T-cell help in the tumor microenvironment enhances rituximab-mediated NK-cell ADCC.

ABSTRACT: Rituximab (RTX) and other monoclonal antibodies (mAbs) that bind directly to malignant cells are of great clinical value but are not effective for all patients. A major mechanism of action of RTX is antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Prior in vitro studies in our laboratory demonstrated that T cells contribute to maintaining the viability and cytotoxic potential of NK cells activated by anti-CD20-coated target B cells. Here, we conducted studies using a novel mouse model and clinical correlative analysis to assess whether T-cell help contribute to RTX-mediated NK-cell ADCC in the tumor microenvironment (TME) in vivo. A humanized mouse model was developed using Raji lymphoma cells and normal donor peripheral blood mononuclear cells that allows for control of T-cell numbers in the lymphoma TME. In this model, NK-cell viability and CD16 and CD25 expression dropped after RTX in the absence of T cells but increased in the presence of T cells. RTX therapy was more effective when T cells were present and was ineffective when NK cells were depleted. In patients with indolent lymphoma, fine needle aspirates were obtained before and ∼1 week after treatment with a RTX-containing regimen. There was a strong correlation between CD4+ T cells as well as total T cells in the pretherapy TME and an increase in NK-cell CD16 and CD25 expression after RTX. We conclude that T-cell help in the TME enhances RTX-mediated NK-cell viability and ADCC.

Bispecific antibody-activated T cells enhance NK cell-mediated antibody-dependent cellular cytotoxicity.

Resistance to anti-cancer monoclonal antibody (mAb) therapy remains a clinical challenge. Previous work in our laboratory has shown that T cell help in the form of interleukin-2 maintains long-term NK cell viability and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). Lack of such T cell help may be a potential mechanism for resistance to mAb therapy. Here, we evaluate whether concomitant treatment with anti-CD3 × anti-cancer bispecific antibodies (bsAbs) can overcome this resistance by enhancing T cell help, and thereby maintaining long-term NK cell-mediated ADCC. Normal donor peripheral blood mononuclear cells were depleted of T cells, replenished with defined numbers of autologous T cells (from 0.75 to 50%) and co-cultured with mono-/bispecific antibody-treated target tumor cells for up to 7 days. At low T cell concentrations, bsAb-activated T cells (mainly CD4+ T cells) were more effective than resting T cells at maintaining NK cell viability and ADCC. Brief (4 h to 2 day) bsAb exposure was sufficient to enhance long-term ADCC by NK cells. These findings raise the hypothesis that local T cell activation mediated by systemic treatment with anti-CD3 X anti-cancer bsAb may enhance the anti-tumor efficacy of monospecific mAbs that mediate their primary therapeutic effect via NK-mediated ADCC.

Monocytes Exposed to Immune Complexes Reduce pDC Type 1 Interferon Response to Vidutolimod.

Vidutolimod, also known as CMP-001, is a virus-like particle composed of the Qß bacteriophage coat protein encasing a TLR9 agonist. Vidutolimod injected intratumorally is showing promise in early phase clinical trials based on its ability to alter the tumor microenvironment and induce an anti-tumor immune response. We previously demonstrated that the in vivo efficacy of vidutolimod is dependent on the presence of anti-Qß antibodies that enhance opsonization and uptake of vidutolimod by TLR9-expressing plasmacytoid dendritic cells (pDCs). Here, we evaluated the effect of immune complexes, including anti-Qß-coated vidutolimod, on induction of Type 1 Interferon production by peripheral blood mononuclear cells in response to vidutolimod and soluble TLR9 agonists. Immune complexes, including but not limited to anti-Qß-coated vidutolimod, indirectly suppressed TLR9-mediated Type 1 Interferon production by pDCs in a monocyte-dependent manner. These findings indicate that anti-Qß-coated vidutolimod has effects in addition to those mediated by TLR9 that could have important clinical implications for understanding the mechanism of action of this exciting new approach to in situ immunization and cancer immunotherapy.

Antibody Opsonization of a TLR9 Agonist-Containing Virus-like Particle Enhances In Situ Immunization.

The immunologic and therapeutic effects of intratumoral (IT) delivery of a novel virus-like particle as a lymphoma immunotherapy were evaluated in preclinical studies with human cells and a murine model. CMP-001 is a virus-like particle composed of the Qß bacteriophage capsid protein encapsulating an immunostimulatory CpG-A oligodeoxynucleotide TLR9 agonist. In vitro, CMP-001 induced cytokine production, including IFN-α from plasmacytoid dendritic cells, but only in the presence of anti-Qß Ab. In vivo, IT CMP-001 treatment of murine A20 lymphoma enhanced survival and reduced growth of both injected and contralateral noninjected tumors in a manner dependent on both the ability of mice to generate anti-Qß Ab and the presence of T cells. The combination of IT CMP-001 with systemic anti-PD-1 enhanced antitumor responses in both injected and noninjected tumors. IT CMP-001 alone or combined with anti-PD-1 augmented T cell infiltration in tumor-draining lymph nodes. We conclude IT CMP-001 induces a robust antitumor T cell response in an anti-Qß Ab-dependent manner and results in systemic antitumor T cell effects that are enhanced by anti-PD-1 in a mouse model of B cell lymphoma. Early-phase clinical evaluation of CMP-001 and anti-PD-1 combination therapy in lymphoma will begin shortly, based in part on these results.

