211At-Labeled Anti-CD45 Antibody as a Nonmyeloablative Conditioning for Canine DLA-Haploidentical Stem Cell Transplantation.

The α-emitter 211At deposits a high amount of energy within a few cell diameters, resulting in irreparable DNA double-strand breaks while minimizing off-target toxicity. We investigated the use of the 211At-labeled anti-CD45 monoclonal antibody (mAb) 211At-CD45-B10 as a nonmyeloablative conditioning regimen for dog-leukocyte-antigen-haploidentical hematopoietic cell transplantation. Methods: Seventeen healthy dogs were injected with either a 0.50 (n = 14) or 0.75 (n = 3) mg/kg dose of anti-CD45 mAb labeled with 211At (8.436-23.199 MBq [0.228-0.627 mCi/kg]) on day -3. Peripheral blood stem cells from dog-leukocyte-antigen-haploidentical donors were given on day 0. Peripheral blood chimerism was calculated by polymerase chain reaction assays, and blood clearance of the radioimmunoconjugate was studied using enzyme-linked immunosorbent assay and radioactivity measurements of serial blood samples. Results: All dogs achieved donor chimerism by day 28 (range, 27%-100%). The hematopoietic engraftment rate was 100%, though engraftment durability was variable. No difference in absorbed dose to blood was seen for the 2 mAb dosing levels studied. Neutropenia (0-29 cells/µL), lymphocytopenia (36-130 cells/µL), and thrombocytopenia (1.5-9 × 103/µL) with prompt recovery were observed. The main adverse nonhematologic event related to 211At-CD45-B10 was mild reversible transaminitis. Graft-versus-host disease was not seen. Twelve of the 17 dogs survived over 30 d, with donor chimerism ranging from 3% to 99%. Conclusion: The results suggest that nonmyeloablative conditioning with 211At-CD45-B10 could be used in haploidentical hematopoietic cell transplantation though with variable engraftment.

Small-scale (sub-organ and cellular level) alpha-particle dosimetry methods using an iQID digital autoradiography imaging system.

Targeted radiopharmaceutical therapy with alpha-particle emitters (αRPT) is advantageous in cancer treatment because the short range and high local energy deposition of alpha particles enable precise radiation delivery and efficient tumor cell killing. However, these properties create sub-organ dose deposition effects that are not easily characterized by direct gamma-ray imaging (PET or SPECT). We present a computational procedure to determine the spatial distribution of absorbed dose from alpha-emitting radionuclides in tissues using digital autoradiography activity images from an ionizing-radiation quantum imaging detector (iQID). Data from 211At-radioimmunotherapy studies for allogeneic hematopoietic cell transplantation in a canine model were used to develop these methods. Nine healthy canines were treated with 16.9-30.9 MBq 211At/mg monoclonal antibodies (mAb). Lymph node biopsies from early (2-5 h) and late (19-20 h) time points (16 total) were obtained, with 10-20 consecutive 12-µm cryosections extracted from each and imaged with an iQID device. iQID spatial activity images were registered within a 3D volume for dose-point-kernel convolution, producing dose-rate maps. The accumulated absorbed doses for high- and low-rate regions were 9 ± 4 Gy and 1.2 ± 0.8 Gy from separate dose-rate curves, respectively. We further assess uptake uniformity, co-registration with histological pathology, and requisite slice numbers to improve microscale characterization of absorbed dose inhomogeneities in αRPT.

Addition of Astatine-211-Labeled Anti-CD45 Antibody to TBI as Conditioning for DLA-Identical Marrow Transplantation: A Novel Strategy to Overcome Graft Rejection in a Canine Presensitization Model: "Radioimmunotherapy to Overcome Transfusion-Induced Sensitization".

In a canine model of presensitization using donor blood transfusions, 100% of historical control dogs receiving 9.2 Gy total body irradiation (TBI) conditioning before dog leukocyte antigen (DLA)-identical marrow grafts had graft rejection. In this presensitization model, we investigated whether the addition of monoclonal antibody (mAb)-based targeted radioimmunotherapy (RIT) with astatine-211 (211At) to TBI could overcome graft rejection. 211At is an alpha-particle-emitting isotope that has a short path length, very high energy, and a short t½ of 7.2 hours, which allowed targeting radiation to the T cells responsible for graft rejection. Normal canine recipients were given three preceding transfusions of unirradiated whole blood on days -24, -17, and -10 before transplant from their DLA-identical marrow donors. 211At-anti-CD45 mAb was administered on day -3, and TBI followed by marrow grafts on day 0. Six of the 7 dogs (86%) achieved sustained engraftment as assessed by 100% donor chimerism in mononuclear cells, granulocytes, and CD3+ T cells. One dog receiving the lowest CD34+ cell content (0.35 × 106 cells/kg) rejected the graft. There were no late rejections in dogs followed up to 1 year. Graft-versus-host disease was seen in one dog. 211At-anti-CD45 mAb in combination with TBI as conditioning was successful in abrogating graft rejection in 86% of dogs in this presensitization model. 211At-anti-CD45 mAb conditioning with TBI may serve as a novel promising strategy to overcome graft rejection in heavily transfused patients with red cell disorders.

