RESUMEN
IL-15 and its receptor α (IL-15Rα) are co-expressed on antigen-presenting cells, allowing transpresentation of IL-15 to immune cells bearing IL-2RßγC and stimulation of effector immune responses. We reported previously that the high-affinity interactions between an IL-15 superagonist (IL-15N72D) and the extracellular IL-15Rα sushi domain (IL-15RαSu) could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes. The IL-15N72D·IL-15RαSuFc complex, also known as ALT-803, is a multimeric complex constructed by fusing IL-15N72D·IL-15RαSu to the Fc domain of IgG1. ALT-803 is an IL-15 superagonist complex that has been developed as a potent antitumor immunotherapeutic agent and is in clinical trials. Here we describe the creation of a novel fusion molecule, 2B8T2M, using the ALT-803 scaffold fused to four single chains of the tumor-targeting monoclonal antibody rituximab. This molecule displays trispecific binding activity through its recognition of the CD20 molecule on tumor cells, stimulation via IL-2RßγC displayed on immune effector cells, and binding to Fcγ receptors on natural killer cells and macrophages. 2B8T2M activates natural killer cells to enhance antibody-dependent cellular cytotoxicity, mediates complement-dependent cytotoxicity, and induces apoptosis of B-lymphoma cells. Compared with rituximab, 2B8T2M exhibits significantly stronger antitumor activity in a xenograft SCID mouse model and depletes B cells in cynomolgus monkeys more efficiently. Thus, ALT-803 can be modified as a functional scaffold for creating multispecific, targeted IL-15-based immunotherapeutic agents and may serve as a novel platform to improve the antitumor activity and clinical efficacy of therapeutic antibodies.
Asunto(s)
Inmunidad Celular/efectos de los fármacos , Interleucina-15/agonistas , Células Asesinas Naturales/inmunología , Linfoma de Células B/tratamiento farmacológico , Proteínas , Proteínas Recombinantes de Fusión , Rituximab , Animales , Línea Celular Tumoral , Femenino , Humanos , Interleucina-15/genética , Interleucina-15/inmunología , Linfoma de Células B/genética , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones SCID , Proteínas/química , Proteínas/genética , Proteínas/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Rituximab/química , Rituximab/genética , Rituximab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Anti-CD20 monoclonal antibodies (mAb) are an important immunotherapy for B-cell lymphoma, and provide evidence that the immune system may be harnessed as an effective lymphoma treatment approach. ALT-803 is a superagonist IL-15 mutant and IL-15Rα-Fc fusion complex that activates the IL-15 receptor constitutively expressed on natural killer (NK) cells. We hypothesized that ALT-803 would enhance anti-CD20 mAb-directed NK-cell responses and antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN: We tested this hypothesis by adding ALT-803 immunostimulation to anti-CD20 mAb triggering of NK cells in vitro and in vivo. Cell lines and primary human lymphoma cells were utilized as targets for primary human NK cells. Two complementary in vivo mouse models were used, which included human NK-cell xenografts in NOD/SCID-γc (-/-) mice. RESULTS: We demonstrate that short-term ALT-803 stimulation significantly increased degranulation, IFNγ production, and ADCC by human NK cells against B-cell lymphoma cell lines or primary follicular lymphoma cells. ALT-803 augmented cytotoxicity and the expression of granzyme B and perforin, providing one potential mechanism for this enhanced functionality. Moreover, in two distinct in vivo B-cell lymphoma models, the addition of ALT-803 to anti-CD20 mAb therapy resulted in significantly reduced tumor cell burden and increased survival. Long-term ALT-803 stimulation of human NK cells induced proliferation and NK-cell subset changes with preserved ADCC. CONCLUSIONS: ALT-803 represents a novel immunostimulatory drug that enhances NK-cell antilymphoma responses in vitro and in vivo, thereby supporting the clinical investigation of ALT-803 plus anti-CD20 mAbs in patients with indolent B-cell lymphoma.