Annexin-V promotes anti-tumor immunity and inhibits neuroblastoma growth in vivo.

The goal of the current study is to determine the effects of blocking phosphatidylserine (PS) on the growth of neuroblastoma in mice. PS, an anionic phospholipid restricted to the cytoplasmic surface of plasma membranes in most cells, is externalized to the surface of apoptotic cells. PS has been shown to induce immune tolerance to self-antigens. PS can also be found on the surface of live cells and in particular tumor cells. Annexin-V (AnV) is a protein that specifically binds and blocks PS. To determine the effects of blocking PS with AnV on tumor growth and immunogenicity, mice were inoculated with AGN2a, a poorly immunogenic murine neuroblastoma that expresses high level of PS on the cell surface. Survival and anti-tumor T cell response were determined. AGN2a were engineered to secrete AnV. Secreted protein effectively blocked tumor PS. 40 % of mice inoculated with AnV-expressing AGN2a cells survived free of tumor, whereas none of the mice inoculated with control cells survived (p = 0.0062). The benefits of AnV were lost when mice were depleted of T cells. The findings suggest that AnV could protect mice from tumor challenge through an immune mediated mechanism. Mice were then immunized with irradiated AnV-secreting or control cells, and challenged with wild-type AGN2a cells. AnV-secreting cell vaccine protected 80 % of mice from AGN2a challenge, while control cell vaccine prevented tumor growth in only 30 % of animals (p = 0.012). ELISPOT analysis demonstrated that AnV-secreting cell vaccine induced a greater frequency of interferon-gamma producing splenic T cells. T cells isolated from mice immunized with AnV-secreting but not control vaccine lysed AGN2a. In summary, AnV blocked PS, enhanced T cell mediated tumor immunity, and inhibited tumor growth.

Characterization of erasin (UBXD2): a new ER protein that promotes ER-associated protein degradation.

Ubiquitin regulator-X (UBX) is a discrete protein domain that binds p97/valosin-containing protein (VCP), a molecular chaperone involved in diverse cell processes, including endoplasmic-reticulum-associated protein degradation (ERAD). Here we characterize a human UBX-containing protein, UBXD2, that is highly conserved in mammals, which we have renamed erasin. Biochemical fractionation, immunofluorescence and electron microscopy, and protease protection experiments suggest that erasin is an integral membrane protein of the endoplasmic reticulum and nuclear envelope with both its N- and C-termini facing the cytoplasm or nucleoplasm. Localization of GFP-tagged deletion derivatives of erasin in HeLa cells revealed that a single 21-amino-acid sequence located near the C-terminus is necessary and sufficient for localization of erasin to the endoplasmic reticulum. Immunoprecipitation and GST-pulldown experiments confirmed that erasin binds p97/VCP via its UBX domain. Additional immunoprecipitation assays indicated that erasin exists in a complex with other p97/VCP-associated factors involved in ERAD. Overexpression of erasin enhanced the degradation of the ERAD substrate CD3delta, whereas siRNA-mediated reduction of erasin expression almost completely blocked ERAD. Erasin protein levels were increased by endoplasmic reticulum stress. Immunohistochemical staining of brain tissue from patients with Alzheimer's disease and control subjects revealed that erasin accumulates preferentially in neurons undergoing neurofibrillary degeneration in Alzheimer's disease. These results suggest that erasin may be involved in ERAD and in Alzheimer's disease.

Suppression of polyglutamine-induced toxicity in cell and animal models of Huntington's disease by ubiquilin.

Expanded polyglutamine (polyQ) tracts are associated with the induction of protein aggregation and cause cytotoxicity in nine different neurodegenerative disorders. Here, we report that ubiquilin suppresses polyQ-induced protein aggregation and toxicity in cells and in an animal model of Huntington's disease. Overexpression of ubiquilin in HeLa cells and primary neurons reduced aggregation of polyQ-containing proteins and cell death induced by overexpression of a green fluorescent protein (GFP)-huntingtin fusion protein containing 74 polyQ repeats [GFP-Htt(Q74)], in a dose-dependent manner. Moreover, overexpression of ubiquilin suppressed oxidative stress-induced cell death in HeLa cell lines stably expressing GFP-Htt(Q74). In contrast, knockdown of ubiquilin expression in these cell lines was associated with increases in DNA fragmentation, caspase activation, GFP-fusion protein aggregation, and cell death. Caenorhabditis elegans lines expressing GFP-Htt fusion proteins in body wall muscle displayed a polyQ repeat length-dependent decrease in body movement compared with wild-type animals. RNA interference of the C. elegans ubiquilin gene exacerbated the motility defect, whereas overexpression of ubiquilin prevented, and could rescue, loss of worm movement induced by overexpression of GFP-Htt(Q55). These results suggest that ubiquilin might be a novel therapeutic target for treating polyQ diseases.