RESUMEN
Sacituzumab govitecan (IMMU-132) is an antibody-drug conjugate (ADC) made from a humanized anti-Trop-2 monoclonal antibody (hRS7) conjugated with the active metabolite of irinotecan, SN-38. In addition to its further characterization, as the clinical utility of IMMU-132 expands to an ever-widening range of Trop-2-expressing solid tumor types, its efficacy in new disease models needs to be explored in a nonclinical setting. Unlike most ADCs that use ultratoxic drugs and stable linkers, IMMU-132 uses a moderately toxic drug with a moderately stable carbonate bond between SN-38 and the linker. Flow cytometry and immunohistochemistry disclosed that Trop-2 is expressed in a wide range of tumor types, including gastric, pancreatic, triple-negative breast (TNBC), colonic, prostate, and lung. While cell-binding experiments reveal no significant differences between IMMU-132 and parental hRS7 antibody, surface plasmon resonance analysis using a Trop-2 CM5 chip shows a significant binding advantage for IMMU-132 over hRS7. The conjugate retained binding to the neonatal receptor, but it lost greater than 60% of the antibody-dependent cell-mediated cytotoxicity activity compared to that of hRS7. Exposure of tumor cells to either free SN-38 or IMMU-132 demonstrated the same signaling pathways, with pJNK1/2 and p21(WAF1/Cip1) upregulation followed by cleavage of caspases 9, 7, and 3, ultimately leading to poly-ADP-ribose polymerase cleavage and double-stranded DNA breaks. Pharmacokinetics of the intact ADC in mice reveals a mean residence time (MRT) of 15.4 h, while the carrier hRS7 antibody cleared at a similar rate as that of the unconjugated antibody (MRT â¼ 300 h). IMMU-132 treatment of mice bearing human gastric cancer xenografts (17.5 mg/kg; twice weekly × 4 weeks) resulted in significant antitumor effects compared to that of mice treated with a nonspecific control. Clinically relevant dosing schemes of IMMU-132 administered either every other week, weekly, or twice weekly in mice bearing human pancreatic or gastric cancer xenografts demonstrate similar, significant antitumor effects in both models. Current Phase I/II clinical trials ( ClinicalTrials.gov , NCT01631552) confirm anticancer activity of IMMU-132 in cancers expressing Trop-2, including gastric and pancreatic cancer patients.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/química , Antígenos de Neoplasias/inmunología , Camptotecina/análogos & derivados , Moléculas de Adhesión Celular/inmunología , Inmunoconjugados/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Antígenos de Neoplasias/metabolismo , Apoptosis/efectos de los fármacos , Camptotecina/química , Camptotecina/farmacocinética , Camptotecina/farmacología , Camptotecina/uso terapéutico , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Inmunoconjugados/uso terapéutico , Irinotecán , Ratones , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epratuzumab, a humanized anti-CD22 antibody, is currently in clinical trials of B-cell lymphomas and autoimmune diseases, demonstrating therapeutic activity in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action remains poorly understood. Here we report for the first time that epratuzumab promptly induces a marked decrease of CD22 (>80%), CD19 (>50%), CD21 (>50%), and CD79b (>30%) on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or SLE patients, and of NHL cells (Daudi and Raji) spiked into normal PBMCs. Although some Fc-independent loss of CD22 is expected from internalization by epratuzumab, the concurrent and prominent reduction of CD19, CD21, and CD79b is Fc dependent and results from their transfer from epratuzumab-opsonized B cells to FcγR-expressing monocytes, natural killer cells, and granulocytes via trogocytosis. The findings of reduced levels of CD19 are implicative for the efficacy of epratuzumab in autoimmune diseases because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients. This was confirmed herein by the finding that SLE patients receiving epratuzumab immunotherapy had significantly reduced CD19 compared with treatment-naïve patients.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Antineoplásicos/farmacología , Linfocitos B/efectos de los fármacos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Fagocitosis/efectos de los fármacos , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Antígenos CD19/genética , Antígenos CD19/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Antígenos CD79/antagonistas & inhibidores , Antígenos CD79/genética , Antígenos CD79/inmunología , Ensayos Clínicos como Asunto , Técnicas de Cocultivo , Expresión Génica/efectos de los fármacos , Granulocitos/citología , Granulocitos/efectos de los fármacos , Granulocitos/inmunología , Humanos , Inmunoterapia , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Fagocitosis/inmunología , Cultivo Primario de Células , Receptores de Complemento 3d/antagonistas & inhibidores , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Lectina 2 Similar a Ig de Unión al Ácido Siálico/genéticaRESUMEN
