Triple Therapy with Mertk and PD1 Inhibition Plus Radiotherapy Promotes Abscopal Antitumor Immune Responses Mauricio S

Published OnlineFirst September 20, 2019; DOI: 10.1158/1078-0432.CCR-19-0795

Translational Cancer Mechanisms and Therapy Clinical Cancer Research Triple Therapy with MerTK and PD1 Inhibition Plus Radiotherapy Promotes Abscopal Antitumor Immune Responses Mauricio S. Caetano1, Ahmed I. Younes1, Hampartsoum B. Barsoumian1, Michael Quigley2, Hari Menon1, Chan Gao2, Thomas Spires2, Timothy P. Reilly2, Alexandra P. Cadena1, Taylor R. Cushman1,3, Jonathan E. Schoenhals1,4, Ailin Li1,5, Quynh-Nhu Nguyen1, Maria Angelica Cortez6, and James W. Welsh1

Abstract

Purpose: Radiotherapy (RT) traditionally has been used in 3 12-Gy fractions), and tumors were monitored for for local tumor control in the treatment of cancer. The recent response. discovery that radiotherapy can have anticancer effects on Results: Thetripletherapysigniﬁcantly delayed abscopal the immune system has led to recognition of its ability to tumor growth, improved survival rates, and reduced num- sensitize the tumor microenvironment to immunotherapy. bers of lung metastases. We further found that the triple þ However, radiation can also prompt adverse immunosup- therapy increased the activated CD8 and NK cells popula- pressive effects that block aspects of systemic response at tions measured by granzyme B expression with upregulation þ þ other tumor sites. Our hypothesis was that inhibition of the of CD8 CD103 tissue-resident memory cells (TRM)within MERproto-oncogenetyrosinekinase(MerTK)incombina- the abscopal tumor microenvironment relative to radiation tion with anti-programmed cell death-1 (a-PD1) check- only. point blockade will enhance immune-mediated responses Conclusions: The addition of a-PD1 þ a-MerTK mAbs to to radiotherapy. radiotherapy could alter the cell death to be more immuno- Experimental design: We tested the efﬁcacy of this triple genic and generate adaptive immune response via increasing therapy (Radiation þ a-PD1 þ a-MerTK mAbs) in 129Sv/Ev the retention of TRM cells in the tumor islets of the abscopal mice with bilateral lung adenocarcinoma xenografts. Prima- tumors which was proven to play a major role in survival of ry tumors were treated with stereotactic radiotherapy (36 Gy non-small cell lung cancer patients.

Introduction Traditionally, the benefit of radiotherapy was seen as resulting from its ability to damage tumor-cell DNA (2), but more Lung cancer is the leading cause of cancer mortality among recently interest has been expressed in its ability to achieve women and men throughout the world (1). At present, the immunogenic cell death (3). To date, studies of this effect have standard of care for advanced nonmetastatic lung cancer is focused mainly on using radiation to "prime" the immune radiotherapy combined with chemotherapy, but this approach system so as to enhance the ability of radio-immunotherapy can be highly toxic and is not effective for controlling metas- combinations to elicit systemic, rather than strictly local, con- tases, the cause of death for most patients with lung cancer. trol. Radiation also has immunomodulatory effects via evoking production of type I IFNs and upregulating MHC-I mole- þ cules (4), which in turn activate CD8 T-effector cells and 1Department of Radiation Oncology, University of Texas MD Anderson Cancer release danger signals such as high-mobility-group box 1 2 Center, Houston, Texas. Bristol-Myers Squibb (BMS), Redwood City, California (HMGB1) and ATP, which can activate macrophages (5), and and Princeton, New Jersey. 3University of Arizona College of Medicine-Phoenix, Phoenix, Arizona. 4University of Texas Southwestern Medical School, Dallas, upregulate Fas molecules on tumors, which leads to their Texas. 5Department of Radiation Oncology, First Hospital of China Medical apoptotic death. Radiation has also been shown to promote University, China. 6Department of Experimental Radiation Oncology, University changes in the inflammatory microenvironment (6, 7) such as of Texas MD Anderson Cancer Center, Houston, Texas. increasing antigen presentation by myeloid cells within the Note: Supplementary data for this article are available at Clinical Cancer tumor stroma, which in turn enhances T-cell–mediated killing Research Online (http://clincancerres.aacrjournals.org/). of tumor cells (8). We recently found that resistance to immune M.S. Caetano and A.I. Younes contributed equally as first authors. checkpoint inhibitors such as anti-PD1 was due in part to downregulation of MHC-I and that radiation was capable of Corresponding Author: James W. Welsh, The University of Texas MD Anderson upregulating MHC-I molecule expression and re-sensitizing Cancer Center, 1515 Holcombe Blvd, Unit 97, Houston, TX 77030. Phone: 713- 563-2447; Fax: 713-563-2366; E-mail: [email protected] tumors to anti-PD1 therapy (4). On the other hand, radiation also has immunosuppressive Clin Cancer Res 2019;XX:XX–XX effects via its upregulation of Tregs (7), myeloid-derived suppres- doi: 10.1158/1078-0432.CCR-19-0795 sor cells (MDSC), and M2 macrophages in the tumor microen- 2019 American Association for Cancer Research. vironment (9, 10). Tregs act to suppress a variety of immune

