) are engineered receptors, which graft a selected specificity onto a cell. Cells modified in this way can be used in immune therapies for treatment of cancers, infections, inflammation, and autoimmune diseases. Strategies for the design and development of chimeric antigen receptors are reviewed in Dotti, et al. , Immunol Rev. 2014 January; 257(1): . doi:10.111 l / imr.12131 (35 pages), which is a specifically incorporated by reference herein in its entirety, as well as Dotti, Molecular Therapy, 22(5):899-890 (2014), Karlsson, et al., Cancer Gene Therapy, 20:386-93 (2013), Charo, et al., Cancer Res., 65(5):2001-8 (2005), Jensen, et al., Immunol Rev., 257(1): 127-144 (2014), Eaton, et al., Gene Therapy, 9:527-35 (2002), Barrett, et al., Anna Rev Med., 65: 333-347 (2014), Cartellieri, et al., Journal of Biomedicine and Biotechnology, Volume 2010, Article ID 956304, 13 pages doi: 10.1155 / 2010 / 956304; and U.S. Published Application Nos. 2015 / 0017120, 2015 / 0283178, 2015 / 0290244, 2014 / 0050709, and 2013 / 0071414.

[0262] The target specificity of the cells expressing a CAR is determined by the antigen recognized by the antibody / ectodomain. In the context of immunotherapy, particularly cancer immunotherapy, numerous antigens, and suitable ectodomains for targeting them, are well known. Unlike the native TCR, the majority of scFv-based CARs recognize target antigens expressed on the cell surface rather than internal antigens that are processed and presented by the cells’ MHC, however, CARs have the advantage over the classical TCR that they can recognize structures other than protein epitopes, including carbohydrates and glycolipids Dotti, et al., ImmunolRev. 2014 January ; 257(1): . doi:10.1111 / imr.l2131 (35 pages) thus increasing the pool of potential target antigens. Exemplary targets include antigens that are only expressed on cancer cells or their surrounding stroma (Cheever, et al., Clin Cancer Res., 15:5323-5337 (2009)), such as the splice variant of EGFR (EGFRvIII), which is specific to glioma cells (Sampson, et al., Semin Immunol., 20(5):267-75 (2008)). However, human antigens meet this requirement, and the majority of target antigens are expressed either at low levels on normal cells (e.g. GD2, CAIX, HER2) and / or in a lineage restricted fashion (e.g. CD 19, CD20).

[0263] Exemplary targets and CARs that target them are known in the art (see, e.g., Dotti, et al., ImmunolRev. 2014 January ; 257(1): . doi:10.1111 / imr.l2131 (35 pages). For example, CAR targets for hematological malignancies include, but are not limited to, CD 19 (e.g., B-cell) (Savoldo, et al., J Clin Invest., 121:1822-1826 (2011), Cooper, et al., Blood, 105:1622-1631 (2005); Jensen, et al., Biol Blood Marrow Transplant (2010), Kochenderfer, et al., Blood, 119:2709-2720 (2012), Brentjens, et al., Molecular Therapy, 17:S157 (2009), Brentjens, et al., Nat Med., 9:279-286 (2003), Brentjens, et al., Blood, 118:4817-4828 (2011), Porter, et al., N Engl J Med., 365:725-733 (2011), Kalos, et al., Sci Transl Med, 3:95ra73 (2011), Brentjens, et al., Sci Transl Med, 5: 177ra38 (2013), Grupp, et al., NEngl J Med (2013)); CD20 (e.g., B-cell)

[0264] 5 (Jensen, et al., Biol Blood Marrow Transplant (2010), Till, et al., Blood, 112:2261-2271 (2008), Wang, et al., Hum Gene Then, 18:712-725 (2007), Wang, et al., Mol Ther., 9:577-586 (2004), Jensen, et al., Biol Blood Marrow Transplant, 4:75-83 (1998)); CD22 (e.g., B-cell) (Haso, et al., Blood, 121:1165-1174 (2013)); CD30 (e.g., B-cell) (Di Stasi, et al., Blood, 113:6392-6402

[0265] (2009), Savoldo, et al., Blood, 110:2620-2630 (2007), Hornbach, et al., Cancer Res. , 58:1116-

