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.

Use of an anti-CD200-blocking antibody improves immune responses to AML in vitro and in vivo.

Treatment of relapsed/resistant acute myeloid leukaemia (AML) remains a significant area of unmet patient need, the outlook for most patients remaining extremely poor. A promising approach is to augment the anti-tumour immune response in these patients; most cancers do not activate immune effector cells because they express immunosuppressive ligands. We have previously shown that CD200 (an immunosuppressive ligand) is overexpressed in AML and confers an inferior overall survival compared to CD200low/neg patients. Here we show that a fully human anti-CD200 antibody (TTI-CD200) can block the interaction of CD200 with its receptor and restore AML immune responses in vitro and in vivo.

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.

Targeting pediatric leukemia-propagating cells with anti-CD200 antibody therapy.

Treating refractory pediatric acute lymphoblastic leukemia (ALL) remains a challenge despite impressive remission rates (>90%) achieved in the last decade. The use of innovative immunotherapeutic approaches such as anti-CD19 chimeric antigen receptor T cells does not ensure durable remissions, because leukemia-propagating cells (LPCs) that lack expression of CD19 can cause relapse, which signifies the need to identify new markers of ALL. Here we investigated expression of CD58, CD97, and CD200, which were previously shown to be overexpressed in B-cell precursor ALL (BCP-ALL) in CD34+/CD19+, CD34+/CD19-, CD34-/CD19+, and CD34-/CD19- LPCs, to assess their potential as therapeutic targets. Whole-genome microarray and flow cytometric analyses showed significant overexpression of these molecules compared with normal controls. CD58 and CD97 were mainly co-expressed with CD19 and were not a prerequisite for leukemia engraftment in immune deficient mice. In contrast, expression of CD200 was essential for engraftment and serial transplantation of cells in measurable residual disease (MRD) low-risk patients. Moreover, these CD200+ LPCs could be targeted by using the monoclonal antibody TTI-CD200 in vitro and in vivo. Treating mice with established disease significantly reduced disease burden and extended survival. These findings demonstrate that CD200 could be an attractive target for treating low-risk ALL, with minimal off-tumor effects that beset current immunotherapeutic approaches.

Clinical Response to Anti-CD47 Immunotherapy Is Associated with Rapid Reduction of Exhausted Bystander CD4+ BTLA+ T Cells in Tumor Microenvironment of Mycosis Fungoides.

Cancer progression in mycosis fungoides, the most common form of cutaneous T-cell lymphoma, occurs in a predictable, sequential pattern that starts from patches and that evolves to plaques and later to tumors. Therefore, unlocking the relationship between the microarchitecture of mycosis fungoides and the clinical counterparts of that microstructure represents important steps for the design of targeted therapies. Using multispectral fluorescent imaging, we show that the progression of mycosis fungoides from plaque to tumor parallels the cutaneous expansion of the malignant CD4+ T cells that express TOX. The density of exhausted BTLA+ CD4+ T cells around malignant CD4+TOX+ cells was higher in tumors than it was in plaques, suggesting that undesired safeguards are in place within the tumor microenvironment that prevent immune activation and subsequent cancer eradication. Overriding the CD47 checkpoint with an intralesional SIRPαFc fusion decoy receptor induced the resolution of mycosis fungoides in patients that paralleled an amplified expansion of NK and CD8+ T cells in addition to a reduction of the exhausted BTLA+ CD4+ T cells that were engaged in promiscuous intercellular interactions. These therapeutic benefits of the CD47 blockade were further unleashed by adjuvant interferon-α, which stimulates cytotoxic cells, underscoring the importance of an inflamed microenvironment in facilitating the response to immunotherapy. Collectively, these findings support CD47 as a therapeutic target in treating mycosis fungoides and demonstrate a synergistic role of interferon-α in exploiting these clinical benefits.

Intralesional TTI-621, a novel biologic targeting the innate immune checkpoint CD47, in patients with relapsed or refractory mycosis fungoides or Sézary syndrome: a multicentre, phase 1 study.

