Takahiro Kamiya

T Lymphocytes Expressing a CD16 Signaling Receptor Exert Antibody-Dependent Cancer Cell Killing

To expand applications for T-cell-based immunotherapy in cancer, we designed a receptor that binds the Fc portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16 (FCGR3A) V158 variant, CD8α hinge, and transmembrane domains, along with signaling domains from CD3ζ and 4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-ζ. After retrovirus-mediated expression in human T cells, CD16V-BB-ζ bound humanized antibodies with higher affinity than a control receptor containing the more common F158 variant. Engagement of CD16V-BB-ζ provoked T-cell activation, exocytosis of lytic granules, and sustained proliferation, with a mean cell recovery after 4-week coculture with Daudi lymphoma cells and rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect. CD16V-BB-ζ T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells in combination with rituximab at a low effector:target ratio, even when assayed on mesenchymal cells. Trastuzumab triggered CD16V-BB-ζ-mediated killing of HER2 (ERBB2)(+) breast and gastric cancer cells; similar results were obtained with an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration of CD16V-BB-ζ T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo. Signaling mediated by 4-1BB-CD3ζ induced higher T-cell activation, proliferation, and cytotoxicity than CD3ζ or FcεRIγ, and the receptor was expressed effectively after mRNA electroporation without viral vectors, facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-ζ as a universal, next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.

Autonomous growth and increased cytotoxicity of natural killer cells expressing membrane-bound interleukin-15.

Natural killer (NK) cell survival and, hence, cytotoxicity requires cytokine support. We determined whether expression of interleukin-15 (IL-15) in a nonsecretory, membrane-bound form could sustain NK cell growth. We linked the human IL15 gene to that encoding CD8α transmembrane domain (mbIL15). After retroviral transduction, human NK cells expressed mbIL15 on the cell surface; IL-15 secretion was negligible. Survival of mbIL15-NK cells without interleukin-2 (IL-2) after 7-day culture was vastly superior to that of mock-transduced NK cells (P < .001, n = 15) and of NK cells expressing nonmembrane-bound IL-15 (P = .025, n = 9); viable mbIL15-NK cells were detectable for up to 2 months. In immunodeficient mice, mbIL15-NK cells expanded without IL-2 and were detectable in all tissues examined (except brain) in much higher numbers than mock-transduced NK cells (P < .001). Expansion further increased with IL-2. The primary mechanism of mbIL15 stimulation was autocrine; it activated IL-15 signaling and antiapoptotic signaling. NK cells expressing mbIL15 had higher cytotoxicity against leukemia, lymphoma, and solid tumor cells in vitro and against leukemia and sarcoma cells in xenograft models. Thus, mbIL15 confers independent growth to NK cells and enhances their antitumor capacity. Infusion of mbIL15-NK cells would allow NK cell therapy without the potential adverse effects of cytokine administration.

EXPANDED AND ACTIVATED NATURAL KILLER CELLS FOR IMMUNOTHERAPY OF HEPATOCELLULAR CARCINOMA

Hepatocellular carcinoma (HCC) is often incurable. Human NK cells with enhanced cytotoxicity against HCC were expanded in vitro, generating efficient killers of HCC in culture and in mice, supporting the infusion of such cells for treatment of HCC. Viral infection of the liver is a major risk factor for hepatocellular carcinoma (HCC). Natural killer (NK) cells recognize virally infected and oncogenically transformed cells, suggesting a therapeutic role for NK-cell infusions in HCC. Using the K562-mb15-41BBL cell line as a stimulus, we obtained large numbers of activated NK cells from the peripheral blood of healthy donors. Expanded NK cells exerted remarkably high cytotoxicity against HCC cell lines, which was generally much higher than that of unstimulated or IL2-activated NK cells. In immunodeficient NOD/scid IL2RGnull mice engrafted with Hep3B, treatment with expanded NK cells markedly reduced tumor growth and improved overall survival. HCC cells exposed for 48 hours to 5 μmol/L of sorafenib, a kinase inhibitor currently used for HCC treatment, remained highly sensitive to expanded NK cells. HCC cell reductions of 39.2% to 53.8% caused by sorafenib in three cell lines further increased to 80.5% to 87.6% after 4 hours of culture with NK cells at a 1:1 effector-to-target ratio. NK-cell cytotoxicity persisted even in the presence of sorafenib. We found that NKG2D, an NK-cell–activating receptor, was an important mediator of anti-HCC activity. We therefore enhanced its signaling capacity with a chimeric NKG2D-CD3ζ-DAP10 receptor. This considerably increased the anti-HCC cytotoxicity of expanded NK cells in vitro and in immunodeficient mice. The NK expansion and activation method applied in this study has been adapted to clinical-grade conditions. Hence, these results warrant clinical testing of expanded NK-cell infusions in patients with HCC, possibly after genetic modification with NKG2D-CD3ζ-DAP10. Cancer Immunol Res; 4(7); 574–81. ©2016 AACR.

