the folate pathway inhibitor pemetrexed pleiotropically ... · translational cancer mechanisms and...

Translational Cancer Mechanisms and Therapy

The Folate Pathway Inhibitor PemetrexedPleiotropically Enhances Effects of CancerImmunotherapyDavid A. Schaer1, Sandaruwan Geeganage2, Nelusha Amaladas1, Zhao Hai Lu2,Erik R. Rasmussen1, Andreas Sonyi1, Darin Chin1, Andrew Capen2, Yanxia Li1,CatalinaM.Meyer2, BonitaD. Jones2, XiaodongHuang1, Shuang Luo2,CarmineCarpenito1,Kenneth D. Roth2, Alexander Nikolayev2, Bo Tan2, Manisha Brahmachary1,Krishna Chodavarapu1, Frank C. Dorsey2, Jason R. Manro2, Thompson N. Doman2,Gregory P. Donoho2, David Surguladze1, Gerald E. Hall1, Michael Kalos1, andRuslan D. Novosiadly1

Abstract

Purpose: Combination strategies leveraging chemothera-peutic agents and immunotherapy have held the promise as amethod to improve benefit for patients with cancer. However,most chemotherapies have detrimental effects on immunehomeostasis and differ in their ability to induce immunogeniccell death (ICD). The approval of pemetrexed and carboplatinwith anti-PD-1 (pembrolizumab) for treatment of non–smallcell lung cancer represents the first approved chemotherapyand immunotherapy combination. Although the clinical datasuggest a positive interaction between pemetrexed-based che-motherapy and immunotherapy, the underlying mechanismremains unknown.

Experimental Design: Mouse tumor models (MC38,Colon26) and high-content biomarker studies (flow cyto-metry, Quantigene Plex, and nCounter gene expressionanalysis) were deployed to obtain insights into the mech-anistic rationale behind the efficacy observed with peme-trexed/anti-PD-L1 combination. ICD in tumor cell lines was

assessed by calreticulin and HMGB-1 immunoassays, andmetabolic function of primary T cells was evaluated bySeahorse analysis.

Results: Pemetrexed treatment alone increased T-cell acti-vation in mouse tumors in vivo, robustly induced ICD inmouse tumor cells and exerted T-cell–intrinsic effects exem-plified by augmented mitochondrial function and enhancedT-cell activation in vitro. Increased antitumor efficacy andpronounced inflamed/immune activation were observedwhen pemetrexed was combined with anti-PD-L1.

Conclusions: Pemetrexed augments systemic intratumorimmune responses through tumor intrinsic mechanismsincluding immunogenic cell death, T-cell–intrinsic mechan-isms enhancing mitochondrial biogenesis leading toincreased T-cell infiltration/activation along with modula-tion of innate immune pathways, which are significantlyenhanced in combination with PD-1 pathway blockade.

See related commentary by Buque et al., p. 6890

IntroductionPD(L)1 inhibitors have markedly changed the therapeutic

landscape in many tumor types including non–small cell lung

cancer (NSCLC), and these agents are becoming standard of careacross an increasing number of tumor types (1, 2). However,clinical benefit from these therapies is limited, and tumor recur-rences are common (3, 4). One strategy to improve the efficacy ofPD(L)1 inhibitors is to combine these agents with tumor-targeting therapies that have the potential for cooperative mech-anistic interactions with immune agents (4, 5). Indeed, numerousclinical trials are underway to evaluate the potential to combineimmune checkpoint inhibitors (ICIs) and chemotherapies (6).

Pemetrexed is an established chemotherapeutic that disruptsthe folate pathway and is part of the standard of care fornonsquamous NSCLC and mesothelioma (7). The front-linetreatment with pemetrexed, carboplatin, and anti-PD-1 (pem-brolizumab) has been evaluated in patients with NSCLC in therandomized KEYNOTE-021G and KEYNOTE-189 trials (8, 9),leading to the accelerated approval of this regimen based onsubstantial increase in progression-free survival and overallresponse rate in the KEYNOTE-021G study. These improve-ments represented the first approval of chemo-immunotherapycombination (10). The rationale to combine chemotherapywith ICIs is based at least in part on the concept of

1Lilly Research Laboratories, Eli Lilly and Company, New York, New York. 2LillyResearch Laboratories, Eli Lilly and Company, Indianapolis, Indiana.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Current address for DA Schaer: Pfizer WRD, Pearl River, New York; M. Kalos:Arsenal Biosciences, South San Francisco, California; and current address forRuslan D. Novosiadly: Bristol-Myers Squibb, Princeton, New Jersey.

D.A. Schaer and S. Geeganage contributed equally to this article.

R.D. Novosiadly is a lead contact.

Corresponding Authors: Ruslan D. Novosiadly, Bristol-Myers Squibb, 3551Lawrenceville Road, Princeton, NJ 08540. Phone: 6465082474; E-mail:[email protected]; andMichael Kalos, Arsenal Biosciences, 571 EcclesAvenue South San Francisco, CA 94080. E-mail: [email protected]

Clin Cancer Res 2019;25:7175–88

doi: 10.1158/1078-0432.CCR-19-0433

�2019 American Association for Cancer Research.

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immunogenic cell death (ICD) that can be a consequence ofthe cytotoxic effects of chemotherapeutic agents on tumorcells (11). ICD involves the release of immune-stimulatingfactors from dying tumor cells that drive antigen cross-presen-tation, T-cell priming, and adaptive immune responseagainst tumors. Cytotoxic agents are not equipotent in theirability to induce ICD; only a few cytotoxic agents (e.g., anthra-cyclines, oxaliplatin) have been demonstrated to induce ICD,whereas most chemotherapeutics induce non-ICD (11). Cyto-toxic agents can also be deleterious to the immune compart-ment by cytotoxic targeting of immune cells (11). The positiveinteraction between pemetrexed-based chemotherapy andimmune checkpoint blockade in KEYNOTE-021 and KEY-NOTE-189 trials may seem counterintuitive given that antifo-late agents (e.g., methotrexate) have been used as immuno-suppressive agents to treat patients with inflammatory condi-tions, and part of their immunosuppressive activity appearsto involve the inhibition of T cells (12–15). Furthermore,recent work has identified one-carbon metabolism, whichincludes the folate and methionine cycles, as a top rankedmetabolic pathway engaged during T-cell activation andsurvival (16, 17).