The antibody-drug conjugate (ADC), IMMU-130, of the moderately cytotoxic topoisomerase I inhibitor, SN-38, and the CEACAM5-targeted humanized antibody (mAb), labetuzumab, was evaluated in model systems of human colon carcinoma and in phase I clinical trials of heavily pretreated patients with metastatic colorectal cancer. The conjugate, designed with a near-homogeneous drug substitution of 7-8 SN-38/mAb and with a linker that released 50% of the drug in â¼20 h, showed significant antitumor effects compared to a nontargeted ADC in human tumor xenografts, which could be augmented in combination with bevacizumab. The advantage of fractionated dosing was demonstrated, with potential implications for the clinical dosing schedule. Biodistribution comparing IMMU-130 with labetuzumab showed that the conjugate cleared somewhat faster from the blood, but this did not affect tumor uptake and retention. The use of an ultrastable linker in the conjugate design abrogated antitumor effects. A tolerability study in rabbits showed a high safety margin, with no-observed-adverse-effect level (NOAEL) corresponding to a cumulative human-equivalent protein dose of 40-60 mg/kg. The preclinical findings appear to be corroborated in two phase I clinical trials, with high tolerability and evidence of antitumor activity, including objective responses. The impact of the ADC design on the utility of IMMU-130, tailored to a poorly internalizing target, is discussed.
Asunto(s)
Camptotecina/análogos & derivados , Inmunoconjugados/química , Inmunoconjugados/uso terapéutico , Animales , Anticuerpos Monoclonales , Antineoplásicos , Bevacizumab/uso terapéutico , Camptotecina/química , Camptotecina/uso terapéutico , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Femenino , Humanos , Inmunoconjugados/farmacocinética , Irinotecán , Ratones , Ratones Desnudos , ConejosRESUMEN
BACKGROUND: Ranpirnase (Rap) is an amphibian ribonuclease with reported antitumor activity, minimal toxicity, and negligible immunogenicity in clinical studies, but the unfavorable pharmacokinetics and suboptimal efficacy hampered its further clinical development. To improve the potential of Rap-based therapeutics, we have used the DOCK-AND-LOCK™ (DNL™) method to construct a class of novel IgG-Rap immunoRNases. In the present study, a pair of these constructs, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, comprising four copies of Rap linked to the CH3 and CK termini of hRS7 (humanized anti-Trop-2), respectively, were evaluated as potential therapeutics for triple-negative breast cancer (TNBC). METHODS: The DNL-based immunoRNases, (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2, were characterized and tested for biological activities in vitro on a panel of breast cancer cell lines and in vivo in a MDA-MB-468 xenograft model. RESULTS: (Rap)2-E1-(Rap)2 was highly purified (>95%), exhibited specific cell binding and rapid internalization in MDA-MB-468, a Trop-2-expressing TNBC line, and displayed potent in vitro cytotoxicity (EC50 ≤ 1 nM) against diverse breast cancer cell lines with moderate to high expression of Trop-2, including MDA-MB-468, BT-20, HCC1806, SKBR-3, and MCF-7. In comparison, structural counterparts of (Rap)2-E1-(Rap)2, generated by substituting hRS7 with selective non-Trop-2-binding antibodies, such as epratuzumab (anti-CD22), were at least 50-fold less potent than (Rap)2-E1-(Rap)2 in MDA-MB-468 and BT-20 cells, both lacking the expression of the cognate antigen. Moreover, (Rap)2-E1-(Rap)2 was less effective (EC50 > 50 nM) in MDA-MB-231 (low Trop-2) or HCC1395 (no Trop-2), and did not show any toxicity to human peripheral blood mononuclear cells. In a mouse TNBC model, a significant survival benefit was achieved with (Rap)2-E1*-(Rap)2 when given the maximal tolerated dose. CONCLUSIONS: A new class of immunoRNases was generated with enhanced potency for targeted therapy of cancer. The promising results from (Rap)2-E1-(Rap)2 and (Rap)2-E1*-(Rap)2 support their further investigation as a potential treatment option for TNBC and other Trop-2-expressing cancers.