RESUMEN
Iodine-131-labeled anti-CD45 antibody has been added to conventional hematopoietic stem cell transplant preparative regimens to deliver targeted radiation to hematopoietic tissues, with the goal of decreasing relapse rates without increasing toxicity. However, higher radiation doses could be delivered to leukemia cells by antibody if the systemic therapy were decreased or eliminated. To examine the ability of (131)I-anti-CD45 antibody to provide sufficient immunosuppression for transplantation across allogeneic barriers, T-cell-depleted BALB.B marrow was transplanted into H2-compatible B6-Ly5(a) mice after (131)I-30F11 (rat antimurine CD45) antibody with or without varying dose levels of total body irradiation (TBI). Groups of five or six recipient mice per (131)I or TBI dose level per experiment were given tail vein injections of 100 microg of (131)I-labeled 30F11 antibody 4 days before marrow infusion, with or without TBI on day 0. Engraftment, defined as > or =50% donor B cells at 3 months posttransplant, was determined by two-color flow cytometric analysis of peripheral blood granulocytes, T cells, and B cells using antibodies specific for donor and host CD45 allotypes and for CD3. Donor engraftment of > or =80% recipient mice was achieved with either 8 Gy of TBI or 0.75 mCi of (131)I-30F11 antibody, which delivers an estimated 26 Gy to bone marrow. Subsequent experiments determined the dose of TBI alone or TBI plus 0.75 mCi of (131)I-30F11 antibody necessary for engraftment in recipient mice that had been presensitized to donor antigens before transplant, a setting requiring more stringent immunosuppression. Engraftment was seen in > or =80% of presensitized recipients surviving after 14-16 Gy of TBI or 12-14 Gy of TBI and 0.75 mCi of (131)I-30F11 antibody. However, only 28 of 69 (41%) presensitized mice receiving 10-16 Gy of TBI alone survived, presumably because of rejection of donor marrow and ablation of host hematopoiesis. In contrast, 29 of 35 (83%) presensitized mice receiving (131)I-30F11 antibody and 10-14 Gy of TBI survived, presumably because the additional immunosuppression provided by estimated radiation doses of 53 Gy to lymph nodes and 81 Gy to spleen from 0.75 mCi of (131)I-30F11 antibody permitted engraftment of donor marrow. These results suggest that targeted radiation delivered by (131)I-anti-CD45 antibody provides sufficient immunosuppression to replace an appreciable portion of the TBI dose used in matched sibling hematopoietic stem cell transplant.
Asunto(s)
Trasplante de Médula Ósea/inmunología , Antígenos H-2/inmunología , Inmunización , Inmunotoxinas/farmacología , Radioisótopos de Yodo/farmacología , Antígenos Comunes de Leucocito/inmunología , Acondicionamiento Pretrasplante , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Trasplante de Células Madre Hematopoyéticas , Inmunotoxinas/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Radioinmunoterapia/métodos , Linfocitos T/citología , Linfocitos T/inmunología , Irradiación Corporal TotalRESUMEN
Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Transducción de Señal , Factores de Transcripción , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Línea Celular Transformada , Células Cultivadas , Citocinas/farmacología , Rayos gamma , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Interleucina-11/farmacología , Leucopoyesis , Ratones , Ratones Endogámicos C57BL , Receptor Notch1 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Timo/inmunología , TransfecciónRESUMEN