The main objective of this work was to obtain mechanisticinsights into the immunostimulatory activity of pemetrexed�PD1 blockade rather than justify clinical development ofpemetrexed/anti-PD(L)1 combinations in NSCLC and othertumor types. We demonstrate that pemetrexed therapyexerts previously unknown immunomodulatory effects thatresult in an immune-permissive tumor microenvironment andimproves the antitumor efficacy of PD(L)1 blockade. Theseresults provide fundamental insights into the mechanismsunderlying the combinatorial activity of pemetrexed andanti-PD-1 therapy, and provide a strong rationale for furtherexploration of combinations of pemetrexed and other folatepathway modulators with immunotherapies.

Materials and MethodsIn vivo tumor studies

Colon26 and MC38 cell lines were purchased from DTP andNCI DCTD Tumor Repository, respectively. Female BALB/c andC57BL/6 mice were purchased from Envigo. All experimentalprocedures were done in accordance with the NIH Guide for Careand Use of Animals and were approved by the InstitutionalAnimal Care and Use Committee.

Metabolic assessments of primary mouse T cellsMouse splenic T cells stimulatedwithCD3/CD28were cultured

in the presence of pemetrexed as indicated. Oxygen consumptionrate (OCR) was analyzed using Seahorse XF Cell Mito Stress TestKit and Seahorse XFe96 instrument (Agilent). Cells were sequen-tially stimulated with oligomycin (1 mmol/L), FCCP (1.5 mmol/L), and rotenone/antimycin A (0.5 mmol/L each) and the sparerespiratory capacity (SRC) was measured as the differencebetween basal OCR values and maximal OCR values obtainedafter FCCP uncoupling. To assess T-cell ability tometabolize fattyacids, XF Palmitate:BSA FAO substrate (Agilent) was incorporatedinto XF Cell Mito Stress Test assay. Wave 2.4 software (Agilent)was used for data acquisition and analysis of Seahorse data.

Tumor cell killing assaySplenocytes from ovalbumin-specific T-cell receptor transgenic

OT-1 mice were incubated in the presence of 0.1 nmol/L ofSIINFEKL peptide and IL2 for 5 days. CD8þ T cells were thenisolated and cultured with B16 tumor cells that had been previ-ously labeled with cell tracer BV421 and pulsed with 100 nmol/Lof SIINFEKL peptide for 2 hours, at a 10:1 effector-to-target ratio.Tumor cell death was analyzed by 7AAD incorporation by flowcytometry after 4 hours of coculture.

In vitro assessment of ICDColon26 and MC38 tumor cell lines were treated with peme-

trexed, carboplatin, paclitaxel, gemcitabine, or doxorubicin. for96 hours followed by analysis of high-mobility group B1(HMGB1) protein and calreticulin (CRT) in culture supernatantsusing commercially available kits (IBL International, Hamburg,Germany, and Cloud Clone Corporation, Katy, TX, respectively).The viability of remaining cells was measured by Cell Titer-Gloassay (Promega) according to manufacturer's protocol.

Gene expression analysisQuantiGene Plex and nCounter gene expression assays were

done as reported previously with slight modifications (18).

Quantification and statistical analysisGroup-wise statistical comparisons were performed as indi-

cated in each figure using standard paired T tests, 1-wayANOVA, or 2-way ANOVA models with Tukey adjustmentper time point, comparing treatment/dose and time point.Additional details are provided in the Supplementary Materialsand Methods.

ResultsPemetrexed exhibits intratumor immunomodulatory effectsin vivo

To characterize the effects of pemetrexed on intratumorimmune response, initial experiments were performed in

Translational Relevance

We describe novel immunomodulatory properties ofpemetrexed, a chemotherapy that targets the folate path-way and is used as standard of care in the frontline treatmentof advanced nonsquamous non–small cell lung cancer andpleural mesothelioma. The combination of pemetrexed-based chemotherapy with anti-PD-1 (pembrolizumab) hasdemonstrated compelling clinical activity in patients withmetastatic NSCLC based on the results of KEYNOTE-189phase III trial, and previously disclosed data fromKEYNOTE-021G phase II trial have led to the acceleratedapproval of this regimen by the FDA. Although this land-mark approval represents the first case of clinical adoptionof chemoimmunotherapy combination in oncology,there are fundamental unanswered questions about whyand how a chemotherapeutic agent such as pemetrexedmight effectively combine with immunotherapy.

This work provides novel data on how pemetrexed pleio-tropically modulates antitumor immunity and provides keyinsights for the development of chemotherapeutic agents incombination with immunotherapies.

Schaer et al.

Clin Cancer Res; 25(23) December 1, 2019 Clinical Cancer Research7176




immunocompetent syngeneic mouse tumor models. To meetour research objective, the models had to meet 2 prerequisites:(i) demonstrate sensitivity to pemetrexed and (ii) responsive-ness to PD(L)1 blockade. We found that MC38 and Colon26colorectal tumor cell lines were sensitive to pemetrexed, where-as Lewis lung carcinoma (LLC), a commonly used lung cancermodel, was pemetrexed-refractory (Supplementary Fig. S1A;ref. 20). Furthermore, although genetically engineered mousemodels (GEMM) of lung carcinoma may sound like a logicalchoice, they are poorly fit for studying effects of immunothera-pies, largely because GEMMs are driven by specific oncogenicevents and do not exhibit high tumor mutational and neo-antigen burden which have emerged as important molecularhallmarks underlying responsiveness of lung tumors to immu-notherapy in humans (21, 22). Because single-agent treat-ment with pemetrexed induced tumor responses across multi-ple tumor types in early clinical trials (7, 23), we rationalizedthat it was appropriate to use tumor models with the rightbiological context irrespective of histology rather than usinglung tumor models with no sensitivity to either pemetrexedor anti-PD(L)1.

