Tumor-Specific T Cell Dysfunction Is a Dynamic Antigen-Driven Differentiation Program Initiated Early during Tumorigenesis.

CD8(+) T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Here we developed a tamoxifen-inducible liver cancer mouse model with a defined oncogenic driver antigen (SV40 large T-antigen) to follow the activation and differentiation of naive tumor-specific CD8(+) T (TST) cells after tumor initiation. Early during the pre-malignant phase of tumorigenesis, TST cells became dysfunctional, exhibiting phenotypic, functional, and transcriptional features similar to dysfunctional T cells isolated from late-stage human tumors. Thus, T cell dysfunction seen in advanced human cancers may already be established early during tumorigenesis. Although the TST cell dysfunctional state was initially therapeutically reversible, it ultimately evolved into a fixed state. Persistent antigen exposure rather than factors associated with the tumor microenvironment drove dysfunction. Moreover, the TST cell differentiation and dysfunction program exhibited features distinct from T cell exhaustion in chronic infections. Strategies to overcome this antigen-driven, cell-intrinsic dysfunction may be required to improve cancer immunotherapy.

EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22.

The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.

Clinical and molecular effects of oral CCR4 antagonist RPT193 in atopic dermatitis: A Phase 1 study.

BACKGROUND: RPT193 is an orally administered small molecule antagonist of the human C-C motif chemokine receptor 4 (CCR4) that inhibits the migration and downstream activation of T-helper Type 2 (Th2) cells. We investigated single- and multiple-ascending doses of RPT193 in healthy subjects, and multiple doses of RPT193 in subjects with moderate-to-severe atopic dermatitis (AD). METHODS: This was a first-in-human randomized, placebo-controlled Phase 1a/1b monotherapy study (NCT04271514) to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and CCR4 surface receptor occupancy in eligible healthy subjects and subjects with moderate-to-severe AD. Clinical efficacy and skin biomarker effects of RPT193 monotherapy were assessed as exploratory endpoints in AD subjects. RESULTS: In healthy (n = 72) and AD subjects (n = 31), once-daily RPT193 treatment was generally well tolerated, with no serious adverse events reported and all treatment-emergent adverse events reported as mild/moderate. In AD subjects, numerically greater improvements in clinical efficacy endpoints were observed with RPT193 monotherapy versus placebo up to the end of the treatment period (Day 29), with statistically significant improvement, compared to Day 29 and placebo, observed 2 weeks after the end of treatment (Day 43) on several endpoints (p < .05). Moreover, significant changes in the transcriptional profile were seen in skin biopsies of RPT193-treated versus placebo-treated subjects at Day 29, which were also significantly correlated with improvements in clinical efficacy measures. CONCLUSIONS: To our knowledge, this is the first clinical study with an oral CCR4 antagonist that showed clinical improvement coupled with modulation of the cutaneous transcriptomic profile in an inflammatory skin disease.

A Potent and Effective Suicidal Listeria Vaccine Platform.

Live-attenuated Listeria monocytogenes has shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenes recombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens. Lm-RIID grows normally in broth but commits suicide inside host cells by inducing Cre recombinase and deleting essential genes flanked by loxP sites, resulting in a self-limiting infection even in immunocompromised mice. Lm-RIID vaccination of mice induces potent CD8+ T cells and protects against virulent challenges, similar to live L. monocytogenes vaccines. When combined with α-PD-1, Lm-RIID is as effective as live-attenuated L. monocytogenes in a therapeutic tumor model. This impressive efficacy, together with the increased clearance rate, makes Lm-RIID ideal for prophylactic immunization against diseases that require T cells for protection.

A Listeria vaccine and depletion of T-regulatory cells activate immunity against early stage pancreatic intraepithelial neoplasms and prolong survival of mice.

