The Folate Pathway Inhibitor Pemetrexed Pleiotropically Enhances Effects of Cancer

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Author Manuscript Published OnlineFirst on August 13, 2019; DOI: 10.1158/1078-0432.CCR-19-0433 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer

immunotherapy

Running title: Immunomodulatory effects of pemetrexed

David A. Schaer1,3, Sandaruwan Geeganage2,3, Nelusha Amaladas1, Zhao Hai Lu2, Erik R.

Rasmussen1, Andreas Sonyi1, Darin Chin1, Andrew Capen2, Yanxia Li1, Catalina M. Meyer2,

Bonita D. Jones2, Xiaodong Huang1, Shuang Luo2, Carmine Carpenito1, Kenneth D Roth2,

Alexander Nikolayev2, Bo Tan2, Manisha Brahmachary1, Krishna Chodavarapu1, Frank Charles

Dorsey2, Jason R. Manro2, Thompson N. Doman2, Gregory P. Donoho2, David Surguladze1,

Gerald E. Hall1, Michael Kalos1,4,* & Ruslan D. Novosiadly1,5,6*

1 Lilly Research Laboratories, Eli Lilly and Company, New York, NY 10016, USA, 2 Lilly

Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA 3These authors contributed equally; Present address: 4 Janssen Pharmaceutical Companies of Johnson and

Johnson, Spring House, PA, USA; 5 Bristol-Myers Squibb, Princeton, NJ, USA

6 Lead Contact

Correspondence:

Ruslan Novosiadly

Bristol-Myers Squibb

3551 Lawrenceville Road

Princeton, NJ 08540

e-mail: [email protected]

Michael Kalos

Janssen Oncology

1400 McKean Road

Spring House, PA 19477

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Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed e-mail: [email protected]

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

ABBREVIATIONS immunogenic cell death (ICD) non-small cell lung cancer (NSCLC) immune checkpoint inhibitor (ICI) analysis of variance (ANOVA) oxygen consumption rate (OCR) spare respiratory capacity (SRC) tumor-draining lymph node (TDLN) enzyme-linked immune absorbent spot (ELISpot) high mobility group B1 (HMGB1) calreticulin (CRT)

Quantigene Plex (QGP) housekeeping genes (HKG)

Lewis Lung carcinoma (LLC) genetically engineered mouse model (GEMM) vascular endothelial growth factor C (VEGF-C) myeloid derived suppressor cell (MDSC) dendritic cell (DC) differentially expressed gene (DEG)

Ingenuity pathway analysis (IPA) sphingosine-1-phosphate receptor 1 (S1P1R) tricarboxylic acid (TCA) ovalbumin (OVA) pattern recognition receptor (PRR)

Toll-like receptor (TLR)

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

3-phosphoglycerophosphate (3-PG) adenosine monophosphate (AMP)

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

CONFLICT OF INTEREST STATEMENT

Authors of this manuscript were full-time employees of Eli Lilly and Company at the time when these studies were conducted

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

WORD COUNT

 120–150-word statement of translational relevance (required) 147  250-word structured abstract 241  5,000 words of text 5,059  6 tables and/or figures 6  50 references 45

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

TRANSLATIONAL RELEVANCE

In this manuscript we describe novel immunomodulatory properties of pemetrexed, a chemotherapy that targets the folate pathway and is used as standard of care in the frontline treatment of advanced non-squamous non-small cell lung cancer and pleural mesothelioma. The combination of pemetrexed-based chemotherapy with anti-PD-1 (pembrolizumab) has demonstrated compelling clinical activity in patients with metastatic NSCLC based on the results of KEYNOTE-189 Phase III trial, and previously disclosed data from KEYNOTE-021G Phase II trial have led to the accelerated approval of this regimen by FDA. While this landmark approval represents the first case of clinical adoption of chemoimmunotherapy combination in oncology, there are fundamental unanswered questions about why and how a chemotherapeutic agent such as pemetrexed might effectively combine with immunotherapy.

This work provides novel data on how pemetrexed pleiotropically modulates anti-tumor immunity and provides key insights for the development of chemotherapeutic agents in combination with immunotherapies.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

ABSTRACT

Purpose: Combination strategies leveraging chemotherapeutic agents and immunotherapy have held the promise as a method to improve benefit to cancer patients. However, most chemotherapies have detrimental effects on immune homeostasis and differ in their ability to induce immunogenic cell death. The approval of pemetrexed and carboplatin with anti-PD-1

(pembrolizumab) for treatment of non-small cell lung cancer, represents the first approved chemotherapy and immunotherapy combination. Although the clinical data suggests a positive interaction between pemetrexed-based chemotherapy and immunotherapy, the underlying mechanism remains unknown.

Experimental Design: Mouse tumor models (MC38, Colon26) and high-content biomarker studies (flow cytometry, Quantigene Plex and nCounter gene expression analysis) were deployed to obtain insights into the mechanistic rationale behind the efficacy observed with pemetrexed/anti-PD-L1 combination. Immunogenic cell death (ICD) in tumor cell lines was assessed by calreticulin and HMGB-1 immunoassays, and metabolic function of primary T cells was evaluated by Seahorse analysis.

Results: Pemetrexed treatment alone increased T cell activation in mouse tumors in vivo, and robustly induced ICD in mouse tumor cells and exerted T cell-intrinsic effects exemplified by augmented mitochondrial function and enhanced T cell activation in vitro. Increased anti-tumor efficacy and pronounced inflamed/immune activation were observed when Pemetrexed was combined with anti-PD-L1.

Conclusions: Pemetrexed augments systemic intra-tumor immune responses through tumor intrinsic mechanisms including immunogenic cell death, T cell-intrinsic mechanisms enhancing mitochondrial biogenesis leading to increased T cell infiltration/activation along with

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed modulation of innate immune pathways, significantly enhanced in combination with PD-1 pathway blockade.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

INTRODUCTION

PD(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 care across an increasing number of tumor types(1, 2). However, clinical benefit from these therapies is limited, and tumor recurrences are common(3, 4). One strategy to improve the efficacy of

PD(L)1 inhibitors is to combine these agents with tumor-targeting therapies that have the potential for co-operative mechanistic interactions with immune agents (4, 5). Indeed, numerous

clinical trials are underway to evaluate the potential to combine immune checkpoint inhibitors

(ICIs) and chemotherapies (6).