Targeted hematopoietic irradiation delivered by 131I-anti-CD45 antibody has been combined with conventional marrow transplant preparative regimens in an effort to decrease relapse. Before increasing the proportion of therapy delivered by radiolabeled antibody, the myeloablative and immunosuppressive effects of such low dose rate irradiation must be quantitated. We have examined the ability of 131I-anti-CD45 antibody to facilitate engraftment in Ly5-congenic and H2-mismatched murine marrow transplant models. Recipient B6-Ly5(a) mice were treated with 30F11 antibody labeled with 0.1 to 1.5 mCi 131I and/or total body irradiation (TBI), followed by T-cell-depleted marrow from Ly5(b)-congenic (C57BL/6) or H2-mismatched (BALB/c) donors. Engraftment was achieved readily in the Ly5-congenic setting, with greater than 80% donor granulocytes and T cells after 0.5 mCi 131I (estimated 17 Gy to marrow) or 8 Gy TBI. A higher TBI dose (14 Gy) was required to achieve engraftment of H2-mismatched marrow, and engraftment occurred in only 3 of 11 mice receiving 1.5 mCi 131I delivered by anti-CD45 antibody. Engraftment of H2-mismatched marrow was achieved in 22 of 23 animals receiving 0.75 mCi 131I delivered by anti-CD45 antibody combined with 8 Gy TBI. Thus, targeted radiation delivered via 131I-anti-CD45 antibody can enable engraftment of congenic marrow and can partially replace TBI when transplanting T-cell-depleted H2-mismatched marrow.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Purgación de la Médula Ósea , Trasplante de Médula Ósea , Terapia de Inmunosupresión , Radioisótopos de Yodo , Antígenos Comunes de Leucocito/inmunología , Absorción , Animales , Supervivencia de Injerto , Antígenos H-2/análisis , Antígenos H-2/inmunología , Radioisótopos de Yodo/farmacocinética , Cinética , Masculino , Ratones , Ratas , Linfocitos T , Irradiación Corporal TotalRESUMEN
BACKGROUND: The organs of laboratory mice used in radioimmunotherapy experiments are relatively small compared to the ranges of high-energy yttrium-90 (Y-90) beta particles. Current Medical Internal Radiation Dose (MIRD) dosimetry methods do not account for beta energy that escapes an organ. A dosimetry model was developed to provide more realistic dose estimates for organs in mice who received Y-90-labeled antibodies by accounting for physical and geometric factors, loss of beta dose due to small organ sizes, and cross-organ doses. METHODS: The dimensions, masses, surface areas, and overlapping areas of different organs of 10 athymic nude mice, each weighing approximately 25 g, were measured to form a realistic geometric model. Major organs in this model include the liver, spleen, kidneys, lungs, heart, stomach, small intestine, large intestine, thyroid, pancreas, bone, marrow, and carcass. A subcutaneous tumor mass also was included in the model. By accounting for small organ absorbed fractions and cross-organ beta doses, the MIRD methodology was extended from humans to mice for beta dose calculations. RESULTS: Absorbed fractions of beta energy were calculated using the Berger's point kernels and the electron transport code EGS4. Except for the tumor and carcass, the self-organ absorbed fractions ranged from 15% to 20% in smaller organs (the marrow and thyroid) to 65%-70% in larger organs (the liver and small intestine). Cross-organ absorbed fractions also were calculated from estimates of the overlapping surface areas between organs. CONCLUSION: The mathematic mouse model presented here provides more realistic organ dosimetry of radiolabeled monoclonal antibodies in the nude mouse, which should, in turn, contribute to a better understanding of the correlation of biodistribution study results and organ-tumor toxicity information.