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Moléculas de Adhesión Celular/metabolismo , Inmunoconjugados/farmacología , Ribonucleasas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antineoplásicos/química , Femenino , Citometría de Flujo , Humanos , Inmunoconjugados/química , Dosis Máxima Tolerada , Ratones , Ratones Desnudos , Terapia Molecular Dirigida , Ribonucleasas/química , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We describe the use of novel bispecific hexavalent Abs (HexAbs) to enhance anticancer immunotherapy. Two bispecific HexAbs [IgG-(Fab)(4) constructed from veltuzumab (anti-CD20 IgG) and milatuzumab (anti-CD74 IgG)] show enhanced cytotoxicity in mantle cell lymphoma (MCL) and other lymphoma/leukemia cell lines, as well as patient tumor samples, without a crosslinking Ab, compared with their parental mAb counterparts, alone or in combination. The bispecific HexAbs have different properties from and are more potent than their parental mAbs in vitro. The juxtaposition of CD20 and CD74 on MCL cells by the HexAbs resulted in homotypic adhesion and triggered intracellular changes that include loss of mitochondrial transmembrane potential, production of reactive oxygen species, rapid and sustained phosphorylation of ERKs and JNK, down-regulation of pAkt and Bcl-xL, actin reorganization, and lysosomal membrane permeabilization, culminating in cell death. They also displayed different potencies in depleting lymphoma cells and normal B cells from whole blood ex vivo and significantly extended the survival of nude mice bearing MCL xenografts in a dose-dependent manner, thus indicating stability and antitumor activity in vivo. Such bispecific HexAbs may constitute a new class of therapeutic agents for improved cancer immunotherapy, as shown here for MCL and other CD20(+)/CD74(+) malignancies.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , Antígenos CD20/inmunología , Antígenos de Diferenciación de Linfocitos B/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/inmunología , Citoesqueleto de Actina/inmunología , Citoesqueleto de Actina/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Línea Celular Tumoral , Citotoxinas/inmunología , Citotoxinas/farmacología , Diseño de Fármacos , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Inmunoterapia/métodos , Lisosomas/inmunología , Lisosomas/metabolismo , Sistema de Señalización de MAP Quinasas/inmunología , Ratones , Ratones SCID , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Pretargeted radioimmunotherapy (PRIT) with bispecific antibodies (bsMAb) and a radiolabeled peptide reduces the radiation dose to normal tissues. Here we report the accuracy of an (111)In-labeled pretherapy test dose for personalized dosing of (177)Lu-labeled IMP288 following pretargeting with the anti-CEA × anti-hapten bsMAb, TF2, in patients with metastatic colorectal cancer (CRC). METHODS: In 20 patients bone marrow absorbed doses (BMD) and doses to the kidneys were predicted based on blood samples and scintigrams acquired after (111)In-IMP288 injection for individualized dosing of PRIT with (177)Lu-IMP288. Different dose schedules were studied, varying the interval between the bsMAb and peptide administration (5 days vs. 1 day), increasing the bsMAb dose (75 mg vs. 150 mg), and lowering the peptide dose (100 µg vs. 25 µg). RESULTS: TF2 and (111)In/(177)Lu-IMP288 clearance was highly variable. A strong correlation was observed between peptide residence times and individual TF2 blood concentrations at the time of peptide injection (Spearman's ρ = 0.94, P < 0.0001). PRIT with 7.4 GBq (177)Lu-IMP288 resulted in low radiation doses to normal tissues (BMD <0.5 Gy, kidney dose <3 Gy). Predicted (177)Lu-IMP288 BMD were in good agreement with the actual measured doses (mean ± SD difference -0.0026 ± 0.028 mGy/MBq). Hematological toxicity was mild in most patients, with only two (10 %) having grade 3-4 thrombocytopenia. A correlation was found between platelet toxicity and BMD (Spearman's ρ = 0.58, P = 0.008). No nonhematological toxicity was observed. CONCLUSION: These results show that individual high activity doses in PRIT in patients with CEA-expressing CRC could be safely administered by predicting the radiation dose to red marrow and kidneys, based on dosimetric analysis of a test dose of TF2 and (111)In-IMP288.