Pemetrexed is an established chemotherapeutic that disrupts the folate pathway and is part of the standard of care for non-squamous NSCLC and mesothelioma (7). The front-line treatment with

pemetrexed, carboplatin and anti-PD-1 (pembrolizumab) has been evaluated in NSCLC patients in the randomized KEYNOTE-021G and -189 trials (8, 9), leading to the accelerated approval of

this regimen based on substantial increase in progression-free survival and overall response rate

in the KEYNOTE-021G study. These improvements represent the first approval of chemo

immunotherapy combination (10). The rationale to combine chemotherapy with ICIs is based at

least in part on the concept of immunogenic cell death (ICD) that can be a consequence of the

cytotoxic effects of chemotherapeutic agents on tumor cells(11). ICD involves the release of

immune-stimulating factors from dying tumor cells that drive antigen cross-presentation, T cell

priming and adaptive immune response against tumors. Cytotoxic agents are not equipotent in

their ability to induce ICD; only a few cytotoxic agents (e.g. anthracyclines, oxaliplatin) have

been demonstrated to induce ICD, while most chemotherapeutics induce non-immunogenic cell

death (11). Cytotoxic agents can also be deleterious to the immune compartment by cytotoxic

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed targeting of immune cells (11). The positive interaction between pemetrexed-based

chemotherapy and immune checkpoint blockade in KEYNOTE-021 and -189 trials may seem

counterintuitive given that antifolate agents (e.g. methotrexate) have been used as

immunosuppressive agents to treat patients with inflammatory conditions, and part of their

immunosuppressive activity appears to involve the inhibition of T cells (12-15). Furthermore,

recent work has identified one-carbon metabolism, which includes the folate and methionine

cycles, as a top ranked metabolic pathway engaged during T cell activation and survival(16, 17).

The main objective of this work was to obtain mechanistic insights into the immunostimulatory

activity of pemetrexed -/+ PD1 blockade rather than justify clinical development of

pemetrexed/anti-PD(L)1 combinations in NSCLC and other tumor types. We demonstrate that

pemetrexed therapy exerts previously unknown immunomodulatory effects that result in an

immune-permissive tumor microenvironment and improves the antitumor efficacy of PD(L)1

blockade. These results provide fundamental insights into the mechanisms underlying the

combinatorial activity of pemetrexed and anti-PD-1 therapy, and provide a strong rationale for

further exploration of combinations of pemetrexed and other folate pathway modulators with

immunotherapies.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

MATERIALS AND METHODS

In vivo tumor studies

Colon26 and MC38 cell lines were purchased from DTP and NCI DCTD Tumor Repository, respectively. Female BALB/c and C57BL/6 mice were purchased from Envigo, Frederick, MD.

All experimental procedures were done in accordance with the NIH Guide for Care and Use of

Animals and were approved by the Institutional Animal Care and Use Committee.

Metabolic assessments of primary mouse T cells

Mouse splenic T cells stimulated with CD3/CD28 were cultured in the presence of pemetrexed as indicated. Oxygen consumption rate (OCR) was analyzed using Seahorse XF Cell Mito Stress

Test kit and Seahorse XFe96 instrument (Agilent, Santa Clara, CA). Cells were sequentially stimulated with oligomycin (1 M), FCCP (1.5 M), and rotenone/antimycin A (0.5 M each) and the spare respiratory capacity (SRC) was measured as the difference between basal OCR values and maximal OCR values obtained after FCCP uncoupling. To assess T cell ability to metabolize fatty acids, XF Palmitate:BSA FAO substrate (Agilent) was incorporated into XF

Cell Mito Stress Test assay. Wave 2.4 software (Agilent) was used for data acquisition and analysis of Seahorse data.

Tumor cell killing assay

Splenocytes from ovalbumin-specific T cell receptor transgenic OT-1 mice were incubated in the presence of 0.1nM of SIINFEKL peptide and IL-2 for 5 days. CD8+ T cells were then isolated

and cultured with B16 tumor cells that had been previously labeled with cell tracer BV421 and

pulsed with 100 nM of SIINFEKL peptide for 2 hours, at a 10:1 effector to target ratio. Tumor

cell death was analyzed by 7AAD incorporation by flow cytometry after 4 hours of coculture.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

In vitro assessment of immunogenic cell death (ICD)

Colon26 and MC38 tumor cell lines were treated with pemetrexed, carboplatin, paclitaxel,

gemcitabine or doxorubicin. for 96 hours followed by analysis of high mobility group B1

(HMGB1) protein and calreticulin (CRT) in culture supernatants using commercially available

kits (IBL International, Hamburg, Germany and Cloud Clone Corporation, Katy, TX respectively). The viability of remaining cells was measured by Cell Titer-Glo assay (Promega) according to manufacturer’s protocol.

Gene expression analysis

QuantiGene Plex and nCounter gene expression assays were done as reported previously with slight modifications (18).

Quantification and statistical analysis

Group wise statistical comparisons were performed as indicated in each figure using standard paired T tests, one-way ANOVA, or two-way ANOVA models with Tukey’s adjustment per

time point, comparing treatment/dose and time point.

Additional details are provided in Supplementary Materials and Methods

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

RESULTS

Pemetrexed exhibits intratumor immunomodulatory effects in vivo

To characterize the effects of pemetrexed on intra-tumor immune response, initial experiments

were performed in immunocompetent syngeneic mouse tumor models. To meet our research

objective, the models had to meet two prerequisites: (1) demonstrate sensitivity to pemetrexed

and (2) responsiveness to PD(L)1 blockade. We found that MC38 and Colon26 colorectal tumor

cell lines were sensitive to pemetrexed whereas Lewis Lung carcinoma (LLC) which is an often

used as a lung cancer model was pemetrexed-refractory (Figure S1A) (20). Furthermore, while

genetically engineered mouse models (GEMMs) of lung carcinoma may sound like a logical

choice, they are poorly fit for studying effects of immunotherapies, largely because GEMMs are

driven by specific oncogenic events and do not exhibit high tumor mutational and neoantigen

burden which have emerged as important molecular hallmarks underlying responsiveness of lung

tumors to immunotherapy in humans (21, 22). Because single agent treatment with pemetrexed

induced tumor responses across multiple tumor types in early clinical trials (7, 23), we

rationalized that it was appropriate to use tumor models with the right biological context

irrespective of histology rather than using lung tumor models with no sensitivity to either

pemetrexed or anti-PD(L)1.

MC38 tumors are modestly responsive to both PD-1 blockade (20) and pemetrexed (Figure 1A).

