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1. WO2020142694 - ERO1-ALPHA INHIBITORS

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[ EN ]

EROl-ALPHA TNHTBTTORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application No. 62/787,968, filed January 3, 2019, the entire contents of which is hereby incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number K12 CA157688 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is directed to modified immune effector cells and a therapy for the treatment of cancer comprising T cells in which expression of EROla or PERK is reduced or eliminated.

All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes and to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

BACKGROUND OF THE INVENTION

In response to antigens, T cells undergo rapid expansion, engaging in up to 15-20 cell divisions, and proliferation is characterized by differentiation to an effector phenotype from a naive state (1). To support the bioenergetic demands of growth and proliferation, naive T cells that primarily engage oxidative phosphorylation (OXPHOS) shift to generate adenosine triphosphate (ATP) via increased OXPHOS and aerobic glycolysis as they become functional effector cells (2,3). Highly differentiated effector T cells rely primarily on glycolysis and are characterized by a loss of mitochondrial integrity (4) that likely accounts for their inability to effectively continue

OXPHOS. The cell-intrinsic mechanisms that consume metabolic energy and impart mitochondrial exhaustion in T cells are unknown.

Programmed cell death protein 1 (PD-1) is expressed on highly differentiated effector T cells that have become exhausted due to chronic antigen exposure (5). In cancers, programmed death-ligand 1 (PD-L1) is expressed by tumor cells to further impair anti-tumor effector function of PD-1+ T cells (6). Checkpoint blockade therapy is a means to reinvigorate effector function of exhausted T cells by inhibition of the PD-1-PD-L1 interaction (7). Unfortunately, anti-PD-1 therapy remains ineffective for the majority of cancer patients due to initial lack of response or loss of durable responses (8-10). In early-stage exhaustion, PD-1+ T cells show diminished OXPHOS and glycolysis, and long-term chronically exhausted PD- 1 hlgh T cells exhibit a dependence on glycolysis due to dysfunctional mitochondria (11). T cell-intrinsic factors that drive PD-1+ CD8+ tumor infiltrating lymphocyte (TIL) metabolic exhaustion in response to tumor antigen are unknown, and simple methods to characterize the metabolic state of PD-1+ tumor infiltrating lymphocytes (TILs) in patient tumors are ill-defined.

T cell expansion initiated by antigen recognition requires T effector cells to greatly increase new protein synthesis and initiate post-translational modifications (12). Imbalances of unfolded and misfolded proteins are detected by endoplasmic reticulum (ER) stress sensors inositol-requiring enzyme- 1 (IREla), protein kinase R-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) (13). The acute arm of the stress response aims to alleviate the burden of unfolded or misfolded proteins in the ER to restore proteostasis. In the face of insurmountable stress, the unfolded protein response (UPR) initiates cell death. PERK mediates the terminal UPR through regulation of transcription factors activating transcription factor 4 (ATF4) and C/EBRa homologous protein (CHOP) which induce downstream target ER oxidoreductase 1 (EROla) (14-17). As a catalyst of disulfide bond formation in the ER, EROla facilitates oxidation-reduction reactions (Redox). Excess EROla produced in response to chronic protein folding induces accumulation of reactive oxygen species (ROS) and impairs survival (15,16). The PERK-specific cell stress response has not been measured in T cells or applied to the rubric of anti-tumor immunity.

SUMMARY OF THE INVENTION

The present invention is directed to a reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture have a reduced expression of EROla or PERK.

The present invention is also directed to a reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture comprise a vector comprising a nucleic acid sequence encoding a protein which knocks out EROla or PERK.

The present invention is also directed to a method of treating a cancer in a subject, comprising administering modified T cells to said subject wherein said modified T cells comprise T cells in which expression of EROla or PERK is reduced or eliminated.

The present invention is also directed to a method for treating a cancer in a mammal, comprising administering to said mammal an EROla inhibitor or PERK inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described below are for illustrative purposes only and are not intended to limit the scope of the invention.

Figure 1. PERK contributes to chronic ER stress in C8+ T effector cells: Naive WT OT-l+ CD8+ T cells were activated and expanded with cognate peptide and harvested at indicated time points. (A-D) PERK ( Eif2ak3 ), ATF4 ( Atf4 ), CHOP ( Ddit3 ), and EROla ( Eroll) gene expression were measured by qPCR and (E) PERK and EROla proteins measured by

immunoblot (5pg, 2min). Data from 4 biological replicates are quantified and represented as SEM; results from students t test performed for naive versus day 7 T cells are displayed.

Experiments were repeated with four different WT animals and immunoblot is representative. Naive PERK KO (OT - 1 xLck-( VNxPERK^) and littermate controls (OT-lxLck-Cre xPERK®) or 7-day expanded T cells were harvested. (F-I) PERK (Eif2ak3), ATF4 (. Atf4 ), CHOP ( Ddit3 ), and EROla {Eroll) gene expression measured by qPCR and (J) immunblot (5pg, lmin) for PERK and EROla proteins. PERK 8min exposure is shown to convey lack of protein expression. Data

from three WT and littermate pairs are quantified and represented as SEM, students t test.

Experiments repeated twice and immunoblot data are representative of four independent experiments. *p<0.05, **p<0.01, ***/?<0.00E

Figure 2. PERK axis impacts CD8+ T effector profiles. Representative oxygen consumption rate (OCR) trace and quantification of spare respiratory capacity (SRC) from day 7 (A) PERK KO or littermate controls or (B) WT and PERK I or (C) WT and EROl I-treated OT-l+ CD8+ T cells measured via Seahorse Bioanalysis. Spare respiratory capacity (SRC) calculated as the difference between initial OCR rate and the maximal OCR rates achieved after FCCP

uncoupling. Data are quantified and represented as SEM, students t test performed for each condition versus control T cells. Experiments were repeated at least 3 times. (D) IFN-g production from day 7 WT, PERK I, or EROl I-treated OT-l+ T cells and WT (littermate) and PERK KO CD8+ T cells. Data from four biological replicates are quantified and represented as SEM, students t test performed for each condition versus WT cells. Individual experiments were repeated 3 times. * p<0.05, **p<0.01, ****p<0.0001. Gene symbol and expression intensity of proteins identified by LC -MS/MS-based shotgun proteomics extracted from the top 100 proteins with greatest enrichment in WT OT-l+ (T eff) compared to (E) PERK KO or (F) EROla I-treated T cells. Biologically functional groups of energy & metabolism, ER transport/ cell stress, oxidative stress/ DNA damage and Redox are shown. Heat maps represent fold increased intensity of proteins from average value of three replicates in each T cell group. Acly (+212.98) in Teff versus PERK KO T cells is represented numerically.

Figure 3. Mitochondrial reactive oxygen species signify mitochondrial exhaustion.

Representative FACS plots of (A) Naive WT OT-l+ CD8+ T cells activated and expanded with cognate peptide or (B) human PBMC expanded in high dose IL-2 (3000U/mL) and CD8+ T cells FACS stained at indicated time points. FACS gates are set from fluorescence minus one controls. Data points represent quantification of five individual mice or human samples and are

represented as SEM, students t test performed for each time point versus To control. Experiments repeated twice. (C) Representative FACS plot with gating from FACS sorts of mtROS/ CD8+ T cells. Lowest 25% - and highest 25% + mtROS populations were collected and (D)

Representative oxygen consumption rate (OCR) trace and quantification of spare respiratory capacity (SRC) from sorted populations at indicated time points measured via Seahorse

Bioanalysis. Spare respiratory capacity (SRC) calculated as the difference between initial OCR rate and the maximal OCR rates achieved after FCCP uncoupling. Data are quantified and represented as SEM, students t test performed for each condition versus control T cells. (E) IFN-g production from sorted subsets at indicated time points. Post-sort purity was >97%. (F)

Representative FACS plot and quantification of mtROS-Annexin co-staining on day 7 WT OT-1+ T cells. FACS gates are set from fluorescence minus one controls. Data are quantified and represented as SEM, students t test. Individual experiments repeated three times. * p<0.05, **** ><0.0001.

Figure 4. PERK axis drives mitochondrial exhaustion and is impaired in memory T cells:

Representative FACS plots and quantification of day 7 WT, PERK I, EROl I-treated and WT (littermate) and PERK KO OT-l+ CD8+ T cells probed for (A) mtROS or (B) CD62L expression. Bar graphs from 4 biological replicates are quantified and represented as SEM, Students t test performed for each condition versus control. FACS gates are set from fluorescence minus one controls. Experiments were repeated at least three times. T effector (Teff) or memory (Tmem) cells were developed and harvested. (C) Representative FACS plot and quantification of mtROS expression (D) quantification of PERK ( Eif2ak3 ), ATF4 ( Atf4 ), CHOP ( Ddit3 ), and EROl a ( Eroll) gene and (E) immunoblot for PERK and EROla proteins. FACS quantification from four biological replicates. Experiment repeated 3 times. Gene expression bar graphs represent average of 3 separate experiments and are shown as SEM, Teff values are expressed relative to respective Tmem values set to 1. Immunoblot is representative data from 3 experiments.

**£><0.01, ***£><0.001, ****£><0.0001.

Figure 5. Inhibition of PERK axis augments T cell-specific tumor control: Individual graphs of mice bearing 7-day B16F1-OVA tumors left untreated (n=5) or treated intravenously with 5xl05 7-day expanded OT-l+ (Teff) (n=8) or PERK KO (n=7) T cells. Tumor size recorded every other day for 3 weeks. Lines represent individual mice. (B) Survival to 45 days or tumor size of 400mm2 was recorded, Log-rank test, **£><0.01 survival proportions of mice treated with Teff (12%) versus PERK KO T cells (86%) (C) Mice bearing 7-day B16F10 melanomas were treated intravenously with 2xl06 7-day expanded Pmel (Teff) or Pmel T cells developed in the presence of (B) PERK inhibitor (PERK I T) or (D) EROla inhibitor (EROla I T). Tumor size

recorded every other day for 3 weeks. Lines represent individual mice. Linear regression of Teff versus PERK KO or inhibitor-treated T cell groups, n=5-6 mice per group, ****/><0.0001.

Experiments repeated twice.

Figure 6. Tumor antigen-specific PD-1+ CD8+ s experience mitochondrial exhaustion:


CD8+ cells were sorted from spleens and tumors of mice bearing 14-day MCA-205-OVA tumors and qPCR was performed to quantify (A) PERK ( Eif2ak3 ), ATF4 ( Atf4 ), CHOP ( Ddit3 ), and EROla ( Eroll) gene expression. Bar graphs represent averages of 4 mice per group and are shown as SEM, experiment repeated twice. Representative FACS plots and quantification of mtROS/PD-l+ CD8+ populations in tumor draining lymph nodes (TDLNs) and tumors (tumor infiltrating lymphocytes (TILs)) harvested from mice bearing 14-day (B) MCA-205-OVA sarcomas or (C) MC38 colon carcinomas. Populations represent gating from CD8+/CD45+ lymphocytes and quadrants are set from PD-1 isotype control expression. Bar graphs represent 4-5 mice per group and are shown as SEM. Individual experiments were repeated 3 times. (D) lxlO6 naive CD45.2 OT-l+ T cells were transferred via tail vein to CD45.1 C57BL/6 mice bearing 7 day established s.c. MCA-205-OVA sarcomas. Tumors were harvested 7 days post transfer. (E) Representative FACS plot overlay and quantification of mtROS/PD-1 co-staining from CD45.1 (gray) or CD45.2 (black) CD8+ cells in TDLNs and tumors. Gates are set from isotype control data. Bar graphs represent 4 mice per group and are shown as SEM. Individual experiments repeated twice. Students / test, **p<0.0 \ , ****p<0.000 \

Figure 7. PERK inhibition reduces CD8+ TIL mtROS and augments anti-PD-1 therapy. (A)

Representative FACS plot and quantification of mtROS/PD-l+ CD8+ T cells from PBMC and tumor of three patients with pleomorphic undifferentiated high grade deep (PU HGD) sarcoma. Gates are set from isotype controls. PERK inhibitor (PERK I) or vehicle control was

administered for 1 week (days 7-14) to mice bearing MCA-205-OVA sarcomas. (B)

Representative FACS plots and quantification of mtROS/PD-1 TILs gated from CD45+/ CD8+ populations. Gates are set from isotype control data. (C) Absolute number of CD45+/PT/ CD8+ TILs calculated per gram of tumor weight. Bar graphs represent 4-5 mice per group and are shown as SEM. Individual experiments repeated twice. Students / test, *p< 0.05, ***/ <0.001 PERK I or vehicle control was administered beginning after 7 days of tumor growth to mice bearing MCA-205-OVA sarcomas and anti-PD-1 or isotype antibody was administered on day

12 and every four days thereafter. Anti-CD8 was administered every 2-3 days beginning 5 days after tumor inoculation. (D) Composite and (E) individual graphs of tumor growth measured every other day for 40 days with complete response (CR) listed per group, composite data represented as SEM. Linear regression of combination measured against anti -PD- 1 therapy, ****/><0.0001. (F) Survival to 41 days or tumor size of 200mm2 was recorded, Log-rank test, **/K0.01 survival proportions of anti-PD-1 therapy (28%) versus combination therapy (100%). Combination experiment repeated twice, anti-CD8 depletion condition performed once.

Figure 8. PERK KO T cells restrict tumor growth. WT or PKO CD8 T cells activated and expanded with OVA peptide for 7 days A) assessed by immunoblot or infused i.v. (5xl05 cells/mouse) into 5Gy irradiated mice bearing 7 day established B 16-OVA melanomas. B) Tumor growth and C) survival were monitored. B, Linear regression C, Log-rank test of survival of WT vs. PKO infusions. 7-8 mice per group. Individual experiments repeated twice.

Throughout proposal *,p< 0.05, **,/><0.01, ***,/><0.001, ****,/><0.0001. Student’ s t test used in data figures unless otherwise noted.

Figure 9. CD8 TILs experience the terminal PERK UPR. A) Representative FACS plot of CD8 T cell infiltrate in spleen and tumor of C57BL/6 mice bearing 14-day established MCA-205 sarcomas with B) long-term in vivo growth kinetics of this tumor model, (n=7 mice). CD8 T cells from spleens and tumors of C57BL/6 mice bearing 14-day established MCA-205 sarcomas were FACS sorted and C) scatter plot bar graph of RT-PCR expression of indicated terminal UPR genes or D) volcano plot of LC -MS/MS proteomic data where differentially expressed (DE) proteins (FDR<0.05) are marked as red with terminal UPR genes annotated are shown. C, Data points are values from 4 individual mice D, T cells from 20 mice were pooled 4 times to create 4 biological replicates for analysis. E) Scatter plot with connecting lines to autologous-matched patient samples of RT-PCR expression of indicated terminal UPR genes in CD8 T cells FACS sorted from PBMC or tumors of HGD PU sarcoma patients.

