Anti-tumor effect of CTLA-4 antibody is independent of checkpoint blockade

Abstract Antibodies targeting CTLA-4 are emerging as an important class of cancer therapeutics. It is assumed that these antibodies cause tumor rejection by blocking negative signaling from the CTLA-4-B7 interactions to enhance the priming of naïve T cells in lymphoid organs. However, recent findings have shown that the effectiveness of CTLA-4 antibody critically depends on the Fc domain and the host Fc receptors. It remains unclear if the blocking function of CTLA-4 antibody is required for its anti-tumor activity. To address this, here we have selected a non-blocking anti-CTLA-4 antibody (D138) and assessed its binding property and antitumor activity in comparison with the therapeutic CTLA-4 antibody ipilimumab. Crystal structures of CTLA-4 complexed with these antibodies show that D138 binds to a distinctly different site to that of ipilimumab on the CTLA-4 surface. D138 binding did not block the association of cells expressing CTLA-4 and B7 whereas ipilimumab did. Subsequent antitumor assay revealed that D138 was similarly effective as ipilimumab in inhibiting tumor growth in mice. This antitumor activity required Fc function for efficacy and was correlated with selective reduction of intratumor regulatory T (Treg) cells, resulting in a significant increase in the ratio of CD8+ over Treg cells. Overall these data clearly demonstrate that blocking CTLA-4-B7 interaction is not required for CTLA-4 antibody mediated antitumor activity, opening prospects of developing non-blocking CTLA-4 antibodies or simple binders towards other Treg surface markers for Treg-targeted immunotherapy.

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Dissociation of Systemic and Local Anti-Tumor Immunity Following Depletion of Regulatory T Cells Limits Therapeutic Activity in Established Tumors.

Blood ◽

10.1182/blood.v110.11.286.286 ◽

2007 ◽

Vol 110 (11) ◽

pp. 286-286

Author(s):

Karl S. Peggs ◽

Sergio A. Quezada ◽

Tyler R. Simpson ◽

James P. Allison

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Immunity ◽

Cd8 T Cells ◽

Clinical Studies ◽

Cell Activity ◽

Tumor Rejection ◽

Tumor Challenge ◽

Treg Depletion ◽

Anti Tumor Activity

Abstract Interference with the inhibitory immunological checkpoints controlling T-cell activation provides new opportunities to augment cancer immunotherapies. CD4+CD25+Foxp3+ T cells (Treg) are important regulators of T cell activity being largely responsible for the maintenance of peripheral self-tolerance. Evidence for their role in fostering immune privilege within tumors has fueled attempts to manipulate their number or function for therapeutic benefit. In pre-clinical tumor models, CD25-directed Treg depletion efficiently synergizes with various immune-based approaches but only when depletion occurs prior or close to the time of tumor challenge. Accordingly, depletion in clinical studies has failed to consistently enhance immunostimulatory strategies. CTLA-4 is a cell-intrinsic inhibitor of T cell activity, and blocking antibodies enhance anti-tumor activity in both pre-clinical and clinical studies. Using in vivo murine models we combined a GM-CSF-secreting cellular vaccine (Gvax), CTLA-4-blockade and CD25-directed Treg depletion (using αCD25 monoclonal antibody either before [prophylactic] or after [therapeutic] tumor challenge) and studied their effects on systemic and local anti-tumor immunity. In contrast to prophylactic Treg depletion, therapeutic depletion failed to promote tumor rejection; this correlated with a lack of accumulation of T-cells within the tumor. Gvax/αCTLA-4 induced systemic accumulation of Treg which was prevented by Treg depletion regardless of its timing. Systemic anti-tumor responses were comparable as shown by similar T cell proliferation profiles and similar numbers of tumor-specific IFN-producing cells, suggesting that failure of therapeutic depletion to enhance rejection was unrelated to depletion of CD25+ effector T cells (Teff). Foxp3-directed depletion (in Foxp3-DTR mice) confirmed these findings. Similar effects in adoptively transferred antigen-specific transgenic CD8+ T cells verify the relevance of these data to tumor-specific T cells. Within the tumor, αCD25 drove mainly CD8+ T cells into cell cycle, compared to mainly CD4+Foxp3− T cells with Gvax/αCTLA-4. Combination had an additive effect, inducing the proliferation of the whole Teff compartment regardless of the timing of αCD25. Intra-tumoral Foxp3+ Treg were in cycle independent of therapy, suggesting a constant turnover. Given the similarities in systemic immunity and proliferative responses of the infiltrating populations regardless of αCD25 timing, but marked differences in the numbers of cells accumulating within the tumor, we focused on the possibility that differences in migration from the vascular compartment might explain our observations. Only prophylactic αCD25 led to expression of endothelial activation markers on tumor vasculature, which directly correlated with intra-tumoral T cell accumulation and tumor rejection. Importantly, systemic anti-tumor activity was transferable from mice receiving therapeutic depletion into tumor-bearing recipients after non-myeloablative conditioning, resulting in activation of the vascular endothelium, T cell infiltration and tumor rejection. Our data demonstrate the potential of vaccination strategies to induce counter-productive immuno-inhibitory host responses and reveal a dichotomy between systemic and local anti-tumor immunity following therapeutic Treg depletion. Finally, they support an alternative strategy for the treatment of established tumors in humans that exploits the augmented systemic immunity induced by vaccination following Treg depletion.