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Translational Relevance Materials and Methods Cell line and antibodies Therapeutics which influence the tumor immune microen- We used 344SQ parental tumor cells (344SQ-P), a murine vironment are of great interest to combine with radiation to metastatic adenocarcinoma NSCLC cell line derived from a further improve its effect on systemic antitumor immune spontaneous subcutaneous metastatic lesion in p53R172HDg/ response. Adding a-PD1þ a-MerTK to radiation significantly þ þ þK-rasLA1/þ mice. This cell line was a generous gift from upregulated CD8 CD103 tissue-resident memory cells Dr. Jonathan Kurie (MD Anderson). Cells were cultured in (T ) at the abscopal tumors, delayed the abscopal tumor RM RPMI1640 supplemented with 100 U/mL penicillin, 100 mg/- growth and extended the survival rate. Therefore, increasing þ þ mL streptomycin, and 10% heat-activated FBS and incubated the percentage of CD8 CD103 T cells would have strong RM at 37Cina5%CO atmosphere. The mAbs were all from clinical application because the presence of these cells would 2 þ þ Bristol Myers Squibb (a-MerTK, 4E9.E6; a-PD1, clone 4H2; increase survival among patients with NSCLC. CD8 CD103 and mouse IgG1 isotype control) and were prepared for injec- T cells mediate tumor-specific memory to treat non–small RM tion by dilution in PBS at pH 7.4. For the depletion studies, cell lung cancer patients, mainly those who develop recurrence þ CD8 cells and NK cells were depleted by anti-CD8 (clone 53– after radiation therapy. 6.7) from BioXcell and anti-Asialo GM1 from Wako Chemicals, respectively.