α-Imaging Confirmed Efficient Targeting of CD45-Positive Cells After 211At-Radioimmunotherapy for Hematopoietic Cell Transplantation.

UNLABELLED: α-radioimmunotherapy targeting CD45 may substitute for total-body irradiation in hematopoietic cell transplantation (HCT) preparative regimens for lymphoma. Our goal was to optimize the anti-CD45 monoclonal antibody (mAb; CA12.10C12) protein dose for (211)At-radioimmunotherapy, extending the analysis to include intraorgan (211)At activity distribution and α-imaging-based small-scale dosimetry, along with immunohistochemical staining. METHODS: Eight normal dogs were injected with either a 0.75 (n = 5) or 1.00 (n = 3) mg/kg dose of (211)At-B10-CA12.10C12 (11.5-27.6 MBq/kg). Two were euthanized and necropsied 19-22 h after injection, and 6 received autologous HCT 3 d after (211)At-radioimmunotherapy, after lymph node and bone marrow biopsies at 2-4 and/or 19 h after injection. Blood was sampled to study toxicity and clearance; CD45 targeting was evaluated by flow cytometry. (211)At localization and small-scale dosimetry were assessed using two α-imaging systems: an α-camera and an ionizing-radiation quantum imaging detector (iQID) camera. RESULTS: (211)At uptake was highest in the spleen (0.31-0.61% injected activity [%IA]/g), lymph nodes (0.02-0.16 %IA/g), liver (0.11-0.12 %IA/g), and marrow (0.06-0.08 %IA/g). Lymphocytes in blood and marrow were efficiently targeted using either mAb dose. Lymph nodes remained unsaturated but displayed targeted (211)At localization in T lymphocyte-rich areas. Absorbed doses to blood, marrow, and lymph nodes were estimated at 3.1, 2.4, and 3.4 Gy/166 MBq, respectively. All transplanted dogs experienced transient hepatic toxicity. Liver enzyme levels were temporarily elevated in 5 of 6 dogs; one treated with 1.00 mg mAb/kg developed ascites and was euthanized 136 d after HCT. CONCLUSION: (211)At-anti-CD45 radioimmunotherapy with 0.75 mg mAb/kg efficiently targeted blood and marrow without severe toxicity. Dosimetry calculations and observed radiation-induced effects indicated that sufficient (211)At-B10-CA12.10C12 localization was achieved for efficient conditioning for HCT.

(211)Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy.

Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a ß-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 ((211)At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×10(6) cells/kg be used with nonmyeloablative doses of (211)At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model.

Safety of treatment with DLA-identical or unrelated mesenchymal stromal cells in DLA-identical canine bone marrow transplantation.

BACKGROUND: Although in vitro and in vivo experiments have suggested that mesenchymal stromal cells (MSC) may have important immunomodulatory functions in allogeneic hematopoietic cell transplantation (HCT), results from clinical studies have been inconsistent. In the current study we investigate the safety of dog leukocyte antigen (DLA) identical or third party unrelated MSC in DLA-identical HCT. RESULTS: There were no differences between treatment groups in depth of granulocyte or platelet nadirs, time to granulocyte or platelet engraftment, rate of acute GVHD or rejection. All dogs tolerated the MSC infusion well, although 2 dogs treated with unrelated MSC were euthanized on day 9 due to complications unrelated to the MSC infusion. While no formation of ectopic tissue was observed, GFP positive signals in bone marrow, spleen or liver were detected at time of necropsy in 75% and 50% of dogs treated with DLA-identical or unrelated MSC, respectively. DISCUSSION: Treatment with DLA-identical or unrelated MSC in high dose DLA-identical HCT is safe, and provides a large animal HCT model in which to investigate immunological mechanisms and optimal treatment strategies for future human trials. METHODS: Fourteen dogs were treated with 920 cGy total body irradiation (TBI) followed by transplantation of marrow from DLA-identical littermates and immunosuppression with cyclosporine. Prior to infusion of marrow, dogs received infusions of DLA-identical MSC from the marrow donor (n = 4), unrelated MSC (n = 4), or culture medium (n = 6), within 1 h of TBI. MSC obtained from relevant donors were ex-vivo expanded and transduced with GFP-retrovirus before infusion.

Reagents for astatination of biomolecules. 6. An intact antibody conjugated with a maleimido-closo-decaborate(2-) reagent via sulfhydryl groups had considerably higher kidney concentrations than the same antibody conjugated with an isothiocyanato-closo-decaborate(2-) reagent via lysine amines.

