Atezolizumab Plus nab-Paclitaxel in the Treatment of Metastatic Triple-Negative Breast Cancer With 2-Year Survival Follow-up: A Phase 1b Clinical Trial.

Importance: The humanized monoclonal antibody atezolizumab targets programmed death-ligand 1 and has demonstrated durable single-agent activity in a subset of metastatic triple-negative breast cancers. To extend the observed activity, combinatorial approaches are being tested with standard cytotoxic chemotherapies known to induce immunogenic tumor cell death. Objective: To examine the safety, tolerability, and preliminary clinical activity of atezolizumab plus nab-paclitaxel in metastatic triple-negative breast cancers. Design, Setting, and Participants: This phase 1b multicohort study enrolled 33 women with stage IV or locally recurrent triple-negative breast cancers and 0 to 2 lines of prior chemotherapy in the metastatic setting from December 8, 2014, to April 30, 2017, at 11 sites in the United States. The median follow-up was 24.4 months (95% CI, 22.1-28.8 months). Interventions: Patients received concurrent intravenous atezolizumab and intravenous nab-paclitaxel (minimum 4 cycles). Main Outcomes and Measures: The primary end point was safety and tolerability. Secondary end points included best overall response rate by Response Evaluation Criteria in Solid Tumors, version 1.1; objective response rate; duration of response; disease control rate; progression-free survival; overall survival; and biomarker analyses. Results: The 33 women had a median age of 55 years (range, 32-84 years) and received 1 or more doses of atezolizumab. All patients (100%) experienced at least 1 treatment-related adverse event, 24 patients (73%) experienced grade 3/4 adverse events, and 7 patients (21%) had grade 3/4 adverse events of special interest. No deaths were related to study treatment. The objective response rate was 39.4% (95% CI, 22.9%-57.9%), and the median duration of response was 9.1 months (95% CI, 2.0-20.9 months). The disease control rate was 51.5% (95% CI, 33.5%-69.2%). Median progression-free survival and overall survival were 5.5 months (95% CI, 5.1-7.7 months) and 14.7 months (95% CI, 10.1-not estimable), respectively. Concurrent nab-paclitaxel neither significantly changed biomarkers of the tumor immune microenvironment (programmed death-ligand 1, tumor-infiltrating lymphocytes, CD8) nor impaired atezolizumab systemic immune activation (expansion of proliferating CD8+ T cells, increase of CXCL10 chemokine). Conclusions and Relevance: In this phase 1b trial for metastatic triple-negative breast cancers, the combination of atezolizumab plus nab-paclitaxel had a manageable safety profile. Antitumor responses were observed, including in patients previously treated with a taxane. Trial Registration: ClinicalTrials.gov identifier: NCT01633970.

Long-term survival follow-up of atezolizumab in combination with platinum-based doublet chemotherapy in patients with advanced non-small-cell lung cancer.

BACKGROUND: Before the availability of immunotherapy, chemotherapy was standard first-line therapy for non-small-cell lung cancer (NSCLC) lacking actionable gene alterations. Preclinical evidence suggests chemotherapy is immunomodulatory, supporting chemotherapy/immunotherapy combinations. Atezolizumab, anti-programmed death ligand-1 (PD-L1) antibody, blocks programmed cell death protein-1 and B7.1 interaction with PD-L1. GP28328 (NCT01633970) assessed atezolizumab with chemotherapy in multiple tumours; we report results for advanced, treatment-naïve NSCLC. METHODS: Patients received atezolizumab plus carboplatin with paclitaxel (Arm C: atezo/cb/pac), pemetrexed (Arm D: atezo/cb/pem, maintenance pemetrexed permitted), or nab-paclitaxel (Arm E: atezo/cb/nab-pac), four-six cycles, then atezolizumab maintenance. Primary end-point was safety; secondary end-points were objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). RESULTS: Seventy-six NSCLC patients were enrolled (n = 25, 25 and 26 for Arms C, D and E, respectively). Common treatment-related grade III/IV adverse events were neutropenia (36% atezo/cb/pac, 36% atezo/cb/pem, 42% atezo/cb/nab-pac) and anaemia (16% atezo/cb/pac, 16% atezo/cb/pem, 31% atezo/cb/nab-pac). Confirmed ORRs were 36% atezo/cb/pac, 68% atezo/cb/pem (one complete response [CR]) and 46% atezo/cb/nab-pac (four CRs). Median PFS was 7.1 months, (95% confidence interval [CI]: 4.2-8.3), 8.4 months (95% CI: 4.7-11) and 5.7 months (95% CI: 4.4-14.8), respectively. Median OS was 12.9 months (95% CI: 8.8-21.3), 18.9 months (95% CI: 9.9-27.4) and 17.0 months (95% CI: 12.7-not evaluable), respectively. CONCLUSION: Atezolizumab with chemotherapy was well tolerated with encouraging efficacy, though the analysis was limited by small numbers. NSCLC chemotherapy combination studies are ongoing. CLINICALTRIALS. GOV IDENTIFIER: NCT01633970.