MC38 tumors are modestly responsive to both PD-1 block-ade (20) and pemetrexed (Fig. 1A). The effect of pemetrexed inMC38 model was consistent with thymidylate synthase inhibi-tion (increased deoxyuridine, dUMP and decreased thymidine,dTMP in the tumor and plasma; Supplementary Fig. S1), assessedbymetabolomics analysis (19) and the highest dose used in thesestudies was determined to be the maximum tolerateddose (7, 24, 25). MC38 tumors were responsive to pemetrexedat 50 and 100 mg/kg (% tumor growth inhibition of 30% and52%, respectively; Fig. 1A). Tumors collected after 14 days ofpemetrexed therapy were analyzed for changes in immune cellfrequencies using flow cytometry. These analyses revealed thatpemetrexed increased the frequency of total intratumoral leuko-cytes (live CD45þ cells) at both doses, with a trend towards anincreased percentage of total CD3þ and cycling (Ki67þ) CD8þ

cells, particularly at 50 mg/kg (Fig. 1B–E). This appeared to bedriven mainly by an increase in Ki67þCD8þ T cells, without anyother significant differences in myeloid cell subsets (Fig. 1F).Molecular analysis of tumor samples using a custom-madeimmune profilingQuantiGene Plex (QGP) gene expression panelrevealed that treatment with pemetrexed at 50 and 100 mg/kgpromoted a T-cell inflamed phenotype, exemplified by upregula-tion of T-cell activation-associated genes including Pdcd1, Cd8b,Prf1, and Gzma (Fig. 1G) (26). The QGP data also suggestactivated vascular endothelium (" Icam1, Vcam1, and chemokineCx3cl1 also known as fractalkine that is induced in endotheliumas result of immune activation) and enhanced interferon (IFN)response (" Irf7) and antigen presentation [upregulated Itgax andZbtb1 associatedwithdendritic cells (DC)]. Beyond these changes,one of the genes most significantly modulated by pemetrexedtreatment was Vegfc, which encodes vascular endothelial growthfactor C (VEGF-C), a key regulator of lymphangiogenesis. VEGF-Cis known to be regulated through the NF-kB pathway and isbelieved to promote T-cell infiltration rather than inhibit antitu-mor immune response (27, 28). Nos2, which encodes induciblenitric oxide synthase, is produced by myeloid-derived suppressorcells (MDSC) and DCs, was downregulated at both dose levels,suggesting that pemetrexed could potentially negatively impactmyeloid cell subsets (Fig. 1G). Although Nos2 displayed down-regulation, we did not observe a significant reduction in CD11bþ

cells by flow cytometric analysis (Fig. 1F). Collectively, theseresults suggest that pemetrexed influences the functionality ratherthan frequency of myeloid cells.

Because pemetrexed is administered in combination withplatinum agents such as carboplatin and cisplatin in front-linetreatment of patients with metastatic NSCLC, we next asked ifcarboplatin had immunomodulatory effects on the tumorimmune microenvironment, and whether the immunomodula-tory effects of pemetrexed were affected by the addition ofcarboplatin. In these experiments, we also evaluated the immu-nomodulatory effects of the chemotherapy doublet of carbopla-tin and paclitaxel, a commonly used treatment option in NSCLC,as well as paclitaxel monotherapy. Mice bearing MC38 tumorswere treated with pemetrexed, paclitaxel, carboplatin, or combi-nation of pemetrexed with carboplatin, or paclitaxel with carbo-platin, at doses designed to model clinical exposures for theseagents. Tumors were harvested 14 days after treatment initiation,and immune-related gene expression changes were evaluated byQGP analysis (Fig. 2A). Pemetrexed monotherapy resulted inupregulation of multiple immune-related genes and induced animmune activation signature indicative of IFNg pathway activa-tion (increased Cd274, Cxcl10, Cxcl11, Psmb8), cytolytic activity(increasedGzma, Prf1), IFN type I response (increased Irf7, Oas3),and activated vascular endothelium (increased Icam1, Vcam1).Paclitaxel monotherapy had a more modest effect on the expres-sion of the gene sets tested, with the immunomodulatory effectmainly associated with moderate upregulation of myeloid cell-related genes (increased Il6, Cxcl1, Ccl2, Ccl3, Ccl4, Timd4).Although carboplatin monotherapy had a weak effect, additionof carboplatin to the pemetrexed regimen appeared to reduce theimmunomodulatory effects of pemetrexed and to a lesser extentpaclitaxel. Cisplatin had a similar effect (Supplementary Fig. S2),suggesting that platinum agents in general can attenuate immu-nomodulatory effects of pemetrexed.

To investigate the breadth of pathways modulated by peme-trexed� carboplatin, we performedNanoString analysis of tumortissues using the nCounter panels spanning key molecular path-ways and cellular compartments of innate and adaptive immu-nity. Consistent with the QGP data, single-agent pemetrexedtreatment significantly modulated the expression of a large num-ber of genes associated with immune response [136 and 133differentially expressed genes (DEG) for immune profiling andmyeloid panel, respectively; Fig. 2B]. Paclitaxel had a quantita-tively weaker effect (31 and 39 DEGs for immune profiling andmyeloid/innate immunity panel, respectively) with a few DEGsshared between pemetrexed and paclitaxel monotherapy groups(Fig. 2B and C). The combination of pemetrexed and carboplatinyielded less prominent gene expression changes compared withpemetrexedmonotherapy (87 and 98DEGs for immune profilingandmyeloid/innate immunity panel, respectively; Fig. 2B and C).Paclitaxel monotherapy and paclitaxel/carboplatin combinationinduced somewhat different immunomodulatory effects, with alimited number of DEGs overlapped between the 2 treatmentgroups (Fig. 2B and C).

Ingenuity pathway analysis (IPA) was used to further explorethe immune-related molecular and/or cellular pathways modu-lated by pemetrexed. These analyses revealed macrophage, DC/NK cell, Th1/Th2 enrichment, and evidence of enhanced inflam-matory response; innate immune activation and increased IFNsignaling inMC38 tumors (Supplementary Table S1). Evaluationof the individual genes associated with these pathways suggested

Immunomodulatory Effects of Pemetrexed

www.aacrjournals.org Clin Cancer Res; 25(23) December 1, 2019 7177




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Figure 1.

Pemetrexed demonstrates antitumor efficacy and increases frequency of tumor-infiltrating lymphocytes in MC38 syngeneic murine tumors. A,Mean tumorvolumes (�SEM) in C57BL/6mice implanted with MC38 cells and treated with 50 or 100mg/kg of pemetrexed dosed intraperitoneally starting 3 days aftertumor implantation (5 days on, 2 days off) for the duration of the experiment. Difference in the tumor volume of treated groups compared with control (%T/C) atDay 16 was only significantly different (P < 0.001) at 100mg/kg (48.4% T/C) and not at 50 mg/kg (70.9% T/C, P¼ 0.17; RM-ANOVA). Mean intratumor leukocyte(B), T-cell (C–E), andmyeloid cell (F) frequencies (�SEM) at Day 17 after tumor implantation. Total leukocytes identified as live CD45þ cells, total T cells: LiveCD45þ, CD3þwith Ki67 percentage taken from CD3þ gate (1-way ANOVA). G,QuantiGene (QGP) gene expression analysis of MC38 tumors (Day 17 after tumorimplantation) after treatment with 50 or 100mg/kg of pemetrexed; volcano plots show DEGs; P values (compared with untreated control) were only listed if thedifferences between the groups reached statistical significance (P < 0.05 by 3-way ANOVA). Representative example of 3 experiments.