BACKGROUND & AIMS: Premalignant lesions and early stage tumors contain immunosuppressive microenvironments that create barriers for cancer vaccines. Kras(G12D/+);Trp53(R172H/+);Pdx-1-Cre (KPC) mice, which express an activated form of Kras in pancreatic tissues, develop pancreatic intraepithelial neoplasms (PanIN) that progress to pancreatic ductal adenocarcinoma (PDA). We used these mice to study immune suppression in PDA. METHODS: We immunized KPC and Kras(G12D/+);Pdx-1-Cre mice with attenuated intracellular Listeria monocytogenes (which induces CD4(+) and CD8(+) T-cell immunity) engineered to express Kras(G12D) (LM-Kras). The vaccine was given alone or in sequence with an anti-CD25 antibody (PC61) and cyclophosphamide to deplete T-regulatory (Treg) cells. Survival times were measured; pancreatic and spleen tissues were collected and analyzed by histologic, flow cytometry, and immunohistochemical analyses. RESULTS: Interferon γ-mediated, CD8(+) T-cell responses were observed in KPC and Kras(G12D/+);Pdx-1-Cre mice given LM-Kras, but not in unvaccinated mice. Administration of LM-Kras to KPC mice 4-6 weeks old (with early stage PanINs), depleted of Treg cells, significantly prolonged survival and reduced PanIN progression (median survival, 265 days), compared with unvaccinated mice (median survival, 150 days; P = .002), mice given only LM-Kras (median survival, 150 days; P = .050), and unvaccinated mice depleted of Treg cells (median survival, 170 days; P = .048). In 8- to 12-week-old mice (with late-stage PanINs), LM-Kras, alone or in combination with Treg cell depletion, did not increase survival time or slow PanIN progression. The combination of LM-Kras and Treg cell depletion reduced numbers of Foxp3(+)CD4(+) T cells in pancreatic lymph nodes, increased numbers of CD4(+) T cells that secrete interleukin 17 and interferon γ, and caused CD11b(+)Gr1(+) cells in the pancreas to acquire an immunostimulatory phenotype. CONCLUSIONS: Immunization of KPC mice with Listeria monocytogenes engineered to express Kras(G12D), along with depletion of Treg cells, reduces progression of early stage, but not late-stage, PanINs. This approach increases infiltration of the lesion with inflammatory cells. It might be possible to design immunotherapies against premalignant pancreatic lesions to slow or prevent progression to PDA.

Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

KBMA Listeria monocytogenes is an effective vector for DC-mediated induction of antitumor immunity.

Vaccine strategies that utilize human DCs to enhance antitumor immunity have yet to realize their full potential. Approaches that optimally target a spectrum of antigens to DCs are urgently needed. Here we report the development of a platform for loading DCs with antigen. It is based on killed but metabolically active (KBMA) recombinant Listeria monocytogenes and facilitates both antigen delivery and maturation of human DCs. Highly attenuated KBMA L. monocytogenes were engineered to express an epitope of the melanoma-associated antigen MelanA/Mart-1 that is recognized by human CD8+ T cells when presented by the MHC class I molecule HLA-A*0201. The engineered KBMA L. monocytogenes induced human DC upregulation of costimulatory molecules and secretion of pro-Th1 cytokines and type I interferons, leading to effective priming of Mart-1-specific human CD8+ T cells and lysis of patient-derived melanoma cells. KBMA L. monocytogenes expressing full-length NY-ESO-1 protein, another melanoma-associated antigen, delivered the antigen for presentation by MHC class I and class II molecules independent of the MHC haplotype of the DC donor. A mouse therapeutic tumor model was used to show that KBMA L. monocytogenes efficiently targeted APCs in vivo to induce protective antitumor responses. Together, our data demonstrate that KBMA L. monocytogenes may be a powerful platform that can both deliver recombinant antigen to DCs for presentation and provide a potent DC-maturation stimulus, making it a potential cancer vaccine candidate.

Suppression of cell-mediated immunity following recognition of phagosome-confined bacteria.