Figure 10. Effector T cells experience the terminal PERK UPR. IL-2 (200U) and IL-15 (50ng/mL) were used to differentiate Pmel CD8 T cells to effector or memory lineage, respectively. A) Volcano plot of 84 UPR genes in Tefif or Tmem cells. Significant genes within the PERK terminal UPR pathway are highlighted in red. B) Volcano plot of LC -MS/MS

proteomic data where differentially expressed (DE) proteins (FDR<0.05) are marked as red. Terminal UPR genes are annotated. A, Gene arrays were performed on 3 biological replicates. B, Data represent analysis of 5 biological replicates. FDR<0.05. C) Immunoblot of Teff and Tmem for indicated proteins, experiment performed 3 times.

Figure 11. PERK-ATF4-ER01a shapes memory T cell formation. IL-2 (200U) and IL-15 (50ng/mL) were used to differentiate OT-1 CD8 T cells to effector or memory lineage, respectively. For ATF4 RNP gene deletion, 3-day ex vivo expanded OT-1 T cells were treated with Atf4 single guide RNA and recombinant Cas9 protein to form RNPs and transfected using the Neon Electroporation System. Naive CD8 T cells from WT, CHOP , and ERO la 7 mice were isolated from spleens and activated and expanded with plate-bound a-CD3 and soluble a-CD28. Scatter plot bar graphs represent A) CD62L expression B) RT-PCR gene expression of Tc/7 and C) Eroll gene expression among UPR T cell groups. Data points represent technical replicates of one representative experiment, performed three times.

Figure 12. EROla impacts CD8 T cell metabolism and tumor control. Representative FACS plot and quantification of mtROS in ex vivo expanded A) WT or EROla CD8 T cells or B)

WT or NAC -treated CD8 T cells. Experiments performed 3 times. C) OT-1 (WT T cells) or OT-1- EROla CD8 T cells (EROla KO T cells) were i.v. infused into 5Gy irradiated mice bearing 7-day established B 16-OVA melanomas. Tumor growth was monitored, linear regression of tumor growth between mice infused with WT and EROla KO T cells used to assess

significance, n=8 mice per group.

Figure 13. mtROS indicate metabolic exhaustion in CD8 T cells. A) Representative histogram of FACS sorting gate of mtROS-/+ subsets. B) Oxygen consumption rate (OCR) trace and quantification of spare respiratory capacity (SRC) in response to injections of FCCP,

Oligomycin, and Rotenone/Antimycin A in FACS sorted populations after indicated days of expansion. C) Quantification of IFN-g secretion from FACS sorted populations after indicated days of expansion. D) Representative FACS plot and quantification of Annexin/mtROS co expression after 7 days of expansion. E) OCR trace and quantification as in (B) of vehicle or EROla inhibitor (EN460, IOmM) treated T cells. Individual experiments performed 3 times.

Figure 14. Tumor antigen specific CD8 TILs are PD-lhigh/mtROS+. A) Cartoon to depict i.v. infusion of 2xl06 naive CD45.2 OT-1 T cells to CD45.1 mice-bearing 7-day established MCA-205-OVA sarcomas. B) Representative FACS plots and quantification of CD45.2 OT-1 CD8 T cells in TDLNs and tumors assessed for expression of mtROS/PD-1 after 7 days in vivo. Red box gated on PD-1 hlgh compartment, dotted line indicates PD-1 designation from isotype control. C) Representative FACS plots and quantification of MCA-205 sarcoma-bearing mice treated with vehicle or PERK inhibitor (PERK I, GSK2606414 50mg/kg b.i.d.) for 7 days, beginning 1 week after tumor inoculation, TILs were assessed for expression mtROS/PD-1. Red circle gated on PD- 1 hlgh and red box gated on PD-llow compartment, dotted line indicates PD-1 designation from isotype control. Data points represent individual mice. D) Representative FACS plots and quantification of mtROS/PD-1 expression in CD8 T cells from PBMC and TIL of HGD PU sarcoma patients. Data points with lines indicate autologous-matched samples. Red box gated on PD- 1 hlgh compartment, dotted line indicates PD-1 designation from isotype control.

Figure 15. PERK inhibition radically augments a-PD-1 therapy. MCA-205 sarcoma-bearing mice treated continuously with vehicle or PERK I as in Figure 14 beginning 7 days after tumor inoculation. Anti -PD-1 therapy or isotype control (200pg) was administered every 3-4 days beginning 13 days post tumor inoculation and anti-CD8 antibody (lOOpg) was administered every other day beginning 5 days post inoculation. A) Tumor growth and B) survival were monitored. 7 mice per group. A, Linear regression and B, Log-rank test of survival of anti -PD-1 vs. PERK I + anti -PD-1 therapy. CR=complete response.

Figure 16. PERK inhibits translation under sarcoma microenvironment stress. OT-1 (WT) or OT-l-PKO (PKO) CD 8 T cells seeded in transwells in co-culture with tumor and T cell were collected 36 hours later and A) western blotting was performed. Representative FACS plots and quantification of protein synthesis relative to cycloheximide (CHX)-treated controls in B) WT and PKO T cells harvested from transwell assay C) WT and PKO CD8 TILs harvested from mice bearing 14-day established MCA-205 sarcomas D) peripheral or TIL CD8 T cells from HGD PU sarcoma patients. Data points represent individual mice or patient values.

Figure 17. Quantification and viability of T cell groups. Littermate OT-1+, PERK KO, or DMSO OT-1+, PERK I or ERO la-inhibitor treated T cells were quantified (A) % live using

automated cell counter and (B) absolute live cell number was recorded. Bar graphs represent 5 separate well counts and are represented with standard deviation, Students t test performed for each condition versus WT control. *p<0.05, **p<0.01.

Figure 18. mtROS+ CD8 TILs are highly activated. Splenic and TIL analysis from mice bearing 14-day established MCA-205-OVA or MC38 tumors. Representative FACS plots and quantification of (A) splenocytes and TILs measured for mtROS/PD-l+ co-expression or (B) CD8+ TILs gated on mtROS+ (gray) or mtROS- (black) populations and measured for CD44 mean fluorescent intensity (MFI). Mice bearing 7-day established MCA-205-OVA or MC38 tumors were treated daily with vehicle or PERK inhibitor (PERK I) and harvested after 7 days of treatment. (C) Quantification of CD44 MFI gated from total CD45/CD8+ TILs. Data are shown as averages of 4-5 mice per group and are represented as SEM. Experiments repeated twice. Students t test, ns=not significant, *p<0.05, **p<0.01, ****p<0.0001.

Figure 19: WT or EROla KO mice (F2 generation) were given B16F1 melanomas subcutaneous and treated with a-PD-1 (200ug/ mouse) after 5 days of tumor growth. Tumor size was measured every other day with calipers and a-PD-1 was administered every 4 days. Tumor size is presented as fold change from initial tumor measurement.

DETAILED DESCIPTION OF THE INVENTION

It is to be understood that the terminology employed herein is for the purpose of describing particular embodiments, and is not intended to be limiting. Further, although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, certain methods, devices and materials are now described.

In one embodiment, the invention provides for a method for treating a cancer in a mammal, comprising administering modified T cells into said mammal wherein said modified T cells comprise T cells in which expression of EROla or PERK is reduced or eliminated. In an embodiment, the mammal is a human.

In one embodiment, provided is a method for treating cancer in a subject comprising

administering to the subject an amount of a EROla inhibitor. It is contemplated that such a method reduces expression of EROla in the subject thereby treating cancer in the subject.

In one embodiment, provided is a method for enhancing an existing cancer therapy by

additionally administering to a subject an amount of a EROla inhibitor. It is contemplated that such a method would enhance the existing cancer therapy, for example, checkpoint inhibitors.

In one embodiment, provided is a method for treating cancer in a subject comprising

administering to the subject an amount of a PERK inhibitor. It is contemplated that such a method reduces expression of PERK in the subject thereby treating cancer in the subject.

In one embodiment, provided is a method for enhancing an existing cancer therapy by

additionally administering to a subject an amount of a PERK inhibitor. It is contemplated that such a method would enhance the existing cancer therapy, for example, checkpoint inhibitors.

In one embodiment, the EROla inhibitor is a EROla antagonist. In some embodiments, the herein the EROla antagonist is:

a. an antibody, or antigen binding fragment of an antibody, that specifically binds to, and inhibits activation of, an EROla receptor, or

b. a soluble form of an EROla receptor that specifically binds to a EROla ligand and inhibits the EROla ligand from binding to the EROla receptor.

Definitions

The articles“a” and“an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article.

A“subject” is a human, and the terms“subject” and“patient” are used interchangeably herein.

The term“treating,” with regard to a subject, encompasses, e.g., inducing inhibition, regression, or stasis of a disease or disorder; or curing, improving, or at least partially ameliorating the disorder; or alleviating, lessening, suppressing, inhibiting, reducing the severity of, eliminating or substantially eliminating, or ameliorating a symptom of the disease or disorder. "Inhibition" of disease progression or disease complication in a subject means preventing or reducing the disease progression and/or disease complication in the subject.

A "symptom" associated with cancer includes any clinical or laboratory manifestation associated with cancer and is not limited to what the subject can feel or observe.

"Administering to the subject" or "administering to the (human) patient" means the giving of, dispensing of, or application of medicines, drugs, or remedies to a subject/patient to relieve, cure, or reduce the symptoms associated with a condition, e.g., a pathological condition. The administration can be periodic administration.

As used herein, "periodic administration" means repeated/recurrent administration separated by a period of time. The period of time between administrations is preferably consistent from time to time. Periodic administration can include administration, e.g., once daily, twice daily, three times daily, four times daily, weekly, twice weekly, three times weekly, four times a week and so on, etc.

As used herein, a "unit dose", "unit doses" and "unit dosage form(s)" mean a single drug administration entity/entities.

As used herein, "effective" or“therapeutically effective” when referring to an amount of a substance, for example an antagonist, refers to the quantity of the substance that is sufficient to yield a desired therapeutic response. In certain embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an antagonist or inhibitor of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibodies to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the antibody or antibodies are outweighed by the therapeutically beneficial effects.

The combination of the invention may be formulated for its simultaneous, separate or sequential administration, with at least a pharmaceutically acceptable carrier, additive, adjuvant or vehicle as described herein. Thus, the combination of the two active compounds may be administered:

• as a combination that is part of the same medicament formulation, the two active

compounds are then administered simultaneously, or

• as a combination of two units, each with one of the active substances giving rise to the possibility of simultaneous, sequential or separate administration.

As used herein, "combination" means an assemblage of reagents for use in therapy either by simultaneous or contemporaneous administration. Simultaneous administration refers to administration of an admixture (whether a true mixture, a suspension, an emulsion or other physical combination) of two or more components. Contemporaneous administration, or concomitant administration refers to the separate administration of two or more components at the same time, or at times sufficiently close together that a synergistic activity relative to the activity of either component alone is observed or in close enough temporal proximately to allow the individual therapeutic effects of each component to overlap.

As used herein, "add-on" or "add-on therapy" means an assemblage of reagents for use in therapy, wherein the subject receiving the therapy begins a first treatment regimen of one or more reagents prior to beginning a second treatment regimen of one or more different reagents in addition to the first treatment regimen, so that not all of the reagents used in the therapy are started at the same time. For example, adding one antagonist therapy to a patient already receiving a different antagonist therapy.

Any known EROla antagonist may be utilized in the practice of the invention, a broad variety of which are known and disclosed in the art. The EROla antagonist preferably neutralizes biological function after binding. The EROla antagonist is preferably a human EROla antagonist. Optionally, the EROla antagonist may be an antibody, such as a monoclonal antibody or fragment thereof; a chimeric monoclonal antibody (such as a human-murine chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble EROla antagonist, including small molecule EROla blocking agents. Optionally, the EROla antagonist is a functional fragment or fusion protein comprising a functional fragment of a monoclonal antibody, such as a Fab, F(ab')2, Fv and preferably Fab. Preferably a fragment is pegylated or encapsulated (e.g. for stability and/or sustained release). The EROla antagonist may also be a camelid antibody. As used herein, EROla antagonists include but are not limited to EROla receptor inhibitors.

Any known PERK antagonist may be utilized in the practice of the invention, a broad variety of which are known and disclosed in the art. The PERK antagonist preferably neutralizes biological function after binding. The PERK antagonist is preferably a human PERK antagonist.

Optionally, the PERK antagonist may be an antibody, such as a monoclonal antibody or fragment thereof; a chimeric monoclonal antibody (such as a human-murine chimeric

monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble PERK antagonist, including small molecule PERK blocking agents. Optionally, the PERK antagonist is a functional fragment or fusion protein comprising a functional fragment of a monoclonal antibody, such as a Fab, F(ab')2, Fv and preferably Fab. Preferably a fragment is pegylated or encapsulated (e.g. for stability and/or sustained release). The PERK antagonist may also be a camelid antibody. As used herein, PERK antagonists include but are not limited to PERK receptor inhibitors.

Any known PD-1 antagonist may be utilized in the practice of the invention, a broad variety of which are known and disclosed in the art. The PD-1 antagonist preferably neutralizes biological function after binding. The PD-1 antagonist is preferably a human PD-1 antagonist. Optionally, the PD-1 antagonist may be an antibody, such as a monoclonal antibody or fragment thereof; a chimeric monoclonal antibody (such as a human-murine chimeric monoclonal antibody); a fully human monoclonal antibody; a recombinant human monoclonal antibody; a humanized antibody fragment; a soluble PD-1 antagonist, including small molecule PD-1 blocking agents. Optionally, the PD-1 antagonist is a functional fragment or fusion protein comprising a functional fragment of a monoclonal antibody, such as a Fab, F(ab')2, Fv and preferably Fab. Preferably a fragment is pegylated or encapsulated (e.g. for stability and/or sustained release). The PD-1 antagonist may also be a camelid antibody. As used herein, PD-1 antagonists include but are not limited to PD-1 receptor inhibitors.

The PD-1 antagonist may be selected, for example, from one or a combination of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab, or a functional fragment thereof.