Asunto(s)
Partículas beta , Inmunotoxinas , Dosificación Radioterapéutica , Radioisótopos de Itrio/análisis , Animales , Ratones , Ratones Desnudos , Modelos BiológicosRESUMEN
The ability to deliver radiation selectively to lymphohematopoietic tissues may have utility in conditions treated by myeloablative regimens followed by bone marrow transplantation. Since the CD45 antigen is the most broadly expressed of hematopoietic antigens, we examined the biodistribution of radiolabeled anti-CD45 monoclonal antibodies in normal mice. Trace 125I or 131I-labeled monoclonal antibodies 30G12 (rat IgG2a), 30F11 (rat IgG2b), and F(ab')2 fragments of 30F11 were injected i.v. at doses of 5 to 1000 micrograms. For both intact antibodies, a higher percentage of injected dose/g (% ID/g tissue) in blood was achieved with higher antibody doses. However, as the dose of antibody was increased, the % ID/g in the target organs of spleen, marrow, and lymph nodes decreased. At doses between 5 and 10-micrograms, % ID/g in these tissues exceeded that in lung, the normal organ with the highest concentration of radiolabel. In contrast, thymus was the only hematopoietic organ in which the % ID/g increased with increasing antibody dose, although at high dose the % ID/g was still far below that achieved in the other hematopoietic organs. Antibody 30F11 F(ab')2 fragments were cleared more quickly than intact antibody from blood and from both target and nontarget organs, although the relationship between increasing antibody dose and decreasing % ID/g in spleen, marrow, and lymph nodes was observed. The time-activity curves for each dose of antibody were used to calculate estimates of radiation absorbed dose to each organ. At the 10-micrograms dose of 30G12, the spleen was estimated to receive a radiation dose that was 13 times more than lung, the lymph nodes 3 to 4 times more, and the bone marrow 3 times more than lung. For each antibody fragment dose, the radiation absorbed dose per MBq 131I administered was lower because the residence times of the fragments were shorter than those of the intact antibody. Thus these estimates suggested that the best "therapeutic ratio" of radiation delivered to target organ as compared to lung was achieved with lower doses of intact antibody. We have demonstrated that radiolabeled anti-CD45 monoclonal antibodies can deliver radiation to lymphohematopoietic tissues with relative selectivity and that the relative uptake and retention in different hematolymphoid tissues change with increasing antibody dose.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Antígenos CD/inmunología , Antígenos de Histocompatibilidad/inmunología , Radioisótopos de Yodo/farmacocinética , Tejido Linfoide/metabolismo , Animales , Médula Ósea/metabolismo , Médula Ósea/efectos de la radiación , Femenino , Fragmentos Fab de Inmunoglobulinas/metabolismo , Radioisótopos de Yodo/sangre , Antígenos Comunes de Leucocito , Leucocitos/metabolismo , Leucocitos/efectos de la radiación , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/efectos de la radiación , Tejido Linfoide/efectos de la radiación , Masculino , Ratones , Ratones Endogámicos AKR , Dosis de Radiación , Bazo/metabolismo , Bazo/efectos de la radiación , Timo/metabolismo , Timo/efectos de la radiación , Distribución TisularRESUMEN
The use of phosphorothioate radioprotectors such as WR2721 in radioimmunotherapy is attractive because radiation delivered to tumors is usually separated in time from that delivered to the marrow and most normal organs, making protection of tumors of little consequence. However, to be effective radioprotectors must provide continuous protection against radiation of varying dose rates. To evaluate the potential of radioprotectors in radioimmunotherapy we treated normal mice with graded amounts of WR2721 in combination with an LD90/30 (26 MBq) of 131I-labeled antibody. A regimen of 15 doses of WR2721, 200 mg/kg prior to antibody infusion followed by 100 mg/kg ip every 4 h for a total of 72 h, was the maximum tolerated dosage schedule. With this schedule, treatment with radioprotectors failed to prolong survival or delay myelosuppression from the 131I-labeled antibody. In contrast, this regimen of radioprotector provided partial protection from a single treatment of 10 Gy total-body radiation given at 0.2 Gy/min. Protection 30 min after the final dose of WR2721 was greater than 3 h after the 14th dose (60 min prior to the final dose). These results suggest that the potential role of phosphorothioate radioprotectors in a radioimmunotherapy is limited because of the difficulty in achieving continuous protection with nontoxic amounts of drug and possibly because of a limited effect on low-dose-rate radiation.