þ cells, including B cells, NK cells, NKT cells, CD4 T helper cells, Mice þ CD8 T-effector cells, monocytes, and dendritic cells, which Eight- to 12-week-old 129Sv/Ev syngeneic female mice were dampen antitumor immune responses (11). In addition to purchased from Taconic Biosciences and bred at a mouse colony Treg-depleting Abs, therapies involving depletion of M2 macro- maintained by the Department of Experimental Radiation Oncol- phages or polarization of macrophages towards the M1 phe- ogy at The University of Texas MD Anderson Cancer Center. All notype also represent a rational therapeutic approach that can animal procedures followed the guidelines of the Institutional be combined with radiotherapy. Another approach is to use Animal Care and Use Committee. an Ab to the MER proto-oncogene tyrosine kinase (MerTK), a macrophage-specificphagocyticreceptor,blockadeofwhich Tumor establishment and treatments suppresses the phagocytosis of apoptotic bodies after radio- Mice were inoculated by subcutaneous injection of 344SQ-P therapy, thereby tilting the balance towards secondary necrosis tumor cells into the right hind leg (to establish primary tumors) and induction of inflammatory and immunogenic cell and the left hind leg (to establish abscopal tumors). Tumor death (12, 13). MerTK is a cell surface receptor of the TAM- growth was monitored twice a week with digital calipers and RTK family (Tyro3/AXL/MerTK receptor tyrosine kinases). Its recorded as the change in average tumor diameter. Primary overexpression on tumor cells has been linked with poor tumors were irradiated with a Cesium source on days 7, 8, and prognosis (14). MerTK initiates efferocytosis by macrophages, 9, when the tumors had reached about 7 mm in diameter. Local which is crucial for the efficient clearance of apoptotic material tumor irradiation involved positioning the mice on a jig that and intracellular antigens (15). MerTK signaling also alters shields the mouse's body except for the leg that is to receive macrophage gene expression so as to suppress inflammatory the designated dose. Tumor-bearing mice were injected intra- cytokine production and polarize macrophages towards the peritoneally with a-PD1 mAb (200 mg/injection) on days 5, 8, wound-healing, anti-inflammatory M2 phenotype (16, 17). 11, and 14; and with a-MerTK mAb (200 mg/injection) on days During engulfment of apoptotic cells, MerTK suppresses the 7, 10, 12, 15, 19, and 22. For the depletion studies, anti-CD8 release of proinflammatorycytokinessuchasIL12,IL6,and (500 mg/injection) and anti-asialo NK depletory were injected TNFbyM1macrophages,partlybydiminishingNF-kB interperitoneally on days 5 and thereafter once/week to main- signaling (18–20); MerTK also enhances the M2 macro- tain the pressure. Mice were euthanized when the average phage-induced production of anti-inflammatory cytokines tumor diameter reached 14 mm in any dimension or became such as IL10, TGFb, and IL4 (17, 21, 22). Therefore, MerTK ulcerated. inhibition promotes re-polarization of macrophages toward the M1 antitumor phenotype and enhances antigen presenta- Tumor processing and flow cytometry tion, a crucial step in T-cell priming and activation. Although Tumors were harvested and tissues digested with 250 mg/mL of previous studies have shown that MerTK antibodies can pro- Liberase (Roche) and incubated for 30 minutes at 37 C while mote antitumor effects when combined with radiation (23), shaking at 105 rpm. FBS was added to stop the digestion reaction, the optimal timing and sequence of radiation with a-MerTK samples were filtered, and TILs were enriched by using Histopa- mAb and a-PD1 mAbs have not been evaluated. Here, we tested que 1077 (Sigma; Catalog No. H8889). Cells were then blocked the efficacy of triple therapy (RT þ a-PD1 þ a-MerTK) in a with anti-CD16/CD32 before being stained for flow cytometry. murine model of non–small cell lung cancer (NSCLC) that Stains included fluorochrome-conjugated anti-CD4 APC (Cata- includes a KrasG12D mutation. We hypothesized that radia- log No. 100412), anti-CD8 PercpCy5.5 (Catalog No. 100734), tion and anti-PD1 can be made more immunogenic by the anti-CD45 Pacific blue (Catalog No. 103126); anti-CD103 BV510 addition of a-MerTK antagonist, and indeed we observed (Catalog No. 121423), anti-PD1 FITC (Catalog No. 135214), improved abscopal tumor control from this triple therapy. We anti-CD49b PE (Catalog No. 108907), anti-CD69 PE-Cy7 (Cat- þ further found this effect to be dependent on CD8 Tcellsand alog No. 104511), anti-Gr1 BV510 (Catalog No. 108437), anti- þ NKcellsandtobemediatedbyincreasednumbersofCD8 CD11b APC Fire750 (Catalog No. 101262), and anti-F4/80 FITC þ fl CD103 tissue-resident memory cells (TRM)atthetumorsite. (Catalog No. 123108) for ow cytometry (LSRII). All antibodies

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were purchased from BioLegend. Flow cytometry data were ana- Results lyzed with FlowJo software. Triple therapy (RT þ a-PD1 þ a-MerTK) has significant abscopal effects in a 344SQ NSCLC tumor model NanoString molecular analysis The schedule for creating the mouse model of 344SQ lung TILs were isolated as described above, and lysed for RNA adenocarcinoma is shown in Fig. 1A. Briefly, mice were first extraction. For quality assurance, a fragment analysis system was injected subcutaneously with 0.5 106 344SQ-P cells in their used to assess nucleic acid fragmentation, with optimal perfor- right hind legs (to establish the primary tumor), and received a mance defined as 50% of samples of more than 300 nucleotides in second subcutaneously injection of 0.1 106 344SQ-P cells in length. Samples were analyzed with the NanoString nCounter the left hind legs (to establish the abscopal tumor). Four a-PD1 Immune Panel by the Genomic & RNA Profiling Core at Baylor doses were given intraperitoneally at 200 mg/dose on days 5, 8, College of Medicine. 11, and 14; high-dose radiation (36 Gy in 3 12-Gy fractions) was given to the primary tumors on days 7, 8, and 9; and 6 IHC staining and analysis doses of a-MERTK were given (also intraperitoneally at 200 mg/ Tumors were harvested 7 days after the last fraction of dose) on days 7, 10, 12, 15, 19, and 22. The radiation dose was radiation. Tumor samples were fixed in 10% neutral buffered picked after optimization for apoptosis rates (Supplementary formalin for 24 hours then washed in PBS at room temperature. Fig. S1). At day 21, half of each treatment group was evaluated Caspase 3 activity, for assessment of apoptosis, was evaluated for tumor growth delay and survival and the other for tumor- using Polink-2 plus HRP anti-Rabbit Detection Kit for IHC infiltrating leukocytes (TIL). (Catalog No. D39–110). The whole process was performed at The triple therapy extended mouse survival beyond day 55 MD Anderson IHC cores. Digital quantification was performed (P < 0.0001; Fig. 1B). The control, a-MerTK-only, and a-MerTK using ImageJ software. þ a-PD1 conditions did not delay growth of the primary (irradiated) tumor (Fig. 1C). The triple therapy significantly Statistical analysis controlled the primary tumors in all mice comparing to All statistical analyses were done with Graph Pad Prism 7 control group. The triple therapy led to substantial growth software. Tumor growth curves were compared with multiple t delay at the abscopal site (Fig. 1D) as compared with RT only P P tests. Mouse survival was analyzed by using the Kaplan–Meier ( < 0.001) and suppressed the number of lung metastases ( < method and compared with log-rank tests. Statistical significance 0.001; Fig. 1E) in this model. was defined as P 0.05. Triple therapy increased M1 macrophages at primary and Study approval abscopal tumor sites Protocols for animal use, treatment, and euthanasia were To explore the mechanism underlying the delay in abscopal approved by the Institutional Animal Care and Use Committee tumor growth from the triple therapy, we collected primary and of The University of Texas MD Anderson Cancer Center. abscopal tumors on day 21 and evaluated them for myeloid cell