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Figure 2.

T-cell–inflamed phenotype induced by pemetrexed is not observed in tumors upon treatment with by paclitaxel or carboplatin. A,QuantiGene Plex (QGP)analysis of immune-related gene expression in MC38 tumors collected at D17 after single-agent treatment starting 3 days after tumor implantation withpemetrexed dosed intraperitoneally (50mg/kg, 5 days on, 2 days off), paclitaxel (10 mg/kg, dosed i.v. once a week), carboplatin (60mg/kg dosed i.p. once every2 weeks), or pemetrexed/carboplatin and paclitaxel/carboplatin combinations; volcano plots show DEGs with P < 0.05 (2-way ANOVA) compared withuntreated control. B,MC38 tumors treated with pemetrexed and/or carboplatin were further subjected to nCounter analysis using PanCancer Immune Profilingand Myeloid panels; summary of DEGs with a number of up- and downregulated genes across treatment groups is shown. C, Venn diagrams visualizing sharedand nonoverlapping DEGs across experimental groups.






macrophage enrichment/reprograming and DC maturation(upregulation of Cd86, Tlr3, Tlr9, Tnf, Il1b, Il1rl1), increased IFNresponse and JAK/STAT signaling (upregulation of Ifnar1, Ifngr1,Jak3, Stat1, Stat2, Ifit3, Ifi35, Isg15, Psmb8, Tap1) and enhanced T-cell signaling mainly driven by increased expression of T-cell–specific transcripts (Ifngr1, Il2ra, Il2rb, Il12rb1, Il21r; Fig. 3). The

same pathway enrichment was identified in pemetrexed mono-therapy and pemetrexed/carboplatin groups; however, combina-tion with carboplatin appeared to qualitatively weaken the effectcompared with pemetrexed monotherapy (SupplementaryTable S1; Fig. 3). As mentioned earlier, paclitaxel-based treat-ments exerted a less prominent immunomodulatory effect, and

Pemetrexed Pemetrexed + Carbopla�n Paclitaxel + Carbopla�n Paclitaxel

19 DEGs 7 DEGs 5 DEGs 1 DEG


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Figure 3.

Macrophage, DC/NK cell, T helper cell, and IFN signaling are among the top ranking ingenuity pathways enriched in pemetrexedmonotherapy and pemetrexed/carboplatin combination groups in MC38 tumors. Volcano plots visualizing DEGs attributable to the top ranking pathways (macrophage, DC/NK cell, T helper cell,IFN signaling) identified by IPA in the pemetrexed-based treatment groups.

Schaer et al.





the IPA results showed a similar trend (SupplementaryTable S1; Fig. 3). Collectively these data indicate that in MC38tumors, treatment with pemetrexed or paclitaxel induced bothqualitatively and quantitatively different immunomodulatoryeffects, and addition of carboplatin appeared to attenuate ratherthan enhance these changes.

Pemetrexed synergizes with PD-1 pathway blockadeThe observed immunomodulatory effects of pemetrexed

prompted us to evaluate pemetrexed in combination with PD(L)1 blockade. To this end, we performed in vivo combinationstudies with anti-PD-L1 antibody in MC38 and Colon26 tumormodels on 2 distinct genetic backgrounds, C57BL/6 and BALB/c,with distinct immunologic Th1 and Th2 profiles, respective-ly (29, 30). In MC38 model, combining pemetrexed with anti-PD-L1 resulted in a modest but statistically significant tumorgrowth delay (Supplementary Fig. S3A). However, Colon26model displayed greater sensitivity to the combination therapy;the combination of pemetrexed and anti-PD-L1 resulted in moresubstantial tumor growth delay accompanied by durableresponses in some animals (Fig. 4A). No combination benefitwas observed in LLC model (Supplementary Fig. S3B).

QGP analysis revealed transient immune-related changes inColon26 tumors after treatment with monotherapies, whereasthe combination effect was most pronounced at a later timepoint (D14 posttreatment, D24 postimplantation; Supplemen-tary Fig. S3C). To further characterize the effects of the peme-trexed/anti-PD-L1combination in Colon26 model, we per-formed nCounter analysis of tumor samples collected atD14 posttreatment, where the differences between groups weremost apparent. Pemetrexed affected the expression of a limitednumber of genes (n ¼ 13), with anti-PD-L1 modulating abroader set of genes (n ¼ 57). Combination treatment alteredthe expression of a large number of genes (n ¼ 198), with themajority (n ¼ 152) uniquely modulated by the combinationtreatment (Fig. 4B). Although pemetrexed treatment predom-inantly resulted in the downmodulation of genes in this model(10/13 genes), the combination therapy of pemetrexed andanti-PD-L1 resulted in the upregulation of a substantial num-ber of genes (173/198), including a large set of genes notsignificantly upregulated by anti-PD-L1 monotherapy.

IPA was used to understand these extensive changes andfurther explore the immune-related molecular and/or cellularpathways modulated by the combination therapy. The path-ways most significantly modulated by the combinationinvolved CD4þ T cell-mediated immunity (Th1/Th2 pathway)and a pathway referred to as "Granulocyte/Agranulocyte Adhe-sion and Diapedesis" (Supplementary Table S2). The latter waslargely driven by genes encoding cell adhesion molecules ("Icam1, Icam2, Pecam, Sell), DC maturation (" H2-Ab1, Cd40,Tlr4, Tlr8), and CXC family chemokines and their receptors ("Cxcl10, Cxcl12, Cxcl13, Cxcl14,Cxcl16, Cxcr4), which have beenassociated with the activated vascular endothelium, leukocytetrafficking, and formation of tertiary lymphoid organs (Fig. 4C;ref. 31).