Therapeutic potential of highly cytotoxic natural killer cells for gastric cancer.

To develop more effective therapies for patients with advanced gastric cancer, we examined the potential of ex vivo expanded natural killer (NK) cells. We assessed the expression of ligands for NK Group 2 Member D (NKG2D, an important NK activation molecule) in primary tumors from 102 patients with gastric cancer by immunohistochemistry and determined their prognostic value. We then examined the in vitro and in vivo cytotoxicity of NK cells from healthy donors and patients with gastric cancer. The cytotoxicity of resting and of interleukin (IL)‐2‐activated NK cells was compared to that of NK cells expanded for 7 days by coculture with the K562‐mb15‐4.1BBL cell line. As a result, the expression of NKG2D ligands in primary tumors was correlated with favorable presenting features and outcomes, suggesting that gastric cancer may be sensitive to NK cell cytotoxicity. Although resting NK cells showed minimal cytotoxicity against gastric cancer cells, K562‐mb15‐4.1BBL‐expanded NK cells were highly cytotoxic and significantly more powerful than IL‐2‐activated NK cells. Cytotoxicity was correlated with NKG2D ligand expression and could be modulated by mitogen‐activated protein kinase and AKT‐PI3 kinase inhibitors. The cytotoxicity of expanded NK cells against HER2‐positive gastric cancer cells could be increased by Herceptin and further augmented by Lapatinib. Finally, expanded NK cells exhibited strong antitumor activity in immunodeficient mice engrafted with a gastric cancer cell line. In conclusion, gastric cancer tumors express NKG2D ligands and are highly susceptible to killing by NK cells stimulated by K562‐mb15‐4.1BBL. These results provide a strong rationale for clinical testing of these NK cells in patients and suggest their use to augment the effects of antibody therapy.

Expanded and armed natural killer cells for cancer treatment.

The capacity of natural killer (NK) cells to recognize and kill transformed cells suggests that their infusion could be used to treat cancer. It is difficult to obtain large numbers of NK cells ex vivo by exposure to cytokines alone but the addition of stimulatory cells to the cultures can induce NK cell proliferation and long-term expansion. Some of these methods have been validated for clinical-grade application and support clinical trials testing feasibility and safety of NK cell administration. Early data indicate that ex vivo expansion of NK cells from healthy donors or from patients with cancer is robust, allowing multiple infusions from a single apheresis. NK cells can transiently expand in vivo after infusion. Allogeneic NK cells are not direct effectors of graft-versus-host disease but this may occur if donor NK cells are infused after allogeneic hematopoietic stem cell transplant, which may activate T cell alloreactivity. NK cells can be directed with antibodies, or engineered using either transient modification by electroporation of mRNA or prolonged gene expression by viral transduction. Thus, expanded NK cells can be armed with activating receptors that enhance their natural anti-tumor capacity or with chimeric antigen receptors that can redirect them towards specific tumor targets. They can also be induced to express cytokines that promote their autonomous growth, further supporting their in vivo expansion. With the implementation of these approaches, expanded and armed NK cells should ultimately become a powerful component of immunotherapy of cancer.

Iodine-131-metaiodobenzylguanidine therapy with reduced-intensity allogeneic stem cell transplantation in recurrent neuroblastoma.

Neuroblastoma is the most common extracranial solid tumor of childhood, and iodine‐131‐metaiodobenzylguanidine (MIBG) therapy is a new approach for grade IV neuroblastoma. We describe the case history of a 3‐year‐old girl with recurrent neuroblastoma who received MIBG therapy with reduced‐intensity allogeneic stem cell transplantation (RIST) because of an extensive bone marrow involvement. The post‐transplant course was uneventful and complete chimerism was obtained. Neither acute nor chronic graft‐versus‐host disease (GVHD) was observed. The patient remained in remission for 3 months after RIST until the second relapse. MIBG therapy combined with RIST warrants further trials. Pediatr Blood Cancer 2008;50:676–678.