Figure 1. Triple therapy (RT þ a-PD1 þ a-MerTK) inhibited tumor growth, improved survival rates, and reduced lung metastases in a mouse model of lung cancer. A, Mice (5 per group) were inoculated in the hind legs with 344SQ non–small cell lung cancer cells, with the right leg considered the primary tumor (and therefore irradiated) and the left leg the abscopal (unirradiated) tumor. Mice were treated with IgG (CTRL), a-MerTK, radiation [RT] (36 Gy in 3 12-Gy fractions), a-PD1þ a-MerTK, RT þ a-PD1, and RTþ a-PD1 þ a-MerTK as shown. Abs were given as intraperitoneal injections of 200 mg/injection. B, Survival of mouse treatment groups from A (, P ¼ 0.0002) RTþ a-PD1 versus RTþ a-PD1 þ a-MerTK. C and D, Tumor growth curves (plotted as average diameters from each mouse at each time point) indicated that triple therapy (RTþ a-PD1 þ a-MerTK) was superior to RT only for suppressing abscopal tumor growth (P ¼ 0.000087). E, Lung metastasis counts showed a decrease with the triple group. All experiments were done twice under the same schedule and with the same numbers of mice (5 per group) to conﬁrm the results.

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þ þ populations including tumor-associated macrophages (TAM) Granzyme B populations. No changes in CD8 cells were found and granulocyte (G)-MDSCs by flow cytometry. The triple therapy in the primary (irradiated) tumors in any treatment condition, but þ did not affect the number of TAMs in the primary tumors the triple therapy led to a significant increase in activated CD8 (Fig. 2A), but greatly increased the number of TAMs at the cells versus RT only group in the abscopal (unirradiated) tumors abscopal tumors relative to the radiation-only condition (P ¼ (P ¼ 0.0072; Fig. 3E and F). Similarly, activated NK cells were only 0.0402; Fig. 2B). Similarly, triple therapy did not affect numbers observed in the abscopal tumors with the triple therapy (P ¼ of G-MDSCs in the primary tumors (Fig. 2C) but greatly decreased 0.0028; Fig. 3G and H). the G-MDSCs in the abscopal tumor relative to radiation-only (P Further investigation at the level of gene expression using ¼ 0.0351; Fig. 2D). Finally, the triple therapy significantly Immuno-NanoString showed that triple therapy led to signif- increased the percentage of M1 macrophages at both the primary icant increases (relative to radiation-only) in Pecam (CD31) tumors (P ¼ 0.0279) and the abscopal tumors (P ¼ 0.0256; Fig. 2E and STAT4 expression at the abscopal tumor sites (Fig. 3I). and F). Abscopal tumors also showed increased expression of the IFN regulator factor-1 (IRF-1), which activates the expression of Triple therapy activated NK cells and cytotoxic T lymphocytes at IFNb (24) and p53 (25), after triple therapy (Fig. 3I). Addi- the abscopal tumor sites tionally, there was significant increase in IL12 and TNFa No differences were found among the various treatment groups expression with the triple therapy compared with no treatment, þ in percentages of CD4 lymphocytes in the tumor microenviron- although IL6 expression was not significantly modulated (Sup- ment at either the primary or abscopal tumor sites (Fig. 3A and B). plementary Fig. S2). þ Similarly, no differences were found in CD8 T cells at the irradiated (primary) tumor sites between the triple therapy and Effects of triple therapy on abscopal tumors depend strongly on þ radiation-only conditions (Fig. 3C), although an increase may the presence of both NK and CD8 T cells þ have been present at the unirradiated (abscopal) tumor sites (P ¼ Next, to evaluate the role of activated CD8 effector T cells and 0.0639; Fig. 3D). activated NK cells in the response to triple therapy, we depleted þ þ Next, we quantified activated cytotoxic CD8 T cells and pan- CD8 cells, NK cells, or both and analyzed the effects on survival NK cells among TILs isolated from the primary and abscopal and tumor control at the primary (irradiated) and abscopal þ þ þ tumors by gating those TILs on CD8 Granzyme B and CD49b (unirradiated) tumor sites. Survival was enhanced in the