To confirm gene expression changes described above, we sub-jected Colon26 tumor samples after pemetrexed and/or anti-PD-L1 treatment to flow cytometry analysis (SupplementaryFig. S4A; Fig. 5). Consistent with the IPA results, we detectedincreased frequency ofCD8þ T cells, CD8þ/CD4þ andCD8þ/Tregratios along with enhanced activation of effector T cells (Ki67þ,

CD4þ Foxp3NEG; Fig. 5A). Because the aforementioned geneexpression data suggested that modulation of the myeloid cellcompartment upon treatment with pemetrexed, we also evaluat-ed the effects of pemetrexed � anti-PD-L1 on myeloid cells. Thecombination treatment resulted in a decreased frequency ofgranulocytic MDSCs (Ly6Gþ) population and a trend towardsgreater DC infiltration along with increased activation phenotypeof macrophages and Ly6Chigh monocytes which displayed higherexpression of MHC class I and II. In addition, the combinationtreatment also resulted inmarkedupregulationofMHCclass II ontumor cells (Fig. 5B). These data indicate that the combinationtreatment promotes antigen-presenting properties of myeloidcells, thus supporting the conclusion that pemetrexed inducesT-cell-permissive changes in the myeloid cell compartment, lead-ing to an activated and "T-cell priming-competent" phenotype.Given that the percentage ofmyeloid cells didnot change, the datasuggest that the effect of pemetrexed on myeloid cells is ratherindirect, andmaybemediated by immunogenic effects (e.g., ICD)on tumor cells. Of note, although the M1 andM2 phenotypes arewell established in mouse macrophage biology, the emergingtranslational data generated on human breast and lung tumorssuggest that the phenotypes of human tumor-associated macro-phages aremuchmore complex and cannot be dichotomized intobinary M1/M2 states (32, 33). Given the lack of clinical relevanceofM1/M2macrophage polarization in human tumor biology, thebona fidemarkers ofM1 andM2macrophages were not pursued inour studies.

To understand if the changes in myeloid cells and subsequentT-cell priming in lymph nodes are required for the antitumoreffects of pemetrexed and anti-PD-L1, we treated Colon26-bear-ing mice with pemetrexed and/or anti-PD-L1 together with thewell-characterized sphingosine-1-phosphate receptor 1 (S1P1R)antagonist (FTY720) to block T-cell egress from lymph nodes(Fig. 5C). Although FTY720 treatment did not have an obviousimpact on either monotherapy, the combination therapy ben-efit was lost after FTY720 treatment. To understand theseobservations in the context of an antigen-specific immuneresponse, we examined the effect of the combination therapyon the frequencies of tumor antigen-specific T cells comparedwith monotherapies in Colon26 model. After 14-day treatmentwith pemetrexed and/or anti-PD-L1, we evaluated the activa-tion status and frequency of tumor-specific T cells in the tumor,tumor-draining lymph node, and spleen using ELISpot andMHC tetramer assays. The results of these experiments indicatethat although a trend towards increased tumor-specific CD8þ T-cell responses was observed in the periphery during the com-bination treatment (as exemplified by IFN-gamma ELISpot andgp70 tetramer assay), no appreciable difference in the frequen-cy of gp70 tetramer positive CD8þ T cells was detected in thetumor. However, a small but statistically significant increase inthe frequency of TNFaþ CD8þ T cells was observed in Colon26tumors after treatment with pemetrexed and anti-PD-L1 sug-gesting that that the increased priming during the combinationtreatment increases the functionality rather than the quantity oftumor-reactive CD8þ T cells (Supplementary Fig. S4B).

Collectively, these data demonstrate the development of anintegrated antitumor immune responsemediated by pemetrexed/anti-PD-L1 combination, and suggest that the underlying mech-anism involves perpetuation of T-cell priming in lymph nodes,presumably through the enhanced antigen presentation functionof myeloid cells.






Pemetrexed induces immunogenic tumor cell deathIncreased antigen presentation and DC maturation gene sig-

natures suggest that pemetrexed treatment may lead to ICD oftumors, activating innate pathways leading to enhanced immuneactivation. To investigate the ability of pemetrexed to induce ICD,we evaluated the extracellular levels of CRT and HMGB1, both ofwhich are specifically released from cells during ICD. Binding ofHMGB1 to Toll-like receptor 4 and CRT to CD91/LRP1 leads to

DC migration and maturation and enhanced antigen presenta-tion and T-cell priming (34). Colon 26 and MC38 tumor cellswere treated with pemetrexed or other chemotherapeutics (car-boplatin, paclitaxel, doxorubicin, or gemcitabine), followed bymeasurement of the extracellular HMGB1 and CRT release(Fig. 5D). Although all agents tested appeared to induce somedegree of ICD, as exemplified by increased CRT and HMGB1release, pemetrexed was the most potent inducer of ICD in both

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Combination of pemetrexed and anti-PD-L1 improves antitumor efficacy andmarkedly enhances T-cell inflamed phenotype in Colon26 syngeneic mouse tumormodel. A,Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with pemetrexed (50 mg/kg5 days on, 2 days off, i.p.) and/or anti-PD-L1 (aPD-L1; 500 mg/mouse, weekly i.p.). Group, individual tumor growth curves and overall survival Kaplan–Meyer curves for single agent and combinationtreatment groups are shown overlaid on top of control group. Difference in the tumor volume of treated groups compared with control (%T/C) at Day 31 (pointwhere at least 50% of controls we present) was significantly different (P < 0.001) for the combination (12.4% T/C) compared with anti-PD-L1 (60.1%) orpemetrexed monotherapy (50.8%; RM-ANOVA), and was shown to be better than additive by Bliss Independence analysis. Overall survival was significantlyincreased with the combination as indicated, with a trend (P¼ 0.07) compared with pemetrexedmonotherapy. B, Colon26 tumors treated with pemetrexedand/or aPD-L1 were further subjected to nCounter PanCancer Immune Profiling; summary of shared and nonoverlapping DEGs with a number of up- anddownregulated genes across treatment groups is shown. C, Volcano plots visualizing DEGs attributable to the top ranking pathways ("Granulocyte/AgranulocyteDiapedesis," "T Helper/T Cell Signaling," "Dendritic Cell Maturation/NK Cell Signaling/DC/NK Crosstalk," and "Cytokine/Chemokine Signaling") identified by IPAin the combination group. Representative example of 3 experiments in Colon26model.

Schaer et al.