Asunto(s)
Antineoplásicos/farmacología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Linfoma de Células B/inmunología , Linfoma de Células B/metabolismo , Proteínas/farmacología , Receptores de IgG/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Granzimas/genética , Granzimas/metabolismo , Humanos , Interferón gamma/biosíntesis , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Ratones , Ratones Noqueados , Perforina/genética , Perforina/metabolismo , Proteínas Recombinantes de Fusión , Rituximab/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
IL15, a potent stimulant of CD8(+) T cells and natural killer (NK) cells, is a promising cancer immunotherapeutic. ALT-803 is a complex of an IL15 superagonist mutant and a dimeric IL15 receptor αSu/Fc fusion protein that was found to exhibit enhanced biologic activity in vivo, with a substantially longer serum half-life than recombinant IL15. A single intravenous dose of ALT-803, but not IL15, eliminated well-established tumors and prolonged survival of mice bearing multiple myeloma. In this study, we extended these findings to demonstrate the superior antitumor activity of ALT-803 over IL15 in mice bearing subcutaneous B16F10 melanoma tumors and CT26 colon carcinoma metastases. Tissue biodistribution studies in mice also showed much greater retention of ALT-803 in the lymphoid organs compared with IL15, consistent with its highly potent immunostimulatory and antitumor activities in vivo. Weekly dosing with 1 mg/kg ALT-803 in C57BL/6 mice was well tolerated, yet capable of increasing peripheral blood lymphocyte, neutrophil, and monocyte counts by >8-fold. ALT-803 dose-dependent stimulation of immune cell infiltration into the lymphoid organs was also observed. Similarly, cynomolgus monkeys treated weekly with ALT-803 showed dose-dependent increases of peripheral blood lymphocyte counts, including NK, CD4(+), and CD8(+) memory T-cell subsets. In vitro studies demonstrated ALT-803-mediated stimulation of mouse and human immune cell proliferation and IFNγ production without inducing a broad-based release of other proinflammatory cytokines (i.e., cytokine storm). Based on these results, a weekly dosing regimen of ALT-803 has been implemented in multiple clinical studies to evaluate the dose required for effective immune cell stimulation in humans.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Antineoplásicos/uso terapéutico , Interleucina-15/uso terapéutico , Proteínas/uso terapéutico , Adyuvantes Inmunológicos/farmacocinética , Adyuvantes Inmunológicos/toxicidad , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Línea Celular Tumoral , Proliferación Celular , Citocinas/metabolismo , Femenino , Humanos , Inmunoterapia , Interleucina-15/farmacocinética , Interleucina-15/toxicidad , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/fisiología , Macaca fascicularis , Melanoma Experimental/tratamiento farmacológico , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Proteínas/farmacocinética , Proteínas/toxicidad , Proteínas Recombinantes de Fusión , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ALT-803, a complex of an interleukin (IL)-15 superagonist mutant and a dimeric IL-15 receptor αSu/Fc fusion protein, was found to exhibit significantly stronger in vivo biologic activity on NK and T cells than IL-15. In this study, we show that a single dose of ALT-803, but not IL-15 alone, eliminated well-established 5T33P and MOPC-315P myeloma cells in the bone marrow of tumor-bearing mice. ALT-803 treatment also significantly prolonged survival of myeloma-bearing mice and provided resistance to rechallenge with the same tumor cells through a CD8(+) T-cell-dependent mechanism. ALT-803 treatment stimulated CD8(+) T cells to secrete large amounts of IFN-γ and promoted rapid expansion of CD8(+)CD44(high) memory T cells in vivo. These memory CD8(+) T cells exhibited ALT-803-mediated upregulation of NKG2D (KLRK1) but not PD-1 (PDCD1) or CD25 (IL2RA) on their cell surfaces. ALT-803-activated CD8(+) memory T cells also exhibited nonspecific cytotoxicity against myeloma and other tumor cells in vitro, whereas IFN-γ had no direct effect on myeloma cell growth. ALT-803 lost its antimyeloma activity in tumor-bearing IFN-γ knockout mice but retained the ability to promote CD8(+)CD44(high) memory T-cell proliferation, indicating that ALT-803-mediated stimulation of CD8(+)CD44(high) memory T cells is IFN-γ-independent. Thus, besides well-known IL-15 biologic functions in host immunity, this study shows that IL-15-based ALT-803 could activate CD8(+)CD44(high) memory T cells to acquire a unique innate-like phenotype and secrete IFN-γ for nonspecific tumor cell killing. This unique immunomodulatory property of ALT-803 strongly supports its clinical development as a novel immunotherapeutic agent against cancer and viral infections.