Figure 2. Triple therapy increased M1 TAMs and suppressed myeloid precursors at the abscopal tumor sites. Tumors were harvested, processed, and analyzed by ﬂow cytometry on day 21. A and B, Percentages of TAMs (CD45þ Gr1intermediate CD11bþ F4/80þ) were analyzed at both primary (A) and abscopal (B) tumor sites. C and D, Percentages of G-MDSCs (CD11bþ Gr-1hi/Ly-6Gþ) were not affected by any of the tested treatments at the primary tumor site (C) but were suppressed after triple therapy at the abscopal sites (D). E and F, Percentages of M1 macrophages (CD38þ cells gated on TAMs) were quantiﬁed at the primary and abscopal sites in each treatment group. The triple therapy increased the proportion of M1 (relative to radiation-only) at both the primary site (P ¼ 0.0279) and at the abscopal site (P ¼ 0.0256).

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Figure 3. Triple therapy promoted activation of NK and CD8þ T cells. Tumors were harvested, processed, and analyzed by ﬂow cytometry. A and B, Triple therapy did not affect percentages of CD4þ T cells at either the primary or the abscopal tumor sites. C and D, Triple therapy did not affect percentages of CD8þ T cells at the primary tumor site (C) or at the abscopal tumor site relative to radiation-only (P ¼ 0.0639) (D). E–H, Similarly, triple therapy did not affect the percentages of either activated CD8þ cells [CD8þGranzyme Bþ (E)] or activated NK cells (G) at the primary tumor sites, but increased the levels of both at the abscopal tumor sites (F) CD8þ cells with P ¼ 0.0072, and (H) NK cells with P ¼ 0.028 relative to radiation-only. I, NanoString heat map analysis showed that triple therapy led to increases in the expression of Pecam (CD31) and STAT4 (relative to radiation-only) at the abscopal tumor sites.

þ þ þ CD8 /NK -proﬁcient condition relative to the CD8 -depletion depletion also suppressed tumor growth at the primary and þ condition (P ¼ 0.0196), the NK-depletion condition (, P ¼ abscopal sites (Fig. 4B and C). Finally, depletion of CD8 and þ þ 0.0074), and the combined CD8 - and NK-depleted condition NK cells abrogated the suppressive effect of triple therapy on the þ þ (P ¼ 0.0064; Fig. 4A). Triple therapy without CD8 and NK number of lung metastases (P ¼ 0.0022; Fig. 4D).

Figure 4. Depletion of NK and CD8þ cells abolished the effect of triple therapy. A, Mice (5 per group) were inoculated with 344SQ cells as described in the text and in Fig. 1A. Mice were given one of 5 treatments: (IgG [CTRL]), triple therapy (RT þ anti-PD1 þ anti-MerTK), triple therapy with CD8-cell depletion, triple therapy with NK-cell depletion, and triple therapy with both NK-cell and CD8-cell depletion. All depletion agents were injected intraperitoneally (200 mg/injection). Mouse survival was recorded and compared between treatment groups with log-rank tests. Survival was enhanced in the CD8 and NK proﬁcient condition relative to the CD8-cell depletion condition (P ¼ 0.0196), the NK-cell depletion condition (P ¼ 0.0074), and the combined CD8- and NK-depleted condition (P ¼ 0.0064). B and C, Measurements of tumor diameters plotted over time show that triple therapy (RT þ a-PD1 þ a-MerTK) inhibited tumor growth to a greater extent than any of the other treatment conditions. D, Depletion of CD8þ cells and NK cells also abrogated the suppressive effect of triple therapy on a number of lung metastases (, P ¼ 0.0022 for triple-therapy vs. triple therapy þ CD8 and NK cells depletion).

