The pivotal role of cytotoxic NK cells in mediating the therapeutic effect of anti-CD47 therapy in mycosis fungoides.

CD47 is frequently overexpressed on tumor cells and is an attractive therapeutic target. The mechanism by which anti-CD47 immunotherapy eliminates cutaneous lymphoma has not been explored. We utilized CRISPR/Cas-9 CD47 knock-out, depletion of NK cells, and mice genetically deficient in IFN-γ to elucidate the mechanism of anti-CD47 therapy in a murine model of cutaneous T cell lymphoma (CTCL). CD47 was found to be a crucial factor for tumor progression since CD47 KO CTCL exhibited a delay in tumor growth. The treatment of CD47 WT murine CTCL with anti-CD47 antibodies led to a significant reduction in tumor burden as early as four days after the first treatment and accompanied by an increased percentage of cytotoxic NK cells at the tumor site. The depletion of NK cells resulted in marked attenuation of the anti-tumor effect of anti-CD47. Notably, the treatment of CD47 WT tumors in IFN-γ KO mice with anti-CD47 antibodies was efficient, demonstrating that IFN-γ was not required to mediate anti-CD47 therapy. We were able to potentiate the therapeutic effect of anti-CD47 therapy by IFN-α. That combination resulted in an increased number of cytotoxic CD107a + IFN-γ-NK1.1 cells and intermediate CD62L + NKG2a-NK1.1. Correlative data from a clinical trial (clinicaltrials.gov, NCT02890368) in patients with CTCL utilizing SIRPαFc to block CD47 confirmed our in vivo observations.

Quantification of a proteotypic peptide from protein C inhibitor by liquid chromatography-free SISCAPA-MALDI mass spectrometry: application to identification of recurrence of prostate cancer.

BACKGROUND: Biomarker validation remains one of the most challenging constraints to the development of new diagnostic assays. To facilitate biomarker validation, we previously developed a chromatography-free stable isotope standards and capture by antipeptide antibodies (SISCAPA)-MALDI assay allowing rapid, high-throughput quantification of protein analytes in large sample sets. Here we applied this assay to the measurement of a surrogate proteotypic peptide from protein C inhibitor (PCI) in sera from patients with prostate cancer. METHODS: A 2-plex SISCAPA-MALDI assay for quantification of proteotypic peptides from PCI and soluble transferrin receptor (sTfR) was used to measure these peptides in 159 trypsin-digested sera collected from 51 patients with prostate cancer. These patients had been treated with radiation with or without neoadjuvant androgen deprivation. RESULTS: Patients who experienced biochemical recurrence of prostate cancer showed decreased serum concentrations of the PCI peptide analyte within 18 months of treatment. The PCI peptide concentrations remained increased in the sera of patients who did not experience cancer recurrence. Prostate-specific antigen concentrations had no predictive value during the same time period. CONCLUSIONS: The high-throughput, liquid chromatography-free SISCAPA-MALDI assay is capable of rapid quantification of proteotypic PCI and sTfR peptide analytes in complex serum samples. Decreased serum concentrations of the PCI peptide were found to be related to recurrence of prostate cancer in patients treated with radiation with or without hormone therapy. However, a larger cohort of patients will be required for unequivocal validation of the PCI peptide as a biomarker for clinical use.

Targeting CD47-SIRPa axis shows potent preclinical anti-tumor activity as monotherapy and synergizes with PARP inhibition.

The objective was to correlate CD47 gene expression with resistance to immune checkpoint inhibitors (ICI) in tumor tissue of gynecological cancer (GC). Further, we sought to assess the efficacy of targeting CD47 pathway alone and in combination in pre-clinical ovarian cancer (OC) models. We performed transcriptomic analyses in GC treated with ICI. Signaling pathway enrichment analysis was performed using Ingenuity Pathway Analysis. Immune cell abundance was estimated. CD47 expression was correlated with other pathways, objective response, and progression-free survival (PFS). Anti-tumor efficacy of anti-CD47 therapy alone and in combination was investigated both in-vitro and in-vivo using cell-line derived xenograft (CDX) and patient-derived xenograft (PDX) models. High CD47 expression associated with lower response to ICI and trended toward lower PFS in GC patients. Higher CD47 associated negatively with PDL1 and CTLA4 expression, as well as cytotoxic T-cells and dendritic cells but positively with TGF-ß, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 significantly enhanced macrophage-mediated phagocytosis of OC cells in-vitro and exhibited potent anti-tumor activity in-vivo in OC CDX and PDX models. In-vitro treatment with PARPi increased CD47 expression. Anti-CD47 led to significantly enhanced in-vitro phagocytosis, enhanced STING pathway and synergized in-vivo when combined with PARP inhibitors in BRCA-deficient OC models. This study provides insight on the potential role of CD47 in mediating immunotherapy resistance and its association with higher TGF-ß, BRD4 and CXCR4/CXCL12 expression. Anti-CD47 showed potent anti-tumor activity and synergized with PARPi in OC models. These data support clinical development of anti-CD47 therapy with PARPi in OC.