Listeria monocytogenes is a facultative intracellular pathogen capable of inducing a robust cell-mediated immune response to sub-lethal infection. The capacity of L. monocytogenes to escape from the phagosome and enter the host cell cytosol is paramount for the induction of long-lived CD8 T cell-mediated protective immunity. Here, we show that the impaired T cell response to L. monocytogenes confined within a phagosome is not merely a consequence of inefficient antigen presentation, but is the result of direct suppression of the adaptive response. This suppression limited not only the adaptive response to vacuole-confined L. monocytogenes, but negated the response to bacteria within the cytosol. Co-infection with phagosome-confined and cytosolic L. monocytogenes prevented the generation of acquired immunity and limited expansion of antigen-specific T cells relative to the cytosolic L. monocytogenes strain alone. Bacteria confined to a phagosome suppressed the production of pro-inflammatory cytokines and led to the rapid MyD88-dependent production of IL-10. Blockade of the IL-10 receptor or the absence of MyD88 during primary infection restored protective immunity. Our studies demonstrate that the presence of microbes within a phagosome can directly impact the innate and adaptive immune response by antagonizing the signaling pathways necessary for inflammation and the generation of protective CD8 T cells.

Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes.

Listeria monocytogenes is a facultative intracellular pathogen that induces a cytosolic signaling cascade resulting in expression of interferon (IFN)-beta. Although type I IFNs are critical in viral defense, their role in immunity to bacterial pathogens is much less clear. In this study, we addressed the role of type I IFNs by examining the infection of L. monocytogenes in BALB/c mice lacking the type I IFN receptor (IFN-alpha/betaR-/-). During the first 24 h of infection in vivo, IFN-alpha/betaR-/- and wild-type mice were similar in terms of L. monocytogenes survival. In addition, the intracellular fate of L. monocytogenes in macrophages cultured from IFN-alpha/betaR-/- and wild-type mice was indistinguishable. However, by 72 h after inoculation in vivo, IFN-alpha/betaR-/- mice were approximately 1,000-fold more resistant to a high dose L. monocytogenes infection. Resistance was correlated with elevated levels of interleukin 12p70 in the blood and increased numbers of CD11b+ macrophages producing tumor necrosis factor alpha in the spleen of IFN-alpha/betaR-/- mice. The results of this study suggest that L. monocytogenes might be exploiting an innate antiviral response to promote its pathogenesis.

Single-Cell Immune Competency Signatures Associate with Survival in Phase II GVAX and CRS-207 Randomized Studies in Patients with Metastatic Pancreatic Cancer.

The identification of biomarkers for patient stratification is fundamental to precision medicine efforts in oncology. Here, we identified two baseline, circulating immune cell subsets associated with overall survival in patients with metastatic pancreatic cancer who were enrolled in two phase II randomized studies of GVAX pancreas and CRS-207 immunotherapy. Single-cell mass cytometry was used to simultaneously measure 38 cell surface or intracellular markers in peripheral blood mononuclear cells obtained from a phase IIa patient subcohort (N = 38). CITRUS, an algorithm for identification of stratifying subpopulations in multidimensional cytometry datasets, was used to identify single-cell signatures associated with clinical outcome. Patients with a higher abundance of CD8+CD45RO-CCR7-CD57+ cells and a lower abundance of CD14+CD33+CD85j+ cells had improved overall survival [median overall survival, range (days) 271, 43-1,247] compared with patients with a lower abundance of CD8+CD45RO-CCR7-CD57+ cells and higher abundance of CD14+CD33+CD85j+ cells (77, 24-1,247 days; P = 0.0442). The results from this prospective-retrospective biomarker analysis were validated by flow cytometry in 200 patients with pancreatic cancer enrolled in a phase IIb study (P = 0.0047). The identified immune correlates provide potential prognostic or predictive signatures that could be employed for patient stratification.

Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4.

BACKGROUND: Checkpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME). METHODS: We developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI. RESULTS: Using a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs. CONCLUSION: Taken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer. STATEMENT OF SIGNIFICANCE: CPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

Novel Piperidinyl-Azetidines as Potent and Selective CCR4 Antagonists Elicit Antitumor Response as a Single Agent and in Combination with Checkpoint Inhibitors.