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Metronomic cyclophosphamide regimen electively depletes CD4+ CD25+ regulatory T cells in patients with advanced solid tumors

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.2561 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 2561-2561

Author(s):

F. Ghiringhelli Esq. ◽

S. Ladoire ◽

L. Zitvogel ◽

B. Chauffert

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cancer Patients ◽

Solid Tumors ◽

Tumor Angiogenesis ◽

Selective Reduction ◽

Absolute Number ◽

Tumor Rejection ◽

Low Doses ◽

Proliferation Capacity

2561 Background: CD4+CD25+ regulatory T cells (Treg) are involved in the prevention of autoimmune diseases and in tumor-induced tolerance. We previously demonstrated in tumor-bearing rodents that low doses of cyclophosphamide (CPM) could significantly decrease both number and suppressive functions of Treg, facilitating vaccine-induced tumor rejection. Repeated low doses of cyclophosphamide, referred to as metronomic treatment aiming at reducing tumor angiogenesis, are used in patients with advanced, chemoresistant tumors. Methods: Six patients with metastatic solid tumors were treated after informed consent with metronomic CPM - 50 mg orally twice a day, with one week on, and one week off, until progression or limiting toxicity. Peripheral blood mononucleated cells (PBMC) were sampled before and after 4 weeks of treatment. Occurrence of blood leucocyte subpopulation and capacity of Treg to inhibit NK and T cells patients functions were measured. Results: metronomic CPM induced a profound and selective reduction of circulating Treg, both in percentage (7.7 ± 0.8 % before versus 3.3 ± 0.8 % after, p < 0.005) and absolute number (25.2 ± 7.5 cells/mm3 before versus 6.1 ± 3.3 cells/mm3 after, p < 0.0006). Inhibitory functions of Treg on conventional T cells and NK cells lead to a restoration of peripheral T cell proliferation - i.e. PBMC from cancer patients exhibited 17 ± 3% T cells proliferation capacity after CD3 CD28 stimulation versus 44 ± 5% (p < 0.01) 1 month after the beginning of the treatment, and versus 45 ± 6% in healthy volunteers- and innate NK killing activities - i.e. PBMC from cancer patients exhibited 9 ± 1% NK killing capacity against K562 target versus 26 ± 5% (p < 0.005) after 4 week treatment. Conclusions: Metronomic CPM has not only effect on tumor angiogenesis, but also strongly curtail immunosuppressive Treg, which could favor a better control of tumor progression. No significant financial relationships to disclose.

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Spontaneous tumor rejection by cbl-b–deficient CD8+ T cells

Journal of Experimental Medicine ◽

10.1084/jem.20061699 ◽

2007 ◽

Vol 204 (4) ◽

pp. 879-891 ◽

Cited By ~ 84

Author(s):

Stefanie Loeser ◽

Karin Loser ◽

Martijn S. Bijker ◽

Manu Rangachari ◽

Sjoerd H. van der Burg ◽

...

Keyword(s):

T Cells ◽

Antitumor Activity ◽

Tumor Cells ◽

Cd8 T Cells ◽

B Cell Lymphoma ◽

Ultraviolet B ◽

Tumor Rejection ◽

T Regulatory Cell ◽

Spontaneous Tumor ◽

B Mice

The concept of tumor surveillance implies that specific and nonspecific components of the immune system eliminate tumors in the early phase of malignancy. Understanding the biochemical mechanisms of tumor immunosurveillance is of paramount significance because it might allow one to specifically modulate spontaneous antitumor activity. We report that inactivation of the E3 ligase Casitas B cell lymphoma-b (Cbl-b) confers spontaneous in vivo rejection of tumor cells that express human papilloma virus antigens. Moreover, cbl-b−/− mice develop significantly fewer ultraviolet B (UVB)–induced skin malignancies and reject UVB-induced skin tumors. CD8+ T cells were identified as key players in the spontaneous tumor rejection response. Loss of Cbl-b not only enhances antitumor reactivity of CD8+ T cells but also occurs in the absence of CD4+ T cells. Mechanistically, cbl-b−/− CD8+ T cells are resistant to T regulatory cell–mediated suppression and exhibit enhanced activation and rapid tumor infiltration. Importantly, therapeutic transfer of naive cbl-b−/− CD8+ T cells is sufficient to mediate rejection of established tumors. Even up to 1 yr after the first encounter with the tumor cells, cbl-b−/− mice carry an “anticancer memory.” These data identify Cbl-b as a key signaling molecule that controls spontaneous antitumor activity of cytotoxic T cells in different cancer models. Inhibition of Cbl-b is a novel approach to stimulate long-lasting immunity against cancer.

