ATF6 activation in tumor cells as an immunotherapy adjuvant or neoadjuvant

Abstract

The present invention provides compositions and methods for the sensitization of tumor cells to immune killing and to improve anti-tumor immunotherapy. The methods of treating cancer include administering to the subject a therapeutically effective amount of an activator of Activating Transcription Factor 6 (ATF6). Methods and compositions for enhancing immune checkpoint blockade therapy in are also provided and include administering an effective amount of an activator of Activating Transcription Factor 6 (ATF6) before, during or after administration of an immune checkpoint blockade therapy.

Description

[0001] ATF6 ACTIVATION IN TUMOR CELLS AS AN IMMUNOTHERAPY ADJUVANT OR NEOADJUVANT

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 574,999 filed on April 5, 2024, and U.S. Provisional Application No. 63 / 670,166 filed on July 12, 2024, the contents of which are incorporated by reference in their entireties.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (16985200166. xml; Size: 12,788 bytes; and Date of Creation: April 4, 2025) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Unleashing the immune anti -tumor response through immune checkpoint blockade (ICB) therapy has been successful in combating many solid-tumor malignancies, including metastatic melanoma. When successful, the anti-tumor response is potent; however, half of melanoma patients fail to respond. ICB responsiveness is dictated by the immune milieu and antigenicity of the tumor microenvironment (TME), with therapy-resistant melanomas exhibiting reduced expression of major histocompatibility complex class I (MHC-I) and increased levels of endoplasmic reticulum (ER) stress. Though the TME is known as immunosuppressive, the specific mechanisms governing metabolic stress and antigen presentation in the context of ICB response are not well characterized. Accordingly, there is a remaining need in the art for new strategies to increase the effectiveness of anti-cancer therapies including ICB for solid tumors to improve clinical outcomes.

[0008] SUMMARY

[0009] Disclosed herein are methods and compositions for the sensitization of tumor cells to immune killing and to improve anti-tumor immunotherapy.

[0010] In some embodiments a method of treating cancer is provided. The method comprises the administration of an Activating Transcription Factor 6 (ATF6) activator to a tumor cell. In some embodiments, the method further comprises administering an immune checkpoint blockade therapy. In some embodiments, a method of enhancing immune checkpoint blockade therapy is provided. The method comprises administering to the subject an effective amount of an activator of Activating ATF6 before, during or after administration of an immune checkpoint blockade therapy.

[0011] In some embodiments, the ATF6 activator comprises a pharmacological activator, a small molecule, a virus, an oligonucleotide, a nanoparticle or combinations thereof.

[0012] In some embodiments, the method further comprises administering an anti-cancer therapy. Anti-cancer therapies comprise immunotherapy, radiation, chemotherapeutic agents, or surgery. In some embodiments, the cancer is a solid tumor.

[0013] Another aspect of the present disclosure provides a pharmaceutical composition to deliver ATF6 to a tumor cell comprising a delivery vehicle and at least one of a polynucleotide encoding an ATF6 polypeptide or an ATF6 polypeptide. The ATF6 polypeptide may be one or more of SEQ ID NOs: 2, 4, 6 or a sequence having at least 90% or 95% identity to one of SEQ ID NOs: 2, 4, or 6. In some embodiments, the polynucleotide encoding ATF6 is selected from the group consisting of SEQ ID NO:1, 3,5 or sequences having 90% or 95% identity to one of SEQ ID NOs: 1, 3, or 5. In some embodiments, the delivery vehicle comprises a viral vector, a nanoparticle, or an exosome.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1. ATF6 activation sensitizes tumors to T cell mediated killing. (A) Schematic of ATF6 mutants; wild-type (WT), dominant negative (DN), and constitutively active (CA). (B) BiP expression illustrating increasing ATF6 activation via mRNA signature, in Vector control, ATF6-WT, and ATF6-CA cells. Asterisks denote p<0.05 determined by mixed-effect model with Geisser-Greenhouse correction and Tukey’s multiple comparisons analysis. (C) Schematic of ATF6 activator AA147, illustrating how it promotes ATF6 activity. (D) BiP expression following AA147 treatment, indicating increased ATF6 activity via qPCR. Asterisks denote p<0.05 determined by mixed-effect model with Geisser-Greenhouse correction and Tukey’s multiple comparisons analysis. (E-F) Flow cytometry measuring pan-MHC-I (H2Kb / H2Db) surface expression, Asterisks denote p<0.05 determined by one-way ANOVA with Tukey’s multiple corrections. (E) Flow cytometry revealed decreased MHC-I with genetic overexpression and increased MHC-I with ATF6 inhibition (DN). (F) Surface MHC-I expression was also significantly reduced following AA147 treatment. (G) Quantitative PCR validated loss of MHC genes with genetic and pharmacologic ATF6 activation, including H2Kb, H2Db, and TAPI . Asterisks denote p<0.05 following a one-way ANOVA and Tukey’s multiple comparisons correction. (H-K) CD8+ T cells isolated from mouse spleens were activated (aCD3 / CD28) for 72 h. Plates are automatically imaged every 2 h and at each time point the total numbers of RFP+ (Target) cells was quantified. Cell count values are normalized to target cell counts at t = 0. Error bars = s.d. of nine unique sites. All graphs represent the effector: target ratio of 4: 1. Bar graphs illustrate the discrepancy in sensitivity to immune killing at the final time point (t=72h). Asterisks denote p<0.05 as determined by either student’s t test or one-way ANOVA and Tukey’s multiple comparisons correction. Closed circles are Vec, closed diamonds are CA, closed upside down triangles are DN, closed squares are Vec +AA147, open circles are Vec + anti-MHC or CA + anti- MHC (H) Vector and ATF6-CA B16.OVA cells cultured with activated T cells illustrate increased sensitivity in ATF6-CA cells, while (I) Vector and ATF6-DN co-culture demonstrate a decreased sensitivity with ATF6 inhibition. (J) Vector with and without AA147 pre-treatment, and ATF6- CA cells and (K) Vector and ATF6-CA B16.OVA cells cultured with OT1 CD8+ (tumor-specific) T cells with and without MHC-I blocking antibodies.

[0016] Figure 2. Genetic and Pharmacologic Models of ATF6 Activation. (A) Visualization of ATF6 constructs in agarose gel, confirming sequencing product. Immunoblots of stable transfected B16 cells, confirming (B) ATF6 activity (BiP) in Vector, ATF6-WT, and ATF6-CA cells, (C) ATF6 expression (FLAG) in B16 Vector, ATF6-CA, and ATF6-DN cells, and (D) ATF6 activity (biP) in B16 Vector, ATF6-CA, and ATF6-DN cells. Two independent suppliers of AA147 were examined for changes in maximal BiP up-regulation, illustrating no meaningful difference (E) SelleckChem, and (F) MedChemExpress. B16 Vector and ATF6-CA cells were cultured with increasing levels of cytotoxic stress proponents. There was no change determined in cellular sensitivity to either (G) Staurosporine, or (H) tumor necrosis factor alpha, determined via Annexin V and DAPI staining with flow cytometry. (I-T) Various cell lines treated with increasing doses of AA147 to reveal the ICso values and validate AA147-mediated increases in BiP expression, key target gene of ATF6 activity. IC50 values and BiP activation were confirmed in cell lines other than Bl 6, including (I- J) YUMM1.7, (K-L) LLC1, (M-N) H460, (O-P) HT-29, (Q-R) MC38, (S- T) MDA-MB-231, covering colon, breast, and lung tumor cells, human or mouse cell lines.