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Triple therapy prolongs the adaptive antitumor immune with digital calipers. Four a-PD1 doses were given intraperi- þ þ response by increasing the numbers of CD8 CD103 TRM cells toneally at 200 mg/dose on days 5, 8, 11, and 14; high-dose We harvested both primary and abscopal tumors from mice on radiation (36 Gy in 3 12-Gy fractions) was given to the primary þ þ day 21, isolated TILs, and gated those cells on CD8 CD103 to tumors on days 7, 8, and 9; and 6 doses of a-MERTK were given identify TRM cells. The triple therapy increased the percentage of (also intraperitoneally at 200 mg/dose) on days 7, 10, 12, 15, þ þ CD8 CD103 cells at the primary tumor site relative to the 19, and 22 following the same experimental timeline. The radiation þ a-PD1 condition (P ¼ 0.0142; Fig. 5A). Triple therapy triple therapy extended mouse survival beyond day 60 (P < þ þ also increased the percentage of CD8 CD103 cells at the 0.0001; Fig. 6A). The control, a-MerTK-only, and a-MerTK þ abscopal tumor site relative to the radiation-only condition (P a-PD1 conditions did not delay growth on both the primary ¼ 0.0456) and relative to the radiation þ a-PD1 condition (P ¼ (irradiated) and abscopal tumor sites (Fig. 6B) whereas the 0.0663; Fig. 5B). triple therapy significantly inhibited the tumor growth mainly þ þ Our discovery of CD8 CD103 TRM cells in the tumor on the abscopal tumor sites (Fig. 6C). We harvested both microenvironment and their expression of PD1 (Supplemen- primary and abscopal tumors from the triple therapy group tary Fig. S3) led us to hypothesize that the effects of the triple on day 60, isolated TILs, and subjected the samples to flow therapy could be enhanced (in terms of extending survival) if cytometryanalysis.Thetripletherapy increased the percentage þ þ we continued the a-PD1 treatment throughout the experiment, of CD8 CD103 cells at both tumor sites relative to no rather than stopping at 4 doses, which presumably would treatment condition (Fig. 6D). protect the TRM cells from exhaustion by blocking the PD1– PDL1 interaction. Indeed, we found that continuing the a-PD1 after the triple therapy extended survival to day 63 (P < 0.0001) Discussion (Fig. 5C). Standard therapy for early-stage lung cancer usually involves some form of local therapy, either radiation or surgical resec- Triple therapy (RT þ a-PD1 þ a-MerTK) promotes significant tion (1). However, the use of radiation is also being explored for abscopal effects in pancreatic tumor model and increases the its ability to elicit systemic responses to control tumors outside þ þ percentage of CD8 CD103 cells the radiation fields (i.e., "abscopal effects"; ref. 26). Abscopal To confirm our 344SQ-P results, we performed an additional responses are rare when radiation is used alone or in combination experiment using another cell line (pancreatic cell line PANC- with chemotherapy, and thus data on survival outcomes for 02)inC57BL/6mice.Themicewereinoculatedbysubcuta- patients whose tumors show abscopal effects are rare as well (27). neous injection of PANC-02 tumor cells into the right hind leg Our prior preclinical (4) and clinical studies (NCT02402920) (to establish primary tumors) and the left hind leg (to establish indicate that combining high (stereotactic) radiation doses with abscopal tumors). Tumor growth was monitored twice a week targeted immune checkpoint blockade such as a-PD1 induces

Figure 5. þ þ Supplementing radiation (RT) with a-MerTK and a-PD1 increases the percentage of CD8 CD103 TRM cells. Tumors were harvested, processed, and analyzed by þ þ þ þ ﬂow cytometry on day 21 for CD8 CD103 (TRM) cells. A and B, Triple therapy led to increases in CD8 CD103 TRM cells at both the primary site [P ¼ 0.0142 vs. RT þ a-PD1 (A)] and the abscopal site [P ¼ 0.0456 vs. RT-only (B)]. C, Maintenance a-PD1 therapy (i.e., beyond the prescribed 4 doses) prolonged mouse survival [log-rank P < 0.0001)].