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Tumor-specific regulatory T cells suppress lytic granule release of adoptively transferred cytotoxic effector T cells and inhibit tumor rejection

Zeitschrift für Gastroenterologie ◽

10.1055/s-0029-1241604 ◽

2009 ◽

Vol 47 (09) ◽

Author(s):

C Bauer ◽

F Marangoni ◽

T Murooka ◽

N Elpak ◽

TR Mempel

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Effector T Cells ◽

Tumor Rejection ◽

Lytic Granule ◽

Cytotoxic Effector ◽

Granule Release

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278 Phase I clinical trial evaluating the safety of ADP-A2M10 SPEAR T-cells in patients with MAGE-A10+ advanced non-small cell lung cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0278 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A303-A303

Author(s):

George Blumenschein ◽

Siddhartha Devarakonda ◽

Melissa Johnson ◽

Victor Moreno ◽

Justin Gainor ◽

...

Keyword(s):

Lung Cancer ◽

T Cells ◽

Antitumor Activity ◽

Small Cell Lung Cancer ◽

Dose Level ◽

Cell Lung Cancer ◽

Lentiviral Vector ◽

Small Cell ◽

Small Cell Lung ◽

Human Dose

BackgroundADP-A2M10 SPEAR T-cells are genetically engineered autologous T-cells that express a high affinity MAGE-A10-specific T-cell receptor targeting MAGE-A10+tumors in the context of HLA-A*02. This trial is now complete (NCT02592577).MethodsThis first-in-human dose escalation trial utilized a modified 3+3 design to evaluate safety and antitumor activity. Eligible patients (pts) were HLA-A*02+ with advanced non-small cell lung cancer (NSCLC) expressing MAGE-A10. Pts underwent apheresis; T-cells were isolated, transduced with a lentiviral vector containing the TCR targeting MAGE-A10, and expanded. Pts underwent lymphodepletion (LD) with varying doses/schedules of fludarabine (Flu) and cyclophosphamide (Cy) prior to receiving ADP-A2M10. ADP-A2M10 was administered at Dose Level (DL) 1= 0.1×109, DL2 0.5–1.2×109, and DL3/Expansion= 1.2–15×109 transduced cells.ResultsAs of Jan 10, 2020, 11 pts (6 male/5 female) with NSCLC (3 squamous cell, 7 adenocarcinoma, 1 adenosquamous) were treated. Five, 3 and 3 pts received cells at DL1, DL2, and DL3/Expansion, respectively. The most frequently reported adverse events ≥ Grade 3 were lymphopenia (11), leukopenia (9), neutropenia (8), anemia (6), thrombocytopenia (5), and hyponatremia (5). Three pts reported CRS (Grades 1, 2, and 4, respectively). One pt received the highest dose of LD (Flu 30 mg/m2 Day 1 4 and Cy 1800 mg/m2 Day 1–2) prior to a second infusion and had a partial response (PR). This pt subsequently developed aplastic anemia and died. Responses included: 1 pt – PR, 3 pts - stable disease, 2 pts – progressive disease, 1 pt - too early to determine, 4 pts - off-study prior to tumor assessment. SPEAR T-cells were detectable in peripheral blood from pts at each dose level, and in tumor tissue from pts at DL1 and DL3.ConclusionsADP-A2M10 SPEAR T-cells have shown acceptable safety and no evidence of toxicity related to off-target binding or alloreactivity. Given the minimal antitumor activity and the discovery that MAGE-A10 expression frequently overlaps with MAGE-A4 expression, the clinical program has closed. Several trials with SPEAR T-cells targeting MAGE-A4 are ongoing (https://bit.ly/35htsZK).Trial RegistrationNCT02592577Ethics ApprovalThe trial was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and was approved by local authorities. An independent ethics committee or institutional review board approved the clinical protocol at each participating center. All the patients provided written informed consent before study entry.