The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg accumulation in the TME has been shown to dampen the antitumor immune response and is thought to be an important driver in tumor immune evasion. Preclinical and clinical data suggest that reducing the Treg population in the TME can potentiate the antitumor immune response of checkpoint inhibitors. We have developed small-molecule antagonists of CCR4, featuring a novel piperidinyl-azetidine motif, that inhibit the recruitment of Treg into the TME and elicit antitumor responses as a single agent or in combination with an immune checkpoint blockade. The discovery of these potent, selective, and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models, is described herein.

Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-Mesothelin (CRS-207) with or without Nivolumab in Patients with Pancreatic Cancer.

PURPOSE: Two studies in previously treated metastatic pancreatic cancer have been completed combining GVAX pancreas vaccine (GM-CSF-secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated Listeria monocytogenes-expressing mesothelin). In the current study, we compared Cy/GVAX followed by CRS-207 with (Arm A) or without nivolumab (Arm B). PATIENTS AND METHODS: Patients with pancreatic adenocarcinoma who received one prior therapy for metastatic disease and RECIST measurable disease were randomized 1:1 to receive treatment on Arm A or Arm B. The primary objective was to compare overall survival (OS) between the arms. Additional objectives included assessment of progression-free survival, safety, tumor responses, CA19-9 responses, and immunologic correlates. RESULTS: Ninety-three patients were treated (Arm A, 51; Arm B, 42). The median OS in Arms A and B were 5.9 [95% confidence interval (CI), 4.7-8.6] and 6.1 (95% CI, 3.5-7.0) months, respectively, with an HR of 0.86 (95% CI, 0.55-1.34). Objective responses were seen in 3 patients using immune-related response criteria (4%, 2/51, Arm A; 2%, 1/42, Arm B). The grade ≥3 related adverse event rate, whereas higher in Arm A (35.3% vs. 11.9%) was manageable. Changes in the microenvironment, including increase in CD8+ T cells and a decrease in CD68+ myeloid cells, were observed in long-term survivors in Arm A only. CONCLUSIONS: Although the study did not meet its primary endpoint of improvement in OS of Arm A over Arm B, the OS was comparable with standard therapy. Objective responses and immunologic changes in the tumor microenvironment were evident.

Novel, Selective Inhibitors of USP7 Uncover Multiple Mechanisms of Antitumor Activity In Vitro and In Vivo.

The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. Thus, USP7 inhibition may decrease oncogene function, increase tumor suppressor function, and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of TP53-mutant cell lines in vitro Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex, polycomb repressive complex 2 (PRC2), and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Furthermore, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer.

Discovery of Potent, Selective, and Orally Bioavailable Inhibitors of USP7 with In Vivo Antitumor Activity.

USP7 is a promising target for cancer therapy as its inhibition is expected to decrease function of oncogenes, increase tumor suppressor function, and enhance immune function. Using a structure-based drug design strategy, a new class of reversible USP7 inhibitors has been identified that is highly potent in biochemical and cellular assays and extremely selective for USP7 over other deubiquitinases. The succinimide was identified as a key potency-driving motif, forming two strong hydrogen bonds to the allosteric pocket of USP7. Redesign of an initial benzofuran-amide scaffold yielded a simplified ether series of inhibitors, utilizing acyclic conformational control to achieve proper amine placement. Further improvements were realized upon replacing the ether-linked amines with carbon-linked morpholines, a modification motivated by free energy perturbation (FEP+) calculations. This led to the discovery of compound 41, a highly potent, selective, and orally bioavailable USP7 inhibitor. In xenograft studies, compound 41 demonstrated tumor growth inhibition in both p53 wildtype and p53 mutant cancer cell lines, demonstrating that USP7 inhibitors can suppress tumor growth through multiple different pathways.

Impact of preexisting vector-specific immunity on vaccine potency: characterization of listeria monocytogenes-specific humoral and cellular immunity in humans and modeling studies using recombinant vaccines in mice.