[0266] 10 1119 (1998)); CD33 (e.g., Myeloid) (Finney, et al., J Immunol., 161:2791-2797 (1998)); CD70 (e.g., B-celVT-cell) (Shaffer, et al., Blood, 117:4304-4314 (2011)); CD123 (e.g„ Myeloid) (Tettamanti, et al., Br J Haematol., 161:389-401 (2013)); Kappa (e.g., B-cell) (Vera, et al., Blood, 108:3890-3897 (2006)); Lewis Y (e.g., Myeloid) (Peinert, et al., Gene Ther., 17:678-686

[0267] (2010), Ritchie, et al., Mol Ther. (2013)); NKG2D ligands (e.g., Myeloid) (Barber, et al., Exp

[0268] 15 Hematol., 36:1318-1328 (2008), Lehner, et al., PLoS One., 7:e31210 (2012), Song, et al., Hum Gene Ther., 24:295-305 (2013), Spear, et al., J Immunol. 188:6389-6398 (2012)); ROR1 (e.g., B-cell) (Hudecek, et al., Clin Cancer Res. (2013)).

[0269] CAR targets for solid tumors include, but are not limited to, B7H3 (e.g., sarcoma, glioma) (Cheung, et al., Hybrid Hybridomics, 22:209-218 (2003)); CAIX (e.g., kidney) (Larners,

[0270] 20 et al., J Clin Oncol., 24:e20-e22. (2006)), Weijtens, et al., Int J Cancer, 77:181-187 (1998)); CD44 v6 / v7 (e.g., cervical) (Hekele, et al., Int J Cancer, 68:232-238 (1996)), Dall, et al., Cancer Immunol Immunother, 54:51-60 (2005); CD171 (e.g., neuroblastoma) (Park, el al., Mol Then, 15:825-833 (2007)); CEA (e.g., colon) (Nolan, et al., Clin Cancer Res., 5:3928-3941 (1999)); EGFRvIII (e.g., glioma) (Bullain, et al., J Neurooncol. (2009), Morgan, et al., Hum Gene Ther.,

[0271] 25 23:1043-1053 (2012)); EGP2 (e.g., carcinomas) (Meier, et al., Magn Reson Med, 65:756-763

[0272] (2011), Ren-Heidenreich, et al., Cancer Immunol Immunother., 51:417-423 (2002)); EGP40 (e.g., colon) (Daly, et al., Cancer Gene Then, 7:284-291 (2000); EphA2 (e.g., glioma, lung) (Chow, et al., Mol Then, 21:629-637 (2013)); ErbB2(HER2) (e.g., breast, lung, prostate, glioma) (Zhao, et al., J Immunol., 183:5563-5574 (2009), Morgan, et al., Mol Then, 18:843-851 (2010),