Asunto(s)
Amifostina/farmacología , Anticuerpos Monoclonales/uso terapéutico , Médula Ósea/efectos de la radiación , Radioisótopos de Yodo/administración & dosificación , Animales , Médula Ósea/efectos de los fármacos , Recuento de Leucocitos/efectos de los fármacos , Recuento de Leucocitos/efectos de la radiación , Ratones , Dosis de Radiación , Organismos Libres de Patógenos Específicos , Irradiación Corporal TotalRESUMEN
Dosimetry and treatment planning for therapeutic infusions of radiolabeled antibodies are usually performed by extrapolation from the biodistribution of trace-labeled antibody. This extrapolation assumes that the biodistribution of high specific activity antibody will be similar to that seen with trace-labeled antibody. However, high doses of radiation result in rapid depletion of lymphoid and hematopoietic cells in lymph nodes, spleen, and marrow with replacement by blood and plasma. If radiolabeled antibody is cleared slowly from blood, this replacement may result in increased radionuclide concentrations in these tissues following infusions of antibody labeled with large amounts of radionuclide. To examine the influence of deposited radiation on the biodistribution of radiolabeled antibody, we treated mice with a constant amount of antibody that was labeled with varying amounts of 131I. Survival was determined in normal specific pathogen-free AKR/Cum mice (Thy1.2+) after infusion of anti-Thy1.1 antibody labeled with 10 to 6500 muCi of 131I, to determine an appropriate range of 131I doses for further study. The dose producing 50% lethality within 30 days following infusion of 131I-labeled antibody was 530 muCi 131I. Biodistribution, bone marrow histology, and dosimetry were subsequently determined after infusion of 500 micrograms of antibody labeled with 10, 250, 500, or 3500 muCi 131I. The amount of 131I did not influence uptake or retention of antibody in blood, liver, lung, or kidney. In contrast, infusion of antibody labeled with 250 to 3500 muCi of 131I led to a dose-related increase in the concentration of 131I in marrow, spleen, lymph node, and thymus. For example, at 96 h after infusion of antibody labeled with 500 or 3500 muCi 131I, concentrations in marrow were 3- to 4-fold higher than after infusion of trace-labeled antibody. The increase in marrow 131I concentrations was associated with depletion of cells and hemorrhage within the marrow space. As a result, estimated mean absorbed doses to marrow, lymph node, spleen, and thymus were 1.2 to 3.1 times higher than would have been predicted from the biodistribution of trace-labeled antibody. These results suggest that the biodistribution of trace-labeled antibody should be an accurate predictor of the behavior of high specific activity antibody in blood and solid organs such as liver and kidney. In contrast, radiation from antibody labeled with large amounts of radionuclide can result in an alteration of the concentration of radiolabeled antibody in rapidly responding tissues such as marrow.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Radioisótopos de Yodo/farmacocinética , Animales , Anticuerpos Monoclonales/administración & dosificación , Antígenos de Superficie/inmunología , Médula Ósea/diagnóstico por imagen , Médula Ósea/patología , Inmunoglobulina G , Infusiones Intravenosas , Radioisótopos de Yodo/administración & dosificación , Masculino , Ratones , Ratones Endogámicos AKR , Cintigrafía , Dosificación Radioterapéutica , Antígenos Thy-1 , Factores de Tiempo , Distribución TisularRESUMEN
We have previously shown that 131I-labeled monoclonal antibodies against the Thy1.1 differentiation antigen can induce the regression of Thy1.1 antigen-positive (Thy1.1+) AKR/J SL2 T cell lymphoma nodules in AKR/Cum (Thy1.2+) mice. In this study, we examined the ability of 131I-labeled anti-Thy1.1 antibodies to eliminate tumor nodules containing variant lymphoma cells that do not express the Thy1.1 antigen (Thy1.1-). AKR/Cum mice were sc inoculated with mixtures of 1-2 X 10(7) AKR/J SL2 cells and varying amounts (0.3%-10%) of Lsp3, a Thy1.1 antigen-negative (Thy1.1-) subclone of the SL2. One week later, when an established tumor nodule was present, mice were treated with 1,500-1,700 microCi of 131I-labeled anti-Thy1.1 antibody. Complete regression of tumor was observed in 11 of 12 (92%) mice inoculated with tumor containing 0.3%-1% antigen-negative cells. In contrast, no complete regressions were observed in mice with only antigen-negative tumor cells treated with 131I-labeled anti-Thy1.1 antibody or in mice inoculated with antigen-positive tumor and treated with an 131I-labeled control antibody. Of the mice receiving mixtures containing 3%-10% antigen-negative cells, five of seven showed complete regression. These results demonstrate that radiolabeled antibodies can eliminate small numbers of antigen-negative tumor cells present within a tumor mass.