Asunto(s)
Antineoplásicos/uso terapéutico , Interleucina-15/agonistas , Mieloma Múltiple/tratamiento farmacológico , Proteínas/uso terapéutico , Receptores de Interleucina-15/uso terapéutico , Animales , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Memoria Inmunológica , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mieloma Múltiple/inmunología , Proteínas Recombinantes de FusiónRESUMEN
IL-15, a promising cytokine for treating cancer and viral diseases, is presented in trans by the IL-15 receptor (IL-15R) alpha-chain to the IL-15Rßγc complex displayed on the surface of T cells and natural killer (NK) cells. We previously reported that an asparagine to aspartic acid substitution at amino acid 72 (N72D) of IL-15 provides a 4-5-fold increase in biological activity compared to the native molecule. In this report, we describe Chinese hamster ovary (CHO) cell expression of a soluble complex (IL-15 N72D:IL-15RαSu/Fc) consisting of the IL-15 N72D superagonist and a dimeric IL-15Rα sushi domain-IgG1 Fc fusion protein. A simple but readily scalable affinity and ion exchange chromatography method was developed to highly purify the complex having both IL-15 binding sites fully occupied. The immunostimulatory effects of this complex were confirmed using cell proliferation assays. Treatment of mice with a single intravenous dose of IL-15N72D:IL-15RαSu/Fc resulted in a significant increase in CD8+ T cells and NK cells that was not observed following IL-15 treatment. Pharmacokinetic analysis indicated that the complex has a 25-h half-life in mice which is considerably longer than <40-min half-life of IL-15. Thus, the enhanced activity of the IL-15N72D:IL-15RαSu/Fc complex is likely the result of the increased binding activity of IL-15N72D to IL-15Rßγc, optimized cytokine trans-presentation by the IL-15RαSu domain, the dimeric nature of the cytokine domain and its increased in vivo half-life compared to IL-15. These findings indicate that this IL-15 superagonist complex could serve as a superior immunostimulatory therapeutic agent.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-15/agonistas , Subunidad alfa del Receptor de Interleucina-15/aislamiento & purificación , Interleucina-15/agonistas , Interleucina-15/aislamiento & purificación , Mamíferos/metabolismo , Recombinación Genética/genética , Animales , Peso Corporal/efectos de los fármacos , Células CHO , Separación Celular , Cromatografía en Gel , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Humanos , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
Human interleukin-15 (hIL-15) and its receptor α (hIL-15Rα) are co-expressed in antigen presenting cells allowing trans-presentation of the cytokine to immune effector cells. We exploited the high-affinity interactions between hIL-15 and the extracellular hIL-15Rα sushi domain (hIL-15RαSu) to create a functional scaffold for the design of multispecific fusion protein complexes. Using single-chain T cell receptors (scTCRs) as recognition domains linked to the IL-15:IL-15Rα scaffold, we generated both bivalent and bispecific complexes. In these fusions, the scTCR domains retain the antigen-binding activity and the hIL-15 domain exhibits receptor binding and biological activity. As expected, bivalent scTCR fusions exhibited improved antigen binding due to increased avidity, whereas fusions comprising two different scTCR domains were capable of binding two cognate peptide/MHC complexes. Bispecific molecules containing scTCR and scCD8αß domains also exhibit enhanced binding to peptide/MHC complexes, demonstrating that the IL-15:IL-15Rα scaffold displays flexibility necessary to support multi-domain interactions with a given target. Surprisingly, functional heterodimeric molecules could be formed by co-expressing the TCR α and ß chains separately as fusions to the hIL-15 and hIL-15RαSu domains. Together, these properties indicate that the hIL-15 and hIL-15RαSu domains can be used as versatile, functional scaffold for generating novel targeted immune molecules.