Colon26 MC38

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Pemetrexed exerts strong immunogenic effect on immune and tumor cells. A and B, Mice bearing Colon26 tumors were treated starting 10 days after tumorimplantation with pemetrexed (50mg/kg5 days on, 2 days off, i.p.) and/or anti-PD-L1 (aPD-L1; 500 mg/mouse, weekly i.p.) and tumors were isolated after14 days of treatment and single cell suspensions were subjected to flow cytometric analysis. A,Mean (�SEM) percentage of indicated T cell (A) and myeloid cell(B) populations, and mean fluorescence intensity (MFI) (�SEM) of MHC-I/II of indicated cell population. Lines indicate groups showing significant difference fromcontrol of P < 0.05 are shown (1-way ANOVA). C,Mice bearing Colon26 tumors were treated with pemetrexed (50mg/kg 5 days on, 2 days off, i.p.) and/or anti-PD-L1 (aPD-L1; 500 mg/mouse, weekly i.p.) as well as FTY720, a well-characterized S1P1R antagonist (q2D until D32), 3 days after starting combination therapy.Individual tumor growth curves for experimental groups are presented. The rate of complete remissions (CR) is indicated, addition of FTY720 to the combinationof aPD-L1 and pemetrexed was shown to be antagonistic (Categorical Response Analysis with Bayesian Ordinal Logistic Regression, see Materials and Methods).D,MC38 and Colon26mouse tumor cells were incubated in the presence of various chemotherapeutic agents (pemetrexed, paclitaxel, carboplatin, gemcitabine,doxorubicin) for 72 hours. Untreated as well as DMSO- and staurosporine-treated cultures were used as controls. ICD was evaluated by measuring extracellularlevels of calreticulin (CRT) and high mobility group box1 (HMGB1) protein. Dose ranges indicated cover the IC50 of each compound. In vivo studies representativeexample of 2 experiments in Colon26.






Colon26 andMC38 cells, particularly across lower concentrationsthat reflect clinical exposure (0.02–0.05 mmol/L). These resultssuggest that the immunomodulatory effects of pemetrexed aremediated, at least in part, by tumor cell-intrinsic mechanismsinvolving ICD.

Pemetrexed exerts direct immunomodulatory effects onactivated T cells in vitro

Purine nucleotide synthesis in general and the folate pathwayin particular depend on metabolic intermediates (e.g., 3-phos-phoglycerophosphate) supplied through glycolysis, and one-carbon metabolism plays a critical role during T-cell activationbecause T cells require high levels of glycolysis andmitochondrialrespiration during the activation and effector phase (17, 35).To evaluate the impact of pemetrexed on T-cell glycolysisand mitochondrial respiration, we used the Seahorse mitochon-dria stress test, using primary T cells activated with anti-CD3/CD28 antibodies and IL2 in the presence of pemetrexedover a broad pharmacologic range spanning clinical exposure(0.004–0.1 mmol/L), and determined extracellular acidificationrates (reflective of glycolysis), as well as basal and maximal OCRand SRC (reflective of mitochondrial respiration; Fig. 6A). Peme-trexed increased both basal and maximal OCR in an inverseconcentration-dependent manner, with the maximum effect at45 hours of stimulus with the lowest concentration tested (0.004mmol/L; ref. 36). SRC (representing the difference between basalandmaximalOCRvalues)wasmarkedly increasedbypemetrexedin a concentration-dependent manner, and this effect was partic-ularly evident at 45 and 70 hours (Fig. 6A). Pemetrexed alsoenhanced OCR when activated T cells were supplemented withfatty acid (palmitate), an effect thatwas abrogatedby etoxomir, aninhibitor of carnitine palmitoyltransferase-1 which blocks fattyacid oxidation (Fig. 6B). Because b-oxidation of fatty acids isdirectly linkedwith the tricarboxylic acid (TCA) cycle, these resultssuggest that pemetrexed may increase metabolic fitness of T cellsthrough the enhancement of mitochondrial function or biogen-esis that increases the bioenergetic reserve of T cells that might becritical for their survival.

Since chemotherapeutic agents including antifolates areknown to have inhibitory effects on T-cell activation andsurvival (16), we next evaluated the direct impact of peme-trexed compared with paclitaxel on T-cell function and activa-tion. Primary human T cells were activated with CD3 and CD28antibodies and exposed to fixed, clinically relevant concentra-tions of pemetrexed (0.05 mmol/L) or paclitaxel (0.2 mmol/L)for various time intervals [days (D) 0–3, 3–9, 0–9] to mimictreatment during different stages of T-cell activation. In vitroactivated T cells showed modest but significant attenuation oftotal proliferation in the presence of pemetrexed, yet this effectwas reversible, and most pronounced when pemetrexed waspresent in the culture medium for the duration of the study,resulting in approximately half the number of total cells com-pared with untreated cells (Fig. 6C). In contrast, the cytotoxiceffect of paclitaxel was detrimental to T-cell proliferation andviability, and paclitaxel-treated T cells did not survive beyondD6 regardless of the duration and timing of exposure (Fig. 6C).Flow cytometry analysis during T-cell expansion revealed thatexposure to pemetrexed enhanced the activation state of T cells,as reflected by significantly increased and sustained surfaceexpression of CD137 and GITR on CD8þ and CD4þ T cellswith continuous pemetrexed exposure (Fig. 6D). The enhanced

T-cell activation state was also accompanied by significantlyincreased mitochondrial content in CD8þ and CD4þ T cells(Fig. 6E). Finally, QGP gene expression analysis revealed peme-trexed-dependent upregulation of IFNg-dependent transcripts(IFNG, CXCL9, CXCL10, CXCL11, IDO1, HLA-DRA), cytolyticgenes (GZMB, PRF1) as well as transcripts encoding costimu-latory receptors CD137, GITR, OX40 (TNFRSF9, TNFRSF18,TNFRSF4; Fig. 6F), and these results were further confirmedusing nCounter analysis (Supplementary Fig. S5).

To assess if the increased metabolic fitness and activationstate of T cells translate to enhanced effector function, wemeasured antigen-dependent tumor cell killing using ovalbu-min (OVA)-specific OT-1 T cells. OT-1 T cells were primed withOVA peptide (SIINFEKL) in the presence or absence of peme-trexed, and their ability to kill tumor targets loaded with OVApeptide was evaluated in vitro. These results demonstrate thatpriming in the presence of pemetrexed resulted in �50%increase in tumor cell killing (�50% and �30% dead tumorcells with pemetrexed and control, respectively). These datatherefore suggest that the enhanced activation state induced bypemetrexed translates into increased effector T-cell functionexemplified by increased cytotoxicity of antigen-specific T cells(Fig. 6G).

