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Background PD-1 and PD-L1 inhibitors have poor efficacy in patients with trastuzumab-resistant advanced HER2-positive breast cancer. Tucatinib is a potent, selective anti-HER2 tyrosine kinase inhibitor with proven clinical benefit in the advanced setting in patients with trastuzumab resistance. We investigated if tucatinib can alter the tumor microenvironment and if this could be harnessed for therapeutic efficacy. Methods We investigated the anti-tumor efficacy and contribution of the immune response of tucatinib using two immunocompetent, HER2-positive murine breast cancer models (trastuzumab-sensitive H2N113; trastuzumab-resistant Fo5) and the efficacy of tucatinib with trastuzumab and PD-1/PD-L1 checkpoint inhibitors. Results In both models, tucatinib significantly inhibited tumor growth and demonstrated dose-dependent efficacy. Ex vivo analysis by flow cytometry of TILs in mice treated with tucatinib showed increased frequency, higher proliferation and enhanced effector function of CD8+ effector memory T cells (TEM). Tucatinib treatment also increased frequency of CD8+PD-1+ and CD8+TIM3+ T cells, CD49+ NK cells, monocytes, and MHC II expression on dendritic cells and macrophages, and a decrease in myeloid derived suppressor cells. Gene expression analysis revealed significant enrichment in pathways associated with immune activation, type I and II interferon response, adaptive immune response and antigen receptor signalling. In vivo, tucatinib and α-PD-L1 or α-PD-1 demonstrated significantly increased efficacy and improved survival of mice compared with tucatinib alone. Conclusion Tucatinib modulates the immune microenvironment favourably and combination treatment with α-PD-L1 or α-PD-1 demonstrated increased efficacy in preclinical HER2-positive tumor models. These findings provide a rationale for investigation of tucatinib and immune checkpoint inhibition in the clinic.

Patients who receive chimeric antigen receptor (CAR)–T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8 + memory T cell progenitors that can become either functional stem-like T (T STEM ) cells or dysfunctional T progenitor exhausted (T PEX ) cells. To that end, we demonstrated that T STEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate T STEM -like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, T STEM -like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4 + T cells during T STEM -like CAR-T cell production. Adoptive transfer of T STEM -like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of T STEM -like CAR-T cells and an increased memory T cell pool. Last, T STEM -like CAR-T cells and anti–programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8 + CAR + T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated T STEM -like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.

Immunotherapies aimed at alleviating the inhibitory constraints on T cells have revolutionized cancer management. To date, these have focused on the blockade of cell-surface checkpoints such as PD-1. Herein we identify protein tyrosine phosphatase 1B (PTP1B) as an intracellular checkpoint that is upregulated in T cells in tumors. We show that increased PTP1B limits T-cell expansion and cytotoxicity to contribute to tumor growth. T cell–specific PTP1B deletion increased STAT5 signaling, and this enhanced the antigen-induced expansion and cytotoxicity of CD8+ T cells to suppress tumor growth. The pharmacologic inhibition of PTP1B recapitulated the T cell–mediated repression of tumor growth and enhanced the response to PD-1 blockade. Furthermore, the deletion or inhibition of PTP1B enhanced the efficacy of adoptively transferred chimeric antigen receptor (CAR) T cells against solid tumors. Our findings identify PTP1B as an intracellular checkpoint whose inhibition can alleviate the inhibitory constraints on T cells and CAR T cells to combat cancer.Significance:Tumors subvert antitumor immunity by engaging checkpoints that promote T-cell exhaustion. Here we identify PTP1B as an intracellular checkpoint and therapeutic target. We show that PTP1B is upregulated in intratumoral T cells and that its deletion or inhibition enhances T-cell antitumor activity and increases CAR T-cell effectiveness against solid tumors.This article is highlighted in the In This Issue feature, p. 587

Movie 2. CAR.OT-I cells co-cultured with MC57-OVA257 cells.

Figure legends for supplementary figures 1, 2 & 3

This figure shows the proliferation and phneotype of tumor lines in response to NECA.