[0273] 30 Pinthus, et al., 114:1774-1781 (2004), Teng, et al., Hum Gene Then, 15:699-708 (2004), Stancovski, et al., J Immunol, 151:6577-6582 (1993), Ahmed, et al., Mol Then, 17:1779-1787 (2009), Ahmed, et al., Clin Cancer Res., 16:474-485 (2010), Moritz, et al., Proc Natl Acad Sci U.S.A., 91:4318-4322 (1994)); ErbB receptor family (e.g., breast, lung, prostate, glioma) (Davies, et al., Mol Med., 18:565-576 (2012)); ErbB3 / 4 (e.g., breast, ovarian) (Muniappan, et al., 68 Cancer Gene Ther., 7:128-134 (2000), Altenschmidt, et al., Clin Cancer Res., 2:1001-1008 (1996)); HLA-A1 / MAGE1 (e.g., melanoma) (Willemsen, et al., Gene Ther., 8:1601-1608 (2001), Willemsen, et al., J Immunol., 174:7853-7858 (2005)); HLA-A2 / NY-ESO-1 (e.g., sarcoma, melanoma) (Schuberth, et al., Gene Ther., 20:386-395 (2013)); FR-a (e.g., ovarian) (Hwu, et al., J Exp Med., 178:361-366 (1993), Kershaw, et al., Nat Biotechnol., 20:1221-1227 (2002), Kershaw, et al., Clin Cancer Res., 12:6106-6115 (2006), Hwu, et al., Cancer Res., 55:3369-3373 (1995)); FAP (e.g., cancer associated fibroblasts) (Kakaria, et al., Mol Ther. (2013)); FAR (e.g., rhabdomyosarcoma) (Gattenlohner, et al., Cancer Res., 66:24-28 (2006)); GD2 (e.g., neuroblastoma, sarcoma, melanoma) (Pule, et al., Nat Med., 14:1264-1270 (2008), Louis, et al., Blood, 118:6050-6056 (2011), Rossig, et al., Int J Cancer., 94:228-236 (2001)); GD3 (e.g., melanoma, lung cancer) (Yun, et al., Neoplasia., 2:449-459 (2000)); HMW-MAA (e.g., melanoma) (Burns, et al., Cancer Res., 70:3027-3033 (2010)); ILllRa (e.g., osteosarcoma) (Huang, et al., Cancer Res., 72:271-281 (2012)); IL13Ro2 (e.g., glioma) (Kahlon, et al., Cancer Res., 64:9160-9166 (2004), Brown, et al., Clin Cancer Res. (2012), Kong, et al., Clin Cancer Res., 18:5949-5960 (2012), Yaghoubi, et al., Nat Clin Pract Oncol., 6:53-58 (2009)); Lewis Y (e.g., breast / ovarian / pancreatic) (Peinert, et al., Gene Ther., 17:678-686 (2010), Westwood, et al., Proc Natl Acad Sci U.S.A., 102:19051-19056 (2005), Mezzanzanica, et al., Cancer Gene Ther., 5:401-407 (1998)); Mesothelin (e.g., mesothelioma, breast, pancreas) (Lanitis, et al., Mol Ther., 20:633-643 (2012), Moon, et al., Clin Cancer Res., 17:4719-4730 (2011)); Muel (e.g., ovarian, breast, prostate) (Wilkie, et al., J Immunol., 180:4901-4909 (2008)); NCAM (e.g., neuroblastoma, colorectal) (Gilham, et al., J Immunother., 25:139-151 (2002)); NKG2D ligands (e.g., ovarian, sacoma) (Barber, el al., Exp Hematol., 36:1318-1328 (2008), Lehner, et al., PLoS One, 7:e31210 (2012), Song, et al., Gene Ther., 24:295-305 (2013), Spear, et al., J Immunol., 188:6389-6398 (2012)); PSCA (e.g., prostate, pancreatic) (Morgenroth, et al., Prostate, 67:1121- 1131 (2007), Katari, et al., HPB, 13:643-650 (2011)); PSMA (e.g., prostate) (Maher, et al., Nat Biotechnol., 20:70-75 (2002), Gong, et al., Neoplasia., 1:123-127 (1999)); TAG72 (e.g., colon) (Hornbach, et al., Gastroenterology, 113:1163-1170 (1997), McGuinness, et al., Hum Gene Ther., 10:165-173 (1999)); VEGFR-2 (e.g., tumor vasculature) (J Clin Invest., 120:3953-3968 (2010), Niederman, et al., Proc Natl Acad Sci U.S. A., 99:7009-7014 (2002)).

[0274] The compositions can be delivered parenterally (e.g., by subcutaneous, intradermal, or intramuscular injection) through the lymphatics, or by systemic administration through the circulatory system (e.g., by intravenous injection or infusion), or by enteral administration (e.g., orally). In some forms, the compositions are delivered non-systemically. In some forms, at least the adjuvant alone or in combination with antigen is delivered locally. In some forms, the compositions are delivered by subcutaneous injection. In some forms, the composition is administered at a site adjacent to or leading to one or more lymph nodes which are close to the site in need of an immune response (i.e., close to a tumor or site of infection). In some forms, the composition is administered in multiple doses at various locations throughout the body. Hie composition can also be administered directly to a site in need of an immune response (e.g., a tumor or site of infection or surrounding area).

[0275] The immune response can be induced, increased, enhanced, maintained, or prolonged by the composition compared to a control.