[0017] Figure 3. Activating ATF6 in vivo improves melanoma anti-tumor immune response. (A-I) C57BL / 6 mice challenged subcutaneously with 5xlO5tumor cells. Tumor volume calculated daily with caliper measurements, where volume = width2* (14 length), and survival events on Kaplan Meier survival curves where death is determined when tumor volume exceeded and remained above 1000mm3. Asterisk denotes p< 005 determined by mixed-effect model with Geisser-Greenhouse correction and Tukey’s multiple comparisons analysis. BL / 6 mice challenged with Vector, WT, and CA B16F10 tumors, showing (A) reduced tumor volume and (B) increased overall survival with ATF6 overexpression. (C) In BL / 6 mice treated with anti-CD8+ depletion antibodies, overall survival of mice challenged with Vector and CA tumors was indistinguishable. BL / 6 mice challenged with Vector, CA, and DN B16 tumors illustrated significant changes in (D- E) tumor development and (F) survival. BL / 6 mice challenged with Vector, CA, and DN MC38 tumors illustrated similar, significant changes in (G-H) tumor development, and (I) overall survival.

[0018] Figure 4. Impact of ATF6-CA in vivo. (A) Treatment schema for in vivo investigation of ATF6 activation. C57BL / 6 mice were challenged with 5xlO5Vector or ATF6-CA tumor cells. 10 days after tumor injection, tumors were harvested. On day 10, there was no change in (B) tumor size, or (C) tumor-infiltrating-lymphocyte profile, determined by flow cytometry. (D) Flow cytometry of Vector and ATF6-CA cells in both B16F10 and MC38 cell lines, illustrating massive difference in basal MHC-I (H2Kb / H2Db) expression.

[0019] Figure 5. Proteomic analysis reveals ATF6-mediated regulation of immune processes. (A) Proteomic investigation of B16 Vector and ATF6-CA cell lines, illustrating differentially expressed proteins via volcano plot. Significant changes in total proteomic signatures reflected by Reactome Pathways analysis as (B) significantly enriched signaling pathways and (C) pathways lost in ATF6-CA cells, compared to the Vector control. (D) Heatmap (z-scores) of differentially expressed genes via RNA sequencing emphasizing the global ATF6-mediated changes in ER- stress proteins and MHC-I antigen presentation. Gene set enrichment analysis highlighting (E) loss of Negative regulation of immune-effector processes signaling and (F) loss of negative regulation of immune response loss in ATF6-CA. (G) Heatmap (z-scores) of differentially expressed proteins shared in negative regulatory pathways, comparing the Vector and ATF6-CA proteomes. (H) Heatmap (z-scores) of transcript abundance from RNA sequencing data of DEPs found in GSEA analysis and Proteomics. (I) qPCR validating LSGAL3 mRNA levels in B16 Vector, CA, and Vector + AA147. Asterisks denote p<0.05, determined by one-way ANOVA with Tukey’s multiple comparisons test. Galectin 3 levels comparing B 16 Vector and ATF6-CA, illustrating loss of (J) protein abundance, (K) mRNA abundance, and (L) abundance in the extracellular medium. Asterisks denote p<0.05 determined by the student’s t test. (M) B 16. OVA Vector, ATF6-CA and Vector treated with NKG2D inhibiting blocking antibodies. Bar graph represents discrepancy in killing at final time point (t=72h). Asterisks denote p<0.05 as determined by one-way ANOVA and Tukey’s multiple comparisons test.

[0020] Figure 6. In Vivo Immunotherapy and AA147 administration. (A) In-vivo model for ATF6 activation and Immunotherapy. (B) Timeline schematic illustrating injection schedule for ATF6 / ICB combination and ATF6 / ACT combination. (C-F) Non-tumor-bearing mice were injected with 10 mg / kg or 100 mg / kg AA147 via I.P. injection, twice daily for 5 days. QPCR for ATF6 target gene BiP illustrating AA147-mediated ATF6 activity in (C) Liver, (D) Heart, (E) Brain, and (F) Lung following 5-day treatment course. B16F10 tumor-bearing C57BL / 6 mice injected twice daily for 5 days with 50 mg / kg AA147 I P., tumors harvested following 5 day treatment course. (G) QPCR revealing increased BiP activation in tumors. Asterisks denote p<0.05 as determined by one-way ANOVA with Sidak multiple comparisons adjustment.

[0021] Figure 7. ATF6 Activation promotes ICB responsiveness in pre-clinical melanoma. C57BL / 6 mice challenged subcutaneously with 5xl05tumor cells. Tumor volume calculated daily with caliper measurements, where volume = width2* (V2 length), and survival events on Kaplan Meier survival curves where death is determined when tumor volume exceeded and remained above 1000mm3. Asterisks denote p<0.05 determined by one-way ANOVA with Sidak multiple comparisons analysis. (A) Tumor volume comparing B16F10 Vector and ATF6-CA + / - ICB therapy. (B) Bar graph illustrating discrepancy in tumor growth at day 24. (C) Kaplan Meier showing overall survival of B16F10 Vector and ATF6-CA + / - ICB treatment. (D) Tumor volume progression of subset of mice in ATF6-CA + ICB group with 4 / 10 mice having curative effect. Cured mice were re-challenged with parental B16F10 melanoma alongside tumor and therapy naive mice. (E) Tumor volume comparing Yumml.7 Vector and ATF6-CA + / - ICB therapy. (F) Bar graph illustrating discrepancy in tumor growth at day 18. (G) Kaplan Meier showing overall survival of Yumml.7 Vector and ATF6-CA + / - ICB treatment. (H) Tumor volume comparing B16F10 Vehicle and AA147-treated tumors + / - ICB therapy. (I) Bar graph illustrating discrepancy in tumor growth at day 18. (J) Kaplan Meier showing overall survival of B16F10 Vehicle and AA147-treated + / - ICB therapy. (K) Tumor volume comparing Yumml.7 Vehicle and AA147- treated tumors + / - ICB therapy. (L) Bar graph illustrating discrepancy in tumor growth at day 18. (M) Kaplan Meier showing overall survival of Yumml .7 Vehicle and AA147-treated + / - ICB therapy.

[0022] Figure 8: ATF6 activation sensitizes melanoma to in vivo adoptive cell transfer. C57BL / 6 mice challenged subcutaneously with 5xlO5B16.0VA tumor cells. 5xl06activated OT- 1 CD8+ T cells adoptive transferred on post-tumor-injection day 7. (A) Tumor volume calculated daily with caliper measurements, where volume = width2* ( 2 length), comparing ATF6-CA and Vector control. (B) Bar graph illustrating discrepancy in tumor growth on day 16 post-tumorinjection. Asterisk denotes p< 005 determined by mixed-effect model with Geisser-Greenhouse correction and Tukey’s multiple comparisons analysis. (C) Kaplan Meier survival curves comparing Vector and ATF6-CA, where death is determined when tumor volume exceeded and remained above 1000mm3.

[0023] DETAILED DESCRIPTION

[0024] The present invention provides compositions and methods for the sensitization of tumor cells to immune mediated killing and to improve anti-tumor immunotherapy. The inventors have shown that ATF6 activation and or over-expression sensitizes tumor cells to immune cell mediated killing. The role of ATF6 in the potentiation of immune-mediated tumor cell killing has not been previously reported.