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102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0102 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A113-A113

Author(s):

Mireia Bachiller García ◽

Lorena Pérez-Amill ◽

Anthony Battram ◽

Alvaro Urbano-Ispizua ◽

Beatriz Martín-Antonio

Keyword(s):

T Cells ◽

Tumor Cell ◽

Cord Blood ◽

Immune Cells ◽

In Vivo Models ◽

Cell Clusters ◽

Cytotoxicity Assays ◽

Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

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721 AT1412, a patient-derived CD9 antibody in preclinical development promoting tumor immune infiltration and inducing tumor rejection

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0721 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A763-A763

Author(s):

Remko Schotte ◽

Julien Villaudy ◽

Martijn Kedde ◽

Wouter Pos ◽

Daniel Go ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Tumor Cells ◽

Tumor Rejection ◽

Human Immune System ◽

Preclinical Development ◽

High Affinity ◽

Human Immune ◽

A375 Melanoma

BackgroundAdaptive immunity to cancer cells forms a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether tumor-targeting antibodies could be isolated from a patient that cured (now 13 years tumor-free) metastatic melanoma following adoptive transfer of ex vivo expanded autologous T cells.MethodsPatient‘s peripheral blood B cells were isolated and tested for the presence of tumor-reactive B cells using AIMM’s immmortalisation technology. Antibody AT1412 was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 binds the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities in cancer and induces ADCC and ADCP by effector cells.ResultsSpontaneous immune rejection of tumors was observed in human immune system (HIS) mouse models implanted with CD9 genetically-disrupted A375 melanoma (A375-CD9KO) tumor cells, while A375wt cells were not cleared. Most notably, no tumor rejection of A375-CD9KO tumors was observed in NSG mice, indicating that blockade of CD9 makes tumor cells susceptible to immune rejection.CD9 has been described to regulate integrin signaling, e.g. LFA-1, VLA-4, VCAM-1 and ICAM-1. AT1412 was shown to modulate CD9 function by enhancing adhesion and transmigration of T cells to endothelial (HUVEC) cells. AT1412 was most potently enhancing transendothelial T-cell migration, in contrast to a high affinity version of AT1412 or other high affinity anti-CD9 reference antibodies (e.g. ALB6). Enhanced immune cell infiltration is also observed in immunodeficient mice harbouring a human immune system (HIS). AT1412 strongly enhanced CD8 T-cell and macrophage infiltration resulting in tumor rejection (A375 melanoma). PD-1 checkpoint blockade is further sustaining this effect. In a second melanoma model carrying a PD-1 resistant and highly aggressive tumor (SK-MEL5) AT1412 together with nivolumab was inducing full tumor rejection, while either one of the antibodies alone did not.ConclusionsThe safety of AT1412 has been assessed in preclinical development and is well tolerated up to 10 mg/kg (highest dose tested) by non human primates. AT1412 demonstrated a half-life of 8.5 days, supporting 2–3 weekly administration in humans. Besides transient thrombocytopenia no other pathological deviations were observed. No effect on coagulation parameters, bruising or bleeding were observed macro- or microscopically. The thrombocytopenia is reversible, and its recovery accelerated in those animals developing anti-drug antibodies. First in Human clinical study is planned to start early 2021.Ethics ApprovalStudy protocols were approved by the Medical Ethical Committee of the Leiden University Medical Center (Leiden, Netherlands).ConsentBlood was obtained after written informed consent by the patient.

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Selective elimination of immunosuppressive T cells in patients with multiple myeloma

Leukemia ◽

10.1038/s41375-021-01172-x ◽

2021 ◽

Author(s):

Mohamed H. S. Awwad ◽

Abdelrahman Mahmoud ◽

Heiko Bruns ◽

Hakim Echchannaoui ◽

Katharina Kriegsmann ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Immune Responses ◽

High Frequency ◽

Surface Markers ◽

Selective Elimination ◽

Related Transcription Factor ◽

Cell Membrane Protein

AbstractElimination of suppressive T cells may enable and enhance cancer immunotherapy. Here, we demonstrate that the cell membrane protein SLAMF7 was highly expressed on immunosuppressive CD8+CD28-CD57+ Tregs in multiple myeloma (MM). SLAMF7 expression associated with T cell exhaustion surface markers and exhaustion-related transcription factor signatures. T cells from patients with a high frequency of SLAMF7+CD8+ T cells exhibited decreased immunoreactivity towards the MART-1aa26–35*A27L antigen. A monoclonal anti-SLAMF7 antibody (elotuzumab) specifically depleted SLAMF7+CD8+ T cells in vitro and in vivo via macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). Anti-SLAMF7 treatment of MM patients depleted suppressive T cells in peripheral blood. These data highlight SLAMF7 as a marker for suppressive CD8+ Treg and suggest that anti-SLAMF7 antibodies can be used to boost anti-tumoral immune responses in cancer patients.

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