The effect of pemetrexed in MC38 model was consistent with thymidylate synthase inhibition

(increased deoxyuridine, dUMP and decreased thymidine, dTMP in the tumor and plasma)

(Figure S1), and the highest dose used in these studies was determined to be the maximum

tolerated dose (7, 24, 25). MC38 tumors were responsive to pemetrexed at 50 and 100 mg/kg (%

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed tumor growth inhibition of 30% and 52% respectively) (Figure 1A). Tumors collected after 14

days of pemetrexed therapy were analyzed for changes in immune cell frequencies using flow

cytometry. These analyses revealed that pemetrexed increased the frequency of total

intratumoral leukocytes (live CD45+ cells) at both doses, with a trend towards an increased

percentage of total CD3+ and cycling (Ki67+) CD8+ cells, particularly at 50 mg/kg (Figure 1B-

E). This appeared to be driven mainly by an increase in Ki67+CD8+ T cells, without any other

significant differences in myeloid cell subsets (Figure 1F). Molecular analysis of tumor tissues using a custom-made immune profiling QuantiGene® Plex (QGP) gene expression panel revealed that treatment with pemetrexed at 50 and 100 mg/kg promoted a T cell inflamed

phenotype, exemplified by upregulation of T cell-specific genes including Cd8b, Prf1 and Gzma

(Figure 1G). The QGP data also suggests activated vascular endothelium (↑ Icam1, Vcam1 and

chemokine Cx3cl1 also known as fractalkine that is induced in endothelium as result of immune activation) and enhanced interferon response (↑ Irf7) and antigen presentation (upregulated Itgax

and Zbtb1 associated with DCs). Beyond these changes, one of the genes most significantly

modulated by pemetrexed treatment was Vegfc which encodes vascular endothelial growth factor

C (VEGF-C), a key regulator of lymphangiogenesis. VEGF-C is known to be regulated through

the NF-kB pathway and is believed to promote T cell infiltration rather than inhibit antitumor

immune response (27, 28). Nos2 which encodes inducible nitric oxide synthase is produced by

myeloid derived suppressor cells (MDSCs) and dendritic cells (DCs), were downregulated at both dose levels, suggesting that pemetrexed could potentially negatively impact myeloid cell subsets (Figure 1G). While Nos2 displayed downregulation, we did not observe a significant reduction in CD11b + cells by flow cytometric analysis (Figure 1F). Collectively, these results

suggest that pemetrexed influences the functionality rather than frequency of myeloid cells.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

Because pemetrexed is administered in combination with platinum agents such as carboplatin and cisplatin in front-line treatment of patients with metastatic NSCLC, we next asked if carboplatin had immunomodulatory effects on the tumor immune microenvironment, and

whether the immunomodulatory effects of pemetrexed were affected by the addition of

carboplatin. In these experiments we also evaluated the immunomodulatory effects of the

chemotherapy doublet of carboplatin and paclitaxel, a commonly used treatment option in

NSCLC, as well as paclitaxel monotherapy. Mice bearing MC38 tumors were treated with

pemetrexed, paclitaxel, carboplatin or combination of pemetrexed with carboplatin, or paclitaxel

with carboplatin, at doses designed to model clinical exposures for these agents. Tumors were

harvested 14 days after treatment initiation, and immune-related gene expression changes were

evaluated by QGP analysis (Figure 2A). Pemetrexed monotherapy resulted in upregulation of

multiple immune-related genes and induced an immune activation signature indicative of

interferon- pathway activation (increased Cd274, Cxcl10, Cxcl11, Psmb8), cytolytic activity

(increased Gzma, Prf1), interferon type I response (increased Irf7, Oas3), and activated vascular

endothelium (increased Icam1, Vcam1). Paclitaxel monotherapy had a more modest effect on the

expression of the gene sets tested, with the immunomodulatory effect mainly associated with

moderate upregulation of myeloid cell-related genes (increased Il6, Cxcl1, Ccl2, Ccl3, Ccl4,

Timd4). While carboplatin monotherapy had a weak effect, addition of carboplatin to the

pemetrexed regimen appeared to reduce the immunomodulatory effects of pemetrexed and to a

lesser extent paclitaxel. Cisplatin had a similar effect (Figure S2) suggesting that platinum

agents in general can attenuate immunomodulatory effects of pemetrexed.

To investigate the breadth of pathways modulated by pemetrexed -/+ carboplatin, we performed nanoString® analysis of tumor tissues using the nCounter panels spanning key molecular

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed pathways and cellular compartments of innate and adaptive immunity. Consistent with the QGP

data, single agent pemetrexed treatment significantly modulated the expression of a large number

of genes associated with immune response (136 and 133 differentially expressed genes [DEGs]

for Immune Profiling and Myeloid panel, respectively) (Figure 2B). Paclitaxel had a

quantitatively weaker effect (31 and 39 DEGs for Immune Profiling and Myeloid/Innate

Immunity panel, respectively) with a few DEGs shared between pemetrexed and paclitaxel

monotherapy groups (Figure 2B, 2C). The combination of pemetrexed and carboplatin yielded

less prominent gene expression changes compared to pemetrexed monotherapy (87 and 98 DEGs

for Immune Profiling and Myeloid/Innate Immunity panel, respectively) (Figure 2B, 2C).

Paclitaxel monotherapy and paclitaxel/carboplatin combination induced somewhat different

immunomodulatory effects, with a limited number of DEGs overlapped between the two

treatment groups (Figure 2B, 2C).

Ingenuity pathway analysis (IPA) was used to further explore the immune-related molecular

and/or cellular pathways modulated by pemetrexed. These analyses revealed macrophage,

DC/NK Cell, Th1/Th2 enrichment and evidence of enhanced inflammatory response; innate

immune activation and increased interferon/interleukin signaling in MC38 tumors (Table S1).

Evaluation of the individual genes associated with these pathways suggested macrophage

enrichment/reprograming and DC maturation (upregulation of Cd86, Tlr3, Tlr9, Tnf, Il1b,

Il1rl1), increased interferon response 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) (Figure

3). The same pathway enrichment was identified in pemetrexed monotherapy and

pemetrexed/carboplatin groups; however, combination with carboplatin appeared to qualitatively

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed weaken the effect compared to pemetrexed monotherapy (Table S1, Figure 3). As mentioned earlier, paclitaxel-based treatments exerted a less prominent immunomodulatory effect, and the

IPA results showed a similar trend (Table S1, Figure 3). Collectively these data indicate that in

MC38 tumors, treatment with pemetrexed or paclitaxel induced both qualitatively and

quantitatively different immunomodulatory effects, and addition of carboplatin appeared to

attenuate rather than enhance these changes.

Pemetrexed synergizes with PD-1 pathway blockade

The observed immunomodulatory effects of pemetrexed prompted us to evaluate pemetrexed in

combination with PD(L)1 blockade. To this end, we performed in vivo combination studies with

anti-PD-L1 antibody in MC38 and Colon26 tumor models on two distinct genetic backgrounds,

C57BL/6 and BALB/c, with distinct immunologic Th1 and Th2 profiles, respectively (29, 30). In

MC38 model, combining pemetrexed with anti-PD-L1 resulted in a modest but statistically significant tumor growth delay (Figure S3A). However, Colon26 model displayed greater

sensitivity to the combination therapy; the combination of pemetrexed and anti-PD-L1 resulted

in more substantial tumor growth delay accompanied by durable responses in some animals

(Figure 4A). No combination benefit was observed in LLC model (Figure S3B).

QGP analysis revealed transient immune-related changes in Colon26 tumors after treatment with

monotherapies, whereas the combination effect was most pronounced at a later time point (D14

post treatment, D24 post implantation) (Figure S3C). To further characterize the effects of the

pemetrexed/anti-PD-L1combination in Colon26 model, we performed nCounter analysis of

tumor samples collected at D14 post treatment where the differences between groups were most

apparent. Pemetrexed affected the expression of a limited number of genes (n=13), with anti-PD-

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

L1 modulating a broader set of genes (n= 57). Combination treatment altered the expression of a large number of genes (n=198), with the majority (n=152) uniquely modulated by the combination treatment (Figure 4B). While pemetrexed treatment predominantly resulted in the downmodulation of genes in this model (10/13 genes), the combination therapy of pemetrexed and anti-PD-L1 resulted in the upregulation of a substantial number of genes (173/198), including a large set of genes not significantly upregulated by anti-PD-L1 monotherapy.

IPA was used to understand these extensive changes and further explore the immune-related

molecular and/or cellular pathways modulated by the combination therapy. The pathways most

significantly modulated by the combination involved CD4+ T cell-mediated immunity (Th1/Th2

pathway) and a pathway referred to as “Granulocyte/Agranulocyte Adhesion and Diapedesis”

(Table S2). The latter was largely 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 been associated with the activated vascular endothelium, leukocyte trafficking and formation of tertiary lymphoid organs (Figure 4C)(31).

To confirm gene expression changes described above, we subjected Colon26 tumor samples after

pemetrexed and/or anti-PD-L1 treatment to flow cytometry analysis (Figure S4A, Figure 5).

Consistent with the IPA results, we detected increased frequency of CD8+ T cells, CD8+/CD4+

and CD8+/Treg ratios along with enhanced activation of effector T cells (Ki67+, CD4+

Foxp3NEG) (Figure 5A). Because the aforementioned gene expression data suggested that

modulation of the myeloid cell compartment upon treatment with pemetrexed, we also evaluated

the effects of pemetrexed -/+ anti-PD-L1 on myeloid cells. The combination treatment resulted in

a decreased frequency of granulocytic MDSCs (Ly6G+) population and a trend towards greater

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

DC infiltration along with increased activation phenotype of macrophages and Ly6Chigh monocytes which displayed higher expression of MHC class I and II. In addition, the combination treatment also resulted in marked upregulation of MHC class II on tumor cells

(Figure 5B). This data indicate that the combination treatment promotes antigen-presenting properties of myeloid cells, thus supporting the conclusion that pemetrexed induces T cell- permissive changes in the myeloid cell compartment, leading to an activated and “T cell priming-competent” phenotype. Given that the percentage of myeloid cells did not change, the data suggests that the effect of pemetrexed on myeloid cells is rather indirect, and may be mediated by immunogenic effects (e.g. ICD) on tumor cells. Of note, while the M1 and M2 phenotypes are well established in mouse macrophage biology, the emerging translational data generated on human breast and lung tumors suggest that the phenotypes of human tumor- associated macrophages are much more complex and cannot be dichotomized into binary M1/M2 states (32, 33) Given the lack of clinical relevance of M1/M2 macrophage polarization in human tumor biology, the bona fide markers of M1 and M2 macrophages were not pursued in our studies.

To understand if the aforementioned changes in myeloid cells and subsequent T cell priming in lymph nodes are required for the antitumor effects of pemetrexed and anti-PD-L1, we treated

Colon26-bearing mice with pemetrexed and/or anti-PD-L1 as well as the well-characterized

sphingosine-1-phosphate receptor 1 (S1P1R) antagonist (FTY720) to block T cell egress from

lymph nodes (Figure 5C). While FTY720 treatment did not have an obvious impact on either

monotherapy, the effect of the combination therapy was lost after FTY720 treatment. To

understand these observations in the context of an antigen-specific immune response, we

examined the effect of the combination therapy on the frequencies of tumor antigen-specific T

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed cells compared to monotherapies in Colon26 model. After 14-day treatment with pemetrexed and/or anti-PD-L1, we evaluated the activation status and frequency of tumor-specific T cells in the tumor, tumor-draining lymph node and spleen using ELISpot and MHC tetramer assays. The results of these experiments indicate that while a trend towards increased tumor-specific CD8+ T cell responses was observed in the periphery during the combination treatment (as exemplified by IFN-gamma ELISpot and gp70 tetramer assay), no appreciable difference in the frequency of

gp70 tetramer positive CD8+ T cells was detected in the tumor. However, a small but

statistically significant increase in the frequency of TNFα+ CD8+ T cells was observed in

Colon26 tumors after treatment with pemetrexed and anti-PD-L1 suggesting that that the

increased priming during the combination treatment increases the functionality rather than the

quantity of tumor-reactive CD8+ T cells (Figure S4B).

Collectively, these data demonstrate the development of an integrated anti-tumor immune

response mediated by pemetrexed/anti-PD-L1 combination, and suggest that the underlying mechanism involves perpetuation of T cell priming in lymph nodes, presumably through the enhanced antigen presentation function of myeloid cells.

Pemetrexed induces immunogenic tumor cell death

Increased antigen presentation and DC maturation gene signatures suggest that pemetrexed

treatment may lead to ICD of tumors, activating innate pathways leading to enhanced immune

activation. To investigate the ability of pemetrexed to induce ICD, we evaluated the

extracellular levels of CRT and HMGB1, both of which are specifically released from cells

during ICD. Binding of HMGB1 to Toll-like receptor 4 and CRT to CD91/LRP1 leads to DC

migration and maturation and enhanced antigen presentation and T cell priming (34). Colon 26

and MC38 tumor cells were treated with pemetrexed or other chemotherapeutics (carboplatin,

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed paclitaxel, doxorubicin or gemcitabine), followed by measurement of the extracellular HMGB1

and CRT release (Figure 5D). While gemcitabine-treated cells did not display any evidence of

ICD in either cell line under these conditions, each of the other agents appeared to induce some

degree of ICD, as exemplified by increased CRT and HMGB1 release. Pemetrexed was the most

potent inducer of ICD in both Colon26 and MC38 cells, particularly across lower concentrations

that reflect clinical exposure (0.02-0.05 M). These results suggest that the immunomodulatory

effects of pemetrexed are mediated, at least in part, by tumor cell-intrinsic mechanisms involving

ICD.

Pemetrexed exerts direct immunomodulatory effects on activated T cells in vitro

Purine nucleotide synthesis in general and the folate pathway in particular depend on metabolic

intermediates (e.g 3-phosphoglycerophosphate) supplied through glycolysis, and one-carbon

metabolism plays a critical role during T cell activation since T cells require high levels of

glycolysis and mitochondrial respiration during the activation and effector phase (17, 35). To evaluate the impact of pemetrexed on T cell glycolysis and mitochondrial respiration, we employed the Seahorse mitochondria stress test, using primary T cells activated with anti-

CD3/CD28 antibodies and IL-2 in the presence of pemetrexed over a broad pharmacologic range spanning clinical exposure (0.004-0.1 M), and determined extracellular acidification rates

(reflective of glycolysis), as well as basal and maximal OCR and SRC (reflective of

mitochondrial respiration) (Figure 6A). Pemetrexed increased both basal and maximal OCR in

an inverse concentration-dependent manner, with the maximum effect at 45 hours of stimulus

with the lowest concentration tested (0.004 M) (36). SRC (representing the difference between

basal and maximal OCR values) was markedly increased by pemetrexed in a concentration-

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed dependent manner, and this effect was particularly evident at 45 and 70 hours (Figure 6A).

Pemetrexed also enhanced OCR when activated T cells were supplemented with fatty acid

(palmitate), an effect that was abrogated by etoxomir, an inhibitor of carnitine

palmitoyltransferase-1 which blocks fatty acid oxidation (Figure 6B). Because beta-oxidation of

fatty acids is directly linked with the tricarboxylic acid (TCA) cycle, these results suggest that

pemetrexed may increase metabolic fitness of T cells through the enhancement of mitochondrial

function or biogenesis that increases the bioenergetic reserve of T cells that might be critical for

their survival.

Since chemotherapeutic agents including antifolates are known to have inhibitory effects on T

cell activation and survival (16), we next evaluated the direct impact of pemetrexed compared to

paclitaxel on T cell function and activation. Primary human T cells were activated with CD3 and

CD28 antibodies and exposed to fixed, clinically relevant concentrations of pemetrexed (0.05

M) or paclitaxel (0.2 M) for various time intervals (days (D) 0-3, 3-9, 0-9) to mimic treatment

during different stages of T cell activation. In vitro activated T cells showed modest but

significant attenuation of total proliferation in the presence of pemetrexed, yet this effect was

reversible, and most pronounced when pemetrexed was present in the culture medium for the duration of the study, resulting in approximately half the number of total cells compared to untreated cells (Figure 6C). In contrast, the cytotoxic effect of paclitaxel was detrimental to T cell proliferation and viability, and paclitaxel-treated T cells did not survive beyond D6 regardless of the duration and timing of exposure (Figure 6C). Flow cytometry analysis during T

cell expansion revealed that exposure to pemetrexed enhanced the activation state of T cells, as

reflected by significantly increased and sustained- surface expression of CD137 and GITR on

CD8+ and CD4+ T cells with continuous pemetrexed exposure (Figure 6D). The enhanced T

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed cell activation state was also accompanied by significantly increased mitochondrial content in

CD8+ and CD4+ T cells (Figure 6E). Finally, QGP gene expression analysis revealed

pemetrexed -dependent upregulation of IFN -dependent transcripts (IFNG, CXCL9, CXCL10,

CXCL11, IDO1, HLA-DRA), cytolytic genes (GZMB, PRF1) as well as transcripts encoding co- stimulatory receptors CD137, GITR, OX40 (TNFRSF9, TNFRSF18, TNFRSF4) (Figure 6F), and these results were further confirmed using nCounter analysis (Figure S5).

To assess if the increased metabolic fitness and activation state of T cells translate to enhanced

effector function, we measured antigen-dependent tumor cell killing using ovalbumin (OVA)-

specific OT-1 T cells. OT-1 T cells were primed with OVA peptide (SIINFEKL) in the presence

or absence of pemetrexed, and their ability to kill tumor targets loaded with OVA peptide was

evaluated in vitro. These results demonstrate that priming in the presence of pemetrexed resulted

in ~ 50% increase in tumor cell killing (~50% and ~30% dead tumor cells with pemetrexed and

control, respectively). This data therefore suggests that the enhanced activation state induced by

pemetrexed translates into increased effector T cell function exemplified by increased

cytotoxicity of antigen-specific T cells (Figure 6G).

Taken together these data reveal that pemetrexed exerts pleiotropic immunomodulatory effects by inducing immunogenic cell death in tumor cells, enhancing the metabolic state of T cells by increasing their oxidative respiration and the mitochondrial content, leading to increased activation and effector function.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

DISCUSSION

Chemotherapeutic agents are part of standard of care treatment in many tumor types and across lines of therapy. A fundamental premise for combining chemo- and immunotherapies is that the chemotherapeutic agents preferentially target tumor cells and do not incapacitate relevant immune functions. The potential for enhanced combinatorial activity of chemotherapy and

immunotherapy is based on the principle that in some cases chemotherapeutic agents may cause

immunogenic tumor cell death, resulting in immune enhancing activities through immune cells,

and/or the tumor microenvironment without incapacitating relevant immune cell function(4, 5,

11).

It is generally believed that folate pathway inhibitors such as methotrexate are

immunosuppressive (12, 13). While the cytotoxic activity of pemetrexed has been attributed to

inhibition of four enzymes in the folate cycle (24), very little is known about how pemetrexed

modulates anti-tumor immunity. Our data indicate that pemetrexed exerts immunomodulatory

effects across multiple pathways and immune cell subsets. These effects were not observed with

other chemotherapeutic agents such as carboplatin or paclitaxel. Furthermore, our data indicate

that carboplatin and cisplatin attenuate, rather than enhance the immunomodulatory effects of

pemetrexed, and suggest that pemetrexed can potentially be combined with ICIs without

platinum agents. This finding might be critical, and additional mechanistic, translational and

clinical studies are needed to further understand the effects of various platinum doublets on

tumor immune microenvironment as well as their combinatorial potential with ICIs.

One hypothesis supporting platinum-based chemotherapy in combination with pemetrexed and

immune checkpoint blockade is that platinum agents could induce somatic mutations in the

tumor cells and, consequently, induce new immunogenic neoantigens. It is therefore possible that

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed adding a platinum agent before pemetrexed/anti-PD(L)1 treatment could potentially enhance the immunomodulatory and antitumor effects through increased priming against these de novo induced neoantigens. The accumulation of somatic mutations in tumor cell DNA requires some time, and syngeneic mouse tumor models have very limited time window that makes them poorly fit for studying effects of cytotoxic agents on tumor mutational burden. It would be worthwhile to test this hypothesis in the clinical setting given that patients with advanced/metastatic NSCLC typically receive front-line chemotherapy every 3 weeks up to 6 cycles.

Gene expression profiling indicated that treatment with pemetrexed -/+ 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 interferon/interleukin signaling that play an important role in priming and establishing an efficient T cell immunity. Additionally, the benefit from the combination treatment was

abrogated when T cell egress from the lymph nodes was blocked by the S1P1R antagonist. These

results strongly support the hypothesis that pemetrexed induces an integrated anti-tumor immune

response by enhancing antigen presentation and T cell priming in tumor-draining lymph nodes.

The gene expression analyses also revealed that pemetrexed monotherapy was accompanied by

activation of vascular endothelium and genes associated with tertiary lymphoid structure

formation; this effect was even more evident when pemetrexed was combined with anti-PD-L1 therapy. It is plausible that the T cell inflamed phenotype observed in pemetrexed-treated tumors might be attributable, at least in part, to enhancement of these pathways, which have the potential to promote T cell trafficking and infiltration.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

The high-content analyses suggested and in vitro data revealed that the immunomodulatory effects of pemetrexed also included direct effects on tumor cells via induction of ICD

exemplified by the extracellular release of HMGB1 and CRT, in a manner superior to other ICD-

inducing chemotherapies. Our data suggest that the immunogenic effects of pemetrexed on

tumor cells exemplified by CRT and HMGB1 release may require lower drug concentrations

compared to the cytotoxic effects. These results may explain the more robust gene expression

changes observed in MC38 tumors after treatment with lower doses (50 mg/kg) of pemetrexed,

and suggest that part of the mechanism of action for pemetrexed might involve increased tumor

immunogenicity followed by the priming and establishment of an anti-tumor T cell response.

It is worth noting that while carboplatin also demonstrated evidence of ICD in mouse tumor cell

lines in vitro, these results did not translate in vivo as exemplified by the gene expression data in

MC38 tumors. A potential explanation of this discrepancy could be due to different effects of

carboplatin on tumor vs immune cells. It is also important to highlight that in MC38 tumors,

treatment with all chemotherapeutic agents tested was accompanied by PD-L1 (encoded by

Cd274) upregulation highlighting the need for PD1 pathway blockade to overcome adaptive

resistance in T cell compartment induced by chemotherapy.

To our knowledge, a positive effect of anti-folates in general and pemetrexed in particular on T

cell biology has not been described previously. While the molecular mechanisms for the T cell-

intrinsic effects have not been fully elucidated, since the folate pathway (which regulates purine

nucleotide synthesis and the TCA cycle) depends on 3-phosphoglycerophosphate (3-PG)

generated through glycolysis(37), it is possible that inhibition of the folate cycle may increase

the abundance of 3-PG and downstream metabolic intermediates required for optimal T cell

activation(37, 38). The enhanced metabolic fitness of T cells exposed to pemetrexed is notable

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed as an immune-enhancing mechanism since activated T cells require adequate mitochondrial mass to support bioenergetic needs required for cytokine production and development of cytotoxic effector function. Indeed, multiple lines of evidence link T cell metabolic state with activation, survival, and intra-tumoral exhaustion (35), and the association between metabolic fitness, activation phenotype and effector function of tumor-reactive T cells has also been demonstrated in the present study. The T cell-intrinsic effects described here can also potentially be

attributable to the unique ability of pemetrexed, relative to other anti-folates, to also inhibit 5-

aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), since blockade of

this enzyme results in the elevated intracellular levels of ZMP, a metabolite structurally related

to AMP that is capable of promoting mitochondrial biogenesis and respiration function via AMP

kinase-mediated mechanisms (39-41). Emerging data indicate that epigenetic and/or metabolic mechanisms rather than immunosuppressive tumor microenvironment play a dominant role in driving intratumoral T cell dysfunction (42-45). The results of the current study, particularly with

regard to the ability of pemetrexed to enhance metabolic fitness and effector function of T cells

have an important biological and translational significance given limited therapeutic options to

revert metabolically exhausted T cells in the tumor.

Collectively, the data from these studies suggest that pemetrexed therapy has the potential to

induce an integrated anti-tumor immune response in tumors. These observations provide

mechanistic rationale for the clinically observed combination activity between pemetrexed and

anti-PD-1 therapy, identify pathways and mechanisms to be explored in translational studies and

highlight the potential for pemetrexed as an important therapeutic modality to be investigated

further in the context of combination immunotherapies. Finally, these studies provide context

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed and direction for the exploration of the immunotherapeutic potential for other tumor-targeting agents currently being used or contemplated for use in the clinic.

ACKNOWLEDGEMENTS

We thank Gregory D. Plowman, Levi Garraway, Ana Oton and Jong Seok Kim (Eli Lilly) for review and helpful discussions during preparation of the manuscript.

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

AUTHOR CONTRIBUTIONS

Experimental design and oversight, data analysis, data interpretation: DAS, SG, NA, ERR, CC,

JRM, TND, YL, GPD, GEH, DS, RDN, MK

Experimental conduct and data collection: NA, ZHL, AS, DC, AC, CMM, BDJ, XH, SL, KDR,

AN, BT, MB, KC, FCD, JRM, TND, YL

Manuscript writing: DAS, SG, RDN, MK

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

FIGURE LEGENDS

Figure 1. Pemetrexed demonstrates anti-tumor efficacy and increases frequency of tumor-

infiltrating lymphocytes in MC38 syngeneic murine tumors

(A) Mean tumor volumes (-/+ SEM) in C57BL/6 mice implanted with MC38 cells and treated with 50 or 100 mg/kg of pemetrexed dosed IP starting 3 days after tumor implantation (5 days on, 2 days off) for the duration of the experiment. Difference in the tumor volume of treated

groups compared to control (%T/C) at Day 16 was only significantly different (P<0.001) at

100mg/kg (48.4% T/C) and not at 50mg/kg (70.9% T/C, p=0.17).(RM-ANOVA). Mean intra-

tumor leukocyte (B), T cell (C-E) and myeloid cell (F) frequencies (-/+SEM) at Day 17 post

tumor implantation. Total leukocytes identified as Live CD45+ cells, total T cells: Live CD45+,

CD3+ with Ki67 percentage taken from CD3+ gate (one-way ANOVA) (G) QuantiGene (QGP)

gene expression analysis of MC38 tumors (Day 17 post tumor implantation) after treatment with

50 or 100 mg/kg of pemetrexed; volcano plots show differentially expressed genes (DEGs); p

values (compared to untreated control) were only listed if the differences between the groups

reached statistical significance (p<0.05 by two-way ANOVA). Representative example of 3 experiments.

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 with

pemetrexed dosed IP (50 mg/kg, 5 days on, 2 days off), paclitaxel (10 mg/kg, dosed IV once a week)), carboplatin (60 mg/kg dosed IP once every two weeks), or pemetrexed/carboplatin and

paclitaxel/carboplatin combinations; volcano plots show differentially expressed genes (DEGs)

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed with p<0.05 (two-way ANOVA) compared to untreated control. (B) MC38 tumors treated with pemetrexed and/or carboplatin were further subjected to nCounter analysis using PanCancer

Immune Profiling and Myeloid panels; summary of DEGs with a number of up- and downregulated genes across treatment groups is shown. (C) Venn diagrams visualizing shared and non-overlapping DEGs across experimental groups.

Figure 3. Macrophage, Dendritic Cell/NK Cell, T Helper Cell and Interferon Signaling are

among the top ranking Ingenuity pathways enriched in pemetrexed monotherapy and

pemetrexed/carboplatin combination groups in MC38 tumors.

Volcano plots visualizing DEGs attributable to the top ranking pathways (Macrophage, Dendritic

Cell/NK Cell, T Helper Cell, Interferon Signaling) identified by Ingenuity Pathway Analysis

(IPA) in the pemetrexed-based treatment groups.

Figure 4. Combination of pemetrexed and anti-PD-L1 improves antitumor efficacy and

markedly enhances T cell inflamed phenotype in Colon26 syngeneic mouse tumor model

(A) Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with

pemetrexed (50 mg/kg5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse, weekly IP). Group, individual tumor growth curves and overall survival Kaplan-Meyer curves for single agent and combination treatment groups are shown overlaid on top of control group.

Difference in the tumor volume of treated groups compared to control (%T/C) at Day 31 (point

where at least 50% of controls we present) was significantly different (P<0.001) for the

combination (12.4% T/C) compared to anti-PD-L1 (60.1%) or pemetrexed monotherapy (50.8%)

(RM-ANOVA), and was shown to be better than additive by Bliss Independence analysis.

Overall survival was significantly increased with the combination as indicated, with a trend

(P=0.07) compared to pemetrexed monotherapy(B) Colon26 tumors treated with pemetrexed

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed and/or PD-L1 were further subjected to nCounter PanCancer Immune Profiling; summary of shared and non-overlapping DEGs with a number of up- and downregulated genes across

treatment groups is shown. (C) Volcano plots visualizing DEGs attributable to the top ranking

pathways (“Granulocyte/Agranulocyte Diapedesis”, “T Helper/T Cell Signaling”, “Dendritic

Cell Maturation/NK Cell Signaling/DC/NK Crosstalk” and “Cytokine/Chemokine Signaling”)

identified by Ingenuity Pathway Analysis (IPA) in the combination group. Representative

example of 3 experiments in Colon26.

Figure 5. Pemetrexed exerts strong immunogenic effect on immune and tumor cells

(A, B) Mice bearing Colon26 tumors were treated starting 10 days after tumor implantation with

pemetrexed (50 mg/kg5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse,

weekly IP)) and tumors were isolated after 14 days of treatment and single cell suspensions

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 from control

of p<0.05 are shown (one-way ANOVA). (C) Mice bearing Colon26 tumors were treated with pemetrexed (50 mg/kg 5 days on, 2 days off, IP) and/or anti-PD-L1 (αPD-L1) (500 ug/mouse,

weekly IP) as well as FTY720, a well-characterized S1P1R antagonist (q2D until D32), three

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

combination of αPD-L1 and pemetrexed was shown to be antagonistic (Categorical Response

Analysis with Bayesian Ordinal Logistic Regression, see methods). (D) MC38 and Colon26

mouse tumor cells were incubated in the presence of various chemotherapeutic agents

(pemetrexed, paclitaxel, carboplatin, gemcitabine, doxorubicin) for 72 hours. Untreated as well

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed as DMSO- and staurosporine-treated cultures were used as controls. ICD was evaluated by measuring extracellular levels of calreticulin (CRT) and high mobility group box1 (HMGB1) protein. Dose ranges indicated cover the IC50 of each compound. In vivo studies representative

example of 2 experiments in Colon26.

Figure 6. 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 and mouse IL-2 in the presence of various concentrations of pemetrexed (0.004-0.1

M), and basal oxygen consumption rates (OCR) were analyzed using Seahorse XFe96

instrument. Cells were stimulated with oligomycin (1 M), FCCP (1.5 M), and rotenone/antimycin A (0.5 M each). Spare respiratory capacity was measured as the difference

between basal OCR values and maximal OCR values obtained after FCCP uncoupling. (B)

Maximal respiration of mouse primary T cells treated with pemetrexed was evaluated in the presence or absence of fatty acid (palmitate, PALM) or inhibitor of fatty acid oxidation

(etoxomir, ETO). (C) Proliferation of primary human T cells stimulated with CD3/CD28 beads in vitro was evaluated in the presence or absence of pemetrexed (0.05 M) or paclitaxel (0.2

M) for the indicated period of time. Total T cell numbers are indicated for various treatment conditions. Note that proliferation could not be assessed in paclitaxel-treated T cells beyond D6 due to a prominent cytotoxic effect. (D, E) Flow cytometry was performed on CD4+ and CD8+

T cell populations throughout the experiment (Day 3, 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 as mean -/+ SD (A,B), SEM (C-E),. Lines show significant differences

between indicated groups with control where p=<0.05 (A,B – one way ANOVA). Asterisk

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed indicates group and time points where differences between treated group and control where significant with p=<0.05 (C-E – one way ANOVA). (F) QGP assay was used to quantify

expression of immune-related genes in T cells treated with pemetrexed in vitro. Volcano plot

shows the p-value (one-way ANOVA) vs. Log2 fold change of pemetrexed compared to

untreated cells. DEGs with p<0.05 are colored pink; DEGs showing greater than twofold change

compared to control are highlighted. Data shown is averages of three donors. (G) Splenocytes

from ovalbumin specific TCR transgenic OT-1 T cells were incubated for 5 days with 0.1nM of

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 for 4hrs. Tumor cell death was

then analyzed by 7AAD incorporation by flow cytometry. Graph shows average tumor cell

killing, and example FACS plots gated on CD45Neg cells show percentage of dead tumor cells

(7AAD+).

