Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination

Abstract

Indolent non-Hodgkin’s lymphomas (iNHLs) are incurable with standard therapy and are poorly responsive to checkpoint blockade. Although lymphoma cells are efficiently killed by primed T cells, in vivo priming of anti-lymphoma T cells has been elusive. Here, we demonstrate that lymphoma cells can directly prime T cells, but in vivo immunity still requires cross-presentation. To address this, we developed an in situ vaccine (ISV), combining Flt3L, radiotherapy, and a TLR3 agonist, which recruited, antigen-loaded and activated intratumoral, cross-presenting dendritic cells (DCs). ISV induced anti-tumor CD8+ T cell responses and systemic (abscopal) cancer remission in patients with advanced stage iNHL in an ongoing trial (NCT01976585). Non-responding patients developed a population of PD1+CD8+ T cells after ISV, and murine tumors became newly responsive to PD1 blockade, prompting a follow-up trial of the combined therapy. Our data substantiate that recruiting and activating intratumoral, cross-priming DCs is achievable and critical to anti-tumor T cell responses and PD1-blockade efficacy.

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Fig. 1: Cross-presentation is required to control lymphoma growth in vivo.
Fig. 2: In situ vaccination with FLt3L, XRT and pICLC induces systemic tumor immunity dependent on cross-presenting dendritic cells.
Fig. 3: In situ vaccination mediates tumor infiltration by CD8+ T cells and augments checkpoint blockade therapy.
Fig. 4: ISV induces TLR3+ DCs and PD1+ T cells in patients with indolent B cell lymphoma.
Fig. 5: In situ vaccination induces systemic tumor regression in patients with indolent B cell lymphoma.

Data availability

The datasets supporting the findings presented in this study are available from the corresponding author upon reasonable request. All requests for data and materials will be promptly reviewed by the Icahn School of Medicine at Mount Sinai to verify whether the request is subject to any intellectual property or confidentiality obligations. Patient-related data were generated as part of a clinical trial and may be subject to patient confidentiality. Any data that can be shared will be released via a Material Transfer Agreement.

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Acknowledgements

We thank the ISMMS human immune monitoring core for help with mass cytometry experiments and Luminex assay; the ISMMS flow cytometry core; and the CCMS animal facility. We also thank N. Sadek and C. Kappauff for technical assistance and N. Bhardwaj for critical review of the manuscript. J.D.B. was supported by a Damon Runyon Cancer Research Foundation Clinical Investigator Award (no. 45367), by an Investigator Studies Program (MSIP) Award (no. 10677), and by a Cancer Research Institute - Clinic & Laboratory Integration Program (CLIP, no. 45367) Award. L.H. was supported by a research fellowship of the German research foundation (DFG, HA 7431/1-1). B.D.B. and M.M. were supported by NIH U19AI128949.

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Contributions

L.H. designed the study, performed and analyzed experiments, interpreted the results and wrote the manuscript. T.U.M. was involved in human sample collection and analysis. R.U. performed and analyzed cell culture experiments. J.S.-A, M.D., S.H. and Y.Z. performed immunohistochemistry. D.O. was involved in human sample collection. M.Y. and H.M. provided CDX-301. A.M.S. provided pICLC. A.H.R. performed and analyzed mass cytometry experiments. B.D.B. and M.M. helped design experiments and wrote the manuscript. J.D.B. conceived the study, supervised the design and execution of the study, interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Joshua D. Brody.

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Competing interests

Research support for these studies was provided by Merck & Co., Celldex Therapeutics, Oncovir, Inc. and Genentech. J.D.B. has received research funding from Acerta Pharma, Bristol Myers Squibb, Genentech, Gilead Sciences, Seattle Genetics, Pharmacyclics and Celgene. M.Y. and H.M. are employed by Celldex Therapeutics. A.M.S. is employed by Oncovir, Inc. The authors declare no other competing financial interests.

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Extended data

Extended Data Fig. 1 Cross-presentation is required to control lymphoma growth in vivo.

a, GFP- and mCherry-expressing tumor cell lines (ratio1:1) were cocultured with JEDI TCs pre-activated with anti-CD3/anti-CD8. Tumor cell killing was analyzed after 3 d. One representative out of three independent experiments is shown. b, A20-GFP lymphoma-bearing Balb/c- and C57Bl/6-Rag1–/– mice were injected with 5 × 103 GFP-specific JEDI CD8+ TCs, and tumors were analyzed for transferred TCs by flow cytometry 2 weeks after transfer. Representative dot plots from n = 9 (Balb/c) or n = 15 (B6) mice from two independent experiments. c, WT mice were inoculated with A20 tumors and tumor were stained for TCs (CD3) and DC (CD11c) after 3 weeks. Representative FACS plots (left), immunofluorescence (middle) and quantification of intratumoral TCs and DC numbers (right). n = 10 from two independent experiments. Data are presented as mean ± s.e.m .