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Figure 6. Triple therapy (RT þ a-PD1 þ a-MerTK) improved survival rates by inhibiting tumor growth in pancreatic carcinoma model. C57BL/6 mice (5 per group) were inoculated in the hind legs with PANC-02 cancer cells and treated with IgG (CTRL), a-MerTK, radiation [RT] (36 Gy in 3 12-Gy fractions), a-PD1þ a-MerTK, RT þ a-PD1, and RTþ a-PD1 þ a-MerTK following the experimental design in Fig. 1A. A, Survival percentages of treatment groups. RTþ a-PD1 versus RTþ a-PD1 þ a-MerTK (, P ¼ 0.0001). B and C, Tumor growth curves (plotted as average diameters of tumors at each time point) indicated that triple therapy (RTþ a-PD1 þ þ þ a-MerTK) suppressed primary (P ¼ 0.0003) and abscopal tumor growth (P ¼ 0.0007). D, Triple therapy increased CD8 CD103 TRM cells at both the primary site (P ¼ 0.004) and the abscopal site (P ¼ 0.01) versus no treatment group. synergetic effects that both control tumor growth at the local level sis (34) and re-polarize M2 macrophages towards M1 phenotype and stimulate a systemic antitumor response by upregulating at both primary and abscopal tumor sites to strengthen antitumor MHC-I molecules on the tumor cells through the induction of immunogenic cell death. We also noted that a-PD1, in combi- type I IFNs (4). nation with a-MerTK, acts to augment long-lasting adaptive Our findings from the current study suggest that adding immunity of radiation by increasing the percentage of tumor- a-MerTK and a-PD1 further augments the adaptive immune- specific memory cells at abscopal tumor sites, which were not mediated effect of radiation by increasing the percentage of subject to the long-term suppressive effect of high-dose þ þ CD8 CD103 TRM cells at the abscopal tumor sites. These radiation (35, 36). cells are a tumor-specific cytotoxic T lymphocyte subpopula- We further found that the significant tumor growth delays, tion that accumulates in the tumor microenvironment (28, 29) improved survival rates, and limitation of metastatic spread and express CD103 [aE (CD103) b7] and CD49a [a1 (CD49a) resulting from triple therapy is mediated by activated NK and þ b1] integrins along with the C-type lectin CD69, which col- CD8 T lymphocytes, as verified in our depletion studies. The þ lectively help these cells to migrate into tumor islets by stability of NK-cell and CD8 T-cell activation at the abscopal þ binding to E-cadherin, leading ultimately to T-cell receptor– metastatic tumor sites is possibly related to the presence of CD8 þ dependent cell killing. Our NanoString molecular analysis CD103 TRM cells. Our phenotyping data of abscopal tumors after results showed that the Pecam (CD31) gene, known to par- the triple therapy showed significant increase in percentages of ticipate in integrin activation (30), was upregulated at the TRM cells that was associated with tumor response and shrinkage. abscopal tumor sites. However, we did not specifically deplete the TRM population that On the other hand, radiation is also known to upregulate PDL1 may reflect direct mechanistic link. Our NanoString data showed þ þ on tumor cells (31). We found here that CD8 CD103 TRM cells significant upregulation in IL12 accompanied with upregulation have high expression of PD1, which provides a rationale for in STAT4 expression. In agreement with our findings, others have þ þ adding a-PD1 at the induction phase (before RT), concurrent shown that IL12 may act on CD8 CD103 TRM cells to produce with RT, and in the maintenance phase (after RT and MerTK IFNg via STAT4 transcription, which enhances antigen-specific blockade). This is consistent with other studies showing that long- immunity (37, 38). term treatment with a-PD1 protects against CD4 and CD8 T-cell The general lack of effect of the triple therapy at the primary exhaustion (32). In our study, we found that continuing a-PD1 as tumor site (relative to the abscopal tumor results) seems to maintenance therapy extended survival time, presumably by validate our previous studies that high-dose radiation upregulates þ þ þ preserving the adaptive immune effects and function of the Tregs (CD4 CD25 ; ref. 39), which are known to suppress CD8 observed TRM cells (33). T cells and immune responses at the primary tumor. Our data also Macrophages expressing MerTK have been shown to engulf showed significant decrease in the percentage of the immuno- apoptotic bodies of dying tumor cells and convert into M2 suppressive G-MDSCs only at the abscopal site, with no effect on phenotype (17). MerTK blockade was essential to shift the apo- the primary tumor site, by adding a-PD1 in combination with ptotic cell death caused by radiation towards secondary necro- a-MerTK to the radiation treatment.

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Figure 7. Anti-MerTK, in combination with a-PD1 and radiation, reprograms radiation-induced cell death and strengthens the antitumor immune response. Triple therapy tilts the balance from apoptotic cell death to secondary necrosis, resulting in repolarization of macrophages to the M1 phenotype, increases activation of NK and þ þ þ CD8 T cells at the abscopal tumor sites, and increases the proportion of CD8 CD103 TRM cells.