[0276] The desired outcome of a prophylactic or therapeutic immune response may vary according to the disease or condition to be treated, or according to principles well known in the art. For example, an immune response against an infectious agent may completely prevent colonization and replication of an infectious agent, affecting “sterile immunity” and the absence of any disease symptoms. However, a treatment of infectious agents may be considered effective if it reduces the number, severity or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or reduces the transmission of an infectious agent. Similarly, immune responses against cancer, may reduce tumor size, or slow tumor growth compared to a control. The stimulation of an immune response against a cancer may be coupled with surgical, chemotherapeutic, radiologic, hormonal and other immunologic approaches in order to affect treatment. Immune responses against cancer or infectious agents may completely treat a disease, may alleviate symptoms, or may be one facet in an overall therapeutic intervention against a disease.

[0277] B. Diseases to Be Treated

[0278] Cancer

[0279] The compositions can be used for treating cancer, by for example, increasing or maintaining an immune response in host against the cancer. The types of cancer that may be treated with the provided compositions and methods include, but are not limited to, the following: bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharyngeal, pancreatic, prostate, skin, stomach, uterine, ovarian, testicular and hematologic.

[0280] Malignant tumors which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer. The tumors can be solid or liquid tumors.

[0281] The compositions can be administered as an immunogenic composition or as part of vaccine, such as prophylactic vaccines, or therapeutic vaccines, which can be used to initiate or enhance a subject’s immune response to a pre-existing antigen, such as a tumor antigen in a subject with cancer.

[0282] The desired outcome of a prophylactic or therapeutic immune response may vary according to the disease, according to principles well known in the art.

[0283] 2. Infectious Diseases

[0284] The compositions are usefol for treating acute or chronic infectious diseases. Thus, the compositions can be administered for the treatment of local or systemic viral infections, including, but not limited to, immunodeficiency (e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV), encephalitis, influenza (e.g., human influenza virus A), coronaviruses, and common cold (e.g., human rhinovirus) viral infections. For example, pharmaceutical formulations including the composition can be administered topically to treat viral skin diseases such as herpes lesions or shingles, or genital warts. The composition can also be administered to treat systemic viral diseases, including, but not limited to, AIDS, influenza, the common cold, or encephalitis.

[0285] In humans, coronaviruses can cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold, while more lethal varieties can cause SARS, MERS, and COVID-19 (i.e., caused by SARS-CoV-2).

[0286] Representative infections that can be treated, include but are not limited to infections cause by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus influenza type B (HIB), Histoplasma, Hyphomicrobium, Legionella, Leishmania, Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Plasmodium vivax, Trypanosoma brucei, Entamoeba histolytica. Toxoplasma gondii, Trichomonas vaginalis and Schistosoma mansoni.

[0287] In some forms, the type of disease to be treated or prevented is a chronic infectious disease caused by a bacterium, virus, protozoan, helminth, or other microbial pathogen that enters intracellularly.

[0288] In particular forms, infections to be treated are chronic infections cause by a hepatitis virus, a human immunodeficiency virus (HIV), a human T-lymphotrophic virus (HTLV), a herpes virus, an Epstein-Barr virus, or a human papilloma virus.

[0289] C. Combination Therapies bi some forms, the compositions are administered in further combination with one or more additional therapeutic agents. The agents can be administered in the same or separate pharmaceutical composition.

[0290] In some forms, the compositions are administered in combination with a conventional therapeutic agent used for treatment of the disease or condition being treated. Conventional therapeutics agents are known in the art and can be determined by one of skill in the art based on the disease or disorder to be treated. For example, if the disease or condition is cancer, the compositions can be co-administered with a chemotherapeutic drug; or if the disease or condition is a bacterial infection, the compositions can be co-administered with an antibiotic.

[0291] For example, when administered as a cancer vaccine, the disclosed compositions may be administered in combination with a checkpoint inhibitor (PD1, CTLA4, HM3, etc.).

[0292] The disclosed invention can be further understood by the following numbered paragraphs:

[0293] 1. A compound including a first ligand that binds to an NR4A transcription factor linked to a second ligand that binds a protein that induces or increases protein degradation.

