RESUMEN
A novel alpha-particle emitting monoclonal antibody construct targeting the external domain of prostate-specific membrane antigen (PSMA) was prepared and evaluated in vitro and in vivo. The chelating agent, N-[2-amino-3-(p-isothiocyanatophen-yl)propyl]-trans-cyclohexane-1, 2-diamine-N,N',N',N'',N''-pentaacetic acid, was appended to J591 monoclonal antibody to stably bind the 213Bi radiometal ion. Bismuth-213 is a short-lived (t 1/2 = 46 min) radionuclide that emits high energy alpha-particles with an effective range of 0.07-0.10 mm that are ideally suited to treating single-celled neoplasms and micrometastatic carcinomas. The LNCaP prostate cancer cell line had an estimated 180,000 molecules of PSMA per cell; J591 bound to PSMA with a 3-nM affinity. After binding, the radiolabeled construct-antigen complex was rapidly internalized into the cell, carrying the radiometal inside. [213Bi]J591 was specifically cytotoxic to LNCaP. The LD50 value of [213Bi]J591 was 220 nCi/ml at a specific activity of 6.4 Ci/g. The potency and specificity of [213Bi]J591 directed against LNCaP spheroids, an in vitro model for micrometastatic cancer, also was investigated. [213Bi]J591 effectively stopped growth of LNCaP spheroids relative to an equivalent dose of the irrelevant control [213Bi]HuM195 or unlabeled J591. Cytotoxicity experiments in vivo were carried out in an athymic nude mouse model with an i.m. xenograft of LNCaP cells. [213Bi]J591 was able to significantly improve (P < 0.0031) median tumor-free survival (54 days) in these experiments relative to treatment with irrelevant control [213Bi]HuM195 (33 days), or no treatment (31 days). Prostate-specific antigen (PSA) was also specifically reduced in treated animals. At day 51, mean PSA values were 104 ng/ml +/- 54 ng/ml (n = 4, untreated animals), 66 ng/ml +/- 16 ng/ml (n = 6, animals treated with [213Bi]HuM195), and 28 ng/ml +/- 22 ng/ml (n = 6, animals treated with [213Bi]J591). The reduction of PSA levels in mice treated with [213Bi]J591 relative to mice treated with [213Bi]HuM195 and untreated control animals was significant with P < 0.007 and P < 0.0136, respectively. In conclusion, a novel [213Bi]-radiolabeled J591 has been constructed that selectively delivers alpha-particles to prostate cancer cells for potent and specific killing in vitro and in vivo.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Bismuto/farmacología , Inmunotoxinas/farmacología , Neoplasias de la Próstata/radioterapia , Radioinmunoterapia , Radioisótopos/farmacología , Partículas alfa/uso terapéutico , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Sitios de Unión , Muerte Celular/efectos de la radiación , Humanos , Inmunotoxinas/inmunología , Inmunotoxinas/metabolismo , Cinética , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/metabolismo , Esferoides Celulares/efectos de la radiación , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
UNLABELLED: The alpha-particle-emitting radionuclides have several physical characteristics that make them attractive candidates for radioimmunotherapy: (a) high linear energy transfer; (b) short path lengths (50-80 microm); and (c) limited ability of cells to repair damage to DNA. This article describes the pharmacokinetic, bioactivity, toxicity and chemical characteristics of alpha-particle-emitting, 213Bi and 212Bi radiometal conjugated HuM195 (anti-CD33) constructs. Conjugation of HuM195 to SCN-CHX-A-DTPA resulted in the attachment of up to 10 chelating ligand molecules per antibody. RESULTS: Radiolabeling efficiency of the CHX-A-DTPA-HuM195 construct with 213Bi was 78%+/-10% (n = 46) after 10 min at specific activities of up to 1110 MBq/mg. The immunoreactivity of the 213Bi-labeled CHX-A-DTPA-HuM195 construct was 84%+/-10% (n = 28) and was independent of the specific activity. The bismuth-labeled CHX-A-DTPA-HuM195 construct was rapidly internalized into the cell in a time-dependent manner ranging from 50% at 1 h to 65% at 24 h. 205Bi/206Bi-labeled constructs were stable for at least 2 d in vitro in the presence of human serum at 37 degrees C. After injection into mice, there was no uptake or loss