Recombinant live-attenuated Listeria monocytogenes is currently being developed as a vaccine platform for treatment or prevention of malignant and infectious diseases. The effectiveness of complex biologic vaccines, such as recombinant viral and bacterial vectors, can be limited by either preexisting or vaccine-induced vector-specific immunity. We characterized the level of L. monocytogenes-specific cellular and humoral immunity present in more than 70 healthy adult subjects as a first step to understanding its possible impact on the efficacy of L. monocytogenes-based vaccines being evaluated in early-phase clinical trials. Significant L. monocytogenes-specific humoral immunity was not measured in humans, consistent with a lack of antibodies in mice immunized with wild-type L. monocytogenes. Cellular immune responses specific for listeriolysin O, a secreted bacterial protein required for potency of L. monocytogenes-derived vaccines, were detected in approximately 60% of human donors tested. In mice, while wild-type L. monocytogenes did not induce significant humoral immunity, attenuated L. monocytogenes vaccine strains induced high-titer L. monocytogenes-specific antibodies when given at high doses used for immunization. Passive transfer of L. monocytogenes-specific antiserum to naïve mice had no impact on priming antigen-specific immunity in mice immunized with a recombinant L. monocytogenes vaccine. In mice with preexisting L. monocytogenes-specific immunity, priming of naïve T cells was not prevented, and antigen-specific responses could be boosted by additional vaccinations. For the first time, our findings establish the level of L. monocytogenes-specific cellular immunity in healthy adults, and, together with modeling studies performed with mice, they support the scientific rationale for repeated L. monocytogenes vaccine immunization regimens to elicit a desired therapeutic effect.

Results from a Phase IIb, Randomized, Multicenter Study of GVAX Pancreas and CRS-207 Compared with Chemotherapy in Adults with Previously Treated Metastatic Pancreatic Adenocarcinoma (ECLIPSE Study).

PURPOSE: Limited options exist for patients with advanced pancreatic cancer progressing after 1 or more lines of therapy. A phase II study in patients with previously treated metastatic pancreatic cancer showed that combining GVAX pancreas (granulocyte-macrophage colony-stimulating factor-secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated Listeria monocytogenes expressing mesothelin) resulted in median overall survival (OS) of 6.1 months, which compares favorably with historical OS achieved with chemotherapy. In the current study, we compared Cy/GVAX + CRS-207, CRS-207 alone, and standard chemotherapy in a three-arm, randomized, controlled phase IIb trial. PATIENTS AND METHODS: Patients with previously treated metastatic pancreatic adenocarcinoma were randomized 1:1:1 to receive Cy/GVAX + CRS-207 (arm A), CRS-207 (arm B), or physician's choice of single-agent chemotherapy (arm C). The primary cohort included patients who had failed ≥2 prior lines of therapy, including gemcitabine. The primary objective compared OS between arms A and C in the primary cohort. The second-line cohort included patients who had received 1 prior line of therapy. Additional objectives included OS between all treatment arms, safety, and tumor responses. RESULTS: The study did not meet its primary efficacy endpoint. At the final study analysis, median OS [95% confidence interval (CI)] in the primary cohort (N = 213) was 3.7 (2.9-5.3), 5.4 (4.2-6.4), and 4.6 (4.2-5.7) months in arms A, B, and C, respectively, showing no significant difference between arm A and arm C [P = not significant (NS), HR = 1.17; 95% CI, 0.84-1.64]. The most frequently reported adverse events in all treatment groups were chills, pyrexia, fatigue, and nausea. No treatment-related deaths occurred. CONCLUSIONS: The combination of Cy/GVAX + CRS-207 did not improve survival over chemotherapy. (ClinicalTrials.gov ID: NCT02004262)See related commentary by Salas-Benito et al., p. 5435.

Clinical Response of Live-Attenuated, Listeria monocytogenes Expressing Mesothelin (CRS-207) with Chemotherapy in Patients with Malignant Pleural Mesothelioma.