Self-specific CD8+ T cells maintain a semi-naive state following lymphopenia-induced proliferation.

Upon transfer into T cell-deficient hosts, naive CD8(+) T cells typically undergo lymphopenia-induced proliferation (LIP, also called homeostatic proliferation) and develop the phenotypic and functional characteristics of memory CD8(+) T cells. However, the capacity of T cells with self-peptide/MHC specificity to respond in this way has not been intensively studied. We examined pmel-1 TCR transgenic CD8(+) T cells that are specific for an epitope from gp100, a protein expressed by melanoma cells and normal melanocytes. Despite their self-specificity, naive pmel-1 cells were inefficient at LIP in typical lymphopenic hosts. In CD132 (common gamma-chain)-deficient hosts, pmel-1 CD8(+) T cells underwent extensive proliferation, but, surprisingly, the majority of these cells retained certain naive phenotypic traits (CD44(low), CD122(low)) rather than acquiring the expected central-memory phenotype. Following LIP, pmel-1 T cells acquired the capacity to control B16F10 tumor growth, but only in common gamma-chain-deficient host mice. Together, these data suggest that LIP does not always favor expansion of self-specific CD8 T cells and that sustained extensive lymphopenia is required for such cells to exhibit tumor control.

Phase I Study of the CD47 Blocker TTI-621 in Patients with Relapsed or Refractory Hematologic Malignancies.

PURPOSE: TTI-621 (SIRPα-IgG1 Fc) is a novel checkpoint inhibitor that activates antitumor activity by blocking the CD47 "don't eat me" signal. This first-in-human phase I study (NCT02663518) evaluated the safety and activity of TTI-621 in relapsed/refractory (R/R) hematologic malignancies. PATIENTS AND METHODS: Patients with R/R lymphoma received escalating weekly intravenous TTI-621 to determine the maximum tolerated dose (MTD). During expansion, patients with various malignancies received weekly single-agent TTI-621 at the MTD; TTI-621 was combined with rituximab in patients with B-cell non-Hodgkin lymphoma (B-NHL) or with nivolumab in patients with Hodgkin lymphoma. The primary endpoint was the incidence/severity of adverse events (AEs). Secondary endpoint included overall response rate (ORR). RESULTS: Overall, 164 patients received TTI-621: 18 in escalation and 146 in expansion (rituximab combination, n = 35 and nivolumab combination, n = 4). On the basis of transient grade 4 thrombocytopenia, the MTD was determined as 0.2 mg/kg; 0.1 mg/kg was evaluated in combination cohorts. AEs included infusion-related reactions, thrombocytopenia, chills, and fatigue. Thrombocytopenia (20%, grade ≥3) was reversible between doses and not associated with bleeding. Transient thrombocytopenia that determined the initial MTD may not have been dose limiting. The ORR for all patients was 13%. The ORR was 29% (2/7) for diffuse large B-cell lymphoma (DLBCL) and 25% (8/32) for T-cell NHL (T-NHL) with TTI-621 monotherapy and was 21% (5/24) for DLBCL with TTI-621 plus rituximab. Further dose optimization is ongoing. CONCLUSIONS: TTI-621 was well-tolerated and demonstrated activity as monotherapy in patients with R/R B-NHL and T-NHL and combined with rituximab in patients with R/R B-NHL.

Targeting CD47 in Sézary syndrome with SIRPαFc.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma, has limited treatment options and rare occurrences of long-term remission, thus warranting research into new treatment approaches. CD47 has emerged as a promising target for multiple tumor types, but its role in SS remains unknown. Here, we show that CD47 is highly expressed on Sézary cells in the peripheral blood and skin, and the high level of CD47 expression correlates with worse overall survival (OS) in patients with SS. We also demonstrate that CD47 expression on Sézary cells is under the influence of interleukin 4 (IL-4), IL-7, and IL-13. Signal regulatory protein αFc (SIRPαFc; TTI-621), a novel CD47 decoy receptor, triggers macrophage-mediated phagocytosis of Sézary cells and, when administered in clinical trial settings, results in significant tumor load reduction. We conclude that inhibition of the CD47-SIRPα signaling pathway has therapeutic benefit for patients with SS. This trial was registered at www.clinicaltrials.gov as #NCT02663518.

Negative regulators in homeostasis of naïve peripheral T cells.

It is now apparent that naïve peripheral T cells are a dynamic population where active processes prevent inappropriate activation while supporting survival. The process of thymic education makes naïve peripheral T cells dependent on interactions with self-MHC for survival. However, as these signals can potentially result in inappropriate activation, various non-redundant, intrinsic negative regulatory molecules including Tob, Nfatc2, and Smad3 actively enforce T cell quiescence. Interactions among these pathways are only now coming to light and may include positive or negative crosstalk. In the case of positive crosstalk, self-MHC initiated signals and intrinsic negative regulatory factors may cooperate to dampen T cell activation and sustain peripheral tolerance in a binary fashion (on-off). In the case of negative crosstalk, self-MHC signals may promote survival through partial activation while intrinsic negative regulatory factors act as rheostats to restrain cell cycle entry and prevent T cells from crossing a threshold that would break tolerance.