The invention will now be further described in the Examples below, which are intended as an illustration only and do not limit the scope of the invention.

EXAMPLES

The disclosure is further illustrated by the following examples, which are not to be construed as limiting this disclosure in scope or spirit to the specific procedures herein described. It is to be understood that the examples are provided to illustrate certain embodiments and that no limitation to the scope of the disclosure is intended thereby. It is to be further understood that resort may be had to various other embodiments, modifications, and equivalents thereof which may suggest themselves to those skilled in the art without departing from the spirit of the present disclosure and/or scope of the appended claims.

Example 1

The experiments below show that the PERK axis contributes to EROla activation in T effector cells. In fact, this axis consumes metabolic energy in T cells and drives a protein profile synonymous with oxidative stress. It is further shown that accumulation of mitochondrial ROS (mtROS) is a hallmark of ER-induced mitochondrial exhaustion, connecting ER biology to metabolic function in T cells. Memory T cells show reduced expression of mtROS and EROla

compared to T effectors. PERK KO, and PERK, or EROla inhibitor-treated T cells exhibited superior tumor control compared to T effectors. Tumor antigen-specific PD-1+ CD8+ TILs acquire mtROS in tumors. In sarcoma patients and a sarcoma mouse model, the experiments below demonstrate that high proportions of PD-1+ CD8+ TILs express mtROS. In vivo treatment with a PERK inhibitor reduced mtROS in PD-1+ CD8+ TILs, increased tumor clearance, and extended animal survival in combination with anti -PD- 1 therapy.

Methods

Mice: T cell-specific deletion of PERK on a C57BL/6J background was accomplished by cross of PERKloxP (Eif2ak3tm l 2U' J) mice with OT-1 (C57BL/6-Tg(TcraTcrb)l lOOMjb/J) or Lck-Cre (B6.Cg-Tg(Lck-icre)3779Nik/J) mice to obtain OT-lxPERKf/f and LckCrexPERKf/w mice, respectively. These lines were crossed to obtain OT 1 Lckcrc PERK^mice and OTl+Lck ere PERK^littermate controls. Lck -Cre, PERKloxP, OT-1, C57BL/6J, Ly5.2 (B6.SJL-Ptprca

Pepcb/BoyJ), and P-mel (B6. CgThy l a/CyTg(TcraTcrb)8Rest/J) mice were obtained from the Jackson Laboratory.

Human Samples: Samples were obtained from normal donor patients undergoing routine non cancer-associated surgery or patients undergoing surgical removal of high grade deep

pleomorphic undifferentiated sarcomas. Blood (8 mL) was collected in EDTA coated tubes and PBMC were isolated via Histopaque-1077 centrifugation (Sigma). Sarcoma tissue was collected on ice and immediately cut into 2mm3 pieces and dissociated to a single cell suspension using Human Tumor Dissociation Kit and gentleMACS dissociator (Miltenyi Biotec) according to manufacturer’s protocol.

RT-PCR and Immunoblot analysis: RNA was isolated with RNeasy Mini Kit (QIAGEN) and single-strand cDNA was made with High Capacity RNA-to-cDNA Kit (Applied Biosystems, Thermo Fisher Scientific). Taqman gene expression assays (Applied Biosystems, Thermo Fisher) were used to perform real-time PCR using the StepOnePlus Real-Time PCR system (Applied Biosystems, Thermo Fisher). Gene expression for PERK (Eif2ak3), ATF4 (Atf4), CHOP (Ddit3), and EROla (Erol) were normalized to Gapdh. For immunoblots cell lysates were prepared in RIPA buffer (Sigma-Aldrich) and probed with PERK (Cell Signaling Technology), EROla (Santa Cruz Biotechnology) antibodies or b-actin (Cell Signaling Technology) loading control.

Cell Sort, FACS Staining & Analysis: Fluorochrome-conjugated monoclonal antibodies and respective isotype controls listed in Table 1 were purchased from ThermoFisher (Ebioscience). Extracellular stains were performed in PBS supplemented with 2% FBS. IFN-g was probed after 4-6 hours of cell restimulation with Cell Stimulation Cocktail (eBioscience) and Golgi Plug Protein Transport Inhibitor (eBioscience). Foxp3/Transcription Factor Intracellular Staining Buffer Set was used according to manufacturer’s protocol (eBioscience). Mitochondrial reactive oxygen species (mtROS) were measured with MitoSOX Red Mitochondrial Superoxide Indicator (3mM) loaded at 37°C for 30 minutes in RT PBS. Extracellular stains were added post dye incubation. For Annexin staining, MitoSOX dye loaded cells were washed and stained for using Annexin V-FITC Apoptosis Detection Kit (eBioscience) according to manufacturers’ protocol. Samples were run directly on a BD Accuri C6 flow cytometer. For FACS sorting of mtROS+ and mtROS cell fractions, samples were stained with MitoSOX Red Mitochondrial Superoxide Indicator and CD8 and sorted on a FACS Aria IIu (BD Biosciences). Further analysis was performed post-sort for confirmation of population purity. For RNA ex vivo gene analysis, lymphocytes were isolated via Histopaque gradient (Sigma) and CD8+ T cell Isolation Kit (Miltenyi) was used to obtain >90% purity from spleens and tumors.

Cell lines: MCA-205-OVA and MC38 (Kerafast Inc.) cell lines were maintained in DMEM 10% FBS. B16F1-OVA and B16F10 were maintained in RPMI complete T cell media. Cell lines were determined to be mycoplasma free (MCA-205-OVA)(MC38)(B16F1-OVA, B16F10). All growth media were supplemented with Plasmocin mycoplasma prophylactic (Invivogen). All cell lines were passaged three times prior to in vivo tumor inoculation, and a fresh vial of cells was prepared for individual experiments.

T cell activation and treatment: Spleens from OT-1 mice were dissociated and RBC lysis was performed using ACK Lysing (Thermo-Fisher Scientific) to achieve a single cell suspension. T cells were activated and expanded for indicated time points from total splenocytes incubated with 1 pg/mL OVA 257-264 (Invivogen) or hgplOO 25-33 (GenScript) peptide in complete T cell media (200U rhIL-2, NCI). Cells were washed and media was changed after 3 days of initial

activation and expansion. For inhibitor treatments, PERK (lOOnM, GSK2606414, Tocris) or EROla inhibitors (IOmM, EN460, Cayman Chemical) were incubated with splenocytes for 10 min prior to introduction of peptide and re-introduced at the d3 cell split. For central memory T cell development, IL-2 was replaced in T cell media with rhIL-15 (50ng/mL, Shenandoah) at the day 3 cell split.

Metabolic & Proteomic Analysis: Oxygen consumption rate (OCR) was measured in non-buffered RS media supplemented with HEPES under basal conditions and in response to 1 mM oligomycin, 1.5 mM FCCP, and 2mM rotenone + ImM Antimycin A using the XFe96

Extracellular Flux Analyzer (Seahorse Bioscience). Cell-Tak (Coming) was used for T-cell adherence.

Liquid Chromatography-Tandem Mass Spectrometry: Proteomics were performed by Bioproximity, LLC. Samples were prepared for digestion using the suspension-trapping (S-trap, Protifi) method. Digested peptides were collected by centrifugation. Peptides were eluted with 80% acetonitrile, 5% ammonium hydroxide and lyophilized in a SpeedVac (Thermo Savant) to remove volatile components. Digestion mixtures were analyzed by UHPLC-MS/MS. LC was performed on an Easy-nLC 1000 UHPLC system (Thermo) interfaced to a quadrupole-Orbitrap mass spectrometer (Q-Exactive HF-X, Thermo Fisher) via nano-electrospray ionization using a source with an integrated column heater (Thermo Easy Spray source).

Data Processing and Library Searching: Tandem mass spectra were searched

using X! Tandem and Open Mass Spectrometry Search Algorithm (OMSSA), requiring expectation value scores of 0.01 or better to be considered a match. Protein intensity values were calculated using OpenMS to measure the area under the curve of identified peptides. Searches were performed on Amazon Web Services-based cluster compute instances using the Proteome Cluster interface which builds species- and genus-specific protein sequence libraries monthly from current UniProtKB distributions. Gene annotations were obtained from Ensembl Release 93 database, Mouse genes (v93GRCm38.p6) data set through the BioMart website. Official Gene Ontology categories to annotate mouse proteins based on corresponding gene symbols were used.

Tumor mouse models and In vivo treatments: For transfer of 7-day expanded OT-l+ or PERK KO T cells, C57BL/6 mice were implanted subcutaneously (s.c.) with 2.5X105 B16F1-OVA tumor cells and 5xl05 T cells were infused via tail vein to 5Gy irradiated mice after 7 days of tumor growth. For transfer of 7-day expanded Pmel or Pmel inhibitor-treated T cells, C57BL/6 mice were implanted subcutaneously (s.c.) with 2.5xl05 B16F10 tumor cells and 2xl06 T cells were infused via tail vein to 5Gy irradiated mice after 7 days of tumor growth. Tumor growth was measured every other day for 3 weeks. For tracking acquisition of mtROS/PD-l+ status in tumor antigen-specific CD8+ TILs, 2.5xl05 MCA-205-OVA were implanted s.c. to Ly5.2 mice. Naive OT-l+ T cells were obtained via Mouse CD8+ T Cell Isolation Kit (Miltenyi Biotec), and lxlO6 CD8+ T cells were transferred via tail vein on day 7 of tumor growth. After 7 days of in vivo expansion, tumor draining lymph nodes (TDLNs) and tumors were harvested. Tumors were processed to single cell suspension using Mouse Tumor Dissociation Kit (Miltenyi Biotec) according to manufacturer’s protocol. For combination therapy experiments, 2.5xl05 MCA-205-OVA were injected s.c. to C57BL/6 mice and tumors were established for 7 days. 50mg/kg PERK Inhibitor (GSK2606414, GlaxoSmithKline) or vehicle was administered twice daily via oral gavage as a suspension of 0.5% hydroxypropylmethyl cellulose + 0.1% Tween-80 in water. 200 pg Anti-PD-1 (RMPl-14) or RatIgG2a isotype control (2 A3) (Bio X Cell) was administered every 4 days after 12 days of initial tumor growth. For CD8a cell depletion, lOOpg Anti-CD8a (53-6.7, Bio X Cell) was administered three times per week after 5 days of tumor growth.

Results

PERK contributes to activation of chronic ER stress in T effector cells: ER activation contributes to mitochondrial stimulation in CD4+ T cells through inositol 1,4, 5 -triphosphate receptor (IP(3)R)-mediated Ca2+ signaling. Inhibition of IP(3)R shifted T cell fate and promoted cell persistence in tumors (18). Continuous disruptions in Ca2+ homeostasis impair proper protein folding and a high burden of misfolded proteins induces chronic stress on the ER. In the face of irresolvable stress, PERK integrates signals from Ca2+ binding protein BiP/grp78 to initiate the terminal UPR through activation of transcription factors ATF4 and CHOP. A key downstream target of ATF4/CHOP is EROla. As a facilitator of Redox reactions, excessive EROla activity can induce oxidative stress and impair cell survival as cells lose capability to scavenge ROS through anti-oxidant systems (17)(19,20). It is examined how the ER stress sensor PERK and the terminal UPR were regulated in differentiating T effector cells. It is hereby found that gene expression of PERK ( Eif2ak3 ), ATF4 ( Atf4 ), CHOP ( Ddit3), and EROla ( Eorll ) were significantly increased over the course of T cell activation and differentiation (Fig. 1A-D). PERK protein content increased in T cells and peaked after 5 days of expansion. EROla protein increased abundance as T cells differentiated (Fig. IE). PERK gene expression peaked 7 days post activation, but protein expression was reduced after the 5 -day time point. The initial data points out a discrepancy between gene and protein regulation of PERK in T cells and raises interesting questions regarding how PERK is post-transcriptionally regulated is needed.

To measure the role of PERK in induction of the terminal UPR in T cells, T cell receptor (TCR) transgenic T cell-specific conditional knock out mice (OT 1 Lckcre PERK^, PERK KO) were created. Genes associated with the chronic PERK response; A 1/4, Ddit3 , and Eroll in naive and 7-day expanded WT and PERK KO T cells were measured. It was found that Eif2ak3 , A if 4,

Ddit3 , and Eroll were significantly reduced in PERK KO T cells (Figs. 1F-I). In line with these data, PERK and EROla proteins were decreased in PERK KO T cells (Fig. 1J). A longer exposure for PERK was performed to convey absence of PERK protein. These data reveal a role for chronic ER stress as a component of differentiating T effector cells.

PERK axis consumes energy in CD8+ T effector cells: Increased spare respiratory capacity (SRC) is a property associated with superior anti-tumor function of T cells (21). The molecular processes that consume T cell metabolic energy are not well understood. Redox reactions for protein folding and disulfide bond formation in the ER are energy consumptive processes that integrate ER activation with mitochondrial bioenergetics (17,19,22). Oxygen consumption rates (OCR) were measured in WT and PERK KO T cells and it was found that PERK KO T cells maintained superior SRC compared to WT cells. To assess a potential contribution of EROla to this phenomenon, T cells were treated with a target-specific PERK (PERK I) (23) or EROla inhibitor (EROl I) (24) across the course of differentiation and expansion. It was found that PERK I, or EROl I-treated T cells preserved metabolic energy compared to WT controls (Fig. 2A-C). Loss of SRC is associated with a reduction in T cell effector function (25). Capacity to secrete IFN-g in the abovementioned T cell groups were measured and it was found that PERK KO, PERK I, or EROl I T cells had increased IFN-g secretion compared to WT controls (Fig.

2D). Inhibitor treatments did not significantly reduce cell numbers and experimental T cell groups exhibited increased viability compared to respective WT controls (Fig. 17).

Activation of chronic PERK UPR mediated by ATF4/CH0P-ER01a signaling has previously been implicated to drive protein synthesis, consume cellular energy, and contribute to death through oxidative cell stress (16,17,19). To gain insight into how PERK and downstream target EROla shape T effector cells, we assayed the proteomes of T effector, PERK KO, and EROla I-treated T cells. Shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the top 100 proteins with greatest intensity expression in T effectors compared to PERK KO or EROla I-treated T cells (See Table 1 below). User-defined categories were used to qualify proteins into biologically functional groups. Proteomics revealed protein groups associated with energy and metabolism, redox reactions and oxidative stress/ DNA damage with increased expression in T effectors compared to PERK KO and EROla I-treated T cell groups (Fig. 2E-F). Data are represented as fold increased expression values in T effectors compared to PERK KO or EROla I-treated T cell groups. Of user-defined categories the greatest overlap in PERK KO and ERO la-treated T cells was in the“ER transport/ cell stress” group (n=6 proteins). 3/7 of redox-associated proteins overlapped as highly expressed in T effectors compared to both PERK KO and EROla I-treated T cells, highlighting lactate dehydrogenases, Thioredoxins, and Glutathione-S-transferases as protein families of potential relevance.