PURPOSE: Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with poor prognosis. CRS-207 is a live-attenuated Listeria monocytogenes engineered to express mesothelin, a tumor-associated antigen highly expressed in MPM. CRS-207 induces antitumor immune responses and increases susceptibility of neoplastic cells to immune-mediated killing. PATIENTS AND METHODS: Patients with unresectable MPM, ECOG 0 or 1, and adequate organ and pulmonary function were enrolled in this multicenter, open-label phase Ib study. They received two priming infusions of 1 × 109 CFU CRS-207, followed by pemetrexed/cisplatin chemotherapy, and CRS-207 booster infusions. Primary objectives were safety and induction of immune response. Secondary/exploratory objectives included tumor response, progression-free survival (PFS), overall survival (OS), immune subset analysis, and gene-expression profiling of tumor. RESULTS: Of 35 evaluable patients, 89% (31/35) had disease control with one complete response (3%), 19 partial responses (54%), and 10 stable disease (29%). The estimated median duration of response was 5.0 months (95% CI, 3.9-11.5). The median PFS and OS were 7.5 (95% CI, 7.0-9.9) and 14.7 (95% CI, 11.2-21.9) months, respectively. Tumor size reduction was observed post-CRS-207 infusion prior to chemotherapy in 11 of 35 (31%) patients. No unexpected treatment-related serious adverse events or deaths were observed. IHC analysis of pre- and post-CRS-207 treatment tumor biopsies revealed possible reinvigoration and proliferation of T cells, increased infiltration of dendritic and natural killer cells, increased CD8:Treg ratio, and a shift from immunosuppressive M2-like to proinflammatory M1-like macrophages following CRS-207 administration. CONCLUSIONS: Combination of CRS-207 and chemotherapy induced significant changes in the local tumor microenvironment and objective tumor responses in a majority of treated patients.

Constitutive Activation of the PrfA regulon enhances the potency of vaccines based on live-attenuated and killed but metabolically active Listeria monocytogenes strains.

Recombinant vaccines derived from the facultative intracellular bacterium Listeria monocytogenes are presently undergoing early-stage clinical evaluation in oncology treatment settings. This effort has been stimulated in part due to preclinical results that illustrate potent activation of innate and adaptive immune effectors by L. monocytogenes vaccines, combined with efficacy in rigorous animal models of malignant and infectious disease. Here, we evaluated the immunologic potency of a panel of isogenic vaccine strains that varied only in prfA. PrfA is an intracellularly activated transcription factor that induces expression of virulence genes and encoded heterologous antigens (Ags) in appropriately engineered vaccine strains. Mutant strains with PrfA locked into a constitutively active state are known as PrfA* mutants. We assessed the impacts of three PrfA* mutants, G145S, G155S, and Y63C, on the immunologic potencies of live-attenuated and photochemically inactivated nucleotide excision repair mutant (killed but metabolically active [KBMA]) vaccines. While PrfA* substantially increased Ag expression in strains grown in broth culture, Ag expression levels were equivalent in infected macrophage and dendritic cell lines, conditions that more closely parallel those in the immunized host. However, only the prfA(G155S) allele conferred significantly enhanced vaccine potency to KBMA vaccines. In the KBMA vaccine background, we show that PrfA*(G155S) enhanced functional cellular immunity following an intravenous or intramuscular prime-boost immunization regimen. These results form the basis of a rationale for including the prfA(G155S) allele in future live-attenuated or KBMA L. monocytogenes vaccines advanced to the clinical setting.

Selective targeting of antitumor immune responses with engineered live-attenuated Listeria monocytogenes.

Improved immunization and ex vivo T-cell culture strategies can generate larger numbers and more potent tumor-specific effector cells than previously possible. Nonetheless, the capacity of these cells to eliminate established tumors is limited by their ability to efficiently enter tumor-bearing organs and mediate their effector function. In the current study, we show that the administration of an engineered organ-homing microbe selectively targets tumor-specific immune responses to metastases within that organ. Specifically, an attenuated Listeria monocytogenes strain, which preferentially infects the liver following systemic administration, dramatically enhances the activity of a cancer vaccine against liver metastases but not metastases in the lung. This enhanced activity results from both local recruitment of innate immune effectors as well as concentration and increased activation of vaccine-induced antitumor T cells within the liver. These findings show a general approach to focus systemic cancer immunotherapies to specific organs bearing tumor metastases by taking advantage of differential tropisms and the proinflammatory nature of microbes.