Asunto(s)
Neoplasias Colorrectales/radioterapia , Medicina de Precisión/métodos , Dosis de Radiación , Radioinmunoterapia , Radiometría/métodos , Adulto , Anciano , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Biespecíficos/uso terapéutico , Antígeno Carcinoembrionario/inmunología , Neoplasias Colorrectales/diagnóstico por imagen , Femenino , Haptenos/inmunología , Compuestos Heterocíclicos con 1 Anillo/administración & dosificación , Compuestos Heterocíclicos con 1 Anillo/farmacocinética , Compuestos Heterocíclicos con 1 Anillo/uso terapéutico , Humanos , Radioisótopos de Indio/administración & dosificación , Radioisótopos de Indio/farmacocinética , Radioisótopos de Indio/uso terapéutico , Lutecio/administración & dosificación , Lutecio/farmacocinética , Lutecio/uso terapéutico , Masculino , Persona de Mediana Edad , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacocinética , Oligopéptidos/uso terapéutico , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Multifunctional antibody-based biologics, such as bispecific antibodies and immunocytokines, can be difficult to produce with sufficient yield and stability, and often exhibit inferior pharmacokinetics. Dock-and-Lock (DNL) is a modular method that combines recombinant engineering with site-specific conjugation, allowing the construction of various complex, yet defined, biostructures with multivalency and multispecificity. The technology platform exploits the natural interaction between two interactive human protein binding domains that are modified to provide covalent fusion. We explored the potential application of a new class of IgG-based DNL modules with an anchor domain fused at the C-terminal end of the kappa light chain (C(k)), instead of the C-terminal end of the Fc. Two C(k)-derived prototypes, an anti-CD22/CD20 bispecific hexavalent antibody, comprising epratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and a CD20-targeting immunocytokine, comprising veltuzumab and four molecules of interferon-α2b, were compared to their Fc-derived counterparts. The Ck-based conjugates exhibited superior Fc-effector functions in vitro, as well as improved pharmacokinetics, stability, and anti-lymphoma activity in vivo. These results favor the selection of DNL conjugates with the C(k)-design for future clinical development.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Inmunoconjugados/uso terapéutico , Interferón-alfa/uso terapéutico , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/química , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Femenino , Humanos , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Interferón alfa-2 , Interferón-alfa/química , Interferón-alfa/farmacocinética , Ratones , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéuticoRESUMEN
The short circulating half-life and side effects of IFNα affect its dosing schedule and efficacy. Fusion of IFNα to a tumor-targeting mAb (mAb-IFNα) can enhance potency because of increased tumor localization and improved pharmacokinetics. We used the Dock-and-Lock method to generate C2-2b-2b, a mAb-IFNα comprising tetrameric IFNα2b site-specifically linked to hL243 (humanized anti-HLA-DR). In vitro, C2-2b-2b inhibited various B-cell lymphoma leukemia and myeloma cell lines. In most cases, this immunocytokine was more effective than CD20-targeted mAb-IFNα or a mixture comprising the parental mAb and IFNα. Our findings indicate that responsiveness depends on HLA-DR expression/density and sensitivity to IFNα and hL243. C2-2b-2b induced more potent and longer-lasting IFNα signaling compared with nontargeted IFNα. Phosphorylation of STAT1 was more robust and persistent than that of STAT3, which may promote apoptosis. C2-2b-2b efficiently depleted lymphoma and myeloma cells from whole human blood but also exhibited some toxicity to B cells, monocytes, and dendritic cells. C2-2b-2b showed superior efficacy compared with nontargeting mAb-IFNα, peginterferonalfa-2a, or a combination of hL243 and IFNα, using human lymphoma and myeloma xenografts. These results suggest that C2-2b-2b should be useful in the treatment of various hematopoietic malignancies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antineoplásicos/uso terapéutico , Antígenos HLA-DR/inmunología , Interferón-alfa/uso terapéutico , Leucemia/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/inmunología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/inmunología , Leucemia/inmunología , Linfoma/inmunología , Ratones , Ratones SCID , Proteínas RecombinantesRESUMEN