Table 1


Mitochondrial ROS are a hallmark of mitochondrial exhaustion in T cells: Persistent EROla activation is bioenergetically costly to cells and OXPHOS may be stimulated in efforts to replenish depleted ATP stores (19). A biomarker of energy loss and aging in T cells that may be associated with chronic stress on the ER consistent with pathogenic EROla activation is established. We reasoned that persistent mitochondrial activation through continued OXPHOS would result in accumulation of mitochondrial reactive oxygen species (mtROS) in T cells. The live cell dye MitoSOX Red detects the superoxide anion, a precursor to the majority of cellular ROS, in mitochondrial membranes and is detectable by flow cytometry (26). kinder normal physiological conditions, detoxification of superoxide occurs through superoxide dismutase-mediated conversion to hydrogen peroxide (27). However, excess ROS is associated with disease and aging (28). mtROS in OT-l+ T cells was measured over the course of T effector cell differentiation. As naive T cells lost CD62L expression, a measure of sternness, mtROS accumulated in T cell mitochondria (Fig. 3 A). Human CD8+ T cells underwent the same pattern over the course of 3 weeks of in vitro expansion (Fig. 3B).

To determine whether mtROS accumulation was a hallmark of energy loss in T cells, day 3 and day 5 mtROS and mtROS+ CD8+ T cell subsets were FACS sorted. The lowest 25% mtROS and highest 25% mtROS+ cells were obtained (Fig. 3C) and SRC among groups was measured. Accumulation of mtROS indicated energy loss in T cells (Fig. 3D). The capacity for IFN-g secretion was next measured among day 5 and 7 expanded mtROS and mtROS+ T cell groups. It was found that IFN-g secretion underwent a similar pattern to SRC among sorted T cell populations (Fig. 3E). In day 7 cells, it was examined whether mtROS+ may indicate early stage apoptosis. It was found that ~8% of CD8+ mtROShlgh T cells harvested on day 7 were in early stage apoptosis (Fig. 3F). This was only detectable in day 7 samples, not in cells harvested at earlier time points. Our data show that mtROS can be used as a hallmark of metabolic energy loss and aging in T cells.

PERK axis contributes to mitochondrial exhaustion of T effector cells: Through EROla activation, oxidative protein folding in the stressed ER has been linked to generation of cellular ROS and inhibition of cell survival (19). However, the specific effects of the PERK axis on mitochondrial activation have not previously been assessed. Given the maintenance of SRC and diminished oxidative stress-associated proteomic signature in PERK KO and EROla I T cells (Fig. 2), it was reasoned that these T cell groups may show reduced mitochondrial activation and low mtROS accumulation. It was found that mtROS was significantly diminished in PERK I, EROl I, and PERK KO T cells compared to T effector controls (Fig. 4A). Indeed, in the abovementioned T cell groups, maintained expression of CD62L was a mark of reduced cell aging (Fig. 4B).

Reacquisition (29) or continued expression of CD62L has been associated with memory T cell formation (25,30). Memory T cells in vitro were developed with IL-15 cytokine conditioning and it was found that memory T cells expressed reduced mtROS compared to T effector controls (Fig. 4C). These data are consistent with maintenance SRC of memory cells (25). We asked if memory T cells experience reduced activation of the chronic PERK axis. We found that Atf4, Ddit3 , and EROll genes were reduced in memory T cells compared to T effectors (Fig. 4D). As expected, EROla protein expression was increased in T effectors, and EROla protein was not highly expressed in memory T cells. Gene expression of Eif2ak3 was not significantly different between T effector and T memory cells, but protein expression of PERK was reduced (Fig. 4D-E). These data, similar to data obtained in Fig. 1, suggest post-transcriptional regulation of PERK in T cells. Together, the data indicate that ER programs may impact T cell lineage fate.

PERK and EROla hinder T cell-mediated tumor control: Memory T cells promote durable anti-tumor responses (30). Adoptive T cell therapy mouse models were used to measure how PERK and EROla impact T cell-specific tumor control. Mice bearing B16Fl-OVA-expressing tumors were left untreated or treated with OT-1 (T eff) or PERK KO T cells and tumor control was measured every other day for 3 weeks. OT-l-PERK KO T cells exhibited significantly greater tumor control compared to T effectors (Fig. 5 A). Survival was significantly extended in tumor-bearing mice treated with PERK KO T cells (Fig. 5B). Although PERK can be acutely protective, the PERK axis can also impair T cell-mediated anti-tumor immunity. To further test how the PERK axis contributes to T cell-specific tumor control, P-mel T cells-conditioned with PERK I or EROla I were transferred to mice bearing pathogenic B16F10 melanomas and tumor growth was monitored for 3 weeks. Both inhibitor treatments of T cells yielded significant increases in tumor control compared to T effectors (Fig. 5C-D).

Tumor antigen-specific PD-1+ CD8+ TILs exhibit mitochondrial exhaustion: It was next examined how the terminal PERK UPR is expressed among endogenous CD8+ TILs. CD8+ cells were sorted from spleens and tumors of mice bearing immunogenic MCA-OVA sarcomas and gene expression was measured between splenocytes and TILs. The chronic PERK signaling axis was significantly upregulated in CD8+ TILs compared to splenic-matched controls (Fig. 6A). We next examined whether mtROS accumulated in CD8+ TILs cells in mice bearing immunogenic tumors. In multiple tumor models, CD8+ TILs accrued mtROS compared to T cells in tumor draining lymph nodes (TDLNs). To expand this finding, it was next examined whether mtROS was a property of exhausted T cells in tumors. Co-expression of mtROS and PD-1 in CD8+ T cells from TDLNs and tumors of mice bearing MCA-205-OVA sarcomas or MC-38 colon carcinomas were measured. In both mouse models mtROS+ cells were localized to the PD-1+ compartment and mtROS expression was significantly increased in TILs compared to TDLNs (Fig. 6B-C). In agreement with gene expression data, splenocytes from MCA-205-OVA tumor bearing mice also expressed reduced mtROS/PD-l+ CD8+ T cells compared to TILs (Fig. 18A).

PD-1+ TILs comprise a pool of T cells enriched for tumor antigen specificity (31) and tumor-specific T cells home to and proliferate in tumors within 7 days of adoptive transfer (32). In view of the above experiments, metabolically exhausted TILs were predicted to be in the tumor antigen-specific T cell compartment. To determine this, naive OT-l+ T cells were transferred to mice bearing 7 day-established MCA-205-OVA sarcomas and mtROS/PD-l+ co-expression among endogenous (CD45.1) was measured and (CD45.2) T cells recovered from tumors after 7 days of expansion (Fig. 6D) were transferred. -70% of transferred CD45.2 CD8+ TILs co expressed mtROS/PD-1 compared to -20-30% in the endogenous polyclonal pool of TILs, and this expression could be visualized in TDLNs (Fig. 6E). Thus, PD-1+ T cells experience mitochondrial depolarization (33), and this shed light on the molecular events within T cells that contribute to mitochondrial exhaustion.

PERK inhibition improves anti-PD-1 therapy: Next, it was determined whether mtROS+ CD8+ TILs could be identified in patients. Bone and soft tissue sarcomas are immunogenic human tumor types, but have shown poor responsiveness to anti-PD-1 and combination checkpoint blockade therapies (34). Tumor tissue from patients undergoing routine surgical removal of high grade deep pleomorphic undifferentiated (HGD PU) sarcomas were obtained.

These patients had not received radiation and had not undergone recent chemotherapy.

Dissociation and analysis of CD8+ TILs from fresh HGD PU sarcomas showed a CD8+ T cell infiltrate in all tumors and FACS analysis of PBMC versus autologous TILs showed a significant increase in mtROS/PD-l+ CD8+ T cells in tumors (Fig. 7A). The data shows that PERK inhibition overcomes mitochondrial exhaustion in T cells in vitro (Fig. 4). MCA-205-OVA tumor-bearing mice were treated with PERK I for 7 days during the time period when T cells become functionally exhausted. Day 14 harvest of TILs showed that PERK I-treated PD-1+

CD8+ TILs expressed significantly reduced mtROS compared to vehicle treated controls (Fig. 7B). In line with these data, PERK I treated mice exhibited increased absolute numbers of CD8+ T cells in tumors (Fig. 7C). mtROS+ TILs comprise the pool of highly activated TILs measured by CD44 (Fig. 18B). A reduction in mtROS+ TILs was concordant with reduced CD44+ expression among PERK I-treated TILs in both MCA-205-OVA and MC-38 tumor models (Fig. 18C). These data suggest that PERK I effects on CD8+ TILs were not due to increased activation associated with reduced formation of immunosuppressive TIL populations or reduced tumor growth. In vivo PERK I treatment augmented anti -PD- 1 therapy as 5/7 combination therapy treated mice achieved complete response compared to 2/7 mice treated with anti -PD- 1 therapy alone. Depletions of CD8+ cells over the course of therapy demonstrated that CD8+ cells were necessary for combination therapy effect (Fig. 7D-E). Combination therapy-treated mice exhibited 100% survival compared to -28% survival in the anti-PD-1 therapy condition (Fig.

7F). Together, our data indicate that targeting ER stress in combination with traditional immunotherapy may improve responses in patients.

Discussion

The ER is at the forefront of programming pro- and anti -tumor immunity (35). ROS in the tumor microenvironment drive MDSC cell death through an ER stress-mediated mechanism. The shortened lifespan of MDSCs promotes their continued expansion in bone marrow (36). In multiple human tumor types, MDSCs in peripheral blood directly correlate with low overall survival rates in patients (37). Similarly, the tumor microenvironment appears to impair tumor-associated dendritic cell (tDC) antigen-presentation, and antigen presentation is enhanced by impairing the IREla-stress pathway in tDCs (38). Lastly, the role of ER stress, and in particular PERK, in augmenting tumor cell development is well defined (39). Together, these data indicate that targeting ER stress pathways in cancer patients may be a formidable strategy to promote anti-tumor immune function and impair immune suppression and tumor cell growth.

Few reports have focused on the role of ER stress in control of T cell anti -tumor immunity, and little attention has been paid to the role of the ER in defining T cell biology. Inhibition of ER activation improves T cell mitochondrial function and aids anti-tumor immunity (18). A proteomic screen yielded increased expression of multiple proteins associated with ER-mitochondrial crosstalk enriched in T effectors compared to PERK KO T cells. A second report found that inhibition of IREla stress signaling led to memory T cell development in response to acute infection (40). These data agree with our data presented here that uncover a role for ER stress signaling a response that affects T cell differentiation and effector cell maturation.

Modulation of ER stress elements alone was able to impact mitochondrial bioenergetics and enhance T cell-specific tumor control. Mechanistically, acute PERK-specific ER stress is protective to cells through momentary attenuation of protein synthesis mediated by

phosphorylation of eukaryotic translation initiation factor 2A (EIF2a) (13). The chronic PERK axis drives protein synthesis, energy consumption, oxidative cell stress and death through activation of transcription factors ATF4 and CHOP (16,17). Though PERK gene and protein expression were reduced in the PERK KO T cells described above, the possibility of remnant PERK expression that may have conferred the protective effect of the acute PERK response combined with reduced lethal effects from the terminal UPR is not discounted. Based on our findings, T cells present a paradigm in which to study the effect of biologically relevant chronic ER stress on cell development and death. The data described herein shows that activation of EROla in rapidly dividing T effectors/TILs governs a cell-intrinsic mechanism that drives energy loss and oxidative stress.

Based on the durability of anti-tumor immunity associated with memory T cells in vivo

(4,21,25,43), it is noted that modulation of the PERK pathway could promote in vivo T cell durability and contribute to long-term efficacy of anti -PD- 1 therapy. Multiple potential benefits of targeting chronic ER stress have been identified in modulation of pro and anti-tumor immunity (35).

Example 2:

Sarcomas are a unique tumor type rife with tumor associated antigens that attract a CD8 TIL infiltrate (25, 26). PD-1 monoclonal antibody therapy targets PD-1-PD-L1 (programmed death-ligand l)-mediated inhibition between T cell and tumor cells, respectively, and is most effective in immunogenic tumor types (37). PD-1+ CD8 TILs are a subset of T cells in tumors enriched for tumor antigen specificity (38). Based on these data, it is surprising that only -20% of sarcoma patients across subtypes respond to anti-PD-1 (a-PD-1) therapy and the therapy is not FDA approved for sarcoma patients (27). CD8 TILs with low to moderate expression of PD-1 are the key subset of TILs that respond to a-PD-1 therapy, as PD- 1 hlgh CD8 TILs are terminally exhausted with poor bioenergetics marked by depolarized mitochondria (39-41). Here the cell response to stress in T cells carried out by an ER stress sensor PERK is studied. In response to acute stress PERK momentarily inhibits cell functions (30). Under repeated stress, PERK carries out a chronic stress response that activates cells to the point of death (17). In vitro , it has been found that the sarcoma microenvironment induces the acute PERK response to protect CD8 T cells. However, the data indicates that in vivo the chronic PERK response drives activation and metabolic exhaustion in PD- 1 hlgh CD8 TILs in human and mouse sarcomas. Inhibition of PERK induced complete and long-term responses to a-PD-1 therapy in sarcoma-bearing mice (1). New PERK inhibitors that bypass pancreatic toxicity are in development (20-22), but first generation PERK inhibitors induce toxicity in animals due to loss of the acute response (11, 13, 31).

Excitingly, the data presented herein suggests that the acute arm of PERK is essential to protect CD8 TILs in sarcoma microenvironments. Herein, it is proposed that new molecular targets in the chronic PERK axis are essential to revive CD8 TILs and restore response to a-PD-1 therapy in sarcoma patients. This hypothesis is a paradigm shift for the field of cancer immunotherapy as we posit for the first time that the chronic ER stress response regulates the efficacy of a-PD-1 therapy in sarcoma patients.