þ þ Increasing the percentage of CD8 CD103 TRM cells would Alpine Immune Sciences, AstraZeneca, Merck, Incyte, and Aileron, have robust clinical application because the presence of these reports receiving commercial research grants from GlaxoSmithKline, Bris- cells would influence survival among patients with NSCLC. tol-Myers Squibb, Merck, Nanobiotix, Mavo Pharma, and Checkmate þ þ Pharmaceuticals, and holds ownership interest (including patents) in CD8 CD103 TRM cells reside in tumor islets, where they Helios Oncology, Molecular Match, OncoResponse, Radscopal, and is listed mediate long-term tumor-specific memory (29); we believe as a co-inventor on patent applications on MP470 and MRX34 regulation thatincreasingthenumbersofthesecellswouldbeofpartic- of PDL1. No potential conflicts of interest were disclosed by the other ular benefit for patients who develop recurrence after radiation authors. plus a-PD1 therapy. We propose the following conceptual framework (Fig. 7) as Authors' Contributions one possible explanation for the results of this study. First, Conception and design: M.S. Caetano, A.I. Younes, T. Spires, T.P. Reilly, radiation initiates the tumor-death process while blockade of T.R. Cushman, J.E. Schoenhals, A. Li, J.W. Welsh MerTK shifts the manner of cell death in the tumor microen- Development of methodology: M.S. Caetano, A.I. Younes, T.P. Reilly, T.R. Cushman, J.W. Welsh vironment from tolerogenic (apoptotic) to nontolerogenic Acquisition of data (provided animals, acquired and managed patients, (immunogenic; Fig. 7), and a-PD1 sustains the resulting þ provided facilities, etc.): M.S. Caetano, A.I. Younes, T.P. Reilly, immune effects. Radiotherapy basically primes the CD8 T- T.R. Cushman, T.R. Cushman, J.E. Schoenhals, J.W. Welsh effector lymphocytes by binding to the upregulated MHC-I Analysis and interpretation of data (e.g., statistical analysis, biostatistics, molecules on the tumor surface. The triple of radiation, anti- computational analysis): M.S. Caetano, A.I. Younes, H.B. Barsoumian, PD1, and anti-MerTK maximizes this priming and leads to M. Quigley, H. Menon, T.P. Reilly, T.R. Cushman, J.W. Welsh þ þ Writing, review, and/or revision of the manuscript: M.S. Caetano, generation of CD8 CD103 T cellsinthetumorisletsat RM A.I. Younes, H.B. Barsoumian, M. Quigley, H. Menon, C. Gao, T.P. Reilly, the abscopal tumor sites. The triple therapy also enhances the þ A.P.Cadena,T.P.Reilly,T.R.Cushman,A.Li,Q.-N.Nguyen,M.Angelica activation of NK and CD8 T cells, mainly at the abscopal Cortez, J.W. Welsh tumor sites. Finally, continuing the anti-PD1 treatment would Administrative, technical, or material support (i.e., reporting or organizing prolong outcomes elicited by the radiation and a-MerTK and data, constructing databases): M.S. Caetano, A.I. Younes, M. Quigley, J.W. Welsh overcome TRM-cell exhaustion. Study supervision: M. Angelica Cortez, J.W. Welsh Other (illustrations/figures): T.P. Reilly Disclosure of Potential Conflicts of Interest M. Quigley, T.E. Spires, and T.P. Reilly are employees/paid consultants Acknowledgments for and hold ownership interest (including patents) in Bristol-Myers Squibb. This work was supported and funded by Bristol-Myers Squibb, and further J.W. Welsh is an employee/paid consultant for Reflexion Medical, Molecular supported by the family of M. Adnan Hamed, the Susan and Peter Goodwin Match, Onco Response, Checkmate Pharmaceuticals, Mavu Pharmaceuticals, Foundation, the Orr Family Foundation (to MD Anderson Cancer Center's

OF8 Clin Cancer Res; 2019 Clinical Cancer Research

RT with MerTK and PD1 Blockade Promotes Abscopal Responses

Thoracic Radiation Oncology program). We also have support from the MD The costs of publication of this article were defrayed in part by the payment of Anderson Knowledge Gap award, Doctors Cancer Foundation Grant, The Lung page charges. This article must therefore be hereby marked advertisement in Cancer Research Foundation, Cancer Center Support (Core) Grant CA016672 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. from the NCI (to The University of Texas MD Anderson Cancer Center). We would like to thank Christine F. Wogan for reviewing and editing the Received March 8, 2019; revised July 10, 2019; accepted September 17, 2019; manuscript. published ﬁrst September 20, 2019.

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www.aacrjournals.org Clin Cancer Res; 2019 OF9

Triple Therapy with MerTK and PD1 Inhibition Plus Radiotherapy Promotes Abscopal Antitumor Immune Responses

Mauricio S. Caetano, Ahmed I. Younes, Hampartsoum B. Barsoumian, et al.

Clin Cancer Res Published OnlineFirst September 20, 2019.

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