PURPOSE: TF12 is a trivalent bispecific antibody that consists of two anti-TROP-2 Fab fragments and one anti-histamine-succinyl-glycine (HSG) Fab fragment. The TROP-2 antigen is found in many epithelial cancers, including prostate cancer (PC), and therefore this bispecific antibody could be suitable for pretargeting in this cancer. In this study, the characteristics and the potential for pretargeted radioimmunoimaging and radioimmunotherapy with TF12 and the radiolabeled di-HSG peptide IMP288 in mice with human PC were investigated. METHODS: The optimal TF12 protein dose, IMP288 peptide dose, and dose interval for PC targeting were assessed in nude mice with s.c. PC3 xenografts. Immuno-positron emission tomography (PET)/CT was performed using TF12/68Ga-IMP288 at optimized conditions. The potential of pretargeted radioimmunotherapy (PRIT) using the TF12 pretargeted ¹77Lu-IMP288 was determined. RESULTS: TF12 and ¹¹¹In-IMP288 showed high and fast accumulation in the tumor [20.4 ± 0.6%ID/g at 1 h post-injection (p.i.)] at optimized conditions, despite the internalizing properties of TF12. The potential for PRIT was shown by retention of 50% of the ¹¹¹In-IMP288 in the tumor at 48 h p.i. One cycle of treatment with TF12 and ¹77Lu-IMP288 showed significant improvement of survival compared to treatment with ¹77Lu-IMP288 alone (90 vs. 67 days, p<0.0001) with no renal or hematological toxicity. CONCLUSION: TROP-2-expressing PC can be pretargeted efficiently with TF12, with very rapid uptake of the radiolabeled hapten-peptide, IMP288, sensitive immuno-PET, and effective therapy.
Asunto(s)
Anticuerpos Biespecíficos/farmacocinética , Compuestos Heterocíclicos con 1 Anillo/farmacocinética , Oligopéptidos/farmacocinética , Tomografía de Emisión de Positrones , Neoplasias de la Próstata/diagnóstico por imagen , Radioinmunoterapia , Radiofármacos/farmacocinética , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias de la Próstata/radioterapia , Unión Proteica , Radiofármacos/uso terapéutico , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Advances in recombinant protein technology have facilitated the production of increasingly complex fusion proteins with multivalent, multifunctional designs for use in various in vitro and in vivo applications. In addition, traditional chemical conjugation remains a primary choice for linking proteins with polyethylene glycol (PEG), biotin, fluorescent markers, drugs, and others. More recently, site-specific conjugation of two or more interactive modules has emerged as a valid approach to expand the existing repertoires produced by either recombinant engineering or chemical conjugation alone, thus advancing the range of potential applications. Five such methods, each involving a specific binding event, are highlighted in this review, with a particular focus on the Dock-and-Lock (DNL) method, which exploits the natural interaction between the dimerization and docking domain (DDD) of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAP). The various enablements of DNL to date include trivalent, tetravalent, pentavalent, and hexavalent antibodies of monospecificity or bispecificity; immnocytokines comprising multiple copies of interferon-alpha (IFNα); and site-specific PEGylation. These achievements attest to the power of the DNL platform technology to develop novel therapeutic and diagnostic agents from both proteins and nonproteins for unmet medical needs.