[0294] 2. The compound of paragraph 1 , wherein the NR4A transcription factor induces or increases T cell exhaustion.

[0295] 3. The compound of paragraph 2, wherein the NR4A transcription factor is NR4A1, NR4A2, and / or NR4A3.

[0296] 4. The compound of paragraphs 2 or 3, wherein the NR4A transcription factor includes an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NOS:l-8.

[0297] 5. The compound of any one of paragraphs 1-4, wherein the ligand that binds to the

[0298] NR4A transcription factor is selected from the group consisting of Amodiaquine, Chloroquine, benzimidazole, SR10098 (compound 12), isoxazolo-pyridinone SR10658 (compound 7a), IP7e (analog of SR10658), imidazopyridine SR24237 (compound 3), C-DIM12 or l,l-bis(3'-indolyl)- l-(p-chlorophenyl)methane, Camptothecin, Celastrol, Isoalantolactone, Cytosporone B, ethyl 2- [2,3,4-trimethoxy-6-(l-octanoyl)phenyl]acetate (TMPA), pentyl 2-(3,5-dihydroxy-2- nonanoylphenyl)acetate (PDNPA), 5,6-dihydroxyindole (DHI), prostaglandin Al (PGA1), DHI, analogs 5-chloroindole and 5-bromoindole, 6-mercaptopurine, benzimidazole scaffolds, and Prostaglandin El and Al.

[0299] 6. The compound of any one of paragraphs 1-5, wherein the protein that induces or increases protein degradation is an E3 ligase.

[0300] 7. The compound of paragraph 6, wherein the E3 ligase is specifically expressed in an immune cell(s), optionally wherein the immune cell(s) include CD8+ T cells.

[0301] 8. The compound of paragraphs 6 and 7, wherein the E3 ligase is selected from the group consisting of KLHL6, KBTB8, CBLL1, CCM2, DAZAP2, LAPTM5, OSTM1, RNF113A, RNF166, RNF4, TNFRSF1B, TRIM22, TRIM34, and TRIM4.

[0302] 9. The compound of any one of paragraphs 1-8, wherein the ligand that binds a protein that induces or increases protein degradation is an E3 ubiquitin ligase binding group.

[0303] 10. The compound of any one of paragraphs 1-9, wherein the first and second ligands are linked by a linker.

[0304] 11. A pharmaceutical compositions including an effective amount of the compound of any one of paragraphs 1-10.

[0305] 12. A method of maintaining or prolonging an immune response by an immune cell including contacting the immune cell with an effective amount of the compound of any one of paragraphs 1-10 to increase degradation of the cellular protein in the cell.

[0306] 13. The method of paragraph 12, wherein the contacting occurs in vitro, ex vivo, or in vivo in a subject in need thereof.

[0307] 14. A method of treating cancer including administering a subject with cancer an effective amount of the compound of any one of paragraphs 1-10.

[0308] 15. The method of paragraph 14, wherein the cancer includes solid and / or liquid tumors.

[0309] 16. A method of treating an infection including administering a subject with an infection an effective amount of the compound of any one of paragraphs 1-10.

[0310] 17. The method of any one of paragraphs 13-16, including oral administration of the compound to the subject. 18. The method of any one of paragraphs 12-16, wherein the compound is administered locally to site in need of a prolonged immune response in the subject.

[0311] 19. The method of any one of paragraphs 12-18, wherein the immune cell is a T cell, optionally a Chimeric Antigen Receptor (CAR) T cell.

[0312] 20. The method of any one of paragraphs 12-19 further including administering the subject an effective amount of T cells, optionally CAR T cells, before, during, and / or after administration of the compound.

[0313] Examples

[0314] Example 1: Degradation of NR4A transcription factors in vitro reduces hallmark exhaustion marker expression.

[0315] Materials and Methods

[0316] CDS T cells from Nr4alfl / flNr4a2 fl / flNr4a3- / - mice were transduced with retrovirus to express Cre and FKBP12F36V-NR4A1, -NR4A2, or -NR4A3 fusion proteins. Then cells were treated with 500nM dTAG-13 for 48 hours and evaluated by flow cytometry.