[0025] Activating transcription factor 6, also known as ATF6, is a protein that, in humans, is encoded by the ATF6 gene and is involved in the unfolded protein response. ATF6 is a constitutively expressed, endoplasmic reticulum (ER) membrane-anchored transcription factor. ATF6 is a key transcriptional activator of the unfolded protein response (UPR), which allows mammalian cells to maintain cellular homeostasis when they are subjected to environmental and physiological stresses that target the ER. The C-terminus of ATF6 is located in the ER lumen and its N-terminal DNA binding domain faces the cytosol. AFT6 plays a key role in the ER stress response by transmitting the ER stress signal across the ER membrane into the nucleus. The induction of new gene expression by ATF6 is an important aspect of the ER stress response. In response to certain stress conditions, ATF6 translocates from the ER to the Golgi. The 90 kDa full- length ATF6 is processed within the Golgi to its active 50 kDa form through sequential cleavage by site-1 and site-2 proteases (SIP and S2P). Proteolytic activation of ATF6 in the ER stress response is a mechanism to regulate membrane-bound factors and is referred to as regulated intramembrane proteolysis. The N-terminal active ATF6 translocates to the nucleus where it binds to ER stress-response elements in ER stress-response genes (ERSRGs). ATF6 is a potent transcriptional activator of ERSRGs. ATF6 can be expressed as at least two isoforms in humans, which both can translocate into the nucleus. ATF6 of the present invention can be any isoform including any one of SEQ ID NOs: 1-6 (with SEQ ID NO: 1, 3 and 5 as DNA sequences and SEQ ID NO: 2, 4 and 6 as the corresponding amino acid sequences of various isoforms).

[0026] Galectin-3 (Gal-3) is a protein that in humans is encoded by the LGALS3 gene. Galectin- 3 is a member of the lectin family, of which 14 mammalian galectins have been identified. Galectin-3 is approximately 30 kDa and contains a carbohydrate-recognition-binding domain of about 130 amino acids that enable the specific binding of P-galactosides. Gal-3 is also a member of the beta-galactoside-binding protein family that plays a role in cell-cell adhesion, cell-matrix interactions, macrophage activation, angiogenesis, metastasis, apoptosis. Galectins play a role in the regulation of the cellular stress response in malignant cells. Gal-3 has been shown to be produced by tumor cells and cells within the tumor microenvironment and is associated with tumor progression and metastasis.

[0027] NKG2D is a crucial activating receptor on NK cells, T cells, and other immune cells, playing a key role in both innate and adaptive immunity. NKG2DLs (NKG2D ligands) are a family of molecules that are typically expressed at low levels in normal cells, but their expression is significantly upregulated in cells experiencing stress, such as those undergoing DNA damage, cellular senescence, or infection. Tumor cells, often under various stressors, including those induced by chemotherapy or other treatments, can also upregulate NKG2DLs. When NK cells encounter tumor cells expressing NKG2DLs, the interaction between NKG2D and its ligands triggers NK cell activation, leading to the release of cytotoxic molecules that kill the target cells.

[0028] Without wishing to be bound by any theory, the inventors demonstrate herein, that activation of ATF6 sensitizes tumor cells to immune cell mediated killing. Further, the inventors demonstrate that ATF6 activation improves tumor cell immune recognition by decreasing tumor cell release of the NKG2D inhibitor galectin-3 (Gal-3) into the tumor microenvironment. One embodiment of the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activator of Activating Transcription Factor 6 (ATF6).

[0029] In some embodiments, the method further comprises administering an immune checkpoint blockade therapy to the subject in need. Blockade of T cell immune checkpoint receptors, can be performed against any such targets, including but not limited to PD-1, PD-L1, TIM-3, LAG-3, CTLA-4, and CSF-1R and combinations of such checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor comprises anti -PD-1 or anti-CTLA-4. The immune checkpoint receptors may be on immune cells such as T cells, monocytes, microglia, and macrophages, without limitation. The agents which assert immune checkpoint blockade may be small chemical entities or polymers, antibodies, antibody fragments, single chain antibodies or other antibody constructs, including but not limited to bispecific antibodies and diabodies.

[0030] Immune checkpoint inhibitors which may be used according to the disclosure are any that disrupt the inhibitory interaction of cytotoxic T cells and tumor cells. These include but are not limited to anti-PD-1 antibody, anti-PD-Ll antibody, anti-CTLA4 antibody, anti- LAG-3 antibody, and / or anti-TIM-3 antibody. Approved checkpoint inhibitors in the U.S. include Atezolizumab, Ipimilumab, Tremelimumab, Cemiplimab, Pembrolizumab, and Nivolumab. Others in Phase 3 clinical trials include tislelizumab. The inhibitor need not be an antibody but can be a small molecule or other polymer. If the inhibitor is an antibody it can be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint known in the art, including but not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. Additionally, CSF-1R blockade may be used in combination or as an alternative to immune checkpoint inhibitor(s), to ensure generation of potent and sustained immunity that effectively eliminates distant metastases and recurrent tumors. Antibodies specific for CSF-1R or drugs that inhibit or blockade CSF-1R may be used for this purpose, including but not limited, to emactuzumab and AMG820. The checkpoint inhibitors are commercially available and known in the art.

[0031] Another aspect of the present invention provides a method of enhancing immune checkpoint blockade therapy. In some embodiments, the method comprises administering an activator of ATF6 and an immune checkpoint inhibitor. As used herein, the term “checkpoint inhibitor(s)” refers to any compound capable of inhibiting and / or disrupting the function and / or expression of cellular checkpoint molecules (e.g., CTLA-4, PD-1, PD-L1, etc.) involved in cell division. The term “checkpoint inhibitor therapy”, “immune checkpoint inhibitor” or “immune checkpoint blockade therapy” may be used interchangeably and refers to the form of cancer immunotherapy that block inhibitory checkpoints and thereby restore immune system function. Such therapies are known by those skilled in the art. In another embodiment, the disclosure provides a method of treating a subject who is refractory to or not responding to immune checkpoint treatment, the method comprising administering an activator of ATF6.

[0032] ATF6 may be activated by any means known in the art. Means of activating ATF6 include, but are not limited to pharmacological activation, activation by small molecules, activation by nanoparticles, activation by oligonucleotides and / or activation by a virus. In some embodiments, the activation of ATF6 may increase the expression of the ATF6 gene or ATF6 protein or provide a constitutively active form of the ATF6 protein by protein delivery or by delivery of an mRNA or DNA construct allowing increased expression of ATF6 or of a constitutively active form of ATF6. An activator of ATF6 may also comprise any compound that increases the function or nuclear localization of ATF6. An activator of ATF6 may also increase the expression or function of a cleaved or N-terminal fragment of ATF6, or the 50kDa form of ATF6.

[0033] In some embodiments, ATF6 is activated by pharmacological compounds including, but not limited to AA147 or AA263 (see US Patent Publication Nos. 2021 / 0093591 and 2021 / 0393557, and Plate L, et al. Small molecule proteostasis regulators that reprogram the ER to reduce extracellular protein aggregation. Elife. 2016 Jul 20;5:el5550. all of which are incorporated herein by reference in their entireties). In some embodiments, ATF6 is activated by a small molecule such as AA147 or AA263. In some embodiments, ATF6 is activated by delivery of an ATF6 polypeptide or a polynucleotide encoding ATF6 (an mRNA encoding ATF6 or a DNA encoding ATF6 and operably connected to a promoter to allow for expression of ATF6 polypeptide once delivered to cells). In these cases, the ATF6 polypeptide or ATF6 polynucleotide may be delivered using a delivery vehicle. Suitable delivery vehicles for delivering a polypeptide or polynucleotide to a cell are known in the art. The delivery vehicle may be a virus, such that the virus can infect a cell and activate ATF6, for example by delivering a compound or a DNA or RNA viral vector or construct to activate ATF6 or yield over-expression of ATF6 in the cell. In some embodiments, a nanoparticle may be used to activate ATF6 via delivery of a small molecule, polypeptide or polynucleotide to increase ATF6 expression or function. In some embodiments, an exosome or liposome may be used to activate ATF6 by delivering a small molecule activator of ATF6 or an ATF6 polypeptide or polynucleotide encoding ATF6. A nanoparticle or exosome may deliver a pharmaceutical compound to activate ATF6 in the tumor cell. A nanoparticle or exosome may also deliver mRNA and or protein to the tumor cell to activate ATF6.