Taken together, these data reveal that pemetrexed exerts pleio-tropic immunomodulatory effects by inducing ICD in tumorcells, enhancing the metabolic state of T cells by increasing theiroxidative respiration and the mitochondrial content, leading toincreased activation and effector function.

DiscussionChemotherapeutic agents are part of standard of care treatment

in many tumor types and across lines of therapy. A fundamentalpremise for combining chemo- and immunotherapies is that thechemotherapeutic agents preferentially target tumor cells anddo not incapacitate relevant immune functions. The potentialfor enhanced combinatorial activity of chemotherapy and immu-notherapy is based on the principle that in some cases chemo-therapeutic agents may cause immunogenic tumor cell death,resulting in immune enhancing activities through immunecells, and/or the tumormicroenvironment without incapacitatingrelevant immune cell function (4, 5, 11).

It is generally believed that folate pathway inhibitors such asmethotrexate are immunosuppressive (12, 13). Although thecytotoxic activity of pemetrexed has been attributed to inhibitionof 4 enzymes in the folate cycle (24), very little is known abouthow pemetrexed modulates antitumor immunity. Our data indi-cate that pemetrexed exerts immunomodulatory effects acrossmultiple pathways and immune cell subsets. These effectswere not observed with other chemotherapeutic agents such ascarboplatin or paclitaxel. Furthermore, our data indicate thatcarboplatin and cisplatin attenuate, rather than enhance theimmunomodulatory effects of pemetrexed, and suggest thatpemetrexed can potentially be combined with ICIs withoutplatinum agents. This finding might be critical, and additionalmechanistic, translational, and clinical studies are needed tofurther understand the effects of various platinum doublets ontumor immunemicroenvironment as well as their combinatorialpotential with ICIs.

One hypothesis supporting platinum-based chemotherapy incombination with pemetrexed and immune checkpoint blockade

Schaer et al.





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Pemetrexed exerts direct effects on primary T cells in vitro. A, T cells isolated from spleens of BALB/c mice were activated with anti-CD3/CD28 antibodies andmouse IL2 in the presence of various concentrations of pemetrexed (0.004–0.1 mmol/L), and basal OCR were analyzed using Seahorse XFe96 instrument. Cellswere stimulated with oligomycin (1 mmol/L), FCCP (1.5 mmol/L), and rotenone/antimycin A (0.5 mmol/L each). SRC was measured as the difference betweenbasal OCR values and maximal OCR values obtained after FCCP uncoupling. B,Maximal respiration of mouse primary T cells treated with pemetrexed wasevaluated in the presence or absence of fatty acid (palmitate, PALM) or inhibitor of fatty acid oxidation (etoxomir, ETO). C, Proliferation of primary human T cellsstimulated with CD3/CD28 beads in vitrowas evaluated in the presence or absence of pemetrexed (0.05 mmol/L) or paclitaxel (0.2 mmol/L) for the indicatedperiod of time. Total T-cell numbers are indicated for various treatment conditions. Note that proliferation could not be assessed in paclitaxel-treated T cellsbeyond D6 due to a prominent cytotoxic effect. D and E, Flow cytometry was performed on CD4þ and CD8þ T-cell populations throughout the experiment (Day3, 6, and 9) to determine cell surface expression of CD137 and GITR indicative of T-cell activation (D) as well as mitochondrial mass (E). Data are shown asmean� SD (A, B), SEM (C–E). Lines show significant differences between indicated groups with control where P� 0.05 (A, B, 1-way ANOVA). Asterisk indicates groupand time points where differences between treated group and control where significant with P� 0.05 (C–E, 1-way ANOVA). F,QGP assay was used to quantifyexpression of immune-related genes in T cells treated with pemetrexed in vitro. Volcano plot shows the P value (1-way ANOVA) vs. log2-fold change ofpemetrexed compared with untreated cells. DEGs with P < 0.05 are colored pink; DEGs showing greater than 2-fold change compared with control arehighlighted. Data shown are averages of 3 donors. G, Splenocytes from ovalbumin-specific TCR transgenic OT-1 T cells were incubated for 5 dayswith 0.1 nmol/Lof SIINFEKL peptide with or without pemetrexed. CD8 T cells were isolated and incubated with OVA loaded B16 tumor cells at a 10:1 effector to target ratio for4 hours. Tumor cell death was then analyzed by 7AAD incorporation by flow cytometry. Graph shows average tumor cell killing, and example FACS plots gatedon CD45Neg cells show percentage of dead tumor cells (7AADþ).






is that platinum agents could induce somatic mutations inthe tumor cells and, consequently, induce new immunogenicneoantigens. It is therefore possible that adding a platinum agentbefore pemetrexed/anti-PD(L)1 treatment could potentiallyenhance the immunomodulatory and antitumor effects throughincreased priming against these de novo induced neoantigens.The accumulation of somatic mutations in tumor cell DNArequires some time, and syngeneic mouse tumor models havevery limited time window that makes them poorly fit forstudying effects of cytotoxic agents on tumor mutational bur-den. It would be worthwhile to test this hypothesis in theclinical setting given that patients with advanced/metastaticNSCLC typically receive front-line chemotherapy every 3 weeksup to 6 cycles.

Gene expression profiling indicated that treatment withpemetrexed � anti-PD-L1 induced macrophage reprograming,DC/NK cell enrichment, and enhanced inflammatory response,activated innate immune mechanisms (PRR/TLR signaling),granulocyte/agranulocyte diapedesis, and IFN signaling thatplay an important role in priming and establishing an efficientT-cell immunity. Additionally, the benefit from the combina-tion treatment was abrogated when T-cell egress from thelymph nodes was blocked by the S1P1R antagonist. Theseresults strongly support the hypothesis that pemetrexedinduces an integrated antitumor immune response by enhanc-ing antigen presentation and T-cell priming in tumor-draininglymph nodes.

The gene expression analyses also revealed that pemetrexedmonotherapy was accompanied by activation of vascular endo-thelium and genes associated with tertiary lymphoid structureformation; this effect was even more evident when pemetrexedwas combined with anti-PD-L1 therapy. It is plausible that the T-cell inflamed phenotype observed in pemetrexed-treated tumorsmight be attributable, at least in part, to enhancement of thesepathways, which have the potential to promote T-cell traffickingand infiltration.