[0317] Results

[0318] A proof-of-concept model system was established in mouse CDS T cells to test whether targeted protein degradation of NR4A TFs could reduce exhaustion-related T cell phenotypes in vitro. In the dTAG-13 model system, PROTAC dTAG-13 binds FKBP12F36Vand recruits the E3 ligase Cereblon, allowing degradation of proteins fused to FKBP12ra6V. Murine NR4A- FKBP12F36Vfusion proteins were expressed in T cells deficient for endogenous Nr4a TFs and results showed that the NR4A-FKBP12F36Vfusion proteins were as effective as the corresponding wildtype NR4A TFs at inducing co-inhibilory receptor expression (PD-1 and TIM3). Treatment with dTAG-13 led to selective, dose-dependent degradation of the NR4A- fusion proteins, and significantly reduced PD-1 and TIM3 expression (Fig. 1A). Upon restimulation, NR4A-FKBP12F36V-expressing T cells produced considerably lower levels of effector cytokines IFNg and TNF compared to control T cells; again, treatment with dTAG-13 substantially reversed this effect (Fig. IB).

[0319] Collectively, these results indicate that cells receiving fusion protein targeting NR4A have a dramatic reduction in co-inhibitory receptor expression while the untreated cells (coral) display hallmarks of ‘terminal exhaustion*. Example 2: dTAG-13 treatment degrades NR4A3 hi vivo.

[0320] Materials and Methods

[0321] CDS T cells from CD45.1 mice were transduced with retrovirus to express Cre and FKBP12F36V-NR4A3-IRES-GFP. 3X106GFP+ T cells were adoptively transferred to RaglKOmice. The next day, mice were treated with a single IP dose of dTAG-13 at 25mg / kg. Spleens were harvested 24 h later and T cells evaluated for NR4A3 expression by anti-V5 tag antibody. Results

[0322] The dTAG-13 model system is well established as a tool for in vitro degradation of target proteins, however, there are limited data supporting its in vivo application. To test the kinetics of protein depletion in vivo, V5-tagged FKBP12F36V-NR4A3-expressing CD8+T cells were adoptively transferred to RaglKOmice and these mice were treated a day later with a single intraperitoneal (IP) injection of 25 mg / kg dTAG-13 or vehicle. Spleens were harvested and assessed for levels of intracellular V5- FKBP12F36V-NR4A3 fusion protein by flow cytometry. By 24 hours after a single dTAG-13 dose, V5-NR4A3 was reduced 4-fold (Fig. 2), indicating that dTAG-13 can substantially reduce NR4A TF protein levels in vivo.

[0323] Example 3: Degradation of NR4A3 in vivo improves T cell anti-tumor function

[0324] Materials and Methods

[0325] Mice were injected with 500k MC38 tumor cells subcutaneously, tumor measurements by digital caliper. Tumor-bearing mice were either WT, heterozygous, or deficient for Nr4a3. One way ANOVA performed for each measurement day, Tukey’s multiple comparison test; * = p<0.05, **p<0.01.

[0326] Results

[0327] In an MC38 tumor model, degradation of NR4A3 in vivo significantly improved T cell anti-tumor function (Fig. 3).

[0328] Additional experiments and results thereof related to Examples 1-3 are illustrated in Figures 4A-8D. Figures 4A-4G show the protein abundance of FKBP12F36V-NR4A fusion proteins decreases with increasing doses of dTAG-13, and this diminishes several features of T cell exhaustion. Figures 5A-5E show PD-1 and TIM3 expression are temporally diminished by degrading FKBP12F36V-NR4A fusion proteins. Figures 6A-6G show PD-lhTIM3+CD8+T cells display distinct tumor killing ability after degrading FKBP12F36V-NR4A fusion proteins. Figures 7A-7D show generation of endogenous NR4A fusion proteins in primary CD8+T cells susceptible to dTAG-mediated degradation. Figures 8A-8D show human 19-28z CD8+CAR T cells improve tumor killing by degrading endogenous NR4A proteins. Example 4: Identification of Immune-specific E3 ligases

[0329] The human genome encodes -600 E3 ligases, of which only a small subset has been explore (-2%). The main effort of the industry remains focused on targeting abundant proteins and oncogenes in tumor cells, and a parallel effort to uncover E3 ligases that are selectively expressed in particular cancers and tissues. This latter goal promises a reduction of off-target activity of the drug and therefore lower drug toxicity, because the degradation activity of the drug is reserved only where the recruited E3 ligase is expressed.