[0034] In some embodiments, the method further comprises administering an anti-cancer therapy. Anti-cancer therapy is administered to treat a cancer. Cancer treatment includes the reducing, repressing, delaying or preventing cancer growth, reduction of tumor volume, and / or preventing, repressing, delaying or reducing metastasis of the tumor. Treating cancer in a subject also includes the reduction of the number of tumor cells within the subject. The term "treatment" can be characterized by at least one of the following: (a) reducing, slowing or inhibiting growth of cancer and cancer cells, including slowing or inhibiting the growth of metastatic cancer cells; (b) preventing further growth of tumors; (c) reducing or preventing metastasis of cancer cells within a subject; and (d) reducing or ameliorating at least one symptom of cancer. Cancer treatment, and anti-cancer therapy includes but is not limited to chemotherapy, radiation, targeted drug therapy, cryoablation, hormone therapy, bone marrow transplant, immunotherapy (including immune checkpoint blockade therapy and adoptive cell therapies, such as CAR T cell-based therapies), and surgery.

[0035] In some embodiments, the anti-cancer therapy or cancer treatment comprises immunotherapy. Immunotherapy is a type of cancer treatment that helps the immune system fight cancer. In some embodiments, immunotherapy may comprise checkpoint inhibitors, cancer vaccines, cytokines and or adoptive cell therapy. Adoptive cell therapy comprises selecting and / or genetically engineering immune cells to be more effective mediators to kill or block continued growth of cancer cells. In some embodiments, the engineered immune cells are the subject's own immune cells. Adoptive cell therapy includes, but is not limited to Chimeric antigen receptor (CAR) cell therapy: immune cells are engineered to express a synthetic receptor that targets specific cancer antigens; T cell receptor (TCR) gene therapy: T cells are modified to express a new TCR that recognizes cancer antigens; and Tumor-infiltrating lymphocyte (TIL) therapy: T cells that have already infiltrated the tumor are isolated and expanded.

[0036] In some embodiments, the adoptive cell therapy comprises a CAR cell therapy. The CAR may be expressed on any immune cell that is able to target a cancer cell. In some embodiments, the CAR is expressed on a T lymphocyte (CAR-T), a macrophage (CAR-M), a natural killer cell (CAR-NK) or a dendritic cell (CAR-DC). In some embodiments, the activation of ATF6 improves immunotherapy. An ATF6 activator may be administered before other anti-cancer therapies and / or immunotherapies, for example as a neoadjuvant therapy. An ATF6 activator may also be administered along with or after other anti -cancer therapies, for example as an adjuvant cancer therapy. In some embodiments, an ATF6 activator may be administered before or with an immune checkpoint inhibitor. In some embodiments, an ATF6 activator may be administered before or together with a CAR-T cell therapy.

[0037] In some embodiments, the activation of ATF6 increases an anti -turn or immune response. In some embodiments, the activation of ATF6 increases immune-mediated tumor cell killing, for example T lymphocyte mediated killing of a tumor cell such as CD8+ T lymphocytes.

[0038] A “subject in need thereof’ as utilized herein may refer to a subject in need of treatment for a disease or disorder associated with a cancer. A subject in need thereof may include a subject having a cancer that is characterized by gross abnormality visible by X-ray, computerized tomography (CT), or magnetic resonance imaging (MRI). In some embodiments, the subject is refractory or unresponsive to cancer treatment. In some embodiments, the subject is unresponsive to treatment with immune checkpoint inhibitors. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.

[0039] Cancer is a group of disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer comprises a cancer associated with decreased ATF6 expression. In some embodiments, the cancer or tumor is MHC-1 deficient as compared to a control cell. A cancer or tumor that is MHC-1 deficient may include a cancer or tumor cell with a reduction of MHC-I antigen presentation or MHC class I expression on the surface of the cell. In some embodiments, activation of ATF6 may decreased the expression of MHC-1 on the cell or cancer cell surface. In some embodiments, a MHC-1 deficient cancer or tumor cell may have decreased MHC-1 expression, for example at least a 10% reduction in MHC-1 expression as compared to a non-cancerous cell of the same type or as compared to a cell where ATF6 is not activated (i.e. a control cell). In some embodiments, an MHC-1 deficient cell has a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in-between reduction in MHC-1 expression as compared to a non-cancerous cell of the same cell type or a cell where ATF6 is not activated (a control cell). In some embodiments, the cancer is being treated with a CAR-T cell therapy, immune checkpoint therapy or other immunotherapy. Cancers may include, but are not limited to melanoma, breast cancer, prostate cancer, hepatocellular carcinoma, lymphoma (including lymphoma sub-types such as germinal center diffuse large B cell lymphoma, activated B cell-like diffuse large B cell lymphoma, follicular lymphoma, cutaneous T cell lymphoma, mantel cell lymphoma, adult T cell leukemia / lymphoma), leukemias, myeloma, ovarian cancer, lung cancer, colorectal cancer, esophageal cancer, bladder cancer, sarcoma, endometrial cancer, glioblastoma (type of brain cancer), and kidney cancer. The cancer may be any form of solid tumor.

[0040] In some embodiments, the activation of ATF6 may decrease gal ectin-3 or the activation of ATF6 may increase the expression of NKG2DL. In some embodiments, the method may further comprise administering a galectin-3 inhibitor or antagonist. In some embodiments, the method may comprise administering a NKG2D agonist. By way of example, and not limitation, galectin- 3 inhibitors include, but are not limited to, TD139, GB0139, GB1211, GR-MD-02, MCP, GCS- 100, GM-CT-01 and prolactin-M. In some embodiments, an NKG2D agonist may comprise an NKG2D ligand such as an engineered immune cell such as a T cell, or an antibody or antibody fragment.

[0041] Methods and compositions described herein, including ATF6 activators can be administered by any means known in the art. Administration may comprise, but is not limited to intratumoral, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration. ATF6 or compositions capable of increasing ATF6 activation may be administered one or more times to a subject in need.

[0042] In another embodiment, the present formulation may also comprise other suitable agents such as a stabilizing delivery vehicle, carrier, support or complex-forming species. The coordinate administration methods and combinatorial formulations of the instant invention may optionally incorporate effective carriers, diluents, excipients, processing agents, or delivery vehicles, to provide improved formulations for delivery of the construct or lymphocyte comprising the construct described herein. As used herein the term “effective amount” refers to the amount or dose of the compound that provides the desired effect. In some embodiments, the effective amount is the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. Suitably the desired effect may be reducing tumor size, volume, or number of cancer cells to effect treatment of cancer or induction of a response to a checkpoint blockade therapy.

[0043] As used herein, “subject” or "patient" refers to both mammals and non-mammals. “Mammals” include any member of the class Mammalia, such as humans, non-human primates (e.g., chimpanzees, other apes and monkey species), farm animals (e.g, cattle, horses, sheep, goats, and swine), domestic animals (e.g., rabbits, dogs, and cats), and laboratory animals (e.g., rats, mice, and guinea pigs). The term “subject” does not denote a particular age or sex. In one embodiment, the subject is a human.