BACKGROUND: Intravenous TTI-621 (SIRPα-IgG1 Fc) was previously shown to have activity in relapsed or refractory haematological malignancies. This phase 1 study evaluated the safety and activity of TTI-621 in patients with percutaneously accessible relapsed or refractory mycosis fungoides, Sézary syndrome, or solid tumours. Here we report the clinical and translational results among patients with mycosis fungoides or Sézary syndrome. METHODS: This multicentre, open-label, phase 1 study was conducted at five academic health-care and research centres in the USA. Eligible patients were aged 18 years or older; had injectable, histologically or cytologically confirmed relapsed or refractory cutaneous T-cell lymphoma (CTCL) or solid tumours; Eastern Cooperative Oncology Group performance status of 2 or less; and adequate haematological, renal, hepatic, and cardiac function. TTI-621 was injected intralesionally in a sequential dose escalation (cohorts 1-5; single 1 mg, 3 mg, or 10 mg injection or three 10 mg injections weekly for 1 or 2 weeks) and in expansion cohorts (cohorts 6-9; 2 week induction at the maximum tolerated dose; weekly continuation was allowed). In cohort 6, patients were injected with TTI-621 in a single lesion and in cohort 7, they were injected in multiple lesions. In cohort 8, TTI-621 was combined with pembrolizumab 200 mg injections per product labels. In cohort 9, TTI-621 was combined with the standard labelled dose of subcutaneous pegylated interferon alpha-2a 90 µg. The primary endpoint was the incidence and severity of adverse events. The study is registered with ClinicalTrials.gov, NCT02890368, and was closed by the sponsor to focus on intravenous studies with TTI-621. FINDINGS: Between Jan 30, 2017, and March 31, 2020, 66 patients with mycosis fungoides, Sézary syndrome, other CTCL, or solid tumours were screened, 35 of whom with mycosis fungoides or Sézary syndrome were enrolled and received intralesional TTI-621 (escalation, n=13; expansion, n=22). No dose-limiting toxicities occurred; the maximum tolerated dose was not established. In the dose expansion cohorts, the maximally assessed regimen (10 mg thrice weekly for 2 weeks) was used. 25 (71%) patients had treatment-related adverse events; the most common (occurring in ≥10% of patients) were chills (in ten [29%] patients), injection site pain (nine [26%]), and fatigue (eight [23%]). No treatment-related adverse events were grade 3 or more or serious. There were no treatment-related deaths. Rapid responses (median 45 days, IQR 17-66) occurred independently of disease stage or injection frequency. 26 (90%) of 29 evaluable patients had decreased Composite Assessment of Index Lesion Severity (CAILS) scores; ten (34%) had a decrease in CAILS score of 50% or more (CAILS response). CAILS score reductions occurred in adjacent non-injected lesions in eight (80%) of ten patients with paired assessments and in distal non-injected lesions in one additional patient. INTERPRETATION: Intralesional TTI-621 was well tolerated and had activity in adjacent or distal non-injected lesions in patients with relapsed or refractory mycosis fungoides or Sézary syndrome, suggesting it has systemic and locoregional abscopal effects and potential as an immunotherapy for these conditions. FUNDING: Trillium Therapeutics.

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.

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

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.

TTI-621 (SIRPαFc), a CD47-blocking cancer immunotherapeutic, triggers phagocytosis of lymphoma cells by multiple polarized macrophage subsets.

Tumor-associated macrophages (TAMs) are heterogeneous and can adopt a spectrum of activation states between pro-inflammatory and pro-tumorigenic in response to the microenvironment. We have previously shown that TTI-621, a soluble SIRPαFc fusion protein that blocks the CD47 "do-not-eat" signal, promotes tumor cell phagocytosis by IFN-γ-primed macrophages. To assess the impact of CD47 blockade on diverse types of macrophages that are found within the tumor microenvironment, six different polarized human macrophage subsets (M(-), M(IFN-γ), M(IFN-γ+LPS), M(IL-4), M(HAGG+IL-1ß), M(IL-10 + TGFß)) with distinct cell surface markers and cytokine profiles were generated. Blockade of CD47 using TTI-621 significantly increased phagocytosis of lymphoma cells by all macrophage subsets, with M(IFN-γ), M(IFN-γ+LPS) and M(IL-10 + TGFß) macrophages having the highest phagocytic response. TTI-621-mediated phagocytosis involves macrophage expression of both the low- and high-affinity Fcγ receptors II (CD32) and I (CD64), respectively. Moreover, macrophages with lower phagocytic capabilities (M(-), M(IL-4), M(HAGG+IL-1ß)) could readily be re-polarized into highly phagocytic macrophages using various cytokines or TLR agonists. In line with the in vitro study, we further demonstrate that TTI-621 can trigger phagocytosis of tumor cells by diverse subsets of isolated mouse TAMs ex vivo. These data suggest that TTI-621 may be efficacious in triggering the destruction of cancer cells by a diverse population of TAMs found in vivo and support possible combination approaches to augment the activity of CD47 blockade.