Atf4 , a driver of T cell activation and exhaustion in cancer immunotherapy. p-eIF2a activation by PERK induces global attenuation of translation (30), but ATF4 is selectively translated (32). Under persistent and pathological stress ATF4 serves as a pro-apoptotic agent that provokes an aberrant translational program and inflammatory response (17). In multiple diseases of chronic inflammation ranging from age and diet-induced obesity, metabolic

syndrome, type 1 and type 2 diabetes, and tumor progression, ATF4 has proven to be the key regulator of inflammation-induced disease. Attenuation of ATF4 relieves age and diet-related obesity (14), ameliorates type 1 and type 2 diabetes-induced vascular retinopathy (15), and reduces proteotoxic stress in tumor cells resultant in diminished disease progression and extended survival (16). Without being bound by theory, the preliminary data suggests that ATF4 is a crucial transcription factor that promotes activation, differentiation, and metabolic exhaustion in CD8 TILs in mouse and human sarcomas. Herein, mice with T cell-specific overexpression of human ATF4 (42) (LckcreRosa26-ATF4loxtg), CD8 TILs from sarcoma patients, and a humanized mouse model of chimeric antigen receptor (CAR) T cell therapy are used to determine whether Atf4 drives exhaustion in CD8 TILs and restricts response to a-PD-1 therapy of sarcoma.

EROla-induced metabolic dysfunction in immunometabolism. A crucial transcriptional target of Atf4 is the enzyme EROla (17). In an effort to relieve a chronic burden of

unfolded/misfolded proteins in the ER lumen, EROla catalyzes protein folding through transfer of electrons to molecular oxygen (18). The consequence of EROla enzymatic activity is generation of free radicals. Initially, the anti-oxidant glutathione acts to detoxify free radicals produced by EROla, but under chronic stress EROla depletes glutathione stores and impairs survival through generation of reactive oxygen species (ROS) (18, 19). Of note, glutathione is essential for inflammatory T cell responses (43). Thus, identification of the crucial enzyme that consumes glutathione in tumor antigen-specific CD8 TILs has widespread implications for cancer immunotherapy. The preliminary data described herein indicates that PD-1+ CD8 TILs generate ROS through EROla resultant in depolarized mitochondria and impaired capacity to generate metabolic energy for response to a-PD-1 therapy (1). This data also shows that EROla /_ T cells exhibit profound tumor control. Herein unique ERO l a mice, metabolomics, and CD8 TILs from sarcoma patients are used to determine if EROla limits response to a-PD-1 therapy by inducing metabolic exhaustion in mouse and human CD8 TILs in sarcomas.

The ER stress field and the acute PERK response. The acute PERK response is a critical protector of CD8 TIL viability. In response to acute cell stress PERK phosphorylates the alpha subunit of eIF2 to inhibit translation and protect cells from death induced by an immediate

abundance of misfolded/ unfolded proteins in the ER lumen (30, 44). Previous toxicity of PERK inhibitors is due to loss of p-eIF2a in pancreatic islets (10-13). Specifically, loss of p-eIF2a mediated attenuation of translation enables accumulation of pro-insulin that overwhelms the processing and secretory capacity of the ER in b-islet cells, culminating in b cell death. Mice with a homozygous mutation at the p-eIF2a phosphorylation site die after birth due to hypoglycemia (11, 12). Without being bound by theory, it is hypothesized that tumor microenvironments induce acute cell stress that leads to PERK-p-eIF2a attenuation of protein translation as a protective measure in CD8 TILs. Creation of PERK-deficient T cells (PKO) revealed that PERK induces expression of p-eIF2a and attenuates translation in CD8 TILs in sarcomas. Herein PKO (LckcrePERK®) mice and mice heterozygous for mutation at the eIF2a phosphorylation site (Eif2aS51A +/ ) (11) are used determine if PERK-p-eIF2a engenders CD8 TIL survival in the sarcoma microenvironment, promoting T cell response to a-PD-1 therapy.

PERK hinders T cell anti-tumor immunity. To measure the contribution of PERK to T cell tumor control TCR transgenic T cell-specific conditional knock out mice (OT-l-Lckcre-PERK®) PKO) were created. The PKO CD8 T cells were devoid of PERK protein (Fig. 8 A). Ex vivo expanded WT or PKO CD8 T cells were transferred to mice bearing B 16-OVA melanomas and measured tumor growth every other day with calipers. Surprisingly, it was found that PKO CD8 T cells exhibit a profound capacity to control tumor growth relative to WT T cells (Fig. 8B) and increase survival in mice bearing melanomas (Fig. 8C) (1). This data demonstrates the unique ability of PERK to severely restrict CD8 T cell tumor control.

CD8 TILs experience the terminal PERK UPR. The MCA-205 sarcoma mouse model is replete with CD8 TILs due to its immunogenic nature, but tumor growth is not controlled in immune-competent mice (45, 46). The data shown in Figure 9 confirms that MCA-205 sarcomas are rife with CD8 TILs (Fig. 9 A) and that tumors grow at a rapid rate after intradermal injection (Fig. 9B). Within the PERK axis, the chronic arm of the PERK response comprised of ATF4, C/EBP homologous protein (CHOP), and EROla is deleterious to cells (17). Given that PERK-deficient T cells control tumor growth (Fig. 8), the chronic PERK response in CD8 TILs in MCA-205 sarcomas was measured. It was found that Atf4, Chop , and Eroll were increased in CD8 TILs relative to autologous-matched splenocytes (Fig. 9C). These data prompted us to

perform LC-MS/MS proteomic analysis between CD8 T cells from spleens and tumors of MCA-205 sarcoma-bearing mice to study differences in protein biology between T cell subsets. Indeed, EROla protein was increased in CD8 TILs of mice bearing MCA-205 sarcomas (Fig. 9D). Analysis of sarcoma patients similarly revealed that the chronic PERK-directed transcription factors, Atf4 and Chop, that induce EROla expression (17) were enhanced in CD8 TILs from untreated high grade deep pleiomorphic undifferentiated (HGD PU) sarcoma patients relative to autologous-matched peripheral CD8 T cells (Fig. 9E). Without being bound by theory, this data suggests that the terminal PERK UPR may shape the biology of CD8 TILs in mouse and human sarcomas.

Effector CD8 T cells experience the terminal PERK UPR. This finding that the terminal PERK UPR affects CD8 TILs prompted the question: How does the chronic PERK axis affect CD8 T cell biology? This axis has not been studied in CD8 T cells. To begin to understand the role of the UPR in T cell biology, 84 common UPR genes were measured in a model of effector and memory T cell development in vitro. The cytokine IL-2 was used to differentiate CD8 T cells to an effector cell phenotype and the cytokine IL-15 to induce memory-like T cell formation (47-49). Comparison of 84 common UPR genes using a gene array led to the striking finding that the terminal UPR comprised of Atf4, Chop , Eroll is the critical UPR axis differentially regulated between effector and memory-like T cells, as effector T cells are enriched for this axis (Fig. 10A). Next, LC-MS/MS proteomic analysis was used between effector (IL-2) and memory-like (IL-15) T cells to study differences in protein biology between T cell subsets. Indeed, it was found that EROla was substantially increased in effectors relative to memory cells (Fig. 10B), and we verified these data by western blotting (Fig. IOC). These data show for the first time that effector T cells experience the terminal UPR.

PERK-ATF4-ER01a shapes memory T cells. T cells with memory traits exhibit superior tumor control relative to effector T cells (50, 51), and patient responses to a-PD-1 therapy are enhanced by the presence of memory T cells in the TIL pool (34). These data are important in the context of cancer immunotherapy because the CD8 TIL pool in sarcoma patients is enriched for effector T cells (26, 52). Thus, strategies to remodel the landscape of endogenous CD8 TILs from an effector to memory state is a critical area of study in cancer immunotherapy (53). Given the predominance of the terminal UPR in CD8 TILs and effector T cells, CHOP KO mice (Jackson Labs) were obtained and CRISPR/Cas9 ribonucleoprotein (RNP) in vitro genome editing was used to delete Atf4 in OT-1 T cells (ATF4 RNP) and unique EROla _/ mice were created to examine the role of PERK, ATF4, CHOP, and EROla in defining CD8 T cell fate. Flow cytometry was used to measure CD62L/CD44 expression in UPR T cells and we found that PERK, ATF4, and EROla -deficient T cells exhibited a memory-like phenotype (Fig. 11 A). Next, RT-PCR was used to measure gene expression of the hallmark stem -like memory T cell transcription factor 7 (Tcf7) (50) in UPR T cells. It was found that PERK, ATF4, and EROla-deficient T cells expressed Tcf7 at levels on par with memory T cells (Fig. 1 IB). ATF4 and CHOP can act in tandem to induce transcription of Eroll (17). To determine the dominant transcription factor of Eroll in T cells, RT-PCR was used to measure Eroll expression in ATF4 RNP and CHOP KO T cells. It was found that ATF4 drives Eroll expression in CD8 T cells (Fig. 11C). These data demonstrate that ATF4 is the dominant transcription factor to induce Eroll (17). Together, our data indicate that PERK-ATF4-ER01a shapes memory T cell properties.

EROla impacts CD8 T cell metabolism and improves tumor control. The data implicates EROla as a candidate enzyme that shapes the biology of CD8 TILs. Thus, a unique ERO la mice was created as described above (Fig. 11). Additionally, EROla has been implicated as an enzyme that induces cell death through generate of ROS (54-56). Further, ROS accumulates in mitochondria (mtROS) of PD- 1 hlgh CD8 TILs and effector T cells, but are substantially reduced in memory T cells (1). mtROS in CD8 T cells from WT and ER01a /_ mice were measured and it was found that EROla induces mtROS generation in CD8 T cells (Fig. 12A). EROla is a critical cell consumer of glutathione (57). It was found that treatment of effector CD8 T cells with N-acetyl cysteine (NAC) to restore cell glutathione substantially diminished mtROS (Fig. 12B). Work by others found that T cells treated with NAC exhibit powerful tumor control relative to effector T cells (58). Thus, the capacity of EROla T cells to control tumor growth was measured. Surprisingly, transfer of OT-1- EROla T cells to mice bearing B16-OVA melanomas revealed that EROla substantially impairs T cell tumor control (Fig. 12C). This data suggests that EROla is a new target to improve the efficacy of CD8 T cells for cancer immunotherapy.

mtROS indicate metabolic exhaustion. FACS was used to sort mtROS and mtROS+ CD8 T cell populations (Fig. 13 A) and levels of mitochondrial ATP were assessed in T cells. It was found that mtROS is an indicator of ATP loss in T cells (Fig. 13B). Moreover, it was found that mtROS+ T cells are primed to produce less IFN-g than mtROS T cells (Fig. 12C), and that mtROS+ T cells experience greater early cell death than mtROS counterparts as measured by co expression with Annexin V (Fig. 13D). T cells treated with an EROla inhibitor (59) expressed replenished mitochondrial ATP (Fig. 13E). These data illustrate that mtROS indicates T cell metabolic exhaustion.

PD-lh,gh CD8 TILs exhibit metabolic exhaustion. In a virus-specific T cell model, Rϋ-1M§ΐ1 CD8 T cells show bioenergetic insufficiencies associated with ATP depletion and mtROS expression that denotes depolarization of mitochondria (39, 41), a hallmark of cell death (60, 61). mtROS/PD-l+ CD8 T cell subsets have not been studied in the context of tumor antigen. Thus, naive OT-1 T cells were infused into CD45.1 mice-bearing MCA-205-OVA sarcomas and measured mtROS/PD-1 expression among transferred tumor antigen-specific CD8 T cells (CD45.2+) in tumor draining lymph nodes (TDLN) and TILs of recipients (Fig. 14A).

Surprisingly, it was found that -40% of transferred CD8 T cells honed to tumors and became PD-lhigh/mtROS+ within 7 days of transfer (Fig. 14B). To measure the potential contribution of the PERK axis to development of PD-lhigh/mtROS+ CD8 TILs MCA-205 sarcoma-bearing mice were treated with vehicle or a PERK inhibitor (62) for 7 days and assessed mtROS/PD-1 expression among endogenous CD8 TILs. PERK inhibition abrogated Rϋ-1M§1i/ihΐK08+ 0ϋ8 TILs and enriched CD8 TILs for a PD-llow/mtROS subset (Fig. 14C). These data are exciting because PD-llow CD8 TILs are the primary responders to a-PD-1 therapy (40). It was found that CD8 TILs in untreated HGD PU sarcoma patients express a vivid Rϋ-1M§1i/ihΐK08+ population (Fig. 14D). Together, this data indicates that tumor antigen-specific CD8 TILs are enriched for PD- 1 hlgh/mtROS expression which indicates metabolic exhaustion.

Example 2.1: Examination of whether ATF 4 promotes aberrant activation and exhaustion in CD8 TILs in mouse and human sarcomas that impairs response to a-PD-1 therapy.

Under persistent and pathological stress the transcription factor ATF4 serves as a pro-apoptotic agent that provokes aberrant translation and inflammatory response (17). In multiple diseases of chronic inflammation ATF4 has proven to be the key regulator of inflammation-induced disease progression (14-16, 63). The data suggests that Atf4 promotes activation and exhaustion in CD8 TILs in a sarcoma mouse model. Sarcoma-bearing mice were treated with a PERK inhibitor (62) combined with a-PD-1 therapy to test the concept that targeting the PERK axis impacts response to a-PD-1 therapy. PERK inhibition radically augmented a-PD-1 therapy in a sarcoma mouse model in a CD8 T cell dependent manner. Sarcoma-bearing mice treated with combination therapy achieved complete responses (Fig. 15 A) and experienced progression-free survival (Fig. 15B). To develop new, specific, non-toxic therapies that surround the PERK axis to improve the power of immunotherapy this experiment will use LckcreRosa26-ATF4loxtg (42) mice with T cell-specific overexpression of human Atf4 (ATF4 OE), CD8 TILs from sarcoma patients, and a humanized model of CAR T therapy to test whether Atf4 drives exhaustion in CD8 TILs and restricts response to a-PD-1 therapy of sarcoma.

Experiment A: Impact of ATF4 in restricting T cell-mediated tumor control through induction of activation and exhaustion of CD8 TILs in sarcoma.

T cell-mediated tumor control in sarcoma. To specifically test the direct role of ATF4 in T cells in sarcomas, MCA-205 sarcoma growth is measured in ATF4 OE and littermate (WT) control mice MCA-205 sarcomas are established intradermally on the right flank of mice and groups will be established as in Table 2. Tumor growth is measured every other day with calipers.: Tumor growth over time is the primary endpoint. Time to sacrifice (TTS) is monitored. It is

contemplated that ATF4 OE mice exhibit greater tumor growth than WT mice as shown by larger tumor size and lower TTS.