Asunto(s)
Ingeniería de Proteínas , Sitios de Unión , Polietilenglicoles/química , Proteínas Recombinantes/químicaRESUMEN
We have generated hexavalent antibodies (HexAbs) comprising 6 Fabs tethered to one Fc of human IgG1. Three such constructs, 20-20, a monospecific HexAb comprising 6 Fabs of veltuzumab (humanized anti-CD20 immunoglobulin G1κ [IgG1κ]), 20-22, a bispecific HexAb comprising veltuzumab and 4 Fabs of epratuzumab (humanized anti-CD22 IgG1κ), and 22-20, a bispecific HexAb comprising epratuzumab and 4 Fabs of veltuzumab, were previously shown to inhibit pro-liferation of several lymphoma cell lines at nanomolar concentrations in the absence of a crosslinking antibody. We now report an in-depth analysis of the apoptotic and survival signals induced by the 3 HexAbs in Burkitt lymphomas and provide in vitro cytotoxicity data for additional lymphoma cell lines and also chronic lymphocytic leukemia patient specimens. Among the key findings are the significant increase in the levels of phosphorylated p38 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by all 3 HexAbs and the notable differences in the signaling events triggered by the HexAbs from those incurred by crosslinking veltuzumab or rituximab with a secondary antibody. Thus, the greatly enhanced direct toxicity of these HexAbs correlates with their ability to alter the basal expression of various intracellular proteins involved in regulating cell growth, survival, and apoptosis, with the net outcome leading to cell death.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD20/inmunología , Antineoplásicos/inmunología , Leucemia/inmunología , Linfoma de Células B/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Leucemia/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/inmunología , FN-kappa B/inmunología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Rituximab , Transducción de SeñalRESUMEN
Bispecific antibody pretargeting is a two-step procedure for selectively delivering radionuclides to tumors. The procedure was developed to solve a number of problems encountered when radionuclides are directly coupled to an IgG, such as slow blood clearance and delayed tumor accretion. While various forms of antibody fragments can reduce blood pool activity and provide faster tumor localization, tumor uptake is reduced considerably. In pretargeting procedures, the radionuclide is attached to a small molecule that quickly traverses the vascular barrier to reach the tumor cells, achieving maximum accretion within 0.5 to 1.0 h. Just as quickly, it is eliminated from the body, thereby minimizing tissue exposure and developing high tumor/tissue ratios very early. In order to capture the radionuclide in the tumor, a bispecific antibody (bsMAb) that binds to the tumor and to the isotope carrier (e.g., a hapten-peptide) is pre-administered some time earlier. The pretargeting procedure has been shown repeatedly to improve tumor localization as compared to directly radiolabeled antibodies, thereby enhancing both imaging and therapy. In this article, we review the progress our group has made toward developing and testing bsMAb pretargeting systems for cancer detection and therapy.
Asunto(s)
Neoplasias/diagnóstico por imagen , Neoplasias/radioterapia , Radioisótopos/uso terapéutico , Animales , Anticuerpos Monoclonales/uso terapéutico , Humanos , Inmunoconjugados/uso terapéutico , RadioinmunodetecciónRESUMEN
Here we describe molecular imaging of cancer using signal amplification of a radiotracer in situ by pretargeting a multivalent, bispecific antibody to carcinoembryonic antigen (CEA), which subsequently also captures a radioactive hapten-peptide. Human colon cancer xenografts as small as approximately 0.15 g were disclosed in nude mice within 1 h of giving the radiotracer, with tumor/blood ratios increased by >or=40-fold (approximately 10:1 at 1 h, approximately 100:1 at 24 h), compared to a (99m)Tc-labeled CEA-specific F(ab') used clinically for colorectal cancer detection, while also increasing tumor uptake tenfold ( approximately 20% injected dose/g) under optimal conditions. This technology could be adapted to other antibodies and imaging modalities.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/radioterapia , Radioinmunoterapia , Cintigrafía , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/inmunología , Antígeno Carcinoembrionario/inmunología , Antígeno Carcinoembrionario/metabolismo , Neoplasias del Colon/inmunología , Femenino , Humanos , Radioisótopos de Yodo/uso terapéutico , Ratones , Ratones Desnudos , Modelos Biológicos , Trasplante de Neoplasias , Tecnecio/farmacocinética , Factores de Tiempo , Distribución Tisular , Trasplante HeterólogoRESUMEN