[0330] Instead of targeting tumor cells, it is believed that protein degradation could be used for ‘cellular reprogramming’, for improving anti-tumor responses by degrading transcription factors that impose T cell exhaustion, for example. However, many of the protein targets of interest in T cells or other immune cells are universally expressed in other tissues. Though their function in these tissues is not necessarily known, it nevertheless increases the likelihood of off-target drug toxicity. Therefore, in an effort to increase the specificity of drug activity, experiments were designed to identify immune-restricted E3 ligases.

[0331] Publicly available RNA sequencing data (Human Protein Atlas) was identified and the datasets were filtered based on an annotated list of human E3 ligases and substrate-recognition subunits (Li, et al., “Genome- Wide and Functional Annotation of Human E3 Ubiquitin Ligases Identifies MULAN, a Mitochondrial E3 that Regulates the Organelle's Dynamics and Signaling,” PLoS ONE, 3(1): el487 (2008), doi.org / 10.1371 / joumal.pone.0001487). The RNA sequencing data consisted of bulk RNA-seq from 40 healthy tissues (i.e., heart, spleen, kidney, etc.) and scRNA-seq from the same tissues, concatenated by taking the maximum expression for each gene in a cell type across all tissues.

[0332] 86% of the E3 ligases were mapped to these datasets (532 of 616, Fig. 11A). Next, the Tau score was calculated for each cell / tissue type in order to assess gene expression specificity (Fig. 11B). A Tau score of 1 means a gene is expressed only in one cell / tissue, whereas a Tau score of 0 is a ubiquitously expressed gene. Both tissues and cells displayed a range of Tau specificities, so analysis was designed to determine whether E3 ligase expression specificity might correlate between the two; indeed, the expression of most of these genes positively correlates between cells and tissues (Fig. 11C).

[0333] To determine whether an E3 ligases or substrate-recognition may be immune specific, E3 ligase genes that were expressed ubiquitously across tissue types (Tau < 0.5, Fig. 11C) were first filtered out, and this filter was expressed (Tau < 0.5) across all 81 cell types. Using hierarchical clustering on the z-score normalized expression values and manual curation of immune cell types (left columns, first segment of top / bottom guide bar a cluster of genes that displayed high expression (box / arrow, selected for further analysis), compared to non-immune cells (center and right columns, second segment of top / bottom guide bar) was detected (Fig. HD). Re-displaying these selected genes in a tau correlation plot again, allowed determination of E3 genes that were consistently specific regardless of cell or tissue space. A stricter cutoff of Tau > 0.6 was used to identify restricted expression (Fig. HE). When illustrated as a heat map, most of these genes displayed restricted expression across tissues (Fig. HF). Two E3 ligase substrate-recognition receptors, KLHL6 and KBTBD8, emerged as specific in both tissues and cells (Fig. HE). These are both only highly expressed in immune related organs: KBTBD8 - bone marrow, lymph node, and tonsil, KLHL6 - appendix, lymph node, and tonsil.

[0334] Gene expression can vary from person to person due to small genetic differences, such as single-nucleotide polymorphisms (SNPs). To investigate if KLHL6 and KBTBD8 were expressed in immune cells across a broader cohort of patients, their expression was examined in healthy donors from the DICE Database. While KBTBD8 was expressed (TPM > 10), its highest expression was naive B cells, and activated CD4 or CDS T cells (Fig. 12A). Meanwhile, KLHL6 has robust expression across almost all immune cell populations, except perhaps NK cells (Fig. 12A). Notably, KLHL6 also had higher expression when T cells (CD4 or CDS) were activated.

[0335] Gene expression and protein expression do not always correlate well, so protein expression of both E3 ligases was investigated in CDS T cells using proteomics. Both KLHL6 and KBTBD8 were detected by mass spectrometry in various activation contexts (Fig. 12B, top), while only KLHL6 was detectible at all timepoints before / after activation (Fig. 12B, bottom).