Extended Data Fig. 2 Flt3L induces recruitment of CD103 precursors.

a,b, Tumor-bearing Balb/c mice were treated with Flt3L and analyzed for DC expansion in the tumor (a) and bone marrow (b). n = 8 mice pooled from two independent experiments. One-way ANOVA with Tukey’s post test. c, Representative dot plots of CD11b+ and CD103+ cells among all intratumoral dendritic cells (one out of four independent experiments) and of IRF8+ and IRF4+ cells among DN dendritic cells in the tumor (one out of two independent experiments). d,e, Intratumoral DC subsets were isolated from Flt3L-treated mice by flow sorting and cultured in the presence of bone marrow feeder cells with or without Flt3L (d) or cocultured with irradiated A20 cells (e). CD103 and XCR1 (d) or CD11b (e) expression was analyzed after 3 d of culture. One representative out of two independent experiments with n = 3 or 4 mice each is shown. f, Splenocytes from Flt3L-treated mice were irradiated with 9 Gy in vitro and analyzed for CD103 expression on DC subsets after 3 d. One representative out of four independent cell culture samples is shown. Data are presented as mean ± s.e.m.

Extended Data Fig. 3 local XRT of the tumor induces maturation and activation of TLR3+ dendritic cells.

a,b, Splenocytes isolated from Flt3L-treated mice were cultured in the presence of supernatant from irradiated A20 cells and either a HMGB1-blocking antibody or TLR agonists. Activation of dendritic cells was analyzed after 48 h. One-way ANOVA with Tukey’s post test. Representative experiment (n = 2 biologically independent cell culture samples) out of three independent shown. c, HMGB1 levels in the serum of irradiated mice (0 Gy: n = 5, 9 Gy: n = 6, 9 Gy no tumor: n = 3; left panel) or supernatants of irradiated A20 cells (24 h: n = 7, 48 h n = 5; right panel). Data pooled from two or three independent experiments, respectively. One-way ANOVA with Tukey’s post test. d,e, Flt3L-treated splenocytes were cultured with irradiated GFP+ A20 cells with or without pIC for 24 h and then cocultured with JEDI splenocytes for 4 d. Activation of GFP-specific JEDI CD8+ TCs was analyzed by proliferation. Data pooled from five independent experiments (n = 10 biologically independent cell culture samples). One-way ANOVA with Tukey’s post test. f, Tumor-bearing WT or Batf3–/– mice were treated with Flt3L and analyzed for intratumoral DC subsets 1 d after the last injection. Representative plots from n = 4 mice per group. Data are presented as mean ± s.e.m.

Extended Data Fig. 4 In situ vaccination induces upregulation of checkpoint molecules and augments checkpoint blockade therapy.

ac, A20-GFP tumor-bearing mice were treated with Flt3L, XRT and pICLC as indicated. 1 × 106 JEDI CD8+ TCs were adoptively transferred 1 d after XRT and intratumoral TCs were analyzed by flow cytometry 7 d after transfer. a, CD25 expression on GFP-specific vs. non-specific CD8+ TCs in the tumor and TdLN of ISV-treated mice. n = 3, unpaired two-tailed t test. b, TIM3 and LAG3 expression on bulk CD8+ TC in the tumor of untreated and ISV-treated mice. n = 3, unpaired two-tailed t test. c, PD1 expression on GFP-specific vs. non-specific CD8+ TCs in the TdLN of untreated and ISV-treated mice. n = 3, unpaired two-tailed t test. d, A20-lymphoma-bearing mice were treated with ISV, and i.t. DCs were analyzed for PD-L1 expression after three injections of pICLC. n = 6 (untreated) or n = 7 (F/X/IC) from two independent experiments, unpaired two-tailed t test. e, A20-lymphoma-bearing mice were treated with ISV and systemic anti-PD1 or isotype control antibody and followed for tumor growth and survival after indicated treatment. n = 18, except Flt3L+XRT+pIC n = 19 and XRT + pIC + anti-PD1 n = 21; two-way ANOVA with Bonferroni post test. f, IFNγ production and CD137 expression by splenic CD8+ TCs isolated from tumor-bearing mice treated as indicated 1 week after the last pICLC injection and cocultured with irradiated tumor cells for 48 h. n = 4 mice per group, one representative experiment out of two independent experiments shown, one-way ANOVA with Tukey’s post test. Data are presented as mean ± s.e.m.

Extended Data Fig. 5 In situ vaccination induces DC expansion and intratumoral TC infiltration in patients with indolent B cell lymphoma.

a,b,11 patients with indolent non-Hodgkin’s B cell lymphoma were treated with intratumoral Flt3L, local XRT of the tumor and intratumoral pICLC. PBMCs collected before the start of treatment and 1 d after the last Flt3L injection were analyzed by CyTOF. a, Relative numbers of DC cell subsets among all live cells. Paired two-tailed t test. b, Relative numbers of classical DC subsets among all cDC. Paired two-tailed t test. c, PBMCs collected before and after Flt3L treatment were stimulated with pIC, and cytokine production in the supernatant was analyzed by Luminex. n = 11, one-way ANOVA with Tukey’s post test. d,e, tumor biopsies from one patient with partial response collected before treatment and 6 months post vaccination were analyzed for presence of lymphoma (κ-LC+) B cells (d) and TCs (e) by flow cytometry. f, Pearson correlation of frequency of DC subsets pretreatment with abscopal BOR (n = 11). Data are presented as mean ± s.e.m.

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Hammerich, L., Marron, T.U., Upadhyay, R. et al. Systemic clinical tumor regressions and potentiation of PD1 blockade with in situ vaccination. Nat Med 25, 814–824 (2019). https://doi.org/10.1038/s41591-019-0410-x

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