Phase 1 study in Japan of siltuximab, an anti-IL-6 monoclonal antibody, in relapsed/refractory multiple myeloma.

Siltuximab, a chimeric monoclonal antibody with high affinity and specificity for interleukin-6, has been shown to enhance anti-multiple myeloma activity of bortezomib and corticosteroid in vitro. We evaluated the safety, pharmacokinetics, immunogenicity, and antitumor effect of siltuximab in combination with bortezomib and dexamethasone in Japanese patients with relapsed or refractory multiple myeloma. This open-label, phase 1, dose-escalating study used two doses of siltuximab: 5.5 and 11.0 mg/kg (administered on day 1 of each 21-day cycle). In total, nine patients were treated. The most common grade 3/4 adverse events, lymphopenia (89 %) and thrombocytopenia (44 %), occurred in patients receiving both doses of siltuximab; however, no dose-limiting toxicities (DLTs) were observed. Following intravenous administration of siltuximab at 5.5 and 11.0 mg/kg, the maximum serum concentration and the area under the curve from 0 to 21 days and from 0 to infinity increased in an approximately dose-proportional manner. Mean half-life, total systemic clearance, and volume of distribution were similar at doses of 5.5 and 11.0 mg/kg. Across both doses, six of the nine patients had complete or partial response (22 and 44 %, respectively). In conclusion, as no DLT was observed, the recommended dose for this combination is 11.0 mg/kg once every 3 weeks. The study is registered at http://www.clinicaltrials.gov as NCT01309412.

Phase-1 study of abiraterone acetate in chemotherapy-naïve Japanese patients with castration-resistant prostate cancer.

Persistent androgen synthesis under castration status in adrenal gland, testes and tumor cells is thought to be one of the major causes of development and progression of castration-resistant prostate cancer (CRPC). Abiraterone acetate (AA), the prodrug of abiraterone, which is an inhibitor of androgen synthesis enzymes, was evaluated for pharmacokinetics, pharmacodynamics, preliminary efficacy and safety in Japanese patients with CRPC in a phase-1, open-label and dose-escalation study. Chemotherapy-naïve Japanese CRPC patients (N = 27) received one of four AA daily doses (250 mg [n = 9], 500 mg [n = 6], 1000 [1 h premeal] mg [n = 6] and 1000 [2 h postmeal] mg [n = 6]) continuously through 28-day treatment cycles. In the first cycle, AA monotherapy was given on days 1-7 for pharmacokinetics, and AA plus prednisone (5 mg twice daily) from days 8 to 28. Of 27 patients, 9 continued treatment with AA until the data cut-off date (18 July 2013). Over the evaluated dose range, plasma abiraterone concentrations increased with dose, with median tmax 2-3 h. At each dose level, mean serum corticosterone concentrations increased, while testosterone and dehydroepiandrosterone sulfate concentrations rapidly decreased following a single AA dose and were further reduced to near the quantification limit on day 8 regardless of the dose. At least 3 patients from each dose-group experienced ≥50% prostate-specific antigen reduction, suggesting clinical benefit from AA in Japanese CRPC patients. AA was generally well-tolerated, and, therefore, the recommended AA dosage regimen in Japanese CRPC patients is 1000 mg oral dose under modified fasting conditions (at least 1 h premeal or 2 h postmeal). This study is registered at ClinicalTrials.gov: NCT01186484.

Identification of NK cell receptor ligands using a signaling reporter system.

NK cell responses are regulated by a balance of inhibitory and activating signals, reflecting the net effect of interactions between receptors and ligands on target and effector cell surfaces. The identification of ligands for orphan NK cell receptors is key to enhancing our understanding of NK cell biology. Here we describe a strategy (protocol) for the identification of ligands for orphan NK cell receptors using signaling reporter cells in combination with a virus rescue system.

Essential role of the Ly49A stalk region for immunological synapse formation and signaling.

NK cells use surface NK receptors to discriminate self from non-self. The NK receptor ligand-binding domain (NKD) has been considered the sole regulator of ligand binding. Using a prototypic murine NK receptor, Ly49A, we show that the membrane proximal nonligand binding ecto-domain (the stalk region) is critical to ligand binding and signaling. The stalk region is required for receptor binding to ligand on target cells (trans interaction), but is dispensable for receptor binding to ligand on the same cell (cis interaction). Also, signaling in a trans manner depends on the stalk region mediating the formation of the immunological synapse. Thus, our data modeling receptor function at the cellular level reveal an essential role for the stalk region as a specific mediator of receptor signal integration, by which NKD-ligand interactions at the interface initiate and deliver information to the spatially separated cytoplasmic domain.

Regulation of the NK cell alloreactivity to bone marrow cells by the combination of the host NK gene complex and MHC haplotypes.