Asunto(s)
Interleucina-15/química , Interleucina-15/inmunología , Receptores de Interleucina-15/química , Receptores de Interleucina-15/inmunología , Proteínas Recombinantes de Fusión/inmunología , Células Cultivadas , Humanos , Interleucina-15/genética , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Interleucina-15/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
IL-15 is an immunostimulatory cytokine trans-presented with the IL-15 receptor alpha-chain to the shared IL-2/IL-15Rbeta and common gamma-chains displayed on the surface of T cells and NK cells. To further define the functionally important regions of this cytokine, activity and binding studies were conducted on human IL-15 muteins generated by site-directed mutagenesis. Amino acid substitutions of the asparagine residue at position 72, which is located at the end of helix C, were found to provide both partial agonist and superagonist activity, with various nonconservative substitutions providing enhanced activity. Particularly, the N72D substitution provided a 4-5-fold increase in biological activity of the IL-15 mutein compared with the native molecule based on proliferation assays with cells bearing human IL-15Rbeta and common gamma-chains. The IL-15N72D mutein exhibited superagonist activity through improved binding ability to the human IL-15Rbeta-chain. However, the enhanced potency of IL-15N72D was not observed with cells expressing the mouse IL-15Ralpha-IL-15Rbeta-gamma(c) complex, suggesting that this effect is specific to the human IL-15 receptor. The enhanced biological activity of IL-15N72D was associated with more intense phosphorylation of Jak1 and Stat5 and better anti-apoptotic activity compared with the wild-type IL-15. IL-15N72D superagonist activity was also preserved when linked to a single-chain TCR domain to generate a tumor-specific fusion protein. Thus, the human IL-15 superagonist muteins and fusions may create opportunities to construct more efficacious immunotherapeutic agents with clinical utility.
Asunto(s)
Sustitución de Aminoácidos , Proliferación Celular/efectos de los fármacos , Interleucina-15/genética , Animales , Apoptosis , Línea Celular , Humanos , Interleucina-15/agonistas , Interleucina-15/farmacología , Subunidad alfa del Receptor de Interleucina-15/metabolismo , Subunidad beta del Receptor de Interleucina-2/metabolismo , Janus Quinasa 1/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Fosforilación , Factor de Transcripción STAT5/metabolismo , Especificidad de la EspecieRESUMEN
We have previously reported that a single-chain T cell receptor/IL-2 fusion protein (scTCR-IL2) exhibits potent targeted antitumor activity in nude mice bearing human tumor xenografts that display cognate peptide/HLA complexes. In this study, we further explore the mechanism of action of this molecule. We compared the biological activities of c264scTCR-IL2, a scTCR-IL2 protein recognizing the aa264-272 peptide of human p53, with that of MART-1scTCR-IL2, which recognizes the MART-1 melanoma antigen (aa27-35). In vitro studies showed that c264scTCR-IL2 and MART-1scTCR-IL2 were equivalent in their ability to bind cell-surface IL-2 receptors and stimulate NK cell responses. In mice, MART-1scTCR-IL2 was found to have a twofold longer serum half-life than c264scTCR-IL2. However, despite its shorter serum half-life, c264scTCR-IL2 showed significantly better antitumor activity than MART-1scTCR-IL2 against p53(+)/HLA-A2(+) tumor xenografts. The more potent antitumor activity of c264scTCR-IL2 correlated with an enhanced capacity to promote NK cell infiltration into tumors. Similar differences in antigen-dependent tumor infiltration were observed with activated splenocytes pre-treated in vitro with c264scTCR-IL2 or MART-1scTCR-IL2 and then transferred into p53(+)/HLA-A2(+) tumor bearing recipients. The data support a model where c264scTCR-IL2 activates immune cells to express IL-2 receptors. Following stable interactions with cell-surface IL-2 receptors, c264scTCR-IL2 fusion molecule enhances the trafficking of immune cells to tumors displaying target peptide/HLA complexes where the immune cells mediate antitumor effects. Thus, this type of fusion molecule could be used directly as a targeted immunotherapeutic or in adoptive cell transfer approaches to activate and improve the anti-cancer activities of immune cells by providing them with pre-selected antigen recognition capability.