of bismuth to mouse tissues, which do not express CD33, or to the kidney, which has avidity for free bismuth. Mice injected intraperitoneally with doses of (213Bi)CHX-A-DTPA-HuM1 95 ranging from 18.5 to 740 MBq/kg showed no toxicity, but at 2590 MBq/kg, two of the three mice died within 2 wk and a third mouse showed significant reductions in white blood cell counts. Mice injected intravenously with doses of (213Bi)CHX-A-DTPA-HuM195 up to 370 MBq/kg exhibited little toxicity, but 666 MBq/kg was above the MTD for mice. Leukemia cell killing in vitro with bismuth-labeled HuM1 95 showed dose- and specific activity-dependent killing of CD33+ HL60 cells; approximately 50% killing was observed when two bismuth atoms (50 fM radiolabeled antibody) were initially bound onto the target cell surface. CONCLUSION: Alpha-emitting antibodies are among the most potent cytotoxic agents known, yet are specific and appear safe in vivo. The physical and biochemical characteristics of the 213Bi isotope and its generation, as well as the biochemistry of the 213Bi-labeled CHX-A-DTPA-HuM195 construct, make it possible to use the constructs safely and feasibly in humans at therapeutic levels.
Asunto(s)
Anticuerpos Monoclonales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Ácido Pentético/análogos & derivados , Partículas alfa , Animales , Células HL-60/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Ácido Pentético/química , Ácido Pentético/inmunología , Ácido Pentético/farmacocinética , Ácido Pentético/toxicidad , Radioinmunoterapia , Proteínas Recombinantes , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
M195, a mouse monoclonal antibody reactive with the early myeloid antigen CD33, has been shown to target leukemia cells in patients and to reduce large leukemic burdens when labeled with 131I. A complementarity-determining region-grafted, humanized version (HuM195) has demonstrated similar targeting of leukemia cells without immunogenicity. We have studied two applications of therapy with 131I-M195. First, to intensify therapy prior to bone marrow transplantation (BMT), we combined 131I-M195 with busulfan and cyclophosphamide. Fifteen patients received first BMT for relapsed or refractory acute myelogenous leukemia or accelerated or blastic chronic myelogenous leukemia; four received second BMT for relapsed chronic or accelerated chronic myelogenous leukemia. Doses of 131I-M195 ranged from 120 to 230 mCi/m2. Few toxicities could be attributed to 131I-M195 therapy, and all patients engrafted. Eighteen patients achieved complete remission. Among those patients receiving first BMT, three have remained in unmaintained remission for 18+ to 29+ months. Six patients relapsed, including one with isolated central nervous system disease 32 months after BMT. Ten patients died in complete remission of transplant-related complications. Second, we studied whether 131I-M195 could reduce minimal residual disease and prolong remission and survival durations safely in patients with relapsed acute promyelocytic leukemia after they attained remission with all-trans-retinoic acid. Seven patients were treated with either 50 or 70 mCi/m2 131I-M195. Toxicity was limited to myelosuppression. As a measure of minimal residual disease, we monitored PML/RAR-alpha mRNA by reverse transcription PCR. Six patients had positive reverse transcription PCR assays prior to receiving 131I-M195; two converted transiently to negative. Median disease-free survival and overall survival of the seven patients were 8 (range, 3-14.5) months and 28 (range, 5.5-43+) months, respectively. This regimen compares favorably with others for relapsed acute promyelocytic leukemia. In an effort to avoid nonspecific cytotoxicity associated with 131I in future trials for minimal residual disease, we have conjugated short-range, alpha particle-emitting radioisotopes to HuM195 using a bifunctional chelate, 2-(p-isothiocyanatobenzyl)-cyclohexyldiethyl-enetriaminep entaacetic acid, with high efficiency and specific activities. 212Bi-HuM195 has demonstrated dose- and specific activity-dependent killing of HL60 cells in vitro. Injection of 213Bi-HuM195 into healthy BALB/c mice produced no effects on weight or viability.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Radioisótopos de Yodo/uso terapéutico , Leucemia Mieloide/radioterapia , Radioinmunoterapia , Adulto , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Trasplante de Médula Ósea , Supervivencia sin Enfermedad , Humanos , Leucemia Mieloide/inmunología , Leucemia Mieloide/cirugía , Ratones , Persona de Mediana Edad , Lectina 3 Similar a Ig de Unión al Ácido SiálicoRESUMEN