[0044] Compositions

[0045] Another aspect of the present disclosure provides a pharmaceutical composition to deliver ATF6 or a means of activating ATF6 to a tumor cell. In some embodiments, the composition comprises a delivery vehicle and at least one of a polynucleotide encoding an ATF6 polypeptide or an ATF6 polypeptide. The composition may further include a pharmaceutically acceptable carrier, diluent and / or excipient. In some embodiments, the polynucleotides encoding ATF6 are selected from the group consisting of SEQ ID NO: 1, 3 or 5 or sequences having at least 80%, 85%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1, 3 or 5. In some embodiments, the ATF6 polypeptide is selected from the group consisting of SEQ ID NO: 2, 4 or 6 or sequences having at least 80%, 85%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2, 4 or 6 . The polynucleotide encoding an ATF6 polypeptide may be an RNA, such as an mRNA or a DNA molecule. If the polynucleotide is a DNA the ATF6 DNA polynucleotide may be operably connected to a promoter to allow for expression of the ATF6 polypeptide after introduction of the polynucleotide into a cell. Those of skill in the art can identify appropriate promoters and means of genetically engineering the polynucleotides encoding ATF6 polypeptides to allow for expression of the polypeptides in a cell. SEQ ID NO: 1 is the nucleotide and SEQ ID NO: 2 is the peptide sequence for ATF6-CA. SEQ ID NO: 3 (NCBI Reference Sequence: NM_007348.4) is the nucleotide, and SEQ ID NO: 4 (NCBI Reference Sequence: NP 031374.2) is the peptide sequence for ATF6 isoform 1. SEQ ID NO: 5 (NCBI Reference Sequence: NM_001410890.1) is the nucleotide and SEQ ID NO: 6 (NCBT Reference Sequence: NP 001397819.1) is the peptide sequence for ATF6 isoform 2.

[0046] In some embodiments, the composition comprises a delivery vehicle. A delivery vehicle may comprise a therapeutic delivery system, including a formulation that enables the introduction of a therapeutic substance into cancer cell. In some embodiments, the delivery vehicle comprises a viral vector, a nanoparticle or an exosome. By way of example, and not limitation, a viral vector delivery vehicle may comprise Adeno-Associated Viruses (AAVs), Adenoviruses, Lentiviruses, Retroviruses, or Herpes Simplex Viruses. By way of example and not limitation, a nanoparticle delivery vehicle may comprise, Lipid nanoparticles, Polymeric nanoparticles, Metal nanoparticles, Dendrimers, Inorganic nanoparticles, or Protein and polysaccharide nanoparticles.

[0047] In some embodiments, the composition is formulated for any type of administration. In some embodiments, the composition is administered intravenously, intramuscularly, intrathecally, intratum orally or intraperitoneal injection. In some embodiments, the composition may be further combined before, during or after additional cancer therapies. In some embodiments, the cancer therapies may comprise immunotherapies such as immune checkpoint inhibitors or adoptive cell therapies.

[0048] Additional definitions

[0049] The present disclosure is not limited to the specific details of construction, arrangement of components, or method steps set forth herein. The compositions and methods disclosed herein are capable of being made, practiced, used, carried out and / or formed in various ways that will be apparent to one of skill in the art in light of the disclosure that follows. The phraseology and terminology used herein is for the purpose of description only and should not be regarded as limiting to the scope of the claims. Ordinal indicators, such as first, second, and third, as used in the description and the claims to refer to various structures or method steps, are not meant to be construed to indicate any specific structures or steps, or any particular order or configuration to such structures or steps.

[0050] 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 facilitate the disclosure and does not imply any limitation on the scope of the disclosure unless otherwise claimed. No language in the specification, and no structures shown in the drawings, should be construed as indicating that any non-claimed element is essential to the practice of the disclosed subject matter.

[0051] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0052] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0053] 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. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. Use of the word “about” to describe a particular recited amount or range of amounts is meant to indicate that values very near to the recited amount are included in that amount, such as values that could or naturally would be accounted for due to manufacturing tolerances, instrument and human error in forming measurements, and the like. All percentages referring to amounts are by weight unless indicated otherwise. In those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g.. “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together ). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0054] No admission is made that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it will be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents forms part of the common general knowledge in the art in the United States or in any other country. Any discussion of the references states what their authors assert, and the applicant reserves the right to challenge the accuracy and pertinence of any of the documents cited herein. All references cited herein are fully incorporated by reference, unless explicitly indicated otherwise. The present disclosure shall control in the event there are any disparities between any definitions and / or description found in the cited references.

[0055] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0056] The following examples are meant only to be illustrative and are not meant as limitations on the scope of the invention or of the appended claims.

[0057] EXAMPLES Example 1: ATF6 activation sensitizes melanoma to ICI therapy independent of MHC-I presentation

[0058] While immune checkpoint blockade (ICB) has emerged as an extremely successful tumor therapy, its efficacy in various solid tumors is dramatically reduced. The high variability in patient response is often attributed to the heterogeneity of tumors and the resulting metabolic and immunosuppressive environments[1]. The pathophysiology of metastatic melanoma, like other solid tumors, is intricately linked to altered metabolic processes and endoplasmic reticulum (ER) stress, which together play a pivotal role in tumor progression and patient survival[2]. We hypothesized that regulators of the adaptive ER stress response mechanism, the unfolded protein response (UPR), could be largely responsible for stress-mediated regulation of antigen presentation, immune recognition, and, ultimately, therapy response. Overcoming mechanisms of immunosuppression within ICB-resistant tumors is essential to improving the efficacy of immunotherapies.

[0059] The UPR is initiated to restore homeostasis under conditions of ER stress, a consequence of tumor-associated increases in protein synthesis required for rapid cell proliferation. Although it is initiated to promote cell survival, it can paradoxically support tumor survival and growth under chronic stress conditions[3, 4]. Though the far-reaching consequences of UPR signaling are not well characterized, the UPR has been implicated in interfering with mechanisms of antigen presentation[5], emphasizing the need to more fully investigate the impact of UPR signaling upon tumor antigenicity in the context of immunotherapy response.

[0060] Activating transcription factor 6 (ATF6) plays a pivotal role in managing ER stress, though its exact mechanism is less underexplored. In contrast to its UPR counterparts, IRE1 and PERK, which buffer ER stress through dampening protein translation, ATF6 increases the capacity or stress tolerance of the ER by increasing chaperones and other adaptive machinery through transcriptional regulation[6]. ATF6-specific UPR signaling is complicated, as it can enable tumors to thrive under otherwise detrimental conditions[7, 8], but has also been associated with more favorable disease states[9].

[0061] MHC-I antigen presentation is a key determinant of immune recognition, and as many solid tumors develop mechanisms to restrict MHC-I expression, there has been a great effort recently to investigate mechanisms of improving immune recognition and the anti-tumor immune response. We have uncovered a previously unrecognized role for ATF6 in tumor sensitization to immune- mediated killing, even in MHC-I deficient environments. This has far-reaching implications in the context of immunotherapy regimens, and investigating these mechanisms further is paramount for improving outcomes for patients currently non-responsive to ICB therapy.