Supplementary Figure 2

This supplementary Figure shows the phenotype and function of CD8+ T cells and tumor cells following A2A blockade single agent therapy.

This supplementray Figure shows the expression of IFNy and Granzyme B on tumor-infiltrating lymphocytes 7 days post anti-PD-1/ anti-PD-1/A2A blockade.

The effect of combination treatment on T cell activation

The lack of targeted therapies available for triple-negative breast cancer (TNBC) patients who fail to respond to first-line chemotherapy has sparked interest in immunotherapeutic approaches. However, trials utilizing checkpoint inhibitors targeting the PD-1/PD-L1 axis in TNBC have had underwhelming responses. Here, we investigated the interplay between type I IFN signaling and the PD-1/PD-L1 axis and tested the impact of combining IFN inducers, as immune activators, with anti–PD-1, to induce an antimetastatic immune response. Using models of TNBC, we demonstrated an interplay between type I IFN signaling and tumor cell PD-L1 expression that affected therapeutic response. The data revealed that the type I IFN-inducer poly(I:C) was an effective immune activator and antimetastatic agent, functioning better than anti–PD-1, which was ineffective as a single agent. Poly(I:C) treatment induced PD-L1 expression on TNBC cells, and combined poly(I:C) and anti–PD-1 treatment prolonged metastasis-free survival in a neoadjuvant setting via the induction of a tumor-specific T-cell response. Use of this combination in a late treatment setting did not impact metastasis-free survival, indicating that timing was critical for immunotherapeutic benefit. Together, these data demonstrated anti–PD-1 as an ineffective single agent in preclinical models of TNBC. However, type I IFN inducers were effective immune activators, and neoadjuvant trials combining them with anti–PD-1 to induce a sustained antitumor immune response are warranted. Cancer Immunol Res; 5(10); 871–84. ©2017 AACR.

This Supplementary Figure shows the expression of CD73 and adenosine receptors by tumor lines.

Supplementary Figure 2. OVA257 peptide dose does not affect effector cell maturation status.

Movie 4. Serial killing of tumor targets by CAR.OT-I effector cells following engagement through the TCR

This document contains the Figure Legends for the Supplementary data Figures.

This Supplememntary Figure shows the effect of combined PD-1 and A2A blockade on in vitro effector functions of OT-I cells.

This supplementary Figure shows the expression of A2A and IFNy on tumor infiltrating lymphocytes following PD-1 blockade.

Movie 3. CAR.OT-I cells co-cultured with MC57-HER2 cells.

Supplementary Figure 1. TCR activated OT-I and CAR. OT-I cells display similar expression levels of cytotoxic granules.

Figures S1 and S2

This supplementary figure shows the phenotype of tumor-infiltrating lymphocytes following PD-1 or PD-1/A2A blockade.

Supplementary data legends

Movie S2

Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8+ T cells coexpressed the clonogenic OT-I TCR, recognizing the H-2Kb–presented ovalbumin peptide SIINFEKL, and an scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T-cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells (“serial killing”), irrespective of the mode of recognition. At low effector:target ratios, the tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of coincubation. However, from 20 to 50 hours, tumor cell death mediated through CAR became attenuated due to CAR downregulation throughout the time course. Our study provides important insights into CAR-T–tumor cell interactions, with implications for single- or dual receptor–focused T-cell therapy. Cancer Immunol Res; 3(5); 483–94. ©2015 AACR.See related commentary by June, p. 470