Intellectual development after treatment in children with acute leukemia and brain tumor

Background:u2002 The influence of central nervous system (CNS)‐directed chemotherapy on intelligence remains controversial. In this study, we investigated the influence of treatment on intellectual development in acute lymphoblastic leukemia (ALL) and brain tumor patients undergoing CNS‐directed treatments.

Congenital dyserythropoietic anemia

Congenital dyserythropoietic anemias (CDAs) are a heterogeneous group of rare hereditary disorders of erythropoiesis characterized by morphologic abnormal erythroblasts in the bone marrow. Three types of the disease are known as type I, II and III, and the variant type of CDA and several minor subgroups of CDA have been also reported since the first classification. Recently, responsible genes for type I (CDAN1) and type II (SEC23B) have been identified and the molecular pathogenesis of the disease is currently being explored. Although CDAs rarely transform to myelodysplastic syndrome or leukemia, the disease is important to understand the mechanism of hemopoiesis in humans.

Acute megakaryoblastic leukemia with acquired trisomy 21 and GATA1 mutations in phenotypically normal children

AbstractGATA1 mutations are found almost exclusively in children with myeloid proliferations related to Down syndrome (DS). Here, we report two phenotypically and cytogenetically normal children with acute megakaryoblastic leukemia (AMKL) whose blasts had both acquired trisomy 21 and GATA1 mutation. Patient 1 was diagnosed with transient abnormal myelopoiesis in the neonatal period. Following spontaneous improvement of the disease, leukemic blasts increased 7xa0months later. He received less intensive chemotherapy, and he is now 6xa0years old in complete remission. Patient 2 was diagnosed with AMKL at the age of 18xa0months. Although he received intensive chemotherapy and a cord blood transplantation, he died without gaining remission. In both cases, trisomy 21 and GATA1 mutation were detected only in leukemic blasts, but not in germline samples. Based on a literature review, we identified reports describing 14 non-DS AMKL with GATA1 mutation and acquired trisomy 21. Of those, 12 cases were diagnosed during the neonatal period, whereas the remaining 2 cases were diagnosed at the age of 22 and 31xa0months, respectively.n Conclusion: These cases suggest that GATA1 mutation may cooperate with the additional chromosome 21 in developing myeloid proliferations even in non-DS patients.

Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies

Effective immunotherapies for T-cell malignancies are lacking. We devised a novel approach based on chimeric antigen receptor (CAR)-redirected T lymphocytes. We selected CD7 as a target because of its consistent expression in T-cell acute lymphoblastic leukemia (T-ALL), including the most aggressive subtype, early T-cell precursor (ETP)-ALL. In 49 diagnostic T-ALL samples (including 14 ETP-ALL samples), median CD7 expression was >99%; CD7 expression remained high at relapse (n = 14), and during chemotherapy (n = 54). We targeted CD7 with a second-generation CAR (anti-CD7-41BB-CD3ζ), but CAR expression in T lymphocytes caused fratricide due to the presence of CD7 in the T cells themselves. To downregulate CD7 and control fratricide, we applied a new method (protein expression blocker [PEBL]), based on an anti-CD7 single-chain variable fragment coupled with an intracellular retention domain. Transduction of anti-CD7 PEBL resulted in virtually instantaneous abrogation of surface CD7 expression in all transduced T cells; 2.0% ± 1.7% were CD7+ vs 98.1% ± 1.5% of mock-transduced T cells (n = 5; P < .0001). PEBL expression did not impair T-cell proliferation, interferon-γ and tumor necrosis factor-α secretion, or cytotoxicity, and eliminated CAR-mediated fratricide. PEBL-CAR T cells were highly cytotoxic against CD7+ leukemic cells in vitro and were consistently more potent than CD7+ T cells spared by fratricide. They also showed strong anti-leukemic activity in cell line- and patient-derived T-ALL xenografts. The strategy described in this study fits well with existing clinical-grade cell manufacturing processes and can be rapidly implemented for the treatment of patients with high-risk T-cell malignancies.