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Contribution of ATF 4 to induce activation and exhaustion of CD8 TILs in sarcomas. To determine whether ATF4 contributes to exhaustion of CD8 TILs, CD8 TILs are profiled in ATF4 OE and littermate controls in mice bearing 14-day established MCA-205 sarcomas as in Table 2. In this tumor model after 14 days of tumor growth, -50% of CD8 TILs express PD-1 and -20%

of these TILs are Rϋ-1M§ΐ1 (Fig. 14C). FACS analysis is used to phenotype PD-1, KLRG-1, TIM-3, CD62L, CD44, and CD69 among CD8 TILs. Isotype and FMO controls are used. The mean fluorescent intensity (MFI) of individual phenotypic marker expression between WT and ATF4 OE mice are quantified and percentage and absolute number of the following phenotypic subgroups: PD-17PD-llow/PD-lMgh/TIM-3/CD8, KLRG-1/CD69/CD62L/CD44/CD8 are examined. It is contemplated that a direct measure of exhaustion in ATF4 OE mice agrees with the preliminary data detailed above. Additionally, it is contemplated that CD8 TILs in ATF4 OE mice exhibit greater proportions of Rϋ-1M§1i/TIM-3+/Oϋ8 TILs as well as KLRG-1 +/CD62L /CD44+/CD8 indicative of terminal exhaustion and terminal differentiation, respectively.

Experiment B: Contribution of ATF4 in CD8 T cell response in a-PD-1 therapy of sarcomas.

To determine the contribution of ATF4 to restrict response to a-PD-1 therapy, the response to a-PD-1 therapy is measured in WT littermates and ATF4 OE mice bearing MCA-205 sarcomas. MCA-205 sarcomas are established and groups are established as in Table 3. After 14 days of tumor growth mice are treated with a-PD-1 antibody or isotype control (200pg/mouse). Tumor growth is measured every other day with calipers. The primary endpoint is Tumor growth over times. TTS is monitored. It is contemplated that a direct measure of response to a-PD-1 therapy suggests that chronic ER stress impairs response to therapy and that ATF4 OE mice exhibit impaired response to a-PD-1 therapy relative to WT mice shown by greater tumor size, fewer complete responses, and reduced TTS.


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Experiment C: ATF4 gene expression between PD-1 and PD-1+ CD8 I II A from sarcoma patients. Peripheral blood and tumor samples are obtained from untreated HGD PU sarcoma patients who have not received radiation or recent chemotherapy. For comparison, healthy normal donor peripheral blood mononuclear cell (PBMC) samples are also obtained. PBMC are obtained by histopaque gradient and tumors are processed to single cell suspension using human tumor dissociation kit (Miltenyi). Total live CD8+ T cells are FACS sorted based on CD8/PD-1+ or CD8/PD-1- expression and PD-1 gating is set from isotype control expression. The

preliminary analysis found that -20% of CD8 TILs in sarcoma patients are PD-1+ with a substantial proportion of these TILs exhibiting PD- 1 hlgh expression (Fig. 14D). TaqMan probes are used to measure Atf4 expression relative to Gapdh. Gene expression of A if 4 among normal CD8+ PBMC, sarcoma patient CD8+ PBMC, and PD-1- and PD-1+ CD8 TILs are compared.

It is contemplated that the measurement of ATF4 in PD-1- and PD-1+ CD8 TILs agrees with the human data described above (Fig. 9E) that assessed ATF4 expression in total CD8 TILs of sarcoma patients. It is further contemplated that Atf4 gene expression is elevated in CD8 TILs relative to healthy donor and sarcoma patient CD8+ PBMC and that Atf4 gene expression is increased in PD-1+ relative to PD-1- CD8 TILs.

Experiment D: Contribution of ATF4 to human T cell tumor control in a humanized mouse model of CAR T cell therapy: Results from CAR T clinical trials show limited durability of therapy in sarcoma patients (25, 27, 64, 65). Lentiviral transduction are used to introduce a CAR specific for the mesothelin antigen (MSLN) (mesoCAR) (66-68) and a ATF ribonucleoprotein (RNP) CRISPR/Cas9 genome editing is used to generate ATF4-/- mesoCARs to determine the contribution of human ATF4 to T cell tumor control of MSLN-expressing Ml 08 tumors in NOD/SCID/g (NSG) mice. This model is relevant for synovial sarcoma patients that ubiquitously express the MSLN antigen (67, 69).

ATF4 RNP Introduction. PBMC from untreated HGD PU sarcoma patients is collected. CD8 T cells are isolated and bead activated (Dynabeads). Atf4 gene deletion will be attained through CRISPR/Cas9 genome editing, the protocol involves annealing of a specific crRNA with tracrRNA. The resulting cr/tracrRNA duplex is complexed with high-fidelity Cas9 protein. The cr/tracrRNA/Cas9 ribonucleoproteins (RNPs) are transfected into T cells using Neon Electroporation System (ThermoFisher). crRNAs are designed and validated in vivo for efficiency targeting Atf4 in human erythroleukemic K562 cells. Genetically engineered animals (70, 71) including our novel ErollemlMusc mice (Figs. 11-12) are used. MesoCAR Introduction: Prior to ATF4 RNP introduction, CD8 peripheral T cells from sarcoma patients are transduced with a lentiviral vector encoding a first-generation CAR that recognizes mesothelin and stimulates the Oϋ3z domain (68). MesoCAR expression will be validated and measured by FACS staining (Jackson ImmunoResearch). Tumor model: NSG mice are s.c. injected with 5x106 Ml 08 tumor cells and 40 days post-M108 establishment mice are i.v. infused with 4x106 CAR T cell groups as in Table 4. Tumor growth is measured every other day with calipers. Tumor growth over time is the primary endpoint. TTS will be monitored. It is contemplated that ATF RNP mesoCARs exhibit better tumor control of human tumors relative to control CAR T.


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Experiments A-D illuminate the crucial transcription factor that induces CD8 TIL activation and exhaustion and describe a completely new axis important in both T and tumor cell (16) fate with potential to transform the efficacy of patient cancer therapy.

An alternative mouse model for LckcreRosa26-ATF4loxtgmice is CD4creRosa26-ATF4loxtgmice. CD4creRosa26-ATF4loxtg mice would induce ATF4 overexpression in T cells in all stages of development, prior to thymic differentiation. In contrast, LckcreRosa26-ATF4loxtgmice utilize dLck-hcre3779 mice (Jackson Labs, 012837) in which ere recombinase expression is driven by the distal promoter of the Lck gene, thus ere expression occurs after positive selection in the thymus. This model enables study of changes affected by gene overexpression in peripheral T cells without having to account for effects of overexpression in T cell thymic development. The present data indicates that PERK-ATF4-ER01a signaling is important in peripheral T cells as they become exhausted in tumor, thus the proposed model best suits the in vivo goals of this experiment. In Experiment A, the possibility exists that LckcreRosa26-ATF4loxtgmice will experience greater tumor control relative to WT mice due to heightened CD8 TIL activation. Our experimental design in Experiment. B it is explore the mechanism of ATF4 overexpression in CD8 TIL phenotype. F or Experiment D, an alternative model to test with importance in sarcoma is disialoganglioside (GD2)-CARs that target the GD2 tumor associated antigen that is ubiquitously expressed in multiple pediatric sarcoma subtypes (72). GD2-CARs coupled with human 143b osteosarcoma can be used to test the efficacy of PERK-deficient CARs to control other sarcoma subtypes (72).

Example 2.2: Examination of whether EROl a induces metabolic dysfunction in the exhausted CD8 TIL pool that limits response to a-PD-1 therapy.

As discussed above, EROla is a notorious PERK-mediated cell death enzyme that impairs survival through generation of free radicals (17, 35). Initially, the anti-oxidant glutathione detoxifies ERO la-induced ROS (57); however, upon glutathione depletion, accumulation of ROS leads to cell death (36, 73). The data indicates that EROla is enhanced at the gene and protein levels in CD8 TILs in a model of T cell exhaustion in sarcoma (Fig. 9). Generation of unique ERO l a mice indicates that EROla enhances ROS in T cells in a manner that is dependent on loss of glutathione. It is established that glutathione is essential for inflammatory T cell responses (43), and we found that ERO la CD8 T cells control tumor growth better than WT cells (Fig. 12). Moreover, ERO l a mice are viable and fertile and do not experience aberrant pathology in a basal state (74). These data demonstrates that EROla is a target to improve the benefits of cancer immunotherapies. Here, EROla mice, metabolomics, and peripheral and tumor infiltrating CD8 T cells from sarcoma patients are used to determine whether EROla limits response to a-PD-1 therapy by inducing metabolic exhaustion in mouse and human CD8 TILs in sarcomas.

Experiment E: Contribution of EROla in shaping glutathione metabolism in CD8 T cells.

In Fig. 12 the data shows that EROla drives mtROS expression in CD8 T cells. Moreover, the data shows that N-acetyl cysteine (NAC) treatment of CD8 T cells is able to clear mtROS accumulation in T cells. Given that NAC is a glutathione precursor, this data agree with the known role of EROla to deplete the anti -oxidant glutathione in cells. Metabolomics are used to measure metabolites associated with the glutathione pathway in ex vivo expanded OT-1 or OT-1 EROla _/ T cells. Due to the number of cells needed for metabolite analysis, these experiments cannot be performed on cells directly ex vivo. Metabolon offers a global metabolomic profiling platform that consists of four independent methods: ultrahigh performance liquid

chromatography/tandem mass spectrometry (UHLC-MS/MS) with positive ion mode electrospray ionization, UPLC -MS/MS with negative ion mode electrospray ionization, and UPLC-MS/MS polar platform. LC-MS is performed on a Waters ACQUITY UPLC and a ThermoScientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a

heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer. Eight (8) biological replicate samples per condition are submitted for analysis for a total of 16 samples. Each sample is accessioned into a LIMS system at Metabolon. The LIMS system encompasses sample accessioning, preparation, instrument analysis and reporting, and advanced data analysis. More than 4,000 commercially available purified standards are registered into LIMS for determination of their analytical characteristics. Peaks are quantified using area under the curve. Subsequent QC and curation processes are designed to ensure accurate identification and to minimize artifacts, mis-assignments, and background noise. Pathways are assigned for each metabolite, allowing discovery of enriched pathways. It is contemplated that oxidized glutathione (GSSG) will be elevated and reduced glutathione (GSH) will be diminished in OT-1 relative to OT-1 EROla T cells. It is also contemplated that changes in these metabolite subgroups will result due to crosstalk between glutathione and methionine/cysteine/taurine metabolism.

Experiment F: Contribution of EROla in restricting T cell-mediated tumor control through induction of metabolic exhaustion in in sarcoma.


Contribution of EROla to restrict T cell-mediated tumor control of CD8 TILs in sarcoma. To determine the contribution of EROla to T cell control of sarcoma, RAGl mice with WT or EROla 7 bone marrow (BM) are reconstituted and growth of MCA-205 sarcomas is measured. BM is isolated, red blood cells lysed, and 5xl06 BM cells from WT or EROla mice is infused via tail vein to sublethally irradiated RAGl 7 mice. Reconstitution among groups is monitored by CD8 populations in PBMC of treatment groups. After reconstitution for ~4 weeks, MCA-205 sarcomas are established intradermally on the right flank of animal groups outlined in Table 5. Tumor growth is measured every other day with calipers. Tumor growth over time is the primary endpoint. TTS will be monitored. It is contemplated that mice bearing EROla 7 BM will exhibit reduced tumor growth and slower TTS.


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Contribution of EROla in inducing metabolic exhaustion in CD8 TILs in sarcoma. A separate group of mice is reconstituted as described immediately above, and MCA-205 sarcomas are

established intradermally on the right flank of treatment groups outlined in Table 5. After 14 days of tumor growth spleens and tumors are harvested and parameters of metabolic exhaustion in CD8 T cells outlined in Fig. 13 are measure. FACS analysis is used to assess mtROS/PD-1 expression, IFN-g production, and Annexin/PI cell death between groups. To measure ATP among CD8 TIL groups, 5-10 mice per group are combined, live CD8 TILs are FACS sorted and plated per well in 96 well Seahorse plates. Oxygen consumption rate in response to injections of Oligomycin, FCCP, and Rotenone/ Antimycin A is measured. The absolute numbers of live CD8 TILs, percentages of mtROS/PD-1 Mgl1 and mtROS-/PD-llow CD8 TILs, IFN- g+ CD8 TILs, Annexin+/PI+ CD8 TILs, are compared and spare respiratory capacity is quantified as in Fig. 13 between groups. It is contemplated that mice bearing WT BM exhibit reduced live, mtROS /PD-llow, and åFN-y + CD8 TILs relative to mice bearing CD8 TILs derived from EROla 7 BM. It is further contemplated that CD8+ Annexin+/PI+ populations are elevated in mice bearing WT BM and SRC reduced relative CD8 TILs derived from ERO l a BM.

Experiment G: Contribution of EROla to CD8 T cell response in a-PD-1 therapy of sarcomas. To determine the contribution of EROla to limit T cell response to a-PD-1, the response to a-PD-1 therapy is measured in RAG 1 mice reconstituted with WT or ERO la BM mice bearing MCA-205 sarcomas as described in Experiment F . After reconstitution for ~4 weeks, MCA-205 sarcomas are established intradermally on the right flank of groups outlined in Table 6. After 14 days of tumor growth mice are treated with a-PD-1 antibody or isotype control (200pg/mouse). Tumor growth is measured every other day with calipers. Tumor growth over time is the primary endpoint. TTS is monitored. It is contemplated that mice bearing EROla BM show less tumor growth and increased response to a-PD-1 therapy.


Experiment H: Eroll gene expression between PD-1 and PD-1+ CD8 TILs from sarcoma patients. As described in Experiment C , Taqman probes are used to measure Eroll expression relative to Gapdh in CD8 T cells sorted from healthy normal donor PBMC, and PBMC and TIL sorted from untreated PU HGD sarcoma patients. Similar to Experiment C, CD8 T cells are FACS sorted based on PD-1 expression. PD-1 gating is set from isotype control expression. It is contemplated that Eroll expression is elevated in CD8 TILs relative to healthy donor and sarcoma patient CD8+ PBMC. It is further contemplated that Eroll gene expression is increased in PD-1+ relative to PD-G CD8 TILs.