Histidine rich glycoprotein and cancer: a multi-faceted relationship.

A better understanding of the interplay between the tumor environment, the immune system, and hemostatic apparatus is essential to effectively improve cancer treatment. Histidine-rich glycoprotein (HRG) is an abundant plasma protein with a wide array of functions. HRG has the ability to bind multiple ligands thereby modulating immunity, cell adhesion, angiogenesis, and thrombosis. Many of these functions are involved in tumor progression and antitumor response. We outline current data on HRG as an important potential player and as a potential future target for cancer therapy.

TGF-ß sensitivity restrains CD8+ T cell homeostatic proliferation by enforcing sensitivity to IL-7 and IL-15.

The pleiotropic cytokine TGF-ß has been implicated in the regulation of numerous aspects of the immune response, including naïve T cell homeostasis. Previous studies found that impairing TGF-ß responsiveness (through expression of a dominant-negative TGF-ß RII [DNRII] transgene) leads to accumulation of memory phenotype CD8 T cells, and it was proposed that this resulted from enhanced IL-15 sensitivity. Here we show naïve DNRII CD8 T cells exhibit enhanced lymphopenia-driven proliferation and generation of "homeostatic" memory cells. However, this enhanced response occurred in the absence of IL-15 and, unexpectedly, even in the combined absence of IL-7 and IL-15, which were thought essential for CD8 T cell homeostatic expansion. DNRII transgenic CD8 T cells still require access to self Class I MHC for homeostatic proliferation, arguing against generalized dysregulation of homeostatic cues. These findings suggest TGF-ß responsiveness is critical for enforcing sensitivity to homeostatic cytokines that limit maintenance and composition of the CD8 T cell pool. (154 words).

Polyfunctional T-cell responses are disrupted by the ovarian cancer ascites environment and only partially restored by clinically relevant cytokines.

BACKGROUND: Host T-cell responses are associated with favorable outcomes in epithelial ovarian cancer (EOC), but it remains unclear how best to promote these responses in patients. Toward this goal, we evaluated a panel of clinically relevant cytokines for the ability to enhance multiple T-cell effector functions (polyfunctionality) in the native tumor environment. METHODOLOGY/PRINCIPAL FINDINGS: Experiments were performed with resident CD8+ and CD4+ T cells in bulk ascites cell preparations from high-grade serous EOC patients. T cells were stimulated with α-CD3 in the presence of 100% autologous ascites fluid with or without exogenous IL-2, IL-12, IL-18 or IL-21, alone or in combination. T-cell proliferation (Ki-67) and function (IFN-γ, TNF-α, IL-2, CCL4, and CD107a expression) were assessed by multi-parameter flow cytometry. In parallel, 27 cytokines were measured in culture supernatants. While ascites fluid had variable effects on CD8+ and CD4+ T-cell proliferation, it inhibited T-cell function in most patient samples, with CD107a, IFN-γ, and CCL4 showing the greatest inhibition. This was accompanied by reduced levels of IL-1ß, IL-1ra, IL-9, IL-17, G-CSF, GM-CSF, Mip-1α, PDGF-bb, and bFGF in culture supernatants. T-cell proliferation was enhanced by exogenous IL-2, but other T-cell functions were largely unaffected by single cytokines. The combination of IL-2 with cytokines engaging complementary signaling pathways, in particular IL-12 and IL-18, enhanced expression of IFN-γ, TNF-α, and CCL4 in all patient samples by promoting polyfunctional T-cell responses. Despite this, other functional parameters generally remained inhibited. CONCLUSIONS/SIGNIFICANCE: The EOC ascites environment disrupts multiple T-cell functions, and exogenous cytokines engaging diverse signaling pathways only partially reverse these effects. Our results may explain the limited efficacy of cytokine therapies for EOC to date. Full restoration of T-cell function will require activation of signaling pathways beyond those engaged by IL-2, IL-12, IL-18, and IL-21.

The antigen-specific CD8+ T cell repertoire in unimmunized mice includes memory phenotype cells bearing markers of homeostatic expansion.

Memory T cells exhibit superior responses to pathogens and tumors compared with their naive counterparts. Memory is typically generated via an immune response to a foreign antigen, but functional memory T cells can also be produced from naive cells by homeostatic mechanisms. Using a recently developed method, we studied CD8 T cells, which are specific for model (ovalbumin) and viral (HSV, vaccinia) antigens, in unimmunized mice and found a subpopulation bearing markers of memory cells. Based on their phenotypic markers and by their presence in germ-free mice, these preexisting memory-like CD44(hi) CD8 T cells are likely to arise via physiological homeostatic proliferation rather than a response to environmental microbes. These antigen-inexperienced memory phenotype CD8 T cells display several functions that distinguish them from their CD44(lo) counterparts, including a rapid initiation of proliferation after T cell stimulation and rapid IFN-gamma production after exposure to proinflammatory cytokines. Collectively, these data indicate that the unprimed antigen-specific CD8 T cell repertoire contains antigen-inexperienced cells that display phenotypic and functional traits of memory cells.