Interferon-alpha (IFN-alpha) has direct inhibitory effects on some tumors and is a potent stimulator of both the innate and adaptive immune systems. A tumor-targeting antibody-IFN-alpha conjugate (mAb-IFN-alpha) could kill by direct actions of the monoclonal antibody (mAb) and IFN-alpha on tumor cells and also potentiate a tumor-directed immune response. The modular Dock-and-Lock method (DNL) was used to generate 20-2b, the first immunocytokine having 4 cytokine (IFN-alpha2b) groups that are fused to the humanized anti-CD20 mAb, veltuzumab. Additional mAb-IFN-alpha constructs, each retaining potent IFN-alpha2b biologic activity, also were produced by DNL. The 20-2b shows enhanced antibody-dependent cellular cytotoxicity compared with veltuzumab but lacks complement-dependent cytotoxicity. The 20-2b inhibits in vitro proliferation of lymphoma cells and depletes them from whole human blood more potently than the combination of veltuzumab and a nontargeting, irrelevant, mAb-IFN-alpha. The 20-2b demonstrated superior therapeutic efficacy compared with veltuzumab or nontargeting mAb-IFN-alpha in 3 human lymphoma xenograft models, even though mouse immune cells respond poorly to human IFN-alpha2b. Targeting IFN-alpha with an anti-CD20 mAb makes the immunocytokine more potent than either agent alone. These findings suggest that 20-2b merits clinical evaluation as a new candidate antilymphoma therapeutic.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Interferón-alfa/farmacología , Linfoma de Células B/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Humanos , Interferón alfa-2 , Interferón-alfa/farmacocinética , Interferón-alfa/uso terapéutico , Masculino , Ratones , Proteínas Recombinantes de Fusión/farmacocinética , Proteínas Recombinantes de Fusión/uso terapéutico , Proteínas RecombinantesRESUMEN
The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD20/inmunología , Linfoma de Células B/terapia , Lectina 2 Similar a Ig de Unión al Ácido Siálico/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Apoptosis , Calcio/metabolismo , Caspasas/metabolismo , Proliferación Celular , Citotoxicidad Inmunológica , Femenino , Humanos , Inmunoterapia , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Ratones , Ratones SCID , Tasa de SupervivenciaRESUMEN
Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.
Asunto(s)
Sustitución de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos CD20/inmunología , Antineoplásicos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Linfoma/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/genética , Antineoplásicos/farmacología , Linfocitos B/inmunología , Línea Celular Tumoral , Chlorocebus aethiops , Proteínas del Sistema Complemento/genética , Proteínas del Sistema Complemento/inmunología , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/farmacología , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Linfoma/tratamiento farmacológico , Linfoma/genética , Ratones , Mutación Missense , Rituximab , Relación Estructura-ActividadRESUMEN
We reported previously the feasibility to radiolabel peptides with fluorine-18 ((18)F) using a rapid one-pot method that first mixes (18)F(-) with Al(3+) and then binds the (Al(18)F)(2+) complex to a NOTA ligand on the peptide. In this report, we examined several new NOTA ligands and determined how temperature, reaction time, and reagent concentration affected the radiolabeling yield. Four structural variations of the NOTA ligand had isolated radiolabeling yields ranging from 5.8% to 87% under similar reaction conditions. All of the Al(18)F NOTA complexes were stable in vitro in human serum, and those that were tested in vivo also were stable. The radiolabeling reactions were performed at 100 degrees C, and the peptides could be labeled in as little as 5 min. The IMP467 peptide could be labeled up to 115 GBq/micromol (3100 Ci/mmol), with a total reaction and purification time of 30 min without chromatographic purification.
Asunto(s)
Aluminio/química , Quelantes/química , Fluoruros/química , Péptidos/química , Animales , Quelantes/síntesis química , Quelantes/farmacocinética , Radioisótopos de Flúor , Humanos , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Ratones , Ratones Desnudos , Estructura Molecular , Distribución TisularRESUMEN
[This corrects the article DOI: 10.18632/oncotarget.4318.].