Movie S1

Immunotherapy is widely accepted as a powerful new treatment modality for the treatment of cancer. The most successful form of immunotherapy to date has been the blockade of the immune checkpoints PD-1 and CTLA-4. Combining inhibitors of both PD-1 and CTLA-4 increases the proportion of patients who respond to immunotherapy. However, most patients still do not respond to checkpoint inhibitors, and prognostic biomarkers are currently lacking. Therefore, a better understanding of the mechanism by which these checkpoint inhibitors enhance antitumor immune responses is required to more accurately predict which patients are likely to respond and further enhance this treatment modality. Our current study of two mouse tumor models revealed that CD4+Foxp3− cells activated by dual PD-1/CTLA-4 blockade modulated the myeloid compartment, including activation of conventional CD103+ dendritic cells (DC) and expansion of a myeloid subset that produces TNFα and iNOS (TIP-DCs). CD4+Foxp3− T cell–mediated activation of CD103+ DCs resulted in enhanced IL12 production by these cells and IL12 enhanced the therapeutic effect of dual PD-1/CTLA-4 blockade. Given the importance of these myeloid subsets in the antitumor immune response, our data point to a previously underappreciated role of CD4+Foxp3− cells in modulating this arm of the antitumor immune response. Cancer Immunol Res; 6(9); 1069–81. ©2018 AACR.

Movie 5. Serial killing of tumor targets by CAR.OT-I effector cells following engagement through the CAR

Supplementary Methods. Time lapse live video (TLLV) microscopy

Chimeric antigen receptor (CAR) T-cell therapy has proven successful in the treatment of hematological malignancies, notably acute lymphoblastic leukemia and B-cell lymphoma. However, the efficacy of CAR T cells against solid tumors is poor, likely due to tumor-associated immunosuppression. Here, we demonstrated that antagonizing the “inhibitor of apoptosis proteins” with the clinical smac-mimetic, birinapant, significantly enhanced the antitumor activity of CAR T cells in a tumor necrosis factor (TNF)-dependent manner. Enhanced tumor cell death occurred independently of the perforin-mediated granule exocytosis pathway, underscoring the cytotoxic potential of CAR T-cell–derived TNF. Combining CAR T-cell therapy with birinapant significantly reduced established tumor growth in vivo, where either therapy alone was relatively ineffective. Using patient biopsy-derived tumoroids, we demonstrated the synergistic potential of combining CAR T-cell therapy with smac-mimetics. Taken together, we identified CAR T-cell–derived TNF as a potent antitumor effector, which can be further harnessed by smac-mimetics.

Immunotherapy is rapidly emerging as a cancer treatment with high potential. Recent clinical trials with anti-CTLA-4 and anti–PD-1/PD-L1 antibodies (mAbs) suggest that targeting multiple immunosuppressive pathways may significantly improve patient survival. The generation of adenosine by CD73 also suppresses antitumor immune responses through the activation of A2A receptors on T cells and natural killer (NK) cells. We sought to determine whether blockade of A2A receptors could enhance the efficacy of anti–PD-1 mAb. The expression of CD73 by tumor cells limited the efficacy of anti–PD-1 mAb in two tumor models, and this was alleviated with concomitant treatment with an A2A adenosine receptor antagonist. The blockade of PD-1 enhanced A2A receptor expression on tumor-infiltrating CD8+ T cells, making them more susceptible to A2A-mediated suppression. Thus, dual blockade of PD-1 and A2A significantly enhanced the expression of IFNγ and Granzyme B by tumor-infiltrating CD8+ T cells and, accordingly, increased growth inhibition of CD73+ tumors and survival of mice. The results of our study indicate that CD73 expression may constitute a potential biomarker for the efficacy of anti–PD-1 mAb in patients with cancer and that the efficacy of anti–PD-1 mAb can be significantly enhanced by A2A antagonists. We have therefore revealed a potentially novel biomarker for the efficacy of anti–PD-1 that warrants further investigation in patients. Because our studies used SYN-115, a drug that has already undergone phase IIb testing in Parkinson disease, our findings have immediate translational relevance for patients with cancer. Cancer Immunol Res; 3(5); 506–17. ©2015 AACR.

This supplementary figure shows the expression of PD-1 on tumor infiltrating immune subsets.

Supplementary Figure 1 - PDF file 73K, Tumor cells express high levels of PD-L1

CARaMEL T cells persist in long term surviving mice.