This paper describes in vitro cytotoxicity experiments with 213Bi- and 225Ac-immunoconjugates on the human epidermoid tumour cell line A431 using a blood group A-reactive murine IgG (2D11) as the specific antibody and MOPC 21 as the control antibody. With both radionuclides, specific cell-killing was achieved. The observed cytotoxicity of 213Bi (T1/2 - 47 min) indicates that this radionuclide is a useful alternative for the alpha-emitter 212Bi in the treatment of blood-borne malignancies. 225Ac-immunoconjugates (T1/2 of 225Ac is 10 days) may be applicable for the treatment of solid tumours, since the daughter radionuclides of 225Ac contribute to the cytotoxic efficacy by a field effect (i.e. toxicity in an area distal from the antibody-binding site). The lack of an adequate chelator for 225Ac is a major drawback.
Asunto(s)
Actinio/toxicidad , Bismuto/toxicidad , Supervivencia Celular/efectos de la radiación , Inmunoconjugados/toxicidad , Radioisótopos/toxicidad , Animales , Especificidad de Anticuerpos , Carcinoma de Células Escamosas , Línea Celular , Relación Dosis-Respuesta en la Radiación , Humanos , Inmunoglobulina G , Cinética , Ratones/inmunología , Ácido Pentético , Células Tumorales CultivadasRESUMEN
In developing and designing radioimmunotherapy, the selection of the isotope is a major factor. This selection depends on a number of criteria and parameters, affecting usefulness and feasibility. Usefulness is directly related to the radiological performance of the ionising radiation in relation to tissue and its morphology, with a major distinction between the effects of alpha and beta-particles (or rays). Usefulness is also directly related to the pharmacodynamic performance of the isotope-carrier (e.g. antibody) complex, where the proper choice of isotope radiodecay halflife is of major importance. Feasibility depends on availability of the components in the isotope-ligand-carrier complex, and also on convenience and safety aspects in the preparation and the handling of the materials as well as in their application in patients. A comparison is made between the various properties of alpha-emitting isotopes that have been proposed over a number of years, concluding that the combination 225Ac- 213Bi deserves serious further attention.
Asunto(s)
Partículas alfa/uso terapéutico , Partículas beta/uso terapéutico , Neoplasias/radioterapia , Radioinmunoterapia , Actinio/uso terapéutico , Bismuto/uso terapéutico , Radioisótopos/uso terapéuticoRESUMEN
This paper proposes the utilization of 225Ac for the alpha-radioimmunotherapy of cancer. The isotope decays with a radioactive half-life of 10 days into a cascade of short-lived alpha- and beta-emitting isotopes. In addition, when indicated by the pharmacokinetic requirements of particular clinical applications, 213Bi, with a radioactive half-life of 47 min, can be chosen as an alternative source of alpha-particles in radioimmunotherapy. This isotope is the last alpha emitter in the 225Ac decay-cascade and can be extracted from a 225Ac source at the bedside of the patient. 225Ac can quasi ad infinitum be obtained from one of its precursors, 229Th, which can be made available by various means. The indications for the use of alpha-particles as an alternative to more traditional classes of radiation are derived from the particle-kinetic characteristics and the radioactive half-life of their source isotope, as well as from the properties of the target-selective carrier moiety for the source isotope. It may be expected that useful applications, complementary to and/or in conjunction with other means of therapy will be identified.