The demonstration that EROla shapes CD8 T cell metabolic fate transforms the concept of immunometabolism and presents a novel molecular target to restore metabolic homeostasis in CD8 TILs to improve the efficacy of standard checkpoint therapy in cancer patients.

The data indicates that EROla CD8 T cells induce superior tumor control relative to WT T cells (Fig. 12). The experimental design in Experiment F determines the contribution of EROla in endogenous CD8 T cells over the course of sarcoma development. Given that the EROla mice are total body knock outs, CD8 7 mice can be reconstituted with WT or EROla 7 BM to produce mice with WT and EROla 7 CD8 T cell compartments, respectively. A similar strategy can be used to address the CD8 T cell-specific contribution of EROla to a-PD-1 therapeutic response in Experiment G.

Example 2.3: Examination of whether the acute PERK-p-eIF2a stress response protects CD 8 TILs and promotes response to a-PD-1 therapy.

In response to acute ER stress, PERK-p-eIF2a attenuates translation to restore protein homeostasis and protect viability (30). Moreover, it is established that PERK toxicities associated with first generation inhibitors in animal models are due to loss of p-eIF2a-mediated attenuation of translation (11, 13). Thus, evidence that the acute arm of the PERK response is necessary to protect CD8 TILs in tumors is critical for the development of new immunotherapies that aim to target the cell stress response. Without being bound by theory, we hypothesize that the sarcoma microenvironment is a form of acute stress and that PERK-p-eIF2a signaling is protective of CD8 TILs in tumors. In a contact-independent transwell tumor microenvironment co-culture assay in which tumor cells and T cells share media without touching, it was found that p-eIF2a was induced in T cells through PERK (Fig. 16A). Using a rigorously validated FACS-based protein synthesis assay in which a fluorescent amino acid analogue of methionine

(homopropargylglycine, HPG) is incorporated into actively translating cells (76-78), it was found that translation is reduced in WT CD8 T cells in the tumor microenvironment through PERK (Fig. 16B). In mice bearing MCA-205 sarcomas, it was found that CD8 TILs in PKO mice

(LC1<67VPERK/ /) continue translation relative to protected WT TILs (Fig. 16C). In multiple untreated HGD PU sarcoma patients, it was found that CD8 TILs restrict translation relative to peripheral CD8 T cells (Fig. 16D). Together, this data indicates that translation in CD8 TILs is repressed through PERK. To develop new, specific, non-toxic therapies that surround the PERK axis to improve the power of immunotherapy this example uses Lck /cPERKi7r (PKO) mice, mice heterozygous for the Eif2aS51A mutation that prevents phosphorylation of the a subunit of eIF2 (Eif2aS51A +/ ), and MCA-205 sarcomas to determine whether PERK-p-eIF2a engenders CD8 TIL survival in the sarcoma microenvironment, promoting response to a-PD-1 therapy.

Experiment I: Measure the contribution of PERK and p-eIF2a to CD8 T cell translation and death in the sarcoma microenvironment.

Contribution of PERK to CD8 T cell translation and death in the sarcoma microenvironment. Translation and cell death is measured in WT and PKO CD8 T cells in response to co-culture with MCA-205 sarcoma cells and in response to treatment with supernatant harvested from MCA-205 sarcomas. Co-culture: Sarcoma cells are seeded in co-culture plates. 24 hours later 3-day ex vivo expanded OT-1 or OT-1 PKO CD8 T cells are added to transwells. Transwells without tumor cells serve as controls. After 36 hours of co-culture T cells are harvested and FACS analysis is used to measure translation and cell death by HPG-assay and Annexin/PI staining, respectively. Cycloheximide (CHX)-treated samples serve as controls for HPG FACS assay and fluorescence minus one controls (FMO) serve as Annexin gating controls. Sarcoma supernatant: MCA-205 sarcomas are established intradermally on the right flank of C57BL/6 mice. 14 day sarcomas will be harvested and cut into ~2mm2 tumor pieces and pieces will be cultured in complete T cell media for 24 hours then supernatants will be pooled and frozen. 3-day ex vivo expanded OT-1 or OT-1 PKO CD8 T cells are cultured for 24 hours in complete T

cell media or sarcoma supernatant media. 24 hours later T cells are harvested and FACS analysis for translation and cell death as described above is performed. In both experiments, lysates are collected to immunoblot p-PERK, PERK, p-eIF2a, and eIF2a with b-actin as loading control. For protein synthesis, MFI of HPG incorporation is divided by MFI of CHX to obtain fold from CHX values in each T cell condition. For cell death, MFI of Annexin and percentage of

Annexin+/PI+ CD8 T cells in each condition is analyzed. It is contemplated that PKO CD8 T cells have a greater increase in translation and cell death in tumor stress conditions relative to WT T cells.

Contribution of p-eIF2a to CD 8 T cell translation and death in the sarcoma microenvironment. Eif2aS51A mutant mice are unable to phosphorylate the a subunit of eIF2 (Eif2aS51A +/ ), but homozygotes die after birth due to hypoglycemia. Heterozygotes are viable and fertile (11). T cells from mice that are heterozygous for the Eif2aS51A mutation (Jackson Labs) are studied. To easily work with T cells in culture Eif2aS51A +/ mutants are crossed to OT-1 mice to obtain OT-1-Eif2aS51A +/ T cells. Co-culture and sarcoma supernatant experiments are carried out as described in Experiment A. Protein synthesis, cell death, and lysates are measured as described in

Experiment A. Protein synthesis and cell death is analyzed as described in Experiment A. It is contemplated that data generated in Eif2aS51A +/ mutants offer a rigorous system to test the contribution of acute ER stress to translation and death of CD8 T cells. It is further contemplated that Eif2aS51A mutant CD8 T cells have a greater increase in translation and cell death in tumor stress conditions relative to WT T cells.

Experiment J: Impact of PERK and p-eIF2a in CD8 T cell translation and death in sarcomas.

Impact of PERK to CD8 T cell translation and death in sarcomas. Translation and cell death among CD8 TILs in WT littermates (Lck /c PERKi7i) and PKO (Lckcrc PERK//r) mice bearing MCA-205 sarcomas are measured. MCA-205 sarcomas are established intradermally on the right flank of groups outlined in Table 7. After 14 days of tumor growth mice are scarified and spleens, non-draining lymph nodes (NDLN), tumor-draining lymph nodes (TDLN), and tumors are processed. FACS analysis is used to measure protein synthesis and cell death of CD8 T cells in various organs using methods and controls described in Experiment A. Analysis of translation and cell death between WT and PKO CD8 T cells in vivo is as described as in Experiment A. It is contemplated that PKO CD8 T cells undergo more translation and death in tumor-associated organs (TDLNs and tumors). Based on memory T cell properties, PKO CD8 T cells are contemplated to exhibit enhanced persistence in non-tumor associated organs such as spleen.


Contribution ofp-eIF2a to CD8 T cell translation and death in sarcomas. RAG 1 mice with

Eif2aS51A + mutant BM are reconstituted using the strategy described in Experiment F. After reconstitution for ~4 weeks, MCA-205 sarcomas are established intradermally on the right flank of groups outlined in Table 8. After 14 days of tumor growth mice are scarified and spleens,

NDLNs, TDLNs, and tumors are processed. FACS analysis is used to measure protein synthesis and cell death of CD8 T cells in various organs using methods and controls described in above.

Analysis of translation and cell death in CD8 T cells from RAGl mice reconstituted with WT or Eif2aS51A +/ mutant BM are as described as in Experiment I. It is contemplated that the data generated in Eif2aS51A +/ mutants offer a rigorous system to test the contribution of acute ER stress to translation and death of CD8 TILs. It is further contemplated that mice bearing

Eif2aS51A +/ mutant BM will exhibit CD8 T cells that undergo more translation and death in tumor-associated organs (TDLNs and tumors) relative to CD8 T cells from mice bearing WT

BM.

Experiment K: Measure the impact of PERK and p-eIF2a to T cell response to a-PD-1 therapy of sarcomas.

Impact of PERK in T cell-mediated response to a-PD-1 therapy in sarcomas. The response to a-PD-1 therapy in WT littermates (Lckcre PERKf/f) and PKO (Lckcrc PERK7 i) mice bearing MCA-205 sarcomas is measured. These experiments further support the combination therapy data using PERK inhibition (Fig. 15). MCA-205 sarcomas are established intradermally on the right flank of groups outlined in Table 7. As in Fig. 15, after 14 days of tumor growth mice are treated with a-PD-1 antibody or isotype control (200pg/mouse). Tumor growth is measured every other day with calipers. Tumor growth over time is the primary endpoint. TTS is

monitored. It is contemplated that PKO mice will exhibit better response to a-PD-1 therapy than WT mice evidenced by reduced tumor size and greater TTS.

Contribution of p-elF 2 a to T cell-mediated response to a-PD-1 therapy in sarcomas. As in Experiment J, T cell-specific Eif2aS51A +/ mutant BM is established in RAG 1 mice.

Reconstitution among groups is monitored by CD8 populations in PBMC of treatment groups. After reconstitution for ~4 weeks, MCA-205 sarcomas are established intradermally on the right flank of groups outlined in Table 9. As in Fig. 15, after 14 days of tumor growth mice are treated with a-PD-1 antibody or isotype control (200pg/mouse). Tumor growth is measured every other day with calipers. Tumor growth over time is the primary endpoint. TTS is monitored. It is contemplated that mice bearing Eif2aS51A+/ BM populations exhibit greater tumor growth and decreased response to a-PD-1 therapy relative to mice bearing WT populations.


For Experiment I, an alternative strategy is to collect supernatant from 2mm2 pieces of untreated human sarcomas and to culture PKO or Eif2aS51A +/ mutant CD8 T cells in the presence of human tumor supernatant. This lends substantial insight into the impact of human tumor microenvironments on the T cell stress response. An alternative mouse model for Lckc/vPERK77 mice is to create CD4crePERKf/f mice. CD4crePERKf/f mice present a global deletion of PERK in T cells that will be affected at all stages of T cell development; whereas the Lckc/vPERK77 mice utilize dLck-hcre3779 mice (Jackson Labs, 012837) in which ere recombinase expression is driven by the distal promoter of the Lck gene, thus ere expression occurs after positive selection in the thymus. This model enables study of changes affected by gene deletion or overexpression in peripheral T cells without having to account for effects of gene deletion or overexpression in T cell thymic development. The data indicates that PERK-ATF4-ER01a signaling is important in peripheral T cells as they become exhausted in tumor. Thus, our current model suits the in vivo goals of this proposal. For the p-eIF2a mouse model, an alternative strategy to study the role of p-eIF2a in CD8 TILs is to transfer OT-l-Eif2aS51A +/ T cells to B16Fl-OVA-bearing mice and to monitor tumor growth relative to OT-1 T cells as in Figs 8 & 12. As in Fig. 14, OT-1 or OT-l-Eif2aS51A +/ CD8 T cells can be transferred to MCA-205-OVA-bearing mice and assess translation and death in CD8 TILs at multiple time points post transfer.

In summary: Immunotherapy is largely ineffective in solid tumor cancer patients (25, 27, 64, 65, 83). In these examples, data indicates that chronic ER stress leaves T cells in a state of metabolic exhaustion in tumors. Additionally, the data presented herein supports the proposition that chronic ATF4 and EROla drive activation and metabolic exhaustion in CD8 TILs which in turn limits the efficacy of a-PD-1 therapy, while acute p-eIF2a protects CD8 T cells under sarcoma microenvironment stress.

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* * *

It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.

The invention will be further described, without limitation, by the following numbered paragraphs:

1. A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture have a reduced expression of EROla.

2. A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture comprise a vector comprising a nucleic acid sequence encoding a protein which knocks out EROla.

The reaction mixture of paragraph 2, wherein the vector is a DNA, RNA, plasmid, lentivirus vector, adenoviral vector, or retrovirus vector.

A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture comprise a nucleic acid molecule that comprises a sequence which knocks out EROla.

A reaction mixture of any one of paragraphs 1-4, wherein the immune effector cells are T cells.

A reaction mixture of any one of paragraphs 1-5, comprising a population of immune effector cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% 1% of EROla expressing T cells.

A method of treating cancer in a subject comprising administering the reaction mixture of any one of paragraphs 1-6 to the subject in need thereof.

A method of treating a cancer in a subject, comprising administering modified T cells to said subject in need thereof wherein said modified T cells comprise T cells in which expression of EROla is reduced or eliminated.

The method of paragraph 7 or 8, wherein the cancer is sarcoma, melanoma, lung cancer, adenocarcinoma, metastatic bone disease or a solid tumor.

The method of any one of paragraphs 7-9, further comprising administering the modified T cells locally to one or more tumors and/or tumor microenvironments of the individual. The method of any one of paragraphs 7-10, further comprising systemically delivering the modified T cells to the subject.

A method of making a modified population of immune effector cells, comprising

a. obtaining a population of immune effector cells,

b. modifying a plurality of the immune effector cells within the population to

eliminate or reduce expression of EROla thereby creating a modified population of immune effector cells.

The method of paragraph 12, wherein the immune effector cells are T cells.

The method of paragraph 12 or 13, wherein the method further provides contacting the plurality of the immune effector cells with CRIPR/Cas9 genes that silence or knock out expression of EROla.

The method of any one of paragraphs 12-14, wherein the population of immune effector cells or the modified population of immune effector cells are contacted with a EROla inhibitor.

The method of paragraph 15, wherein the EROla inhibitor is EROl Inhibitor II (EN460). The method of any one of paragraphs 12-16, wherein the population of immune effector cells or the modified population of immune effector cells are contacted with N-acetyl cysteine (NAC).

The method of any one of paragraphs 12-17, wherein the population of the modified immune effector cells is expanded for a period of 8 days or less, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day or less..

The method of any one of paragraphs 12-18, wherein the population of immune effector cells are obtained from a subject afflicted with cancer.

The method of paragraph 19, wherein the cancer is a sarcoma or a tumor.

The method of any one of paragraphs 12-20, wherein the population of cells is cryopreserved after the appropriate expansion period.

The method of any one of paragraphs 7-11 and 19-21, wherein the subject is a human. A method for treating a cancer in a mammal, comprising administering to said mammal an EROla inhibitor.

The method of paragraph 23, wherein the EROla inhibitor reduces or eliminates the expression of EROla.

The method of any one of paragraphs 7-11 and 19-24, further comprising administering a checkpoint inhibitor.

The method of paragraph 25, wherein the checkpoint inhibitor is ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab-rwlc.