TTI-621 (SIRPαFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Antitumor Activity and Minimal Erythrocyte Binding.

Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47-SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication.Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails.Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo, TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies.Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068-79. ©2016 AACR.

Disruption of T cell suppression in chronic lymphocytic leukemia by CD200 blockade.

CD200 plays a key role in regulating the immune system and has been shown to be upregulated on the surface of different tumors including chronic lymphocytic leukemia. In this study we addressed the effects of CD200 over-expression in CLL cells on autologous T cells in a mixed lymphocyte reaction system. We used native and CD40 ligand (CD40L)-stimulated CLL cells as antigen-presenting cells (APCs) to expand autologous T cells of 14 patients. T cell proliferation over 3 weeks of in vitro culture was significantly enhanced compared to control cells when using CD40L-stimulated APCs and the anti-CD200 antibody 1B9 (p=0.0004). CD200 blockade was further shown to stimulate antigen-specific T cell responses towards the CLL-associated antigen fibromodulin (p=0.04). Finally, the number of CD4+/CD25high/FOXP3+ T cells (T(reg)) was significantly decreased adding anti-CD200 antibody (p=0.04). In summary, CD200 blockade may provide therapeutic benefits in CLL by augmenting an antigen-specific T cell response with suppression of regulatory T cells.

CD200-Fc, a novel antiarthritic biologic agent that targets proinflammatory cytokine expression in the joints of mice with collagen-induced arthritis.

OBJECTIVE: The CD200 receptor (CD200R) is an inhibitory receptor expressed by myeloid cells that is postulated to play an important role in regulation of the immune system. The purpose of this study was to evaluate the efficacy of a soluble ligand of CD200R in established collagen-induced arthritis (CIA) in mice and to analyze changes in cytokine expression following therapy in order to understand its primary mechanism of action. METHODS: Arthritis was induced in DBA/1 mice, and CD200-Fc fusion protein, an isotype control monoclonal antibody, or TNFR-Fc fusion protein was administered over a period of 10 days (total of 4 doses). Cytokine expression in the joint was assessed by flow cytometry, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction. RESULTS: CD200-Fc significantly reduced the severity of established arthritis at the clinical and histologic levels. The therapeutic effect of CD200-Fc at 1 mg/kg was comparable with that of TNFR-Fc at 4 mg/kg. CD200R was found to be expressed in arthritic synovia and in lymph nodes, yet no changes in T cell cytokine levels (interferon-gamma, interleukin-5 [IL-5], IL-10, IL-17) were detected after CD200-Fc therapy. There was no evidence of an expansion of forkhead box P3-positive regulatory T cells or a change in serum anticollagen IgG1 and IgG2a levels. However, administration of CD200-Fc markedly decreased the expression of messenger RNA for tumor necrosis factor alpha, IL-1beta, IL-10, and matrix metalloproteinase 13 in the joint to the same extent as administration of TNFR-Fc. CONCLUSION: CD200-Fc is an effective therapeutic agent in established CIA that targets proinflammatory cytokine expression in the joint without any obvious systemic immunosuppressive effects. Our findings indicate that CD200-Fc has considerable potential as a novel therapeutic agent in rheumatoid arthritis in humans.

Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response.

2F5 is one of the few broadly neutralizing monoclonal antibodies against type 1 Human Immunodeficiency Virus (HIV-1). It recognizes the amino acid sequence ELDKWAS in gp41. We have previously identified a number of immunotargeting 2F5-reactive candidate immunogens. Three of them (designated H-BT1-3) have the ELDKWAS sequence constrained at beta-turn sites within the immunoglobulin heavy chain. Two others (L-CT and L-CTx3) have the sequence attached at the C-terminus of the immunoglobulin light chain with minimal conformational constraints. In the present investigation, the H-BTs were found to bind 2F5 with up to 10-fold higher affinities than their unconstrained counterpart. When used as immunogens, immunogen-specific antibodies were induced with or without adjuvant, confirming the immunotargeting potential of these immunogen constructs. While HIV-1 gp160 cross-reactive antibodies were induced, virus neutralization was not detected. Thus, factors other than 2F5 binding affinity may have a critical role to play in the design of a 2F5-based vaccine.