We are investigating the use of an (211)At-labeled anti-CD45 monoclonal antibody (mAb) as a replacement of total body irradiation in conditioning regimens designed to decrease the toxicity of hematopoietic cell transplantation (HCT). As part of that investigation, dose-escalation studies were conducted in dogs using (211)At-labeled anticanine CD45 mAb, CA12.10C12, conjugated with a maleimido-closo-decaborate(2-) derivative, 4. Unacceptable renal toxicity was noted in the dogs receiving doses in the 0.27-0.62 mCi/kg range. This result was not anticipated, as no toxicity had been noted in prior biodistribution and toxicity studies conducted in mice. Studies were conducted to understand the cause of the renal toxicity and to find a way to circumvent it. A dog biodistribution study was conducted with (123)I-labeled CA12.10C12 that had been conjugated with 4. The biodistribution data showed that 10-fold higher kidney concentrations were obtained with the maleimido-conjugate than had been obtained in a previous biodistribution study with (123)I-labeled CA12.10C12 conjugated with an amine-reactive phenylisothiocyanato-CHX-A″ derivative. The difference in kidney concentrations observed in dogs for the two conjugation approaches led to an investigation of the reagents. SE-HPLC analyses showed that the purity of the CA12.10C12 conjugated via reduced disulfides was lower than that obtained with amine-reactive conjugation reagents, and nonreducing SDS-PAGE analyses indicated protein fragments were present in the disulfide reduced conjugate. Although we had previously prepared closo-decaborate(2-) derivatives with amine-reactive functional groups (e.g., 6 and 8), a new, easily synthesized, amine-reactive (phenylisothiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with coadministered (125)I- and (211)At-labeled CA12.10C10 conjugated with 10. In that study, lower kidney concentrations were obtained for both radionuclides than had been obtained in the earlier study of the same antibody conjugated with 4 after reduction of disulfide bonds.

Durable donor engraftment after radioimmunotherapy using α-emitter astatine-211-labeled anti-CD45 antibody for conditioning in allogeneic hematopoietic cell transplantation.

To reduce toxicity associated with external γ-beam radiation, we investigated radioimmunotherapy with an anti-CD45 mAb labeled with the α-emitter, astatine-211 ((211)At), as a conditioning regimen in dog leukocyte antigen-identical hematopoietic cell transplantation (HCT). Dose-finding studies in 6 dogs treated with 100 to 618 µCi/kg (211)At-labeled anti-CD45 mAb (0.5 mg/kg) without HCT rescue demonstrated dose-dependent myelosuppression with subsequent autologous recovery, and transient liver toxicity in dogs treated with (211)At doses less than or equal to 405 µCi/kg. Higher doses of (211)At induced clinical liver failure. Subsequently, 8 dogs were conditioned with 155 to 625 µCi/kg (211)At-labeled anti-CD45 mAb (0.5 mg/kg) before HCT with dog leukocyte antigen-identical bone marrow followed by a short course of cyclosporine and mycophenolate mofetil immunosuppression. Neutropenia (1-146 cells/µL), lymphopenia (0-270 cells/µL), and thrombocytopenia (1500-6560 platelets/µL) with prompt recovery was observed. Seven dogs had long-term donor mononuclear cell chimerism (19%-58%), whereas 1 dog treated with the lowest (211)At dose (155 µCi/kg) had low donor mononuclear cell chimerism (5%). At the end of follow-up (18-53 weeks), only transient liver toxicity and no renal toxicity had been observed. In conclusion, conditioning with (211)At-labeled anti-CD45 mAb is safe and efficacious and provides a platform for future clinical trials of nonmyeloablative transplantation with radioimmunotherapy-based conditioning.

Immunomodulatory effects induced by cytotoxic T lymphocyte antigen 4 immunoglobulin with donor peripheral blood mononuclear cell infusion in canine major histocompatibility complex-haplo-identical non-myeloablative hematopoietic cell transplantation.

BACKGROUND AIMS. Previously, cytotoxic T lymphocyte antigen 4 (CTLA4) immunoglobulin (Ig) has been shown to allow sustained engraftment in dog leukocyte antigen (DLA)-identical hematopoietic cell transplant (HCT) after non-myeloablative conditioning with 100 cGy total body irradiation (TBI). In the current study, we investigated the efficacy of pre-transplant CTLA4-Ig in promoting engraftment across a DLA-mismatched barrier after non-myeloablative conditioning. METHODS. Eight dogs were treated with CTLA4-Ig and donor peripheral blood mononuclear cells (PBMC) prior to receiving 200 cGy TBI followed by transplantation of granulocyte-colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells from DLA haplo-identical littermates with post-grafting immunosuppression. A control group of six dogs was conditioned with 200 cGy only and transplanted with grafts from DLA haplo-identical littermates followed by post-grafting immunosuppression. RESULTS. In vitro and in vivