Combines Effectively with Anti−PD-L1 Treatment and Can Augment Antitumor Responses

This information is current as Rafael Cubas, Marina Moskalenko, Jeanne Cheung, of September 28, 2021. Michelle Yang, Erin McNamara, Huizhong Xiong, Sabine Hoves, Carola H. Ries, Jeong Kim and Stephen Gould J Immunol published online 12 September 2018 http://www.jimmunol.org/content/early/2018/09/11/jimmun

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 12, 2018, doi:10.4049/jimmunol.1800275 The Journal of Immunology

Chemotherapy Combines Effectively with Anti–PD-L1 Treatment and Can Augment Antitumor Responses

Rafael Cubas,* Marina Moskalenko,* Jeanne Cheung,* Michelle Yang,* Erin McNamara,* Huizhong Xiong,* Sabine Hoves,† Carola H. Ries,† Jeong Kim,* and Stephen Gould*

Immunotherapy with checkpoint inhibitors has proved to be highly effective, with durable responses in a subset of patients. Given their encouraging clinical activity, checkpoint inhibitors are increasingly being tested in clinical trials in combination with che- motherapy. In many instances, there is little understanding of how chemotherapy might influence the quality of the immune re- sponse generated by checkpoint inhibitors. In this study, we evaluated the impact of chemotherapy alone or in combination with anti–PD-L1 in a responsive syngeneic tumor model. Although multiple classes of chemotherapy treatment reduced immune cell numbers and activity in peripheral tissues, chemotherapy did not antagonize but in many cases augmented the antitumor activity

mediated by anti–PD-L1. This dichotomy between the detrimental effects in peripheral tissues and enhanced antitumor activity Downloaded from was largely explained by the reduced dependence on incoming cells for antitumor efficacy in already established tumors. The effects of the various were also agent specific, and synergy with anti–PD-L1 was achieved by different mechanisms that ultimately helped establish a new threshold for response. These results rationalize the combination of chemotherapy with immunotherapy and suggest that, despite the negative systemic effects of chemotherapy, effective combinations can be obtained through distinct mechanisms acting within the tumor. The Journal of Immunology, 2018, 201: 000–000. http://www.jimmunol.org/

mmunotherapy with checkpoint inhibitors has proved to be for the establishment of antitumor immunity by reducing the highly effective in certain indications with durable clinical function and number of effector T cells (13–15). Additionally, I responses for a subset of individuals. However, the majority of chemotherapies may impact different stages of the immune re- patients still show reduced or no clinical benefit (1–5). Clinical sponse, including differential effects on T cell priming in draining responses to checkpoint inhibitors like anti-PD1/PD-L1 are gen- lymph nodes (dLNs) or T cell effector functions in tumor. There- erally observed in cancers with increased tumor mutational bur- fore, combining chemotherapy with checkpoint inhibitors could den, pre-existing immunity, and higher expression of PD-L1, have various effects, depending on the balance between the bene- although this is not always the case (6–8). In an effort to broaden ficial and antagonistic effects of chemotherapy on components of by guest on September 28, 2021 the number of responding individuals, overcome resistance to the immune system. The summation of these factors will eventually single-agent therapy, and extend the duration of response, a determine whether a particular combination therapy will show en- combination of chemotherapy with checkpoint inhibitors is cur- hanced combinatorial activity or reduced responses. Therefore, a rently being tested in multiple clinical trials. Combining check- better understanding of the potential interactions between specific point blockade with standard of care chemotherapy could also chemotherapies and immune cell subsets and how these interplay bring the benefits of immunotherapy into earlier lines of treatment with checkpoint inhibitors could yield valuable insights into future in which the cancer-immune set point might have a lower combinatorial modalities. threshold for clinical response (9). Given that checkpoint inhibitors like anti–PD-L1 are being tested Some chemotherapies can augment antitumor responses through in earlier lines of treatment concurrent with chemotherapy, we the induction of immunogenic cell death (10–12) or through the wanted to explore the effects of such combinatorial approaches on depletion of immunosuppressive cell subsets such as myeloid- the antitumor activity and immune modulation mediated by anti– derived suppressive cells or T regulatory cells (Tregs). Alterna- PD-L1 treatment in a responsive syngeneic tumor model. This ap- tively, other chemotherapies are hypothesized to be detrimental proach enabled us to assess the pharmacodynamics of different chemotherapies on various immune cell components in different *Genentech, South San Francisco, CA 94080; and †Roche Pharmaceutical Re- tissues and how these influenced anti–PD-L1 responses. We found search and Early Development, Roche Innovation Center Munich, 82377 Penzberg, that most chemotherapeutic agents reduced T cell numbers and Germany activity in peripheral tissues such as blood and dLNs but did not ORCIDs: 0000-0002-3450-9054 (R.C.); 0000-0001-7969-2449 (J.C.); 0000-0002- antagonize responses in the tumor tissue, which in many cases was 4953-7234 (S.H.); 0000-0002-1855-8745 (C.H.R.); 0000-0002-4255-383X (S.G.). augmented in combination with anti–PD-L1. This disconnect be- Received for publication February 26, 2018. Accepted for publication August 13, 2018. tween the effects in peripheral tissues and antitumor activity was partly due to responses in established tumors relying mostly on Address correspondence and reprint requests to Dr. Rafael Cubas, Genentech, 1 DNA Way, MS-50, South San Francisco, CA 94080. E-mail address: [email protected] infiltrating T cells at the time of treatment initiation. Additionally, The online version of this article contains supplemental material. synergy with anti–PD-L1 was observed through different mecha- Abbreviations used in this article: ARG1, arginase 1; CIT, checkpoint inhibitor; CR, nisms that ultimately led to establishing a new threshold for anti- complete response; CTX, ; dLN, draining lymph node; GZMB, tumor activity. Overall, our results suggest that chemotherapy can granzyme B; MHC-II, MHC class II; TAM, tumor-associated macrophage; TIL, effectively combine with checkpoint inhibitors like anti–PD-L1 tumor-infiltrating lymphocyte; Treg, T regulatory cell. with various effects that may be chemotherapy specific and de- Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 pendent on individual tumor microenvironmental components.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800275 2 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT

Materials and Methods A total of 0.1 million (100 ml) MC38 cells were then implanted s.c. in the ∼ Animal study oversight right unilateral thoracic area. Tumors were monitored for 14 d until they measured between 130 and 250 mm3, at which time mice were then ran- All animal studies were reviewed and approved by Genentech’s Institu- domized into treatment groups based upon their tumor’s volume. The next tional Animal Care and Use Committee. Mice whose tumors exceeded day, treatment was initiated with either isotype control Ab (gp120 mIgG1) acceptable size limits (2000 mm3) or became ulcerated were euthanized or anti–PD-L1 mIgG1 (clone 6E11) at an initial loading dose of 10 mg/kg and removed from the study. All individuals participating in animal care i.v., followed by 5 mg/kg i.p. thereafter twice a week for 10 d (pharma- and use are required to undergo training by the institution’s veterinary codynamic studies) or 3 wk (efficacy studies). Chemotherapies were also staff. Any procedure, including handling, dosing, and sample collection, administered the day after group assignment at the dose, routes, and mandates training and validation of proficiency under the direction of the schedules depicted in Table I. Tumor volumes and body weights were veterinary staff prior to performing procedures in experimental in vivo measured twice per week up to the end of the study. Tumor volumes were studies. All animals were dosed and monitored according to guidelines measured in two dimensions (length and width) using Ultra-Cal IV calipers from the Institutional Animal Care and Use Committee on study protocols (Fred V. Fowler, Newton, MA), and volume was calculated using the approved by Genentech’s Laboratory Animal Resource Committee. following formula: tumor size (mm3) = (length 3 width2) 3 0.5. Mice body weights were measured using an Adventura Pro AV812 scale (Ohaus, Mice Pine Brook, NJ). Abs were diluted in histidine buffer [20 mM histidine acetate, 240 mM sucrose, and 0.02% polysorbate 20 (pH 5.5)]. For Eight- to ten-week-old female C57BL/6 mice were obtained from Charles FTY720 studies, stock solution was made by dissolving FTY720 (Cayman River Laboratories (Hollister, CA). Only animals that appeared to be Chemical, Ann Arbor, MI) in ethanol at 50 mg/ml and stored at 280˚C. healthy and that were free of obvious abnormalities were used for the Immediately before administration, the stock solution was diluted in PBS studies. For pharmacodynamic studies (tissue collection followed by flow and orally administered by gavage at 1 mg/kg every day for 21 d. cytometry analysis), IFN-g–YFP reporter mice (IFNG.IRES.eYFP.ki.B6N) For CD8 depletion studies, once tumors reached an average size of 3 were used. IFN-g–YFP reporter mice are of BL/6J background and have an ∼190 mm , mice were randomized into treatment groups, and CD8 de- Downloaded from IRES–EYFP cassette knocked in to the endogenous IFN-g locus 39 to the pletion was started by dosing an anti-CD8–depleting Ab (clone ATCC- stop codon. IFN-g promoter activity thus results in expression of a bicis- 2.43) or control rat IgG2b at 10 mg/kg i.p. on days 0, 3, 8, and 15. Isotype tronic mRNA encoding both the endogenous IFN-g gene and EYFP. For (gp120 mIgG1) or anti–PD-L1 mIgG1 (clone 6E11) treatment was initiated 2/2 tm1Fwa the RAG study, female B6.129S6-Rag2 N12 (RAGN12-F) mice on day 1, and the Abs were dosed at 10 mg/kg i.v., followed by 5 mg/kg i.p. from Taconic Biosciences (Albany, NY) were used. twice a week for 3 wk. For studies with anti–CSF-1R, female C57BL/6N mice (6 wk of age; Abs Charles River Laboratories, Sulzfeld, Germany) were inoculated s.c. into the right flank with MC38 (106 cells). Tumor growth was monitored by http://www.jimmunol.org/ For animal studies, murine IgG1 anti–PD-L1 clone 6E11 and murine IgG1 perpendicular caliper measurement, and tumor volume was calculated by anti-gp120 isotype control Abs were used. Abs were stored in 20 mM using the following formula: V = (length 3 width2) / 2. Group allocation histidine acetate, 240 mM sucrose, and 0.02% polysorbate 20 (pH 5.5) and and treatment started on day 7, and the average tumor size was 120 mm3. diluted in PBS prior to use. Animals received 30 mg/kg of either mIgG1 (clone MOPC-21; Bio X Cell) For flow cytometry analysis, the following fluorochrome-conjugated Abs or anti-CSF-1R [clone 2G2 (16)] i.p.; treatment with these Abs was con- were used: anti-mouse CD45 (clone 30-F11, 1:100), anti-mouse I-A/E (clone tinued weekly for 4 wk maximum. On day 9, anti-PD-L1 (6E11; Gen- M5/114.15.2, 1:5000), anti-mouse CD11b (clone M1/70, 1:100), anti-mouse entech) was given, starting with a loading dose of 10 mg/kg i.v. and CD11c (clone N418, 1:50), anti-mouse Thy1.2 (clone 30-H12, 1:200), anti- continued with 5 mg/kg i.p. every 3 to 4 d (total of seven administrations) mouse CD19 (clone 6D5, 1:500), anti-mouse Ly-6C (clone HK1.4, 1:800), in the presence of mIgG1 or anti-CSF-1R Ab. Monotherapies of mIgG1 and anti-mouse ICOS (clone C398.4A, 1:100) were purchased from BioLegend.

and anti-CSF-1R groups received additional matching volumes of by guest on September 28, 2021 Anti-mouse CD4 (clone RM4-5, 1:100), anti-mouse CD8 (clone 53-6.7, 1:100), saline on the days of PD-L1 Ab administration. Mice were graphically anti-mouse CXCR3 (clone CXCR3-173, 1:200), anti-mouse SiglecF (clone censored when tumor volume reached $700 mm3 (Kaplan–Meier E50-2440, 1:50), anti-mouse Ly-6G (clone 1A8, 1:200), and anti-mouse PD-L1 plots), n = 10 mice per group. All procedures were performed upon (clone 10F.9G2, 1:200) were all from BD Biosciences (San Jose, CA). Anti- approval by the Regierung Oberbayern, Weilheim (approval number: mouse Ki-67 (clone SolA15, 1:100), anti-mouse CD62L (clone Mel14, 55.2-1-54-2531.2-32-10). 1:80), anti-mouse CD206 (clone MR6F3 1:200), anti-mouse F4/80 (clone BM8, 1:50), and anti-mouse CD44 (clones IM7, 1:200) were from eBioscience. Anti- Flow cytometry human granzyme B (GZMB) (cross-reacts with mouse) (clone MHGB05, 1:100) was purchased from Life Technologies. LIVE/DEAD Fixable Dead Cell To generate single-cell suspensions, tumors were collected, minced into Stain from Life Technologies was used to gate on live cells. 2- to 4-mm pieces, and digested for 30 min using the mouse Tumor Dis- sociation Kit from Miltenyi (Miltenyi Biotec, Auburn, CA). Tumor ho- Chemotherapy agents mogenates were then filtered through a 70-mm nylon filter (Corning) and washed twice with RPMI 1640 medium. After the last wash, cells were was purchased from LC Laboratories (Woburn, MA), dissolved in resuspended in staining buffer (PBS + 0.5% FCS + 5 mM EDTA) and 1–2 50% Cremophor EL and 50% ethanol at 20 mg/ml to be stored at 4˚C, and million cells were transferred to 96-well V-bottom plates. Cells were then then further diluted in saline immediately before administration. Oxali- surface stained for 30 min at 4˚C and washed twice. For intracellular platin was purchased from Winthrop (Bridgewater, NJ) and diluted in staining, cells were then fixed and permeabilized using the eBioscience water immediately before administration. from Hospira Foxp3 Fix/Perm buffer (Thermo Fisher Scientific, Waltham, MA). (Lake Forest, IL), from TEVA Pharmaceuticals (Sellersville, PA), and from Fresenius Kabi USA (Lake Zurich, IL) were di- Statistical analysis luted in saline immediately before administration. from ChemShuttle (Hayward, CA) and cyclophosphamide (CTX) from Baxter All data were presented as means 6 SD. Comparisons between treatment Healthcare (Deerfield, IL) were dissolved in saline immediately before groups were generated using nonparametric, Mann–Whitney U tests. Prism administration. For dosing concentration, route, and regimen, please see 6.0 (GraphPad) was used to process all the statistical analyses. Supplemental Table II. Cell culture Results Anti–PD-L1 treatment modulates CD8+ T cell responses MC38 murine colon adenocarcinoma cells were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 To start delineating how different chemotherapies affect the an- medium plus 1% L-glutamine with 10% FBS (HyClone, Waltham, MA). titumor activity mediated by anti–PD-L1, we made use of the Cells in log-phase growth were harvested, washed once with HBSS, MC38 tumor model that partially responds to treatment (Fig. 1A, counted, and resuspended in 50% HBSS and 50% Matrigel (BD Biosci- ences) at 1 3 106 cells/ml for injection into mice. 1B). We first characterized the pharmacodynamic changes medi- ated by anti–PD-L1 treatment in tumor, dLNs, and blood. Anti– Syngeneic tumor studies PD-L1 treatment increased the number of CD8+ T cells in both MC38 cells were harvested in log-phase growth and resuspended in HBSS tumor and dLNs 9 d after treatment initiation, consistent with containing Growth Factor Reduced Matrigel (BD Biosciences) at a 1:1 ratio. the pharmacodynamic effects reported in patients treated with The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Anti–PD-L1 treatment modulates CD8+ T cell responses in dLNs and tumor tissue of MC38 tumor-bearing mice. C57BL/6 mice were inoculated s.c. with MC38 tumor cells, and when tumors reached a volume of ∼190 mm3 (day 0), mice with similarly sized tumors were randomized and enrolled into the study. The next day (day 1), mice were treated with anti–PD-L1 or isotype control Ab twice a week for 3 wk. Tissues were collected on day 10 for subsequent analysis by flow cytometry, or tumors were allowed to grow for up to 60 d to assess tumor growth. (A) Outline depicting the murine tumor 3 studies. (B) Tumor volume (cubic millimeter) of control- or anti–PD-L1–treated mice shown on a log2 scale. Tumor volumes below 32 mm (dotted line) represent CRs. Tumor efficacy plots are the compilation of five independent experiments. (C) Absolute number of CD8+ T cells in tumor tissue and dLNs of control- and anti–PD-L1–treated mice. (D) Frequency of ICOS+ CD8+ T cells in dLNs. (E) Frequency of intratumoral IFN-g+, ICOS+, Ki-67+, and GZMB+ CD8+ T cells following treatment with anti–PD-L1 or isotype control Ab. (F) Frequency of intratumoral Tregs and the CD8/Treg ratio following treatment. Flow cytometry results are the compilation of nine independent experiments. Established MC38 tumors were treated (Figure legend continues) 4 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT

Table I. Chemotherapy dosing, formulation, route, and regimen

IP, intraperitoneal; IV, intravenous; Q4D, once every 4 d; QW, once weekly. *Diluted in saline to 12.5% prior to dosing. **Diluted in saline to 2.5% prior to dosing.

MPDL3280A (2) (Fig. 1C). We also observed a significant in- netic studies (data not shown). The particular dosing schedule was crease in the activation level of CD8+ T cells in dLNs as measured determined to approximate clinical exposure, and the mouse Downloaded from by expression of the activation marker inducible T cell cos- regimen was selected based on clinical use (once weekly in hu- timulator (ICOS) (Fig. 1D). To investigate the induction of IFN-g mans was equivalent to once every 4 d in mice, and once every 3 expression, an effector cytokine linked to the PD1/PD-L1 axis wk in humans was equivalent to once weekly in mice) (Table I). (2, 17, 18), we used IFN-g–YFP reporter mice to circumvent the Carboplatin treatment was initiated 1 d after tumors became need to restimulate T cells ex vivo (see Materials and Methods). established (Fig. 2A). We observed that carboplatin treatment Consistent with anti–PD-L1 data from the clinic, the frequency of significantly decreased the number of CD8+ and CD4+ T cells in IFN-g+ CD8+ T cells in the tumor increased after treatment dLNs, although these trends were appreciable but NS in tumors http://www.jimmunol.org/ (Fig. 1E). Additionally, we observed an increase in the frequency (Fig. 2B). There was also a significant decrease in the frequency of of activated intratumoral CD8+ T cells expressing ICOS, the activated ICOS+ CD8+ T cells in dLNs but no change in the proliferation marker (Ki-67), and the cytolytic marker GZMB phenotype of these cells in tumor tissue following carboplatin (Fig. 1E). Furthermore, anti–PD-L1 did not appear to affect the treatment (Fig. 2C). However, there was a trend toward increased overall frequency of Tregs in the tumor tissue, but there was an frequency of Tregs, which altered the CD8/Treg ratio (Fig. 2D), overall increase in the CD8/Treg ratio given the increase in CD8+ whereas no change was observed in the myeloid compartment T cells (Fig. 1F). We did not observe any difference in the number (data not shown). Together, these results suggest that the effects of of intratumoral neutrophils, monocytes, or macrophages following carboplatin on immune cells in the tumor were significantly muted by guest on September 28, 2021 treatment (data not shown). These results demonstrate that anti– when compared with dLNs and did not significantly affect intra- PD-L1 treatment significantly modulates CD8+ T cell responses in tumoral CD8+ T cell numbers or phenotype. peripheral and tumor tissue by increasing the number of activated Given that carboplatin treatment had an apparent detrimental effector CD8+ T cells, leading to tumor growth inhibition and effect on dLNs, we wanted to further investigate whether these complete responses (CRs) in a subset of mice. effects would translate into diminished anti–PD-L1–mediated re- To further corroborate that CD8+ T cells are required for the sponses in the combination setting. Interestingly, carboplatin not antitumor activity mediated by anti–PD-L1 treatment, we treated only showed single-agent activity when compared with the control mice harboring established MC38 tumors with anti–PD-L1 in group (tumor growth inhibition of 71% relative to the control the presence of a CD8-depleting Ab. Twenty-four hours after anti- group and 23 time to progression of 9 d versus 3.5 d, respec- CD8 treatment, CD8+ T cells were significantly depleted in blood tively) but also showed enhanced combinatorial activity with anti– and present at almost undetectable levels (Fig. 1G). Importantly, PD-L1, resulting in 30% CRs (Fig. 2E). Analysis of the immune depletion of CD8+ T cells completely abolished the activity of infiltrate revealed that combination treatment did not significantly anti–PD-L1, demonstrating that CD8+ T cells are critical in affect the phenotype of CD8+ T cells in tumors (Fig. 2F). Sur- driving the response to treatment (Fig. 1H). prisingly, combination treatment still led to a significant reduction in the frequency of activated CD8+ T cells in dLNs despite the Carboplatin-induced inhibition of T cell responses in dLNs is strong combinatorial activity observed (Fig. 2G). These results led spared in tumor tissues us to speculate that immune changes in peripheral tissues do not Given that carboplatin is used for the treatment of a wide range of significantly affect the antitumor responses in established tumors cancers, we wanted to further investigate the effects of this che- and response is likely dependent on direct changes to the tumor- motherapy in combination with anti–PD-L1. To determine whether infiltrating lymphocytes (TILs). carboplatin suppressed T cell responses, we focused on CD8+ To further investigate the disconnect between the detrimental T cell numbers and phenotype as these cells drive the antitumor effects of chemotherapy in peripheral tissues (dLNs and blood) and activity mediated by anti–PD-L1 in MC38 tumors. The dose for increased antitumor responses, we treated established MC38 carboplatin was selected to approximate clinical exposure (area tumor-bearing mice with carboplatin alone or in combination with under the curve mM 3 h) after running individual pharmacoki- anti–PD-L1 in the presence or absence of FTY720 (fingolimod),

with anti-CD8 on day 0 to deplete CD8+ T cells. (G) Number of CD8+ T cells per ml of blood on day 2 and day 6 of study. (H) Tumor volume (cubic millimeter) of control or anti–PD-L1– treated mice in the presence or absence of CD8 depletion. (n = 10 mice per group). **p , 0.01, ***p , 0.001, ****p , 0.0001. NS, not significant. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. Carboplatin-induced inhibition of T cell responses in dLNs is spared in tumor tissues. (A) Outline of chemotherapy tumor studies. (B) Effect of carboplatin on the absolute number of CD8+ and CD4+ T cells in dLNs (top) and tumor tissue (bottom). (C) Effect of carboplatin on the frequency of ICOS+ CD8+ T cells in dLNs (top) and IFN-g+, ICOS+, Ki-67+, and GZMB+ CD8+ T cells in the tumor tissue (bottom). (D) Effect of carboplatin on the CD8/Treg ratio in treated mice. (E) Tumor volume (cubic millimeter) of control-, carboplatin-, anti–PD-L1–, and (Figure legend continues) 6 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT an immune modulator that inhibits T cell egress from lymphoid CTX is a strong modulator of CD8+ T cell responses organs (19). Treatment with FTY720 significantly decreased the in the tumor tissue and combines effectively with number of circulating T cells in the blood of treated mice but had anti–PD-L1 treatment only a partial effect on the activity of either agent alone or in Because immune modulation by chemotherapy is agent specific, combination (Fig. 3A, 3B). These results suggest that, at least in we wanted to investigate the activity of additional chemotherapies mice that harbor already established MC38 tumors, the antitumor with well-known immune modulatory effects like CTX. CTX has activity is majorly dependent on TILs at the time of treatment been shown to enhance dendritic cell expansion and cross- initiation. presentation, stimulate NK and CD8+ T cells, deplete Tregs, and help modulate the myeloid compartment (20–24). When tested as Effect of chemotherapy on immune cells is not class specific single agent, CTX significantly reduced the frequency of activated Having observed that carboplatin had no antagonistic effect on the ICOS+ CD8+ T cells in dLNs without decreasing their absolute antitumor activity mediated by anti–PD-L1 and actually en- numbers (Supplemental Table I). However, the frequency of Ki- hanced responses, we wanted to determine whether this was a 67+ CD8+ T cells in the tumor tissue was significantly reduced, carboplatin-specific effect or related to -based chemo- whereas their activation state was preserved. Interestingly, in the therapy. To evaluate this, we performed single-agent and com- combination setting, CTX significantly increased the frequency bination efficacy studies with two additional platinum analogs, and number of activated CD8+ T cells in the tumor as measured by cisplatin and . In contrast to carboplatin, neither cis- IFN-g, ICOS, and GZMB expression without significantly re- + + platin nor oxaliplatin decreased the number of CD8 or CD4 ducing the level of Ki-67–expressing cells (Fig. 5A, 5B). Not

+ + Downloaded from T cells, but both agents decreased the frequency of ICOS CD4 surprisingly, these changes translated into significantly enhanced + or CD8 T cells in dLNs, respectively (Fig. 4A). Similar to antitumor activity, resulting in 90% CRs in the combination set- carboplatin, cisplatin and oxaliplatin did not show an antago- ting despite only modest single-agent CTX activity (Fig. 5C). This + nistic effect on the phenotype of intratumoral CD8 Tcellswith pronounced effect on CD8+ T cell activation in the tumor was not + + oxaliplatin, increasing the frequency of ICOS CD8 T cells in observed with the other chemotherapies analyzed and was quite tumor when given as single agent (data not shown). Both cis- striking given the detrimental effect on activated CD8+ T cells in platin and oxaliplatin displayed lower antagonistic effects on dLNs and the reduction in Ki-67–expressing cells in the tumor http://www.jimmunol.org/ T cells at peripheral sites when compared with carboplatin when given as single agent. Additional changes in immune cell (Supplemental Table I). subsets are summarized in Supplemental Table II. These results When these agents were combined with anti–PD-L1 treatment, suggest that CTX is indeed a strong modulator of T cell responses + + oxaliplatin significantly decreased the frequency of ICOS CD8 with observed beneficial effects in the tumor tissue when com- T cells in dLNs, but this was not the case in tumor tissue bined with anti–PD-L1 treatment, but similar to the other che- (Fig. 4B, 4C). Contrary to carboplatin, cisplatin significantly motherapies, it could antagonize responses in secondary lymphoid + + decreased the number of CD8 and CD4 T cells in dLNs in the organs. combination setting and also led to a reduced number and acti- + vation of T cells in blood (Supplemental Table II). Oxaliplatin, Gemcitabine antagonizes intratumoral CD8 T cells but still by guest on September 28, 2021 contrary to carboplatin and cisplatin, led to a decrease in IFN-g+ enhances antitumor responses CD8+ T cells in the tumor, with GZMB levels also trending down Gemcitabine is another potent immune modulator with known in the combination setting (Fig. 4C). Despite these changes, myelosuppressive effects (25–28). Similar to CTX, treatment with neither agent antagonized the activity of anti–PD-L1 and, on the gemcitabine showed a trend toward decreasing the frequency of contrary, enhanced antitumor responses (Fig. 4D, 4E). These ICOS+ CD8+ T cells in dLNs with a significant decrease on the results suggest that the effect of chemotherapy, at least for these level of intratumoral Ki-67–expressing cells (Supplemental Table I). platinum-based agents, is not class-specific, but rather each There was no change in the number of CD8+ and CD4+ T cells in treatment has a particular effect on immune cell subsets in dif- dLNs following treatment, and, unlike combination with CTX, ferent tissues. combination with gemcitabine did not show enhancement in Given the wide use of , such as paclitaxel and docetaxel, the frequency of activated CD8+ T cells in the tumor but rather for the treatment of various cancers, we also wanted to determine significantly decreased the frequency of Ki-67– and GZMB- the effect of these chemotherapies on the antitumor activity me- expressing cells (Fig. 6A). This was accompanied by a decrease diated by anti–PD-L1. Similar to platinum-based chemotherapies, in the absolute number of intratumoral CD8+ T cells (Fig. 6B) that we observed a more pronounced and antagonistic effect in dLNs led to a decrease in the CD8/Treg ratio (Fig. 6C). Given these and blood when these taxanes were given as a single agent or in antagonistic effects on CD8+ T cells in both dLNs and tumor combination with anti–PD-L1, with minimal effects observed in tissue, we did not expect gemcitabine to combine effectively with TILs (Supplemental Tables I–II). Additionally, paclitaxel and anti–PD-L1 treatment. However, despite modest single-agent ac- docetaxel led to differential effects in peripheral tissues without a tivity, combination with gemcitabine led to enhanced antitumor generalized class-specific effect, and, when combined with anti– responses, resulting in 40% CRs in the combination setting (Fig. PD-L1, these chemotherapies did not antagonize antitumor re- 6D), demonstrating that gemcitabine significantly improves the sponses (Supplemental Fig. 1). From these results, it is evident activity of anti–PD-L1 treatment despite reducing the levels of that the effects of chemotherapy are agent specific and do not activated intratumoral CD8+ T cells. appear to generally antagonize the activity of anti–PD-L1, largely To demonstrate that the activity of gemcitabine in combination preserving the phenotype of TILs. with anti–PD-L1 was driven by T cells, we evaluated antitumor

3 combination-treated mice shown on a log2 scale (n = 10 mice per group). Tumor volumes below 32 mm (dotted line) represent CRs (limit of detection). (F) Frequency of IFN-g+,ICOS+,Ki-67+,andGZMB+ CD8+ T cells in tumors of anti–PD-L1– or combination-treated mice. (G) Effect of anti–PD-L1 treatment alone or in combination with carboplatin on the frequency of ICOS+ CD8+ T cells in dLNs. BIWx3, twice a week for 3 wk; NS, not significant. The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Antitumor activity in established MC38 tumors is mostly dependent on intratumoral T cells at the time of treatment initiation. Mice with established MC38 tumors were treated with control, carboplatin, anti–PD-L1, or combination treatment in the presence or absence of FTY720 treatment. Forty-eight hours and nine days after treatment initiation, blood was collected from treated mice to measure the number of T cells in circulation. (A) Number of T cells per microliter of blood. (B) Tumor volumes of control- or FTY720-treated groups (n = 10 mice per group). Tumor volumes below 32 mm3 (dotted line) represent CRs. *p , 0.05, **p , 0.01. 8 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Effect of chemotherapy on immune cells is not class specific. (A) Frequency of ICOS+ CD8+ (top) and CD4+ T cells (bottom) in dLNs following treatment with cisplatin or oxaliplatin as single agent. (B) Frequency of ICOS+ CD8+ T cells in dLNs of anti–PD-L1– or combination-treated mice with cisplatin (top) or oxaliplatin (bottom). (C) Frequency of IFN-g+, ICOS+, Ki-67+, and GZMB+ CD8+ T cells in tumor tissue following combination treatment with cisplatin (left) or oxaliplatin (right). (D) Tumor volumes (cubic millimeter) of control-, cisplatin-, anti–PD-L1–, or combination-treated mice. (E) Tumor volumes (cubic millimeter) of control-, oxaliplatin-, anti–PD-L1–, or combination-treated mice (n = 10 mice per group). Tumor volumes below 32 mm3 (dotted line) are considered CRs. *p , 0.05. NS, not significant. The Journal of Immunology 9 Downloaded from

FIGURE 5. CTX enhances CD8+ T cell responses and antitumor activity in combination with anti–PD-L1. (A) Absolute number of intratumoral CD8+ and CD4+ T cells following combination treatment with CTX. (B) Frequency of IFN-g+, ICOS+, Ki-67+, and GZMB+ CD8+ T cells in the tumor tissue C following treatment with anti–PD-L1 alone or in combination with CTX. ( ) Tumor volumes (cubic millimeter) of control-, CTX-, anti–PD-L1–, or http://www.jimmunol.org/ combination-treated mice (n = 10 mice per group). Tumor volumes below 32 mm3 (dotted line) are considered CRs. For flow cytometry data, tumors were harvested on day 9 after treatment initiation. *p , 0.05, **p , 0.01. NS, not significant. responses in immunodeficient RAG2/2 mice that lack mature effects, gemcitabine alone or in combination with anti–PD-L1 T cells and B cells. As shown in Supplemental Fig. 2A, anti– led to a significant decrease in the frequency of TAMs PD-L1 treatment had no activity in RAG2/2 mice, whereas gem- (Fig. 7D). citabine treatment alone or in combination showed only partial To further corroborate that in the MC38 tumor model mac- activity (driven by gemcitabine), which was short lived as tumors rophages have a suppressive phenotype, we subdivided the quickly regrew, resulting in no CRs as was observed in wild-type macrophage population based on MHC class II (MHC-II) and by guest on September 28, 2021 mice. To further corroborate that the combination efficacy was mannose receptor (CD206) expression (Fig. 7A). MHC-II ex- dependent on CD8+ T cells despite their lower numbers following pression is normally associated with a more proinflammatory, gemcitabine treatment, we depleted CD8+ T cells in established M1-like phenotype, whereas CD206 expression is associated tumors from wild-type mice. As expected, anti–PD-L1 activity was with suppressive, M2-like macrophages (29–31). By using lost, and single-agent gemcitabine and combination treatment these two markers, we were able to subdivide the TAM pop- showed only partial activity, which was quickly followed by tumor ulation into three main subsets: an M1-like subset (CD2062 regrowth, demonstrating a lack of effective tumor control in the MHC-II+), an intermediate subset (CD206+MHC-II+), and an absence of CD8+ T cells (Supplemental Fig. 2B). These results M2-like subset (CD206+MHC-II2); we determined that most corroborate that despite the effect of gemcitabine on CD8+ T cells, macrophages showed an intermediate and M2-like phenotype in these cells are still required to drive the antitumor activity when established MC38 tumors (Fig. 7E). Additionally, this enriched combined with anti–PD-L1. CD206+ TAM population also expressed the highest levels of arginase 1 (ARG1), an immunosuppressive enzyme that Gemcitabine reduces the number of suppressive processes L-arginine into L-ornithine and urea and can pro- intratumoral macrophages foundly suppress T cell responses (31–33). Altering the mac- The strong antitumor activity of gemcitabine in combination with rophage population in the MC38 tumor model could therefore anti–PD-L1 was unexpected, given its antagonistic effect on have beneficial consequences for antitumor activity by affecting intratumoral CD8+ T cells. This led us to speculate that gemci- a major immunosuppressive component in the tumor microen- tabine must be affecting a major immunosuppressive component vironment. Indeed, gemcitabine treatment alone or in combi- in the tumor microenvironment that compensated for the reduced nation with anti–PD-L1 significantly decreased the number of numbers of activated CD8+ T cells while maintaining robust an- intratumoral CD206+MHC-II2 macrophages relative to anti–PD-L1 titumor activity. In addition to direct antitumor effects, gemcita- treatment (Fig. 8A). This effect on TAMs could have important bine is known to be myelosuppressive and could potentially be implications for antitumor activity as the relative abundance of reducing the numbers of tumor-associated myeloid cells. At the CD206+ macrophages was correlated with tumor burden (Fig. 8B). time of treatment initiation, established MC38 tumors were mostly The effect of gemcitabine on TAMs could therefore outweigh some infiltrated by macrophages defined as CD11b+CD11c2/intLy-6G2 of its negative impacts on CD8+ T cells by allowing fewer activated Ly-6C2F4/80+ cells (Fig. 7A, 7B). These tumor-associated mac- CTLs to effectively control tumor growth. Together, these results rophages (TAMs) also expressed the highest levels of PD-L1 demonstrate that combination treatment with gemcitabine, although relative to the other cell subsets (Fig. 7C) and are likely it reduces the number of activated CD8+ T cells, also reduces the main contributors to PD1/PD-L1–mediated suppression in the number of suppressive TAMs in the tumor, thereby allowing fewer tumor tissue. Consistent with its known myelosuppressive activated CD8+ T cells to effectively control tumor growth. 10 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT Downloaded from http://www.jimmunol.org/

+ +

FIGURE 6. Gemcitabine antagonizes the effect of anti–PD-L1 on intratumoral CD8 T cells but enhances antitumor responses. (A) Frequency of IFN-g , by guest on September 28, 2021 ICOS+,Ki-67+,andGZMB+ CD8+ T cells in the tumor tissue following treatment with anti–PD-L1 alone or in combination with gemcitabine. (B) Absolute number of intratumoral CD8+ and CD4+ T cells following combination treatment with gemcitabine. (C) CD8/Treg ratio in tumors of anti–PD-L1– or combination- treated mice. (D) Tumor volumes (cubic millimeter) of control-, gemcitabine-, anti–PD-L1–, or combination-treated mice (n = 10 mice per group). Tumor volumes below 32 mm3 (dotted line) are considered CRs. For flow cytometry data, tumors were harvested on day 9 after treatment initiation. *p , 0.05, **p , 0.01.

Targeting tumor macrophages with anti-CSF1R combines Discussion effectively with anti–PD-L1 treatment Chemotherapies can have various effects on the immune system. Given that the MC38 tumor model is highly enriched in macro- Some agents can have immunosuppressive side effects by directly phages with a suppressive phenotype, we hypothesized that using depleting immune effector cells or by inhibiting their function (11), other approaches to target this population while preserving CD8+ whereas others can enhance the immunogenic properties of tumor T cell functionality should help augment the activity of anti–PD- cells, thus favoring T cell immunosurveillance over suppression L1 treatment. To test this hypothesis and to further corroborate (10, 12, 23, 34). Chemotherapies can also modulate different that the improved activity following gemcitabine treatment is immune cell subsets, conferring enhanced antitumor activity (22, partly due to its effect on TAMs, we treated MC38 tumor-bearing 26, 27). In this study, we show that combining anti–PD-L1 with mice with anti–PD-L1 alone or in combination with anti-CSF1R different chemotherapy agents did not antagonize responses in to deplete macrophages (16). Because gemcitabine not only tar- established MC38 tumors and in many cases augmented antitumor gets TAMs but also has a plethora of effects on other immune cell activity. Despite apparent antagonistic effects on T cells in pe- components and tumor cells, we were not expecting to replicate ripheral tissues like dLNs and blood, the various chemotherapies the effects observed with gemcitabine combination, but we were tested in this study did not significantly affect TILs. expecting to observe combinatorial activity with anti–PD-L1 This differential effect could be due to various factors, including mediated by the depletion of suppressive TAMs. Indeed, anti- differences in drug exposure and other local mediated effects (35). CSF1R treatment combined effectively with anti–PD-L1, leading Similar differences between local and systemic effects of che- to increased tumor control and delaying tumor growth relative to motherapy have been observed in a glioblastoma murine tumor either treatment alone (Fig. 8C). These results highlight that in the model in which local chemotherapy enhanced the activity of anti- particular immune contexture of this tumor model, targeting PD1, but systemic chemotherapy led to lymphodepletion in dLNs TAMs, which represent a major immunosuppressive component of and peripheral blood (36). Another possible explanation is that the tumor microenvironment, augments tumor killing and that chemotherapies affect T cells differently based on their differen- gemcitabine could indeed be conferring improved antitumor re- tiation state. In the MC38 tumor model, most infiltrating CD8+ sponses by significantly decreasing the population of suppressive T cells have an effector phenotype (CD62LloCD44hi/lo), whereas TAMs in the tumor tissue. most cells in dLNs are naive (CD62L+CD44lo) (data not shown). The Journal of Immunology 11 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Gemcitabine treatment reduces the number of intratumoral macrophages in MC38 tumor-bearing mice. (A) Gating strategy used to define tumor-associated intermediate macrophages (black), mature macrophages (red), neutrophils (orange), and monocytes (blue) based on Ly-6C and Ly-6G expression and TAM subsets based on CD206 and MHC-II expression. (B) Frequency of different intratumoral cell subsets in the MC38 tumor model at the time to treatment initiation (percentage of total CD45+ gated cells). Macrophages shown in red. (C) Expression levels of PD-L1 on different intratumoral immune cell subsets at the time of treatment initiation (macrophages in red). Frequency (top) and mean fluorescence intensity (MFI) (bottom). (D) Frequency of macrophages in the tumor tissue and spleen following treatment with gemcitabine alone or in combination with anti–PD-L1. (E) Ratio of TAM subsets in MC38 tumors based on CD206 and MHC-II expression and their levels of ARG1 expression. *p , 0.05, **p , 0.01. 12 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Gemcitabine reduces the number of suppressive intratumoral macrophages. (A) Absolute number of total macrophages as well as CD206 and MHC-II TAM subsets following treatment with gemcitabine alone or in combination with anti–PD-L1. (B) Correlation between tumor burden and the relative abundance of CD206+MHC-II+/2 TAMs. (C) C57BL/6 mice were inoculated s.c. with MC38 tumor cells. Once tumors were established, animals received treatment with either mIgG1 isotype control, anti–CSF-1R, or anti–PD-L1 Abs. Tumor volumes (cubic millimeter) of treated mice (n = 10 mice per group). *p , 0.05, **p , 0.01.

Even though the chemotherapies tested in this study are antineo- reveal that even though none of the chemotherapies tested plastic, it is possible that the effect of these agents is more pro- in combination with anti–PD-L1 led to a reduction in antitumor nounced in cells undergoing priming, activation, and expansion at activity, the effect of these chemotherapies at peripheral sites peripheral sites rather than tumor-infiltrating effector cells that are could affect subsequent responses, and this will likely depend on less proliferative (37–39). how durable the antagonistic effects are and how fast the numbers Despite most chemotherapies having an antagonistic effect on and phenotype of T cells can rebound following chemotherapy CD8+ T cells in dLNs and blood, this effect did not translate into treatment. reduced antitumor activity. CD8+ T cells drive anti–PD-L1 re- It was also evident from our studies that the effect of chemo- sponses in the MC38 tumor model, so decreasing CD8+ T cell therapy was agent specific and not class specific. This was dem- numbers and activation state should have translated into reduced onstrated by differences in the pharmacodynamic regulation at both antitumor activity. However, by blocking lymph node egress with peripheral and tumor tissue for carboplatin, cisplatin, and oxali- FTY720 (fingolimod) (19), we showed that tumor responses to platin. Despite acting through DNA platination, which leads to anti–PD-L1 in established tumors were not majorly dependent on Pt-DNA lesions that can accumulate and result in (40), incoming cells from the periphery but rather relied mostly on TILs these drugs had various effects on tumor and immune cells. Cis- at the time of treatment initiation. This finding suggests that when platin showed a more pronounced inhibition of CD8+ and CD4+ working with established syngeneic tumor models of short dura- T cell numbers in blood and dLNs in combination with anti–PD- tion, the peripheral effect of a particular treatment might not be L1, whereas carboplatin and oxaliplatin decreased the frequency reflected in the overall antitumor activity. Therefore, our results of activated T cells in dLNs, with only oxaliplatin significantly The Journal of Immunology 13 reducing the frequency of IFN-g+ CD8+ T cells in the tumor. whether targeting TAMs could help modify the threshold for an- These chemotherapies, however, combined effectively with anti– titumor activity in the MC38 tumor model and enhance responses PD-L1 treatment with carboplatin and oxaliplatin, showing more to anti–PD-L1, we treated tumor-bearing mice with anti-CSF1R, robust combinatorial activity. which has been shown to strongly reduce the number of tumor The effects of chemotherapy are therefore drug specific and de- macrophages (16). Targeting TAMs with anti-CSF1R led to an pendent on multiple factors. In this study, we focused on the effects enhancement in anti–PD-L1–mediated activity, corroborating that of chemotherapy on various immune cell subsets in peripheral and in this particular tumor model, depleting macrophages, which tumor tissue and explored how both antagonistic and beneficial represent a major immunosuppressive population, can help aug- effects could balance out to establish a new threshold for antitumor ment antitumor responses. response as observed with gemcitabine treatment. However, addi- Our results suggest that the effects of combining checkpoint tional factors not investigated in this study are likely involved in inhibitors (CIT) such as anti–PD-L1 with chemotherapy will de- driving the overall combinatorial activity of chemotherapy, such as pend on the specific tumor-immune contexture at the time of the effects of drugs on tumor cell death (tolerogenic versus im- treatment initiation. The antagonist effect of some chemotherapies munogenic) or microregional effects of chemotherapy on the tumor at peripheral sites might affect the level of new Ag presentation, tissue-like changes in interstitial fluid pressure, vasculature, and priming, and repopulation of tumor immune cell subsets, but these desmoplastic architecture. Multiple studies have suggested that cells might recover between dosing cycles. Thus, the detrimental different chemotherapies can result in immunogenic cell death, effect of chemotherapy at peripheral sites will likely be only leading to the release of damage-associated molecular pattern transient, and recruitment of new T cells to the tumor tissue could molecules like high mobility group box 1 protein (HMGB1), cal- still be achieved. In the MC38 tumor model, none of the che- Downloaded from reticulin, ATP, and heat shock proteins (HSPs) that can sensitize motherapies tested antagonized the activity of anti–PD-L1, and in tumors to immune checkpoint blockade (10, 12, 34). Although some many cases they significantly improved responses as observed for chemotherapies tested in this study could have resulted in immu- carboplatin, oxaliplatin, CTX, and gemcitabine. The effects of nogenic cell death, we focused our efforts on identifying the sub- these chemotherapy combinations were agent specific, and the sequent immunological changes and started delineating how these pharmacodynamic changes were distinct for each of the chemo-

could affect overall antitumor responses. therapies. Overall, our data support the combination of CIT agents http://www.jimmunol.org/ Certain chemotherapies, such as CTX and gemcitabine, have with chemotherapy in earlier lines of treatment, but further un- been shown to have various effects on specific immune cell pop- derstanding the effects specific chemotherapies have on tumor and ulations (20, 26, 41, 42). These two chemotherapies showed strong immune cell subsets as well as other microenvironmental com- combinatorial activity with anti–PD-L1, whereas their single- ponents, such as stromal and endothelial cells, will improve the agent effect was only modest and mostly delayed tumor growth. selection of such agents for combination treatment. Our results Both chemotherapies negatively affected T cells in dLNs and also provide some initial evidence for the potential of chemo- blood, but their effect in TILs was quite distinct. Combination therapy combinations that may not have been previously realized with CTX led to a clear enhancement in CD8+ T cell numbers and and that are not commonly used as standard of care but that should activation state, although gemcitabine decreased CD8+ T cell be further investigated because of their effects on specific immune by guest on September 28, 2021 numbers as well as the frequency of GZMB- and Ki-67– cell populations. This could result in a more personalized ap- expressing cells. The MC38 tumor model is well infiltrated by proach to chemotherapy–CIT combinations by trying to target CD8+ T cells, and increasing their number and activity was ex- specific tumor-immune contextures when possible. pected to further enhance the response to anti–PD-L1, which is in itself already augmenting the activity of CTLs. For gemcitabine, Acknowledgments the strong combinatorial effect with anti–PD-L1 was more sur- We thank the core dosers at Genentech as well as the cell line core group for prising, given that activity in this tumor model is dependent on their contribution to this project. We also thank Marcia Belvin for useful + CD8 T cells. Upon further analysis, it was evident that even discussions of the data. though gemcitabine reduced the number of CD8+ T cells, its effect on decreasing suppressive CD206+MHCIIlo macrophages proba- Disclosures bly balanced out the decrease in CTL numbers. In the MC38 tu- All authors are employees and stock holders of Genentech or Roche. mor model, TAMs represent the major immune infiltrate, and these cells show a suppressive phenotype (CD206+ARG1+) (43). By reducing this inhibitory barrier, gemcitabine treatment was References able to bolster antitumor responses despite reducing the number of 1. Rosenberg, J. E., J. Hoffman-Censits, T. Powles, M. S. van der Heijden, A. V. Balar, A. Necchi, N. Dawson, P. H. O’Donnell, A. Balmanoukian, CTLs. This likely led to the establishment of a lower response Y. Loriot, et al. 2016. Atezolizumab in patients with locally advanced and threshold, allowing fewer CTLs to effectively control tumor metastatic urothelial carcinoma who have progressed following treatment with growth. This is likely why other chemotherapies that were largely platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet 387: 1909–1920. muted in their tumor effect were still able to enhance antitumor 2. Herbst, R. S., J.-C. Soria, M. Kowanetz, G. D. Fine, O. Hamid, M. S. Gordon, activity. Even though few significant pharmacodynamic changes J. A. Sosman, D. F. McDermott, J. D. Powderly, S. N. Gettinger, et al. 2014. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in were observed for some of these chemotherapies in the tumor, cancer patients. Nature 515: 563–567. there were possibly enough small changes that translated into a 3. Hodi, F. S., S. J. O’Day, D. F. McDermott, R. W. Weber, J. A. Sosman, more favorable response threshold. It is important to point out that J. B. Haanen, R. Gonzalez, C. Robert, D. Schadendorf, J. C. Hassel, et al. 2010. Improved survival with ipilimumab in patients with metastatic melanoma. N. the strong combinatorial activity of gemcitabine is likely tumor Engl. J. Med. 363: 711–723. specific and, as observed for the MC38 tumor model, dependent 4. Topalian, S. L., F. S. Hodi, J. R. Brahmer, S. N. Gettinger, D. C. Smith, on the balance between tumor-infiltrating populations and how D. F. McDermott, J. D. Powderly, R. D. Carvajal, J. A. Sosman, M. B. Atkins, et al. 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in these are affected by chemotherapy treatment. In other tumor cancer. N. Engl. J. Med. 366: 2443–2454. models that do not share this level of infiltrating CTLs together 5.Powles,T.,J.P.Eder,G.D.Fine,F.S.Braiteh,Y.Loriot,C.Cruz,J.Bellmunt, H.A.Burris,D.P.Petrylak,S.-L.Teng,etal.2014.MPDL3280A(anti-PD- with a suppressive macrophage population, the effect of gemci- L1) treatment leads to clinical activity in metastatic bladder cancer. Nature tabine combination could be antagonistic. To further corroborate 515: 558–562. 14 CHEMOTHERAPY COMBINES EFFECTIVELY WITH ANTI–PD-L1 TREATMENT

6. Rizvi, N. A., M. D. Hellmann, A. Snyder, P. Kvistborg, V. Makarov, J. J. Havel, 24. Moschella, F., M. Valentini, E. Arico`, I. Macchia, P. Sestili, M. T. D’Urso, W. Lee, J. Yuan, P. Wong, T. S. Ho, et al. 2015. Cancer immunology. Mutational C. Alessandri, F. Belardelli, and E. Proietti. 2011. Unraveling cancer chemo- landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer. immunotherapy mechanisms by gene and protein expression profiling of re- Science 348: 124–128. sponses to cyclophosphamide. Cancer Res. 71: 3528–3539. 7. Schumacher, T. N., and R. D. Schreiber. 2015. Neoantigens in cancer immu- 25. Nowak, A. K., B. W. S. Robinson, and R. A. Lake. 2002. Gemcitabine exerts a notherapy. Science 348: 69–74. selective effect on the humoral immune response: implications for combination 8. Tumeh, P. C., C. L. Harview, J. H. Yearley, I. P. Shintaku, E. J. M. Taylor, chemo-immunotherapy. Cancer Res. 62: 2353–2358. L. Robert, B. Chmielowski, M. Spasic, G. Henry, V. Ciobanu, et al. 2014. PD-1 26. Suzuki, E., V. Kapoor, A. S. Jassar, L. R. Kaiser, and S. M. Albelda. 2005. blockade induces responses by inhibiting adaptive immune resistance. Nature Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor 515: 568–571. cells in tumor-bearing animals and enhances antitumor immune activity. Clin. 9. Chen, D. S., and I. Mellman. 2017. Elements of cancer immunity and the cancer- Cancer Res. 11: 6713–6721. immune set point. Nature 541: 321–330. 27. Bruchard, M., G. Mignot, V. Derange`re, F. Chalmin, A. Chevriaux, F. Ve´gran, 10. Zitvogel, L., L. Galluzzi, M. J. Smyth, and G. Kroemer. 2013. Mechanism of W. Boireau, B. Simon, B. Ryffel, J. L. Connat, et al. 2013. Chemotherapy- action of conventional and targeted anticancer therapies: reinstating immuno- triggered cathepsin B release in myeloid-derived suppressor cells activates the surveillance. Immunity 39: 74–88. Nlrp3 inflammasome and promotes tumor growth. Nat. Med. 19: 57–64. 11. Zitvogel, L., L. Apetoh, F. Ghiringhelli, and G. Kroemer. 2008. Immunological 28. Mundy-Bosse, B. L., G. B. Lesinski, A. C. Jaime-Ramirez, K. Benninger, aspects of cancer chemotherapy. Nat. Rev. Immunol. 8: 59–73. M. Khan, P. Kuppusamy, K. Guenterberg, S. V. Kondadasula, A. R. Chaudhury, 12. Bracci, L., G. Schiavoni, A. Sistigu, and F. Belardelli. 2014. Immune-based K. M. La Perle, et al. 2011. Myeloid-derived suppressor cell inhibition of the mechanisms of cytotoxic chemotherapy: implications for the design of novel IFN response in tumor-bearing mice. Cancer Res. 71: 5101–5110. and rationale-based combined treatments against cancer. Cell Death Differ. 21: 29. Martinez, F. O., L. Helming, and S. Gordon. 2009. Alternative activation of 15–25. macrophages: an immunologic functional perspective. Annu. Rev. Immunol. 27: 13. Chan, O. T., and L. X. Yang. 2000. The immunological effects of taxanes. 451–483. Cancer Immunol. Immunother. 49: 181–185. 30. Wang, N., H. Liang,, and K. Zen, 2014. Molecular mechanisms that influence the 14. Weinblatt, M. E., J. S. Coblyn, D. A. Fox, P. A. Fraser, D. E. Holdsworth, macrophage M1–M2 polarization balance. Front Immunol. 5: 614. D. N. Glass, and D. E. Trentham. 1985. Efficacy of low-dose in 31. Gabrilovich, D. I., S. Ostrand-Rosenberg, and V. Bronte. 2012. Coordinated rheumatoid arthritis. N. Engl. J. Med. 312: 818–822. regulation of myeloid cells by tumours. Nat. Rev. Immunol. 12: 253–268. Downloaded from 15. Weiner, H. L., and J. A. Cohen. 2002. Treatment of multiple sclerosis with 32. Biswas, S. K., and A. Mantovani. 2010. Macrophage plasticity and interaction cyclophosphamide: critical review of clinical and immunologic effects. Mult. with lymphocyte subsets: cancer as a paradigm. Nat. Immunol. 11: 889–896. Scler. 8: 142–154. 33. Munder, M. 2009. Arginase: an emerging key player in the mammalian immune 16. Ries,C.H.,M.A.Cannarile,S.Hoves,J.Benz,K.Wartha,V.Runza,F.Rey-Giraud, system. Br. J. Pharmacol. 158: 638–651. L. P. Pradel, F. Feuerhake, I. Klaman, et al. 2014. Targeting tumor-associated mac- 34. Pfirschke, C., C. Engblom, S. Rickelt, V. Cortez-Retamozo, C. Garris, F. Pucci, rophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer T. Yamazaki, V. Poirier-Colame, A. Newton, Y. Redouane, et al. 2016. Immu- Cell 25: 846–859. nogenic chemotherapy sensitizes tumors to checkpoint blockade therapy.

17. Peng, W., C. Liu, C. Xu, Y. Lou, J. Chen, Y. Yang, H. Yagita, W. W. Overwijk, Immunity 44: 343–354. http://www.jimmunol.org/ G. Lize´e, L. Radvanyi, and P. Hwu. 2012. PD-1 blockade enhances T-cell mi- 35. Poulin, P., Y.-H. Chen, X. Ding, S. E. Gould, C. E. Hop, K. Messick, J. Oeh, and gration to tumors by elevating IFN-g inducible chemokines. Cancer Res. 72: B. M. Liederer. 2015. Prediction of drug distribution in subcutaneous xenografts of 5209–5218. human tumor cell lines and healthy tissues in mouse: application of the tissue 18. Zou, W., J. D. Wolchok, and L. Chen. 2016. PD-L1 (B7-H1) and PD-1 pathway composition-based model to antineoplastic drugs. J. Pharm. Sci. 104: 1508–1521. blockade for cancer therapy: Mechanisms, response biomarkers, and combina- 36. Mathios, D., J. E. Kim, A. Mangraviti, J. Phallen, C.-K. Park, C. M. Jackson, tions. Sci. Transl. Med. 8: 328rv4. T. Garzon-Muvdi, E. Kim, D. Theodros, M. Polanczyk, et al. 2016. Anti-PD-1 19. Matloubian, M., C. G. Lo, G. Cinamon, M. J. Lesneski, Y. Xu, V. Brinkmann, antitumor immunity is enhanced by local and abrogated by systemic chemo- M. L. Allende, R. L. Proia, and J. G. Cyster. 2004. Lymphocyte egress from therapy in GBM. Sci. Transl. Med. 8: 370ra180. thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 37. Cui, W., and S. M. Kaech. 2010. Generation of effector CD8+ T cells and their 427: 355–360. conversion to memory T cells. Immunol. Rev. 236: 151–166. 20. Schiavoni, G., A. Sistigu, M. Valentini, F. Mattei, P. Sestili, F. Spadaro, 38. Masopust, D., V. Vezys, A. L. Marzo, and L. Lefranc¸ois. 2001. Preferential lo-

M. Sanchez, S. Lorenzi, M. T. D’Urso, F. Belardelli, et al. 2011. Cyclophos- calization of effector memory cells in nonlymphoid tissue. Science 291: 2413– by guest on September 28, 2021 phamide synergizes with type I interferons through systemic dendritic cell 2417. reactivation and induction of immunogenic tumor apoptosis. Cancer Res. 71: 39. Sallusto, F., D. Lenig, R. Fo¨rster, M. Lipp, and A. Lanzavecchia. 1999. Two 768–778. subsets of memory T lymphocytes with distinct homing potentials and effector 21. Bracci, L., F. Moschella, P. Sestili, V. La Sorsa, M. Valentini, I. Canini, functions. Nature 401: 708–712. S. Baccarini, S. Maccari, C. Ramoni, F. Belardelli, and E. Proietti. 2007. Cy- 40. Johnstone, T. C., G. Y. Park, and S. J. Lippard. 2014. Understanding and im- clophosphamide enhances the antitumor efficacy of adoptively transferred im- proving platinum anticancer drugs--phenanthriplatin. Anticancer Res. 34: 471– mune cells through the induction of cytokine expression, B-cell and T-cell 476. homeostatic proliferation, and specific tumor infiltration. Clin. Cancer Res. 13: 41. Le, D. T., and E. M. Jaffee. 2012. Regulatory T-cell modulation using cyclo- 644–653. phosphamide in vaccine approaches: a current perspective. Cancer Res. 72: 22. Ghiringhelli, F., C. Menard, P. E. Puig, S. Ladoire, S. Roux, F. Martin, E. Solary, 3439–3444. A. Le Cesne, L. Zitvogel, and B. Chauffert. 2007. Metronomic cyclophospha- 42. Bauer, C., A. Sterzik, F. Bauernfeind, P. Duewell, C. Conrad, R. Kiefl, S. Endres, mide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T A. Eigler, M. Schnurr, and M. Dauer. 2014. Concomitant gemcitabine therapy and NK effector functions in end stage cancer patients. Cancer Immunol. negatively affects DC vaccine-induced CD8(+) T-cell and B-cell responses but Immunother. 56: 641–648. improves clinical efficacy in a murine pancreatic carcinoma model. Cancer 23. Schiavoni, G., F. Mattei, T. Di Pucchio, S. M. Santini, L. Bracci, F. Belardelli, Immunol. Immunother. 63: 321–333. and E. Proietti. 2000. Cyclophosphamide induces type I interferon and augments 43. Mantovani, A., F. Marchesi, A. Malesci, L. Laghi, and P. Allavena. 2017. the number of CD44(hi) T lymphocytes in mice: implications for strategies of Tumour-associated macrophages as treatment targets in oncology. Nat. Rev. Clin. chemoimmunotherapy of cancer. Blood 95: 2024–2030. Oncol. 14: 399–416.

Supplemental Table 1

Effect in peripheral tissues (Chemotherapy vs. Control)

Effect in tumor tissue (Chemotherapy vs. Control)

No significant difference

Significant increase

Significant decrease

Supplemental Table 2

Effect in peripheral tissues (Combination vs. Anti-PD-L1)

Effect in tumor tissue (Combination vs. Anti-PD-L1)

No significant difference

Significant increase

Significant decrease

Anti-PD-L1 + Pacli. 4096 Control 4096 Paclitaxel 4096 Anti-PD-L1 4096 2048 2048 2048 2048 1024 1024 1024 1024 512 512 512 512 256 256 256 256 128 128 128 128 64 64 64 64 32 32 32 32 16 16 16 16 Tumor Volume (mm3) Volume Tumor 8 8 8 8 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 Day Day Day Day

Control Docetaxel Anti-PD-L1 Anti-PD-L1 + Docetax. 4096 4096 4096 4096 2048 2048 2048 2048 1024 1024 1024 1024 512 512 512 512 256 256 256 256 128 128 128 128 64 64 64 64 32 32 32 32 16 16 16 16 8 8 8 8 Tumor Volume (mm3) Volume Tumor 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 Day Day Day Day

Supplementary Figure 1. Paclitaxel and Docetaxel do not antagonize the activity of anti-PD-L1 in MC38 tumor bearing mice. C57BL/6 mice were inoculated subcutaneously with MC38 tumor cells, then enrolled into the study when tumor volumes reached ~190 mm3 (Day 0). Treatment initiated on Day 1 and continued for 3 weeks. Mice were monitored for up to 50 days. (Top) Tumor volume (mm3) of control, paclitaxel, anti-PD- L1 and combination treated mice. (Bottom) Tumor volume (mm3) of control, docetaxel, anti-PD-L1 and combination treated mice. N=10 mice/group. Tumor volumes below 32 mm3 (dotted line) are considered complete responses.

Control Anti-PD-L1 4096 4096 2048 2048 ) ) 1024 1024 512 512 mm3 mm3 ( ( 256 256

128 128 me me u u l l 64 64 o o V V

32 32 RAG-/- mice 16 16 8 8 0 10 20 30 40 50 0 10 20 30 40 50 Day Day

Gemcitabine Anti-PD-L1 + Gemcit. 4096 4096 2048 2048 ) 1024 ) 1024 512 512 mm3 mm3 ( 256 ( 256

128 128 me me u u l 64 l 64 o o V 32 V 32 16 16 8 8 0 10 20 30 40 50 0 10 20 30 40 50 Day Day

Control Anti-PD-L1 4096 4096 2048 2048 ) ) 1024 1024 512 512 mm3 mm3 ( ( 256

256

128 128 me me u u l l 64 64 o o V V 32 32 CD8 depleted mice 16 16 8 8 0 10 20 30 40 50 0 10 20 30 40 50 Day Day

Gemcitabine Anti-PD-L1 + Gemcit.

4096 4096 2048 2048 ) ) 1024 1024 512 512 mm3 mm3 ( ( 256 256

128 128 me me u u l l 64 64 o o V V 32 32 16 16 8 8 0 10 20 30 40 50 0 10 20 30 40 50 Day Day

Supplementary Figure 2. Anti-tumor activity of gemcitabine in combination with anti- PD-L1 requires CD8+ T cells. (A) Tumor volume (mm3) of control, anti-PD-L1, gemcitabine and combination treated RAG-/- mice. (B) Established MC38 tumors were treated with anti-CD8 on Day 0 to deplete CD8+ T cells. Treatment with gemcitabine and anti-PD-L1 started the next day (Day 1). Tumor volume (mm3) of control, anti-PD-L1, gemcitabine and combination treated mice in the absence of CD8+ T cells. N=10 mice/group. Tumor volumes below 32 mm3 (dotted line) are considered complete responses.