Supplementary Figure 3. Pano induced pancreatic tumor cell apoptosis.

Supplementary Data from Toll-Like Receptor Triggering and T-Cell Costimulation Induce Potent Antitumor Immunity in Mice

Supplementary Figure 2 - PDF file 20K, Expression of PD-1 is upregulated on CAR T cells following antigen stimulation with e0771-Her2+ tumor cells

Supplementary Figure 2. Pancreatic cells are resistant to VV-gp100 oncolysis but sensitive to CAR T cell-mediated cytotoxicity.

Supplemental Figure Legend, Supplementary Figure 1 Flow cytometric analysis of HER2 expression on AU565pv and BT474 tumor cells; Supplementary Figure 2 Co-treatment with panobinostat and trastuzumab does not alter splenic NK cell activation/maturation status or frequency in AU565pv tumor-bearing SCID mice; Supplementary Figure 3 Panobinostat-mediated enrichment of interferon-gamma response and chemokine associated gene signatures; Supplementary Figure 4 SCID mice bearing AU565pv tumors were treated with PBS/DMSO (Vehicle), DMSO/trastuzumab (10 mg/kg), PBS/panobinostat (15 mg/kg) or panobinostat and trastuzumab (combination); Supplementary Figure 5 Panobinostat treatment modulates expression of both the activation receptor CD69 and Fc-receptors CD16/32 on IL-2 activated mouse and human NK cells; Supplementary Figure 6 Co-treatment of panobinostat and trastuzumab attenuates HER2 expression in BT474 and BT474-MyrAKT tumor cells; Supplementary Figure 7 Panobinostat is more effective than doxorubicin at supporting the curative activity of anti-HER2 therapy in Her2/neu transgenic mice bearing established rHER2+ H2N113 mammary tumors; Supplementary Figure 8 Rejection of Bcl-2 over-expressing AU565pv tumors in SCID mice treated with the combination therapy.

upplementary Figure 3 - PDF file 17K, Gr1+ cells express PD-1 and PD-L1

Figure S7 shows the gene expression profile of macrophages and other immune cell subsets in cancer patients

Purpose:Response rates to immune checkpoint blockade (ICB; anti-PD-1/anti-CTLA-4) correlate with the extent of tumor immune infiltrate, but the mechanisms underlying the recruitment of T cells following therapy are poorly characterized. A greater understanding of these processes may see the development of therapeutic interventions that enhance T-cell recruitment and, consequently, improved patient outcomes. We therefore investigated the chemokines essential for immune cell recruitment and subsequent therapeutic efficacy of these immunotherapies.Experimental Design:The chemokines upregulated by dual PD-1/CTLA-4 blockade were assessed using NanoString-based analysis with results confirmed at the protein level by flow cytometry and cytometric bead array. Blocking/neutralizing antibodies confirmed the requirement for key chemokines/cytokines and immune effector cells. Results were confirmed in patients treated with immune checkpoint inhibitors using single-cell RNA-sequencing (RNA-seq) and paired survival analyses.Results:The CXCR3 ligands, CXCL9 and CXCL10, were significantly upregulated following dual PD-1/CTLA-4 blockade and both CD8+ T-cell infiltration and therapeutic efficacy were CXCR3 dependent. In both murine models and patients undergoing immunotherapy, macrophages were the predominant source of CXCL9 and their depletion abrogated CD8+ T-cell infiltration and the therapeutic efficacy of dual ICB. Single-cell RNA-seq analysis of patient tumor-infiltrating lymphocytes (TIL) revealed that CXCL9/10/11 was predominantly expressed by macrophages following ICB and we identified a distinct macrophage signature that was associated with positive responses to ICB.Conclusions:These data underline the fundamental importance of macrophage-derived CXCR3 ligands for the therapeutic efficacy of ICB and highlight the potential of manipulating this axis to enhance patient responses.

Supplementary Figure Legends

Supplementary Figure 4. Pano did not enhance the cytotoxicity of CARaMEL cells nor oncolysis of VV-gp100.