[0062] Results and Discussion

[0063] ATF6 activation sensitizes tumors to T cell mediated killing

[0064] ER stress is commonly induced in vitro with Tunicamycin and Thapsigargin[10, 11], which make no distinction between the three sensors of the UPR and can provoke confounding stress responses. Though ER stress has been studied in tumor cells as a consequence of the solid tumor microenvironment, the role of ATF6-specific signaling is largely unclear. We sought to investigate ATF6-specific signaling in melanoma, using both genetic and pharmacologic models. To specifically overexpress ATF6, we engineered a series of B16.F10 murine melanoma cell lines which stably express the 90kDa native form of ATF6 (ATF6-WT), the 50kDa constitutively active form of ATF6 (ATF6-CA), and the 50kDa form with a DNA-binding mutation (ATF6-DN), which functions as a dominant-negative

[0012] (Fig. 1A). These models successfully activate ATF6 expression and activity, indicated by induction of HSPA5 (BiP), a known effector of ATF6 (Fig. 2A-D). As anticipated, ATF6 was activated in a step-wise pattern across the native ATF6-WT and constitutively active ATF6-CA (Fig. IB). To increase the rigor and translational potential of this work, we next utilized a small molecule that selectively activates the ATF6 arm of the UPR, AA147. This compound was initially identified in a high-throughput screen of small molecule ER proteostasis regulators that specifically and preferentially activate the ATF6 arm of the adaptive UPR

[0013] (Fig. 1C). AA147 successfully activated ATF6 in B16F10 melanoma cells, in vitro, as demonstrated by significant elevations in BiP expression (Fig. ID). For experimental consistency, we validated AA147 from two independent sources, which demonstrated similar ECso values and toxicity thresholds in vitro (Fig. 2E-F).

[0065] Profound ER stress is known to interfere with processes that utilize ER-mediated localization and export, including the reduction of MHC-I antigen presentation of various cancer cells

[0014] . To confirm these changes, we examined pan-MHC-I surface expression (H2Kb / H2Db) via flow cytometry. B16 melanoma constitutively expressing active ATF6 (B16.CA) had significantly blunted MHC-I expression, with an expected elevation in the DNA-binding mutant (B16:DN) (Fig. IE). The ATF6-mediated loss of MHC-I was robust, even with IFNy treatment (Fig. 2C). Likewise, AA147 treatment significantly reduced MHC-I expression in B16 melanoma (Fig. IF). Though ATF6 is not known to directly regulate the transcription of antigen presentation machinery (APM), including MHC genes, we identified significant reductions in mRNA levels of MHC-I genes (H2Kb,H2Db) and TAPI with AA147 treatment and ATF6 over-expression (Fig. 1G).

[0066] In the context of immune-mediated tumor control, these robust changes in tumor antigenicity have the potential to significantly impact tumor sensitivity to immune killing. To assess the functional role of ATF6-specific activation on CD8+ T cell killing, we engineered B16F10.OVA cell lines to express the active ATF6 (B16.0VA.CA) and the dominant-negative DNA-binding mutant (B16.0VA.DN). The B16F10.OVA cell lines express the SIINFEKL peptide from ovalbumin on MHC-I molecules. When combined with OVA CD8+ T cells, which are transgenic for the SIINFEKL-specific TCR, this is extremely valuable for elucidating MHC-I- mediated changes on immune tumor control[15f Using this model of MHC-I restricted killing, ATF6-CA cells demonstrated significantly more sensitivity to CD8+ T cell control (Fig. 1H). Further, ATF6-DN cells were significantly less sensitive to immune killing (Fig. II), indicating that ATF6 can disrupt these critical immune interactions. Pre-treating Bl 6. OVA cells with AA147 revealed a similar phenotype, sensitizing the control cells to levels indistinguishable from ATF6- CA (Fig. 1J)

[0067] These data give us confidence in our genetic and pharmacologic models of ATF6 activation, but raises significant questions about the mechanism of ATF6-mediated immune-tumor control. To assess cellular sensitivity, we performed dose-responses of staurosporine and tumor necrosis factor alpha (TNF-a) exposure. There was no discernable difference between the sensitivity of B16F10 Vector and ATF6-CA cells, determined via flow cytometry for apoptosis markers Annexin V and DAPI (Fig. 2 G-H). To expand the rigor and implications for AA147- mediated ATF6 activation, we examined multiple tumor cell lines. AA147 dose-response curves and qPCR revealed successful induction of BiP expression at doses well below the IC50 values for each respective cell line, further validating AA147 administration as a pharmacologic mechanism of ATF6 activation (Fig. 21- T).

[0068] Interested to further elucidate the role of MHC-I presentation in the ATF6 mechanism of immune control, we then introduced pan-MHC blocking antibodies into the B16F10.OVA and OT1 co-culture. Reduced MHC-I availability in vitro did not significantly impact the discrepancy between Vector and ATF6-CA cells, with ATF6 activity continuing to promote robust sensitivity to immune mediated killing (Fig. IK). Though the STINFEKL / 0T1 model of in vitro killing utilizes MHC-I restricted killing, this indicates that the impact of ATF6 extends beyond classic immune responsiveness.

[0069] ATF6 activation in vivo improves anti -turn or immune response

[0070] Though the impact of ATF6 signaling on immune-mediated killing was convincing in vitro, we sought to explore the impact of immune control in our pre-clinical model of metastatic melanoma. C57BL / 6 mice were challenged subcutaneously with 5xl05B16F10 melanoma cells, either ATF6-WT, ATF6-CA or the Vector control (Fig. 4A). Shockingly, this phenomenon was sustained in vivo, with ATF6 overexpression attenuating the development of subcutaneous tumors and promoting increased overall survival (Fig. 3A,B), while the ATF6-WT tumors were indistinguishable from the control. As MHC-I antigens are the chief recognition signal for cytotoxic CD8+ T cells, we were interested to determine the in vivo role of CD8+ T cells in the ATF6-mediated immune control phenomenon. C57BL / 6 mice were depleted of CD8+ T cells and challenged, as before, with either B16 Vector or ATF6-CA tumors. In vivo depletion revealed a central role for CD8+ T cells in the immune sensitivity of ATF6-CA melanoma, removing any significant difference between Vector and ATF6-CA tumors (Fig. 3C). To further assess the immune involvement in the ATF6-mediated phenotype, we challenged a small cohort of mice with Vector and ATF6-CA B16F10 tumors and harvested on Day 10, before discrepancies in tumor volume had developed (Fig. 4B). After tumor dissociation, we assessed tumor-infiltrating lymphocytes via flow cytometry, which revealed no difference in CD4+ T cells, CD8+ T cells, and NK cells (Fig. 4C). Together, these data suggest a mechanism of heightened CD8+ T cell killing independent of infiltration, and these findings support ATF6 activation in the context of immunotherapies, including ICB therapy.

[0071] To investigate the impact of ATF6 inhibition on in vivo tumor development, we performed additional experiments utilizing the dominant negative, ATF6-DN cells. As before, BL / 6 mice were subcutaneously challenged with Bl 6. C A and B16.DN tumors. B16.CA tumors continued to develop more slowly (Fig. 3D,E) and had a significantly prolonged survival, when compared to B16.DN and the vector control (Fig. 3F). B16F10 melanoma is a prototypical model of murine melanoma, however, it is not the perfect model. We next created ATF6 mutants of MC38 cells, a murine adenocarcinoma which, among many differences, expresses much more basal MHC-I (Fig. 4D). Though MC38.DN tumors seemingly developed more slowly early, they rapidly passed the MC38.CA tumors, which remained significantly blunted (Fig. 3G,FT). MC38 CA tumors, similar to Bl 6, had a significant increase in the overall survival (Fig. 31). Collectively, these data indicate a robust, anti-tumor phenotype with ATF6 activation and suggest an immune tumor control mechanism that may not depend on classical MHC-I tumor antigenicity.