RESUMEN
UNLABELLED: Small biomolecules are typically radiolabeled with (18)F by binding it to a carbon atom, a process that usually is designed uniquely for each new molecule and requires several steps and hours to produce. We report a facile method wherein (18)F is first attached to aluminum as Al(18)F, which is then bound to a chelate attached to a peptide, forming a stable Al(18)F-chelate-peptide complex in an efficient 1-pot process. METHODS: For proof of principle, this method was applied to a peptide suitable for use in a bispecific antibody pretargeting method. A solution of AlCl(3).6H(2)O in a pH 4.0 sodium-acetate buffer was mixed with an aqueous solution of (18)F to form the Al(18)F complex. This was added to a solution of IMP 449 (NOTA-p-Bn-CS-d-Ala-d-Lys(HSG)-d-Tyr-d-Lys(HSG)-NH(2)) (NOTA-p-Bn-CS is made from S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid; HSG is histamine-succinyl-glycine) and heated to 100 degrees C for 15 min. In vitro and in vivo stability and targeting ability of the Al(18)F-IMP 449 were examined in nude mice bearing LS174T human colonic tumors pretargeted with an anti-CEACAM5 bispecific antibody (TF2). RESULTS: The radiolabeled peptide was produced in 5%-20% yield with an estimated specific activity of 18,500-48,100 GBq (500-1,300 Ci)/mmol. The Al(18)F-IMP 449 was stable for 4 h in serum in vitro, and in animals, activity isolated in the urine 30 min after injection was bound to the peptide. Nonchelated Al(18)F had higher tissue uptake, particularly in the bones, than the chelated Al(18)F-IMP 449, which cleared rapidly from the body by urinary excretion. Tumor uptake was 30-fold higher with TF2-pretargeted Al(18)F-IMP 449 than with the peptide alone. Dynamic PET showed tumor localization within 30 min and rapid and thorough clearance from the body. CONCLUSION: The ability to bind highly stable Al(18)F to metal-binding ligands is a promising new labeling method that should be applicable to a diverse array of molecules for PET.
Asunto(s)
Radioisótopos de Flúor , Marcaje Isotópico , Tomografía de Emisión de Positrones/métodos , Animales , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Espectrometría de Masas , Ratones , Distribución TisularRESUMEN
UNLABELLED: We determined whether therapeutic responses using a bispecific antibody that pretargeted (90)Y-hapten-peptide radioimmunotherapy or a directly radiolabeled, humanized, (90)Y-anti-CD20 IgG (veltuzumab) could be improved by combining these treatments with unlabeled humanized antibodies against CD22 (epratuzumab), CD74 (milatuzumab), or veltuzumab. METHODS: Nude mice bearing established subcutaneous Ramos human Burkitt lymphoma were treated with antibodies alone or in combination with pretargeted radioimmunotherapy (PT-RAIT) or radioimmunotherapy, and tumor growth was monitored. Biodistribution studies examined the effect that predosing with unlabeled veltuzumab had on radioimmunotherapy and PT-RAIT targeting. RESULTS: None of the unconjugated antibodies was effective against established and rapidly growing xenografts, but PT-RAIT, at approximately 30% of its maximum tolerated dose, and radioimmunotherapy alone, at its maximum tolerated dose, were able to arrest growth and even entirely ablate tumors in some animals. Only combinations with veltuzumab improved therapeutic responses, most significantly when a veltuzumab regimen (weekly, 1.0 mg followed by 3 x 0.5 mg) was initiated 1 wk after PT-RAIT or (90)Y-veltuzumab. Biodistribution data indicated that when unlabeled veltuzumab (1.0 or 0.25 mg) was administered in advance of the radiolabeled veltuzumab or bispecific antibody injection, tumor uptake was significantly reduced ((111)In-veltuzumab, 47% and 25%, respectively; (111)In-hapten-peptide, 74% and 49%, respectively). Despite an approximately 50% decrease in radioactivity uptake in the tumor, antitumor responses were not diminished significantly for (90)Y-veltuzumab, and in the case of PT-RAIT responses were improved. However, higher amounts of predosed veltuzumab reduced the effects of PT-RAIT. CONCLUSION: These studies suggest that administering unlabeled anti-CD20 IgG therapy after the radioactivity dose provides the best efficacy and that the amount of unlabeled anti-CD20 IgG administered as a predose to anti-CD20-targeted radionuclide therapy should be minimized.