RESUMEN
Outcomes in acute myeloid leukemia (AML) remain unsatisfactory, and novel treatments are urgently needed. One strategy explores antibodies and their drug conjugates, particularly those targeting CD33. Emerging data with gemtuzumab ozogamicin (GO) demonstrate target validity and activity in some patients with AML, but efficacy is limited by heterogeneous drug conjugation, linker instability, and a high incidence of multidrug resistance. We describe here the development of SGN-CD33A, a humanized anti-CD33 antibody with engineered cysteines conjugated to a highly potent, synthetic DNA cross-linking pyrrolobenzodiazepine dimer via a protease-cleavable linker. The use of engineered cysteine residues at the sites of drug linker attachment results in a drug loading of approximately 2 pyrrolobenzodiazepine dimers per antibody. In preclinical testing, SGN-CD33A is more potent than GO against a panel of AML cell lines and primary AML cells in vitro and in xenotransplantation studies in mice. Unlike GO, antileukemic activity is observed with SGN-CD33A in AML models with the multidrug-resistant phenotype. Mechanistic studies indicate that the cytotoxic effects of SGN-CD33A involve DNA damage with ensuing cell cycle arrest and apoptotic cell death. Together, these data suggest that SGN-CD33A has CD33-directed antitumor activity and support clinical testing of this novel therapeutic in patients with AML.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Benzodiazepinas/química , Resistencia a Antineoplásicos , Inmunoconjugados/química , Leucemia Mieloide Aguda/tratamiento farmacológico , Lectina 3 Similar a Ig de Unión al Ácido Siálico/química , Animales , Apoptosis , Ciclo Celular , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacología , Cisteína/genética , Dimerización , Diseño de Fármacos , Células HEK293 , Células HL-60 , Humanos , Leucemia Mieloide Aguda/inmunología , RatonesRESUMEN
PURPOSE: Leukemias with MLL gene rearrangement are associated with a poor prognosis. Natural killer (NK) cell therapy is a potential treatment, but leukemia cells may be resistant. Here, we sought to determine the susceptibility of MLL-rearranged leukemia cells to NK cell lysis and to develop a novel immunotherapeutic approach to optimize NK cell therapy, including the use of an antibody against leukemia-associated antigen and the elimination of killer-cell immunoglobulin-like receptor (KIR)-mediated inhibition. EXPERIMENTAL DESIGN: Three MLL-rearranged leukemia cell lines (RS4;11, SEM, and MV4-11) and primary leukemia blasts were assessed for surface phenotype and susceptibility to NK cell lysis with or without antibodies against CD19 (XmAb5574), CD33 (lintuzumab), or KIR ligands. RESULTS: All three cell lines were resistant to NK cell lysis, had some inhibitory KIR ligands and protease inhibitor-9, and expressed low levels of NKG2D activating ligands and adhesion molecules. After treatment with XmAb5574 or lintuzumab, MLL-rearranged leukemia cells were efficiently killed by NK cells. The addition of pan-major histocompatibility complex class I antibody, which blocked inhibitory KIR-HLA interaction, further augmented degranulation in all three KIR2DL1, KIR2DL2/3, and KIR3DL1 subsets of NK cells based on the rule of missing-self recognition. A mouse model showed a decreased rate of leukemia progression in vivo as monitored by bioluminescence imaging and longer survival after antibody treatment. CONCLUSION: Our data support the use of a triple immunotherapy approach, including an antibody directed against tumor-associated antigen, KIR-mismatched NK cell transplantation, and inhibitory KIR blockade, for the treatment of NK cell-resistant MLL-rearranged leukemias.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos HLA-B/metabolismo , Células Asesinas Naturales/trasplante , Leucemia/terapia , Proteína de la Leucemia Mieloide-Linfoide/genética , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas de Cocultivo , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de IgG/genética , Receptores KIR/metabolismo , Translocación Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite major advances in the treatment of non-Hodgkin lymphoma (NHL), including the use of chemotherapeutic agents and the anti-CD20 antibody rituximab, the majority of patients eventually relapse, and salvage treatments with non-cross-resistant compounds are needed to further improve patient survival. Here, we evaluated the antitumor effects of the microtubule destabilizing agent monomethyl auristatin E (MMAE) conjugated to the humanized anti-CD19 antibody hBU12 via a protease-sensitive valine-citrulline (vc) dipeptide linker. hBU12-vcMMAE induced potent tumor cell killing against rituximab-sensitive and -resistant NHL cell lines. CD19 can form heterodimers with CD21, and high levels of CD21 were reported to interfere negatively with the activity of CD19-targeted therapeutics. However, we observed comparable internalization, intracellular trafficking, and drug release in CD21(low) and CD21(high), rituximab-sensitive and -refractory lymphomas treated with hBU12-vcMMAE. Furthermore, high rates of durable regressions in mice implanted with these tumors were observed, suggesting that both rituximab resistance and CD21 expression levels do not impact on the activity of hBU12-vcMMAE. Combined, our data suggest that hBU12-vcMMAE may represent a promising addition to the treatment options for rituximab refractory NHL and other hematologic malignancies, including acute lymphoblastic leukemia.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígenos CD19/inmunología , Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Inmunoconjugados/uso terapéutico , Linfoma no Hodgkin/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Animales , Anticuerpos Monoclonales de Origen Murino , Western Blotting , Línea Celular Tumoral , Supervivencia Celular , Citrulina/química , Citrulina/metabolismo , Dimerización , Dipéptidos/metabolismo , Femenino , Citometría de Flujo , Dosificación de Gen , Humanos , Técnicas para Inmunoenzimas , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/metabolismo , Lisosomas , Ratones , Ratones SCID , Oligopéptidos/metabolismo , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/inmunología , Receptores de Complemento 3d/metabolismo , Rituximab , Valina/química , Valina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bone metabolism requires tightly coupled activities exhibited by two unique cell populations, the bone-resorbing osteoclasts and the bone-forming osteoblasts. Imbalance in the function of these two cell types can result in osteoporosis, a condition characterized by loss in bone integrity and of bone mass. We developed a human bone cell culture model that allows the in vitro study of bone formation and osteoclastogenesis and employed this bone model for the screening and pharmacological analyses of protein and small molecule therapeutics. The cytokines, interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF), play an intricate role in osteoclastogenesis in this system. Neutralizing antibodies to IL-6 and GM-CSF decreased the formation of osteoclast-like cells. SP500263, an early lead compound from a novel class of selective estrogen receptor modulators (SERMs), was more efficacious than estrogen and comparable to raloxifene in blocking cytokine production and formation of osteoclast-like cells. Our research demonstrates the usefulness of the in vitro co-culture model in the dissection of molecular events relevant to bone metabolism and provides greater insight into a potential novel role for cytokines in bone resorption. Furthermore, representatives of the SP500263 family of SERMs may be effective as therapeutics for the treatment of osteoporosis.