[0072] Proteomic analysis reveal ATF6-mediated negative immune regulation.

[0073] To understand the changes mediated by specific ATF6 overexpression and activation, we utilized quantitative tandem mass tag (TMT) mass-spectrometry analysis, investigated changes between B16 ATF6-WT, ATF6-CA, and the vector control (Fig. 5A). Differential Pathway analysis with Reactome revealed robust increases in gene expression and regulation (Fig. 5B), further indicating ATF6-mediated transcriptional regulation. This also demonstrated reductions in interferon signaling and immune system pathways, suggesting a systemic immunomodulatory role of ATF6 signaling (Fig. 5C). Individual protein changes revealed, as anticipated, successful induction of the ER stress response, specifically in the ATF6-CA cells. This also confirmed dysregulation of antigenicity, with a significant and robust reduction in APM proteins, specifically those involved with MHC-I antigen presentation (Fig. 5D).

[0074] The simultaneous loss of MHC-I and stimulation of T cell-mediated killing demonstrated with ATF6 overexpression suggests a mechanism of immune killing not dependent upon MHC-I antigen presentation. To uncover additional regulatory elements of the immune control that could contribute to this phenomenon, we performed Gene Set Enrichment Analysis (GSEA). This highlighted pathways of negative immune regulation (Fig. 5E,F). As we have previously demonstrated a robust reduction of immune anti-tumor activity in vitro and in vivo, we identified the group that contributed most to the differential pathway analysis (Fig. 5G). To identify genes of interest responsible for ATF6-mediated changes, we examined RNA-sequencing data to reveal 4 potential ATF6 targets in the curated list of differential proteins (Fig. 5H). Among others, Galectins were identified as significantly downregulated in ATF6-CA cells, relative to the Vector control. The lectin-binding protein Galectin-3 (Gal3) is a known immunosuppressive molecule of both innate and adaptive immune responses. We confirmed the loss of mRNA signature via qPCR in both ATF6-CA and AA147-treated cells (Fig. 51). Upon further examination, Gal3 was globally reduced in ATF6-CA cells: proteome levels and mRNA signature (Fig. 5J,K). Though Gal3 is not exported by canonical vesicular export, it is infamously promiscuous in the extracellular space and reported to interfere with the immune synapse[16, 17]. An ELISA was used to assess the levels of Gal3 in the extracellular medium, revealing significantly less Gal3 in the ATF6-CA medium (Fig. 5L)

[0075] Among the host of immunosuppressive consequences of extracellular Gal3, it is suspected to interfere with anti-tumor activity mediated by NKG2D

[0017] , a signaling axis in T cells recently implicated in the immune control of MHC-I deficient tumors

[0018] . Though NKG2D is a lymphocyte receptor classically involved in innate immunity, it is expressed on a variety of immune cells, including CD8+ T cells, where it can promote tumor cell cytolysis. Introduction of NKG2D blocking antibodies similarly blunted ATF6-mediated immune sensitivity, implicating the NKG2D axis in the ATF6-mediated tumor control (Fig. 5M). Together, this provides substantial evidence for a mechanism of ATF6-mediated immune control, and supports the dysregulation of Gal-3 as mechanism of interference with the NKG2D immune axis.

[0076] ATF6 activation promotes ICB responsiveness in pre-clinical models of melanoma

[0077] Though the exact mechanism of this ATF6-mediated tumor sensitization to immune killing is still unclear, we have demonstrated a robust phenotype in which ATF6 can promote anti-tumor immune activity. These data support the development of ATF6-based therapeutics and specifically, we see potential for utilizing ATF6 activation to improve tumor responsiveness to immune checkpoint blockade. To assess this relationship, C57BL / 6 mice were given subcutaneous injections of 5xlO5B16F10 melanoma cells, as before, with and without the combination with an ICB regimen (Fig. 6A). As previously reported, combination immune checkpoint blockade was given via intraperitoneal (IP) injections: a-PDl on days 7,9,11,13,15,17,19 and a-CTLA4 on days 7,10,13,16,19 (Fig. 6B). Control Vector and ATF6-CA tumors demonstrated a similar phenotype as before, however, the addition of ICB therapy had a profound impact on tumor development (Fig. 7A,B). Remarkably, this combination had a profound impact on the overall survival, completely curing a subset of mice (Fig. 7C).

[0078] Though improving the ICB response of poorly immunogenic tumor cells, like B16F10[19,20], speaks to the potency and therapeutic potential of ATF6 activation. To assess the longevity of this response and any potential immune memory, after fifty days, mice cured of tumors were rechallenged with parental B16F10 melanoma alongside tumor and treatment-naive mice. Tumors in naive mice progressed rapidly, whereas 75% of the previously cured mice never developed a tumor (Fig. 7D). To further examine the impact of ATF6 overexpression on other melanoma cell lines, we repeated the ICB combination experiment with mice challenged with 5xlCP YUMM1.7 melanoma cells, which harbor more translation ally relevant mutations (BrafV600E / wt, Pten ' , Cdkn2 ). This revealed an extremely similar phenotype, with ATF6 activation and ICB therapy synergistically promoting reduced tumor development (Fig. 7E-F) and a significant improvement in overall survival (Fig. 7G). This is highly supportive of utilizing ATF6-activating agents to improve the efficacy of ICB against melanoma.

[0079] The profound in vivo anti -turn or response of genetic ATF6 activation in melanoma and the similar in vitro phenotype we consistently demonstrated with AA147 treatment gave us confidence that pharmacologic ATF6 activation would provide similar results. Though the in vivo administration of AA147 has been reported, it is not wide-spread. For an initial safety profile, and to confirm ATF6 activation at the tumor site, we treated non tumor-bearing BL / 6 mice with twice- daily injections of AA147 for 5 days, at either 10 mg / kg or 100 mg / kg. Though highly variable, this demonstrated successful up-regulation of ATF6-target gene, BiP in various organs following the 5 day treatment course (Fig. 6C-F). We then administered AA147 injections at the median dose, 50 mg / kg, to B 16F 10 tumor-bearing mice and further demonstrated successful up-regulation of BiP specifically within the tumors (Fig. 6G). To assess the impact of AA147 treatment combined with combination ICB therapy (aCTLA4 / aPDl), BL / 6 were challenged, as before with B16F10 melanoma. Though the AA147 alone had little impact, when combined with ICB, it conveyed a significant delay in tumor growth, and an increase in overall survival (Fig. 6H-J). Though, like most human melanomas, Bl 6F 10 melanoma is poorly responsive, it is not necessarily representative of patient melanomas. Therefore, we repeated this model of pre-clinical melanoma, utilizing AA147 and combination ICB in mice bearing YUMM1.7 melanoma tumors. Again, in combination with ICB therapy, AA147 dramatically blunted tumor development and significantly prolonged the overall survival (Fig. 7K-M).