Schaer & Geeganage 2019 Immunomodulatory effects of pemetrexed

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GAuthor Manuscript Published OnlineFirst on AugustPemetrexed 13, 2019; DOI: 10.1158/1078-0432.CCR-19-0433 50 mg/kg Pemetrexed 100 mg/kg Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. p value p value

(-) (+) (-) (+)

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A Pemetrexed Paclitaxel Carboplatin Pemetrexed + Carboplatin Paclitaxel + Carboplatin p value p value

Erik Rasmussen Nelusha Amaladas (-) (+) Ruslan Novosiadly

Log2 Fold Change (Treatment vs Vehicle) B Differentially Expressed Genes vs Untreated Control (p<0.05) Pemetrexed + Paclitaxel + DEG Pemetrexed vs. Paclitaxel vs. nCounter Panel Carboplatin vs. Carboplatin vs. Criteria Control Control Control Control

Immune Panel p<0.05 136 (129↑, 7↓) 87 (83↑, 4↓) 31 (27↑, 4↓) 20 (15↑, 5↓) (n=458 genes FC +/- 1.5 and pass QC) p<0.05 93 (90↑, 3↓) 46 (45↑, 1↓) 14 (11↑, 3↓) 11 (7↑, 4↓)

Myeloid Panel p<0.05 133 (122↑, 11↓) 98 (93↑, 5↓) 39 (38↑, 1↓) 48 (44↑, 4↓)

(n=522Author Manuscript genes Published OnlineFirst on August 13, 2019; DOI: 10.1158/1078-0432.CCR-19-0433 Author manuscripts have been peer reviewedFC +/-and accepted1.5 and for publication but have not yet been edited. pass QC) p<0.05 83 (80↑, 3↓) 56 (55↑, 1↓) 27 (26↑, 1↓) 30 (29↑, 1↓)

C Immune Panel (p<0.05) Myeloid Panel (p<0.05)

Pemetrexed 108 28 3 Paclitaxel Pemetrexed 103 30 9 Paclitaxel 136 (129↑, 7↓) 31 (27↑, 4↓) 133 (122↑, 11↓) 39 (38↑, 1↓)

Pemetrexed + Pemetrexed + Pemetrexed Pemetrexed 63 73 14 Carboplatin 73 60 38 Carboplatin 136 (129↑, 7↓) 133 (122↑, 11↓) 87 (83↑, 4↓) 98 (93↑, 5↓)

Paclitaxel + Paclitaxel + Paclitaxel Paclitaxel 23 8 12 Carboplatin 25 14 34 Carboplatin 31 (27↑, 4↓) 39 (38↑, 1↓) 20 (15↑, 5↓) 48 (44↑, 4↓)

Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Macrophage Enrichment Figure 3 43 (41↑, 2↓) DEGs 25 ↑ DEGs 4 ↑ DEGs 6 (4↑, 2↓) DEGs Pemetrexed Pemetrexed + Carboplatin Paclitaxel Paclitaxel + Carboplatin p-value p-value

Log2 Fold Change (Treatment vs Vehicle) Dendritic Cell/Natural Killer Cell Enrichment 32 (31↑, 1↓) DEGs 14 ↑ DEGs 2↑ DEGs 1 ↓ DEGs Pemetrexed Pemetrexed + Carboplatin Paclitaxel Paclitaxel + Carboplatin p-value p-value

Log2 Fold Change (Treatment vs Vehicle) T Helper Cell Enrichment 32 (30↑, 2↓) DEGs 15 ↑ DEGs 4 (3↑, 1↓) DEGs 4 (2↑, 2↓) DEGs Author Manuscript Published OnlineFirst on August 13, 2019; DOI: 10.1158/1078-0432.CCR-19-0433 Author manuscripts have Pemetrexedbeen peer reviewed and accepted for publication but have not yetPemetrexed been edited. + Carboplatin Paclitaxel Paclitaxel + Carboplatin p-value p-value

Log2 Fold Change (Treatment vs Vehicle)

Interferon Signaling Enrichment 19 ↑ DEGs 7 ↑ DEGs 5 ↑ DEGs 1 ↓ DEG Pemetrexed Pemetrexed + Carboplatin Paclitaxel Paclitaxel + Carboplatin p-value p-value Interferon Signaling Enrichment with Immune Panel Comparison of leading edge genes from GSEA and Ingenuity differentially expressed genes (DEGs) Log2 Fold Change (Treatment vs Vehicle)

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2 5 0 2 5 0 2 5 0 2 5 0 * 2 / 1 0 C R s 0 0 0 0 0 0 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 5 5 6 0 6 5 P e m e t r e x e d : D a y P o s t T u m o r I m p l a n t a t i o n DaysD a posty P o s tumort T u m o challenger I m p l a n t a t i o n D a y P o s t T u m o r I m p l a n t a t i o n a n t i - P D - L 1 : D a y P o s t T u m o r I m p l a n t a t i o n D a y P o s t T u m o r I m p l a n t a t i o n B C Pemetrexed PD-L1 Pemetrexed + αPD-L1 Pemetrexed PD-L1 Granulocyte/Agranulocyte Diapedesis n=13 (3 , 10 ) n=57 (46 , 11 ) 5 0 15 4 4 38 Author Manuscript Published OnlineFirst on August 13, 2019; DOI: 10.1158/1078-0432.CCR-19-0433 Author manuscripts152 have been peer reviewed and accepted for publication but have not yet been edited. Combination T Helper Cell Signaling (Pemetrexed + αPD-L1) n=198 (173 , 25 )

Dendritic Cell Maturation NK Cell Signaling p value p value DC/NK Crosstalk

Cytokine/Chemokine Signaling

Log2 Fold Change (Treatment vs Vehicle)

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N o P e p t i d e + O V A P e p t i d e

The folate pathway inhibitor pemetrexed pleiotropically enhances effects of cancer immunotherapy

David A Schaer, Sandaruwan Geeganage, Nelusha Amaladas, et al.

Clin Cancer Res Published OnlineFirst August 13, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-0433

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/08/13/1078-0432.CCR-19-0433.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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