Asunto(s)
Inmunoterapia/métodos , Interleucina-2/uso terapéutico , Melanoma Experimental/tratamiento farmacológico , Receptores de Antígenos de Linfocitos T/uso terapéutico , Proteínas Recombinantes de Fusión/farmacología , Animales , Antígenos de Neoplasias/inmunología , Femenino , Citometría de Flujo , Semivida , Humanos , Inmunohistoquímica , Interleucina-2/inmunología , Interleucina-2/metabolismo , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Antígeno MART-1 , Melanoma Experimental/inmunología , Ratones , Ratones Desnudos , Proteínas de Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína p53 Supresora de Tumor/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We previously have generated a single-chain T cell receptor-cytokine fusion protein (264scTCR/IL-2) comprising interleukin-2 genetically linked to a soluble HLA-A2.1-restricted TCR recognizing a peptide of human p53 protein. In this report, we show that 264scTCR/IL-2 inhibits the growth of primary tumors derived from the A375 (p53+/HLA-A2.1+) human melanoma and exhibits significantly better antitumor activity than recombinant human IL-2 alone. Moreover, treatment with 264scTCR/IL-2 results in tumor growth retardation in mice bearing large A375 tumors and other p53+/HLA-A2.1+ human tumors but does not affect tumor outgrowth of HLA-A2.1-negative tumors. This suggests that antigen targeting plays a substantial role in the efficacy of 264scTCR/IL-2 against p53+/HLA-A2+ tumors. Further, the antitumor activity of 264scTCR/IL-2 was found to be likely mediated by NK cell activation and tumor infiltration. A biologically active chimeric version of the molecule (c264scTCR/IL-2) also exhibits favorable pharmacokinetic properties required of a clinical candidate for this novel class of potent antitumor activities and targeted anticancer immunotherapeutics.
Asunto(s)
Antineoplásicos/uso terapéutico , Interleucina-2/uso terapéutico , Receptores de Antígenos de Linfocitos T/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Proteína p53 Supresora de Tumor/inmunología , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Femenino , Células HT29 , Humanos , Interleucina-2/genética , Interleucina-2/inmunología , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Solubilidad , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/uso terapéuticoRESUMEN
The amplified fragment length polymorphism (AFLP) technique was used to assess genetic variation across the genera of 2 Morone and 3 Thunnus species. The AFLP profiles from 23 primers revealed higher levels of polymorphism in each of 3 Thunnus species than in either of the 2 Morone species. However, extensive variation (20 of 23 primers) was observed between the 2 Morone species, but much less variation was observed among the 3 Thunnus species. In addition, comparisons of banding patterns indicated that albacore is divergent from both Atlantic northern bluefin tuna and yellowfin tuna. This result is consistent with the findings of several previous studies employing either allozymes or mitochondrial DNA. Overall, these results demonstrate that AFLP is a useful technique for the assessment of both intraspecies and interspecies variation of fish. Furthermore, the species-specific patterns produced by AFLP can be used for the identification of closely related species.
RESUMEN
Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass.
RESUMEN
Diplacus aurantiacus contains large amounts of a leaf phenolic resin, an important deterrent to a leaf-eating caterpillar, Euphydryas chalcedona. The resin can also retard water loss during drought. Furthermore, the leaf resin content differs among plants and populations. This study investigates the existence of heritable variation (h2 ) in resin production and tests for a genetic correlation (rG ) between carbon allocation to secondary metabolites and growth rate, as well as with three other vegetative traits. Nine dam and 10 sire plants were chosen randomly at a field site and used to generate 78 full-sib families (19 half-sib families) by crossing all males to all females in a factorial design. Heritability was estimated in two ways, and genetic correlations were estimated by three methods. We found: (1) the heritability of resin production estimated by the regression of offspring on sires was significantly greater than zero (hs2=0.32, P<0.01); (2) the maternal variance in resin content was significantly greater than zero (21.3% of total phenotypic variance); (3) significant negative genetic correlation between resin content and growth rate was observed from two of three methods and was consistent with the phenotypic correlation; and (4) the cost of resin could be assessed quantitatively. The genetic cost of 1 mg in resin is equivalent to 25 mg of dry shoot-biomass growth, but the phenotypic cost is only 2.1 mg. This study indicates that carbon allocation to these secondary metabolites may respond to natural selection, and the phenotypic cost of resin production has a genetic basis in D. aurantiacus. This trade-off suggests that once selection occurs, increased phenolic resin production may result in decreased growth, or vice versa.