[0080] ATF6 activation sensitizes tumors to adoptive cell transfer in vivo

[0081] After demonstrating the robust anti-tumor effects of ATF6 activation and improving the response of immune checkpoint blockade therapy, we then sought to examine the impact of ATF6 activation on the effectiveness of adoptive cell therapies (ACT). ACT, especially the use of chimeric antigen receptor (CAR) T cells, has had remarkable success in treating hematologic malignancies; however, the efficacy against solid tumors has been underwhelming. Recent advancements have led to the first approved ACT for melanoma patients, leading us to examine the impact of ATF6 activation in this context. C57BL / 6 mice were challenged with subcutaneous injection of Vector and ATF6-CA Bl 6. OVA tumors. Activated, tumor-specific (OT-1) CD8+ T cells were adoptively transferred via tail-vein injection on post-injection day 5, 5xl06T cells per mouse. ATF6-CA tumors had significantly blunted growth in response to ACT, compared to the vector control (Fig.8A-B), and had a profound impact on improving the overall survival (Fig. 8C). Discussion

[0082] Innate and acquired resistance to immunotherapies is a major contributor to advanced disease and poor prognoses of solid tumors, including melanoma. For immune checkpoint inhibitors (ICIs), the chief obstacle has been identified repeatedly as alterations driven by the tumor microenvironment (TME)[21, 22], which facilitate mechanisms that dampen the host anti-tumor response and promote immune evasion l23b Mechanisms to combat reduced tumor antigenicity and acquired ICI resistance are still underexplored. In this study we reveal a mechanism of ER stress that promotes immune control of tumors, independent of MHC-I antigenicity and a putative therapeutic agent to improve the immunotherapy response in solid tumors.

[0083] Because endoplasmic reticulum (ER) stress is elevated in solid tumors and classically interrupts ER-mediated trafficking, including MHC-I antigen presentation, we sought to examine the impact of ER stress on melanoma. Specifically, we anticipated the pro-survival arm of the unfolded protein response (UPR), mediated by activating transcription factor 6 (ATF6), to be most responsible for conveying stress tolerance to rapidly developing solid tumors. Therefore, we expected ATF6 inhibition to blunt tumor adaptation to the TME and promote cell death. Genetic and pharmacologic activation of ATF6, in conjunction with literature evidence, blunted MHC-I antigen presentation; however, this also revealed an ATF6-mediated sensitivity to immune- mediated killing. A simultaneous loss of MHC-I presentation and stimulation of CD8+ T cell mediated killing suggests a powerful anti-tumor response that is not dependent on classical MHC- I / TCR signaling.

[0084] In addition to confirming ATF6 activation had no impact on cellular sensitivity or viability, we shockingly demonstrated this phenotype in vivo, where murine melanoma and adenocarcinoma cells which over-expressed ATF6 had blunted tumor growth and prolonged overall survival. Though the exact mechanism of this MHC-I - independent anti-tumor cascade is still unclear, our data does suggest that ATF6 is promoting ‘bystander-killing’, wherein CD8+ T cells identify stressed cells by non-MHC-I ligands. We leveraged proteomics, RNA-sequencing, and gene set enrichment analysis to identify key players of immunomodulation in ATF6-OE cells, revealing dysregulation of Galectin expression and the NKG2D-NKG2DL axis. Galectins are promiscuous carbohydrate-binding proteins that have been implicated in interfering with the immune synapse, and interrupting the NKG2D axis. NKG2D is a lectin-like receptor present on CD8+ T cells which recognizes ligands classically involved in the innate immune response of NK cells. By inhibiting NKG2D signaling in vitro we removed any immune sensitivity of ATF6-CA cells, implicating NKG2D signaling in this ATF6-mediated phenomenon.

[0085] Combination of ATF6-OE with an ICI regimen in a pre-clinical mouse model revealed a profound increase in treatment responsiveness, even demonstrating a curative effect in some of the mice, which demonstrated a memory response. These results were repeated with a more translationally relevant model of murine melanoma, Yumml.7 (BRAFV600E,CDKN2A' ', Pten"). To extend these findings further, we administered ATF6-activator, AA147, in vivo. AA147 combination with ICB treatment revealed similarly profound responses in an otherwise non- responsive model, indicating the potential therapeutic impact of ATF6 activation. We also demonstrate that ATF6 activation can improve the response of adoptive cell transfers, a newly approved therapy option for advanced melanomas.

[0086] These studies have uncovered mechanisms with therapeutic potential, particularly in combination with immune checkpoint inhibitors (ICIs). These findings emphasize ATF6 activation as a putative therapy to enhance the solid tumor response of immune checkpoint inhibitors, and other immunotherapies.

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Claims

CLAIMSWhat is claimed:

1. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an activator of Activating Transcription Factor 6 (ATF6).

2. The method of claim 1, further comprising administering an immune checkpoint blockade therapy.

3. A method of enhancing immune checkpoint blockade therapy in a subject in need thereof, the method comprising administering to the subject an effective amount of an activator of Activating Transcription Factor 6 (ATF6) before, during or after administration of an immune checkpoint blockade therapy.

4. The method of any one of the previous claims, wherein the ATF6 activator comprises a pharmacological activator, a small molecule, a virus, an oligonucleotide, a nanoparticle or combinations thereof.

5. The method of claim 4, wherein the pharmacological activator comprises AA147 or AA263.

6. The method of any one of the previous claims, wherein the ATF6 activator increases the expression of ATF6.

7. The method of any one of the previous claims, wherein the method further comprises administering an anti-cancer therapy.

8. The method of claim 7, wherein the anti-cancer therapy comprises immunotherapy, radiation, chemotherapeutic agents, or surgery.

9. The method of claim 8, wherein the immunotherapy comprises adoptive cell therapy.

10. The method of claim 9, wherein the adoptive cell therapy comprises administering an immune cell expressing a chimeric antigen receptor (CAR).11 . The method of claim 10, wherein the adoptive cell therapy comprises a CAR T cell, a CAR macrophage cell or a CAR natural killer cell.

12. The method of claim 7, wherein the anti-cancer therapy comprises an immune checkpoint inhibitor and wherein the immune checkpoint inhibitor is optionally selected from the group consisting of inhibitors of PD-1, PD-L1, LAG-3, and CTLA-4.

13. The method of any one of the previous claims, wherein the cancer is a solid tumor.

14. The method of any one of the previous claims, wherein the cancer is selected from the group consisting of melanoma, breast cancer, prostate cancer, hepatocellular carcinoma, lymphoma, leukemias, myeloma, ovarian cancer, lung cancer, colorectal cancer, esophageal cancer and kidney cancer.

15. The method of any one of the previous claims, wherein the cancer is MHC-I deficient as compared to a control cell.

16. The method of any one of the previous claims, wherein the method further comprises administering a galectin-3 inhibitor and or a NKG2D agonist.

17. The method of any one of the previous claims, wherein the ATF6 activator is administered intravenously or intratum orally.

18. A pharmaceutical composition to deliver ATF6 to a tumor cell comprising a delivery vehicle and at least one of a polynucleotide encoding an ATF6 polypeptide or an ATF6 polypeptide, wherein the ATF6 polypeptide is selected from the group consisting of SEQ ID NOs: 2, 4 or 6 or sequences with at least 95% identity to SEQ ID NO: 2, 4 or 619. The pharmaceutical composition of claim 18, wherein the polynucleotide encoding ATF6 is selected from the group consisting of SEQ ID NO: 1, 3 or 5, or sequences with at least 95% identity to SEQ ID NO: 1, 3 or 5.

20. The pharmaceutical composition of claim 18 or 19, wherein the polynucleotide is an RNA or messenger RNA.21 . The pharmaceutical composition of claim 18 or 19, wherein the polynucleotide is DNA and the polynucleotide encoding the ATF6 polypeptide is operably connected to a promoter.

22. The pharmaceutical composition of any one of claims 18-21, further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

23. The pharmaceutical composition of any one of claims 18-22, wherein the delivery vehicle comprises a viral vector, a nanoparticle, or an exosome.

24. The pharmaceutical composition of claim 23, wherein the viral vector is an adenovirus associated vector, a retroviral vector, a lentiviral vector, a herpesvirus vector25. The pharmaceutical composition of any one of claims 18-24, wherein the composition is formulated for intravenous, or intratumoral delivery.

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