The method of paragraph 25, wherein the checkpoint inhibitor is an programmed cell death protein 1 (PD-1) antagonist or a T-lymphocyte-associated protein 4 (CTLA-4) antagonist.

The method according to paragraph 27, wherein the PD-1 or CTLA-4 antagonist is:

a. an antibody, or antigen binding fragment of an antibody, that specifically binds to, and inhibits activation of, an PD-1 or CTLA-4 receptor, or

b. a soluble form of an PD-1 or CTLA-4 receptor that specifically binds to a PD-1 or CTLA-4 ligand and inhibits the PD-1 or CTLA-4 ligand from binding to the PD-1 or CTLA-4 receptor.

The method according to paragraph 27, wherein the PD-1 ligand is PD-L1 or PD-L2. The method according to paragraph 27, wherein the PD-1 antagonist is a PD-1 monoclonal antibody.

The method of paragraph 27, wherein the PD-1 antagonist is nivolumab,

pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

The method of paragraph 27, wherein the CTLA-4 antagonist is ipilimumab or tremelimumab.

The method according to any one of paragraphs 25-32, wherein the administration of the modified T cells or the EROltx inhibitor precedes the administration of the checkpoint inhibitor.

The method according to any one of paragraphs 25-32, wherein the administration of the checkpoint inhibitor precedes the administration of the modified T cells or the EROla inhibitor.

The method according to any one of paragraphs 25-32, wherein the modified T cells or the EROla inhibitor is administered adjunctively to the checkpoint inhibitor.

The method according to any one of paragraphs 25-32, wherein the checkpoint inhibitor is administered adjunctively to the modified T cells or the EROla inhibitor.

The method of any one of paragraphs 25-36, wherein the checkpoint inhibitor is administered daily, more often than once daily or less often than once daily.

The method according to paragraph 37, wherein the checkpoint inhibitor is administered once every 3 days, once every week, once every 2 weeks, once every 3 weeks or once every 4 weeks.

The method according to any one of paragraphs 27-36, wherein the PD-1 antagonist is nivolumab and the amount of the nivolumab administered to the subject is 3 mg/kg body weight every 3 weeks, 240 mg every 2 weeks or 480 mg every 4 weeks.

The method according to any one of paragraphs 27-36, wherein the PD-1 antagonist is pembrolizumab and the amount of the pembrolizumab administered to the subject is 200 mg every 3 weeks.

The method according to any one of paragraphs 27-36, wherein the PD-1 antagonist is avelumab and the amount of the avelumab administered to the subject is 800 mg every 2 weeks.

The method according to any one of paragraphs 27-36, wherein the PD-1 antagonist is durvalumab and the amount of the durvalumab administered to the subject is 10 mg/kg body weight every 2 weeks.

The method according to any one of paragraphs 27-36, wherein the PD-1 antagonist is cemiplimab and the amount of the cemiplimab administered to the subject is 250 mg every 3 weeks.

The method according to any one of paragraphs 27-36, wherein the PD-1 antagonist is atezolizumab and the amount of the atezolizumab administered to the subject is 840 mg every 2 weeks, 1200 mg every 3 weeks or 1680 mg every 4 weeks.

The method according to any one of paragraphs 25-44, wherein the subject is receiving checkpoint inhibitor therapy prior to initiating modified T cells therapy or ERO la inhibitor therapy.

The method according to any one of paragraphs 25-44, wherein the subject is receiving modified T cells therapy or ERO la inhibitor therapy prior to initiating checkpoint inhibitor therapy.

The method according to any one of paragraphs 25-46, where in the subject is receiving a first therapy for at least 8 weeks, at least 10 weeks, at least 24 weeks, at least 28 weeks, at least 48 weeks or at least 52 weeks prior to initiating a second therapy.

The method according to any one of paragraphs 25-47, wherein periodic administration of the modified T cells and/or EROla inhibitor and/or the checkpoint inhibitor continues for at least 3 days, for at least 30 days, for at least 42 days, for at least 8 weeks, for at least 12 weeks, for at least 24 weeks or for at least 6 months.

The method according to any one of paragraphs 25-48, wherein each of the amount of the modified T cells or EROla inhibitor when taken alone, and the amount of the checkpoint inhibitor when taken alone is effective to treat the subject.

The method according to any one of paragraphs 25-48, wherein either the amount of the modified T cells or the EROla inhibitor when taken alone, the amount of the checkpoint

inhibitor when taken alone, or each such amount when taken alone is not effective to treat the subject.

The method according to any one of paragraphs 25-48, wherein either the amount of the modified T cells or the ERO la inhibitor when taken alone, the amount of the checkpoint inhibitor when taken alone, or each such amount when taken alone is less effective to treat the subject.

The method of any one of paragraphs 23-51, wherein the EROla inhibitor is EROl Inhibitor II (EN460).

The method according to any one of paragraphs 23-52, wherein the subject or mammal is a human patient.

A EROla inhibitor for use as an add-on therapy or in combination with a checkpoint inhibitor in treating a subject afflicted with cancer.

A checkpoint inhibitor for use as an add-on therapy or in combination with an EROla inhibitor in treating a subject afflicted with cancer.

Use of an amount of a checkpoint inhibitor and an amount of an EROla inhibitor in the preparation of a combination for treating a subject afflicted with cancer wherein the checkpoint inhibitor and the EROla inhibitor are prepared to be administered

simultaneously, contemporaneously or concomitantly.

A combination of ERO 1 a inhibitor and an checkpoint inhibitor for use in the manufacture of a medicament.

The combination according to paragraph 57, wherein the medicament is for the treatment, prevention, or alleviation of a symptom of cancer.

The combination according to paragraph 57, wherein the cancer is sarcoma, melanoma, lung cancer, adenocarcinoma, metastatic bone disease or a solid tumor

The combination according to paragraph 57, wherein the EROla inhibitor is EROl Inhibitor II (EN460).

A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture have a reduced expression of PERK. A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture comprise a vector comprising a nucleic acid sequence encoding a protein which knocks out PERK.

The reaction mixture of paragraph 62, wherein the vector is a DNA, RNA, plasmid, lentivirus vector, adenoviral vector, or retrovirus vector.

A reaction mixture comprising a population of immune effector cells, wherein a plurality of the cells of the population in the reaction mixture comprise a nucleic acid molecule that comprises a sequence which knocks out PERK.

A reaction mixture of any one of paragraphs 61-64, wherein the immune effector cells are T cells.

A reaction mixture of any one of paragraphs 61-65, comprising a population of immune effector cells containing less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% 1% of PERK expressing T cells.

A method of treating cancer in a subject comprising administering the reaction mixture of any one of paragraphs 61-66 to the subject in need thereof.

A method of treating a cancer in a subject, comprising administering modified T cells to said subject in need thereof wherein said modified T cells comprise T cells in which expression of PERK is reduced or eliminated.

The method of paragraph 67 or 68, wherein the cancer is sarcoma, melanoma, lung cancer, adenocarcinoma, metastatic bone disease or a solid tumor.

The method of any one of paragraphs 67-69, further comprising administering the modified T cells locally to one or more tumors and/or tumor microenvironments of the individual.

The method of any one of paragraphs 67-70, further comprising systemically delivering the modified T cells to the subject.

A method of making a modified population of immune effector cells, comprising

a. obtaining a population of immune effector cells,

b. modifying a plurality of the immune effector cells within the population to

eliminate or reduce expression of PERK thereby creating a modified population of immune effector cells.

The method of paragraph 72, wherein the immune effector cells are T cells.

The method of paragraph 72 or 73, wherein the method further provides contacting the plurality of the immune effector cells with CRIPR/Cas9 genes that silence or knock out expression of PERK.

The method of any one of paragraphs 72-74, wherein the population of immune effector cells or the modified population of immune effector cells are contacted with a PERK inhibitor.

The method of paragraph 75, wherein the PERK inhibitor is 7-methyl-5-(l-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-lH-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), GSK2656157, AMG PERK 44, or LDN-0070977.

The method of any one of paragraphs 72-76, wherein the population of immune effector cells or the modified population of immune effector cells are contacted with N-acetyl cysteine (NAC).

The method of any one of paragraphs 72-77, wherein the population of the modified immune effector cells is expanded for a period of 8 days or less, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day or less..

The method of any one of paragraphs 72-78, wherein the population of immune effector cells are obtained from a subject afflicted with cancer.

The method of paragraph 79, wherein the cancer is a sarcoma or a tumor.

The method of any one of paragraphs 72-80, wherein the population of cells is cryopreserved after the appropriate expansion period.

The method of any one of paragraphs 67-71 and 79-81, wherein the subject is a human. A method for treating a cancer in a mammal, comprising administering to said mammal an PERK inhibitor.

The method of paragraph 83, wherein the PERK inhibitor reduces or eliminates the expression of PERK.

The method of any one of paragraphs 67-71 and 79-84, further comprising administering a checkpoint inhibitor.

The method of paragraph 85, wherein the checkpoint inhibitor is ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab-rwlc.

The method of paragraph 85, wherein the checkpoint inhibitor is an programmed cell death protein 1 (PD-1) antagonist or a T-lymphocyte-associated protein 4 (CTLA-4) antagonist.

The method according to paragraph 87, wherein the PD-1 or CTLA-4 antagonist is:

a. an antibody, or antigen binding fragment of an antibody, that specifically binds to, and inhibits activation of, an PD-1 or CTLA-4 receptor, or

b. a soluble form of an PD-1 or CTLA-4 receptor that specifically binds to a PD-1 or CTLA-4 ligand and inhibits the PD-1 or CTLA-4 ligand from binding to the PD-1 or CTLA-4 receptor.

The method according to paragraph 87, wherein the PD-1 ligand is PD-L1 or PD-L2. The method according to paragraph 87, wherein the PD-1 antagonist is a PD-1 monoclonal antibody.

The method of paragraph 87, wherein the PD-1 antagonist is nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, or atezolizumab.

The method of paragraph 87, wherein the CTLA-4 antagonist is ipilimumab or tremelimumab.

The method according to any one of paragraphs 85-92, wherein the administration of the modified T cells or the PERK inhibitor precedes the administration of the checkpoint inhibitor.

The method according to any one of paragraphs 85-92, wherein the administration of the checkpoint inhibitor precedes the administration of the modified T cells or the PERK inhibitor.

The method according to any one of paragraphs 85-92, wherein the modified T cells or the PERK inhibitor is administered adjunctively to the checkpoint inhibitor.

The method according to any one of paragraphs 85-92, wherein the checkpoint inhibitor is administered adjunctively to the modified T cells or the PERK inhibitor.

The method of any one of paragraphs 85-96, wherein the checkpoint inhibitor is administered daily, more often than once daily or less often than once daily.

The method according to paragraph 97, wherein the checkpoint inhibitor is administered once every 3 days, once every week, once every 2 weeks, once every 3 weeks or once every 4 weeks.

The method according to any one of paragraphs 87-96, wherein the PD-1 antagonist is nivolumab and the amount of the nivolumab administered to the subject is 3 mg/kg body weight every 3 weeks, 240 mg every 2 weeks or 480 mg every 4 weeks.

The method according to any one of paragraphs 87-96, wherein the PD-1 antagonist is pembrolizumab and the amount of the pembrolizumab administered to the subject is 200 mg every 3 weeks.

The method according to any one of paragraphs 87-96, wherein the PD-1 antagonist is avelumab and the amount of the avelumab administered to the subject is 800 mg every 2 weeks.

The method according to any one of paragraphs 87-96, wherein the PD-1 antagonist is durvalumab and the amount of the durvalumab administered to the subject is 10 mg/kg body weight every 2 weeks.

The method according to any one of paragraphs 87-96, wherein the PD-1 antagonist is cemiplimab and the amount of the cemiplimab administered to the subject is 250 mg every 3 weeks.

The method according to any one of paragraphs 87-96, wherein the PD-1 antagonist is atezolizumab and the amount of the atezolizumab administered to the subject is 840 mg every 2 weeks, 1200 mg every 3 weeks or 1680 mg every 4 weeks.

The method according to any one of paragraphs 85-104, wherein the subject is receiving checkpoint inhibitor therapy prior to initiating modified T cells therapy or PERK inhibitor therapy.

The method according to any one of paragraphs 85-104, wherein the subject is receiving modified T cells therapy or PERK inhibitor therapy prior to initiating checkpoint inhibitor therapy.

The method according to any one of paragraphs 85-106, where in the subject is receiving a first therapy for at least 8 weeks, at least 10 weeks, at least 24 weeks, at least 28 weeks, at least 48 weeks or at least 52 weeks prior to initiating a second therapy.

The method according to any one of paragraphs 85-107, wherein periodic administration of the modified T cells and/or PERK inhibitor and/or the checkpoint inhibitor continues for at least 3 days, for at least 30 days, for at least 42 days, for at least 8 weeks, for at least 12 weeks, for at least 24 weeks or for at least 6 months.

The method according to any one of paragraphs 85-108, wherein each of the amount of the modified T cells or PERK inhibitor when taken alone, and the amount of the checkpoint inhibitor when taken alone is effective to treat the subject.

The method according to any one of paragraphs 85-108, wherein either the amount of the modified T cells or the PERK inhibitor when taken alone, the amount of the checkpoint inhibitor when taken alone, or each such amount when taken alone is not effective to treat the subject.

The method according to any one of paragraphs 85-108, wherein either the amount of the modified T cells or the PERK inhibitor when taken alone, the amount of the checkpoint inhibitor when taken alone, or each such amount when taken alone is less effective to treat the subject.

The method of any one of paragraphs 83-111, wherein the PERK inhibitor is 7-methyl-5-(l-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-lH-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), GSK2656157, AMG PERK 44, or LDN-0070977. The method according to any one of paragraphs 83-112, wherein the subject or mammal is a human patient.

A PERK inhibitor for use as an add-on therapy or in combination with a checkpoint inhibitor in treating a subject afflicted with cancer.

A checkpoint inhibitor for use as an add-on therapy or in combination with an PERK inhibitor in treating a subject afflicted with cancer.

Use of an amount of a checkpoint inhibitor and an amount of an PERK inhibitor in the preparation of a combination for treating a subject afflicted with cancer wherein the checkpoint inhibitor and the PERK inhibitor are prepared to be administered

simultaneously, contemporaneously or concomitantly.

A combination of PERK inhibitor and an checkpoint inhibitor for use in the manufacture of a medicament.

The combination according to paragraph 117, wherein the medicament is for the treatment, prevention, or alleviation of a symptom of cancer.