The high-content analyses suggested and in vitro data revealedthat the immunomodulatory effects of pemetrexed also includeddirect effects on tumor cells via induction of ICD exemplified bythe extracellular release ofHMGB1 andCRT, in amanner superiorto other ICD-inducing chemotherapies. Our data suggest that theimmunogenic effects of pemetrexed on tumor cells exemplifiedby CRT and HMGB1 release may require lower drug concentra-tions compared with the cytotoxic effects. These results mayexplain the more robust gene expression changes observed inMC38 tumors after treatment with lower doses (50 mg/kg) ofpemetrexed, and suggest that part of the mechanism of action forpemetrexed might involve increased tumor immunogenicity fol-lowed by the priming and establishment of an antitumor T-cellresponse.

It is worth noting that although carboplatin also demon-strated evidence of ICD in mouse tumor cell lines in vitro, theseresults did not translate in vivo as exemplified by the geneexpression data in MC38 tumors. A potential explanation ofthis discrepancy could be due to different effects of carboplatinon tumor versus immune cells. It is also important to highlightthat in MC38 tumors, treatment with all chemotherapeuticagents tested was accompanied by PD-L1 (encoded by Cd274)upregulation highlighting the need for PD1 pathway blockadeto overcome adaptive resistance in T-cell compartment inducedby chemotherapy.

To our knowledge, a positive effect of anti-folates in generaland pemetrexed in particular on T-cell biology has not beendescribed previously. Although the molecular mechanismsfor the T-cell-intrinsic effects have not been fully elucidated,because the folate pathway (which regulates purine nucleotidesynthesis and the TCA cycle) depends on 3-phosphoglyceropho-sphate (3-PG) generated through glycolysis (37), it is possible thatinhibition of the folate cycle may increase the abundanceof 3-PG and downstream metabolic intermediates requiredfor optimal T-cell activation (37, 38). The enhanced metabolicfitness of T cells exposed to pemetrexed is notable as animmune-enhancing mechanism because activated T cellsrequire adequate mitochondrial mass to support bioenergeticneeds required for cytokine production and development ofcytotoxic effector function. Indeed, multiple lines of evidencelink T-cell metabolic state with activation, survival, and intra-tumoral exhaustion (35), and the association between meta-bolic fitness, activation phenotype, and effector function oftumor-reactive T cells has also been demonstrated in thepresent study. The T-cell-intrinsic effects described here canalso potentially be attributable to the unique ability of peme-trexed, relative to other anti-folates, to also inhibit 5-aminoi-midazole-4-carboxamide ribonucleotide formyltransferase(AICARFT), because blockade of this enzyme results in theelevated intracellular levels of ZMP, a metabolite structurallyrelated to AMP that is capable of promoting mitochondrialbiogenesis and respiration function via AMP kinase-mediatedmechanisms (39–41). Emerging data indicate that epigeneticand/or metabolic mechanisms rather than immunosuppressivetumor microenvironment play a dominant role in drivingintratumoral T-cell dysfunction (42–45). The results of thisstudy, particularly with regard to the ability of pemetrexed toenhance metabolic fitness and effector function of T cells havean important biological and translational significance givenlimited therapeutic options to revert metabolically exhausted Tcells in the tumor.

Collectively, the data from these studies suggest that peme-trexed therapy has the potential to induce an integratedantitumor immune response in tumors. These observationsprovide mechanistic rationale for the clinically observed com-bination activity between pemetrexed and anti-PD-1 therapy,identify pathways and mechanisms to be explored in trans-lational studies and highlight the potential for pemetrexedas an important therapeutic modality to be investigatedfurther in the context of combination immunotherapies.Finally, these studies provide context and direction for theexploration of the immunotherapeutic potential for othertumor-targeting agents currently being used or contemplatedfor use in the clinic.

Disclosure of Potential Conflicts of InterestD.A. Schaer, S.Geeganage, E.R. Rasmussen, K.Chodavarapu, J.R.Manro,G.P.

Donoho, and M. Kalos hold ownership interest (including patents) in Eli Lillyand Company. R. Novosiadly holds ownership interest (including patents) inEli Lilly and Bristol-Myers Squibb. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design:D.A. Schaer, S. Geeganage, F.C. Dorsey, D. Surguladze,M. Kalos, R.D. NovosiadlyDevelopment of methodology: D.A. Schaer, N. Amaladas, Z.H. Lu,E.R. Rasmussen, Y. Li, S. Luo, C. Carpenito, G.E. Hall, M. Kalos, R.D. Novosiadly

Schaer et al.





Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Amaladas, Z.H. Lu, A. Sonyi, D. Chin, A. Capen,Y. Li, C.M. Meyer, B.D. Jones, S. Luo, A. Nikolayev, B. Tan, F.C. Dorsey,G.P. Donoho, D. Surguladze, G.E. HallAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.A. Schaer, S. Geeganage, N. Amaladas, Z.H. Lu,E.R. Rasmussen, A. Sonyi, D. Chin, A. Capen, Y. Li, C. Carpenito, K.D. Roth,A. Nikolayev, K. Chodavarapu, J.R. Manro, T.N. Doman, D. Surguladze,G.E. Hall, M. Kalos, R.D. NovosiadlyWriting, review, and/or revision of themanuscript:D.A. Schaer, S. Geeganage,N. Amaladas, E.R. Rasmussen, D. Chin, C. Carpenito, M. Brahmachary,F.C. Dorsey, J.R. Manro, G.P. Donoho, M. Kalos, R.D. NovosiadlyAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. Amaladas, X. Huang, J.R. Manro,T.N. Doman, D. Surguladze, G.E. Hall, M. Kalos

Study supervision: D.A. Schaer, S. Geeganage, A. Capen, G.P. Donoho,D. Surguladze, M. Kalos, R.D. Novosiadly

AcknowledgmentsWe thank Gregory D. Plowman, Levi Garraway, Ana Oton, and Jong Seok

Kim (Eli Lilly) for review and helpful discussions during preparation of themanuscript.

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 3, 2019; revised May 31, 2019; accepted August 7, 2019;published first August 13, 2019.

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2019;25:7175-7188. Published OnlineFirst August 13, 2019.Clin Cancer Res David A. Schaer, Sandaruwan Geeganage, Nelusha Amaladas, et al. Effects of Cancer ImmunotherapyThe Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances

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