[0336] These data provide evidence for immune-specific E3 ligase substrate recognition domains. Both KLHL6 and KBTBD8 are substrate recognition receptors that form complexes as part of the Cullin-RING ligases (CRL), specifically forming a complex with CUL3. Although KLHL6 and KBTBD8 share similar functional protein domains (BTB, Back, and Kelch domains), there is substantial structural and pocket variability among the Kelch domains - even compared to other similarly annotated E3 ligase substrate recognition receptors (BTB-Kelch) - indicating a tractable druggable surface for small molecule development.

[0337] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0338] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific forms of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0339] 78

Claims

We claim:

1. A compound comprising a first ligand that binds to an NR4A transcription factor linked to a second ligand that binds a protein that induces or increases protein degradation.

2. The compound of claim 1, wherein the NR4A transcription factor induces or increases T cell exhaustion.

3. The compound of claim 2, wherein the NR4A transcription factor is NR4A1, NR4A2, and / or NR4A3.

4. The compound of claim 3, wherein the NR4A transcription factor comprises an amino acid sequence with at least 70% sequence identity to any one of SEQ ID NOS:l-8.

5. The compound of claim 4, wherein the ligand that binds to the NR4A transcription factor is selected from the group consisting of Amodiaquine, Chloroquine, benzimidazole, SR10098 (compound 12), isoxazolo-pyridinone SR10658 (compound 7a), IP7e (analog of SR10658), imidazopyridine SR24237 (compound 3), C-DIM12 or l,l-bis(3'-indolyl)- l-(p-chlorophenyl)methane, Camptothecin, Celastrol, Isoalantolactone, Cytosporone B, ethyl 2- [2,3,4-trimethoxy-6-(l-octanoyl)phenyl]acetate (TMPA), pentyl 2-(3,5-dihydroxy-2- nonanoylphenyl)acetate (PDNPA), 5,6-dihydroxyindole (DHI), prostaglandin Al (PGA1), DHI, analogs 5-chloroindole and 5-bromoindole, 6-mercaptopurine, benzimidazole scaffolds, and Prostaglandin El and Al.

6. The compound of claim 5, wherein the protein that induces or increases protein degradation is an E3 ligase.

7. The compound of claim 6, wherein the E3 ligase is specifically expressed in an immune cell(s), optionally wherein the immune cell(s) comprise CD8+ T cells.

8. The compound of claim 6, wherein the E3 ligase is selected from the group consisting of KLHL6, KBTB8, CBLL1, CCM2, DAZAP2, LAPTM5, OSTM1, RNFU3A, RNF166, RNF4, TNFRSF1B, TRIM22, TRIM34, and TRIM4.

9. The compound of claim 5, wherein the ligand that binds a protein that induces or increases protein degradation is an E3 ubiquitin ligase binding group.

10. The compound of claim 8, wherein the first and second ligands are linked by a linker.

11. A pharmaceutical compositions comprising an effective amount of the compound of any one of claims 1-10.

12. A method of maintaining or prolonging an immune response by an immune cell comprising contacting the immune cell with an effective amount of the pharmaceutical composition of claim 11 to increase degradation of the cellular protein in the cell.

13. The method of claim 12, wherein the contacting occurs in vitro, ex vivo, or in vivo in a subject in need thereof.

14. A method of treating cancer comprising administering a subject with cancer an effective amount of the pharmaceutical composition of claim 11.

15. The method of claim 14, wherein the cancer comprises solid and / or liquid tumors.

16. A method of treating an infection comprising administering a subject with an infection an effective amount of the pharmaceutical composition of claim 11.

17. The method of claim 12, comprising oral administration of the compound to the subject.

18. The method of claim 12, wherein the compound is administered locally to site in need of a prolonged immune response in the subject19. The method of claim 12, wherein the immune cell is a T cell, optionally aChimeric Antigen Receptor (CAR) T cell.

20. The method of claim 12 further comprising administering the subject an effective amount of T cells, optionally CAR T cells, before, during, and / or after administration of the compound.

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