Host NK cells can reject MHC-incompatible (allogeneic) bone marrow cells (BMCs), suggesting their effective role for graft-vs leukemia effects in the clinical setting of bone marrow transplantation. NK cell-mediated rejection of allogeneic BMCs is dependent on donor and recipient MHC alleles and other factors that are not yet fully characterized. Whereas the molecular mechanisms of allogeneic MHC recognition by NK receptors have been well studied in vitro, guidelines to understand NK cell allogeneic reactivity under the control of multiple genetic components in vivo remain less well understood. In this study, we use congenic mice to show that BMC rejection is regulated by haplotypes of the NK gene complex (NKC) that encodes multiple NK cell receptors. Most importantly, host MHC differences modulated the NKC effect. Moreover, the NKC allelic differences also affected the outcome of hybrid resistance whereby F1 hybrid mice reject parental BMCs. Therefore, these data indicate that NK cell alloreactivity in vivo is dependent on the combination of the host NKC and MHC haplotypes. These data suggest that the NK cell self-tolerance process dynamically modulates the NK cell alloreactivity in vivo.

Protection from lethal infection by adoptive transfer of CD8 T cells genetically engineered to express virus-specific innate immune receptor.

CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3zeta cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.

Arrested natural killer cell development associated with transgene insertion into the Atf2 locus.

Natural killer (NK) cell development in the bone marrow is not fully understood. Following lineage commitment, these cells appear to advance through a series of developmental stages that are beginning to be characterized. We previously reported a selective deficiency of NK cells in a C57BL/6 mouse with a transgenic construct consisting of the cDNA for the Ly49A major histocompatibility complex (MHC) class 1-specific inhibitory receptor driven by the granzyme A gene. This mouse has few NK cells in peripheral tissues with relative preservation of other immune cells, including T and B cells. Herein we demonstrate that these mice have an accumulation of NK cells with an immature phenotype in the bone marrow, consistent with a block at a previously proposed stage in normal NK-cell development. The phenotype is associated with transgenic insertion into Atf2, the gene for the basic leucine zipper (bZIP) transcription factor family member ATF-2. Although analysis of Atf2-null NK cells shows no defect, the transgenic mice express abnormal truncated Atf2 transcripts that may mediate a repressor effect because ATF2 can heterodimerize with other bZIP molecules. The defect is cell intrinsic, suggesting that certain bZIP molecules play significant roles in NK-cell development.

Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors.

The natural killer (NK) gene complex (NKC) encodes orphan lectin-like NK cell receptors that may explain uncharacterized NK cell specificities. Unlike other NKC-encoded receptors that recognize molecules with major histocompatibility complex (MHC) class I folds, here we show that mouse Nkrp1d and Nkrp1f bind specific C-type lectin-related (Clr) molecules. Nkrp1d mediated inhibition when recognizing Clrb, a molecule expressed in dendritic cells and macrophages. Nkrp1 (official gene name, Klrb1) and Clr are intertwined in a genetically conserved NKC region showing recombination suppression, reminiscent of plant self-incompatibility loci. Thus, these findings broaden the 'missing-self' hypothesis from solely involving MHC class I to including related NK cell receptors for lectin-like ligands, and reflect genetic strategies for biological self-recognition processes in other species.

Recognition of a virus-encoded ligand by a natural killer cell activation receptor.

Natural killer (NK) cells express inhibitory and activation receptors that recognize MHC class I-like molecules on target cells. These receptors may be involved in the critical role of NK cells in controlling initial phases of certain viral infections. Indeed, the Ly49H NK cell activation receptor confers in vivo genetic resistance to murine cytomegalovirus (MCMV) infections, but its ligand was previously unknown. Herein, we use heterologous reporter cells to demonstrate that Ly49H recognizes MCMV-infected cells and a ligand encoded by MCMV itself. Exploiting a bioinformatics approach to the MCMV genome, we find at least 11 ORFs for molecules with previously unrecognized features of predicted MHC-like folds and limited MHC sequence homology. We identify one of these, m157, as the ligand for Ly49H. m157 triggers Ly49H-mediated cytotoxicity, and cytokine and chemokine production by freshly isolated NK cells. We hypothesize that the other ORFs with predicted MHC-like folds may be involved in immune evasion or interactions with other NK cell receptors.

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice.

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.

In vivo developmental stages in murine natural killer cell maturation.

Natural killer (NK) cells develop in the bone marrow, but their in vivo stages of maturation, expansion and acquisition of receptors that guide target cell specificity are not well defined. We describe here such stages of development. We also show that developing NK cells actively proliferate at a phenotypically distinguishable immature stage after they have acquired expression of Ly49 and CD94-NKG2 receptors. These studies provide a developmental framework for NK cell maturation in vivo and suggest the possible involvement of the Ly49 and CD94-NKG2 receptors themselves in modulating expansion of NK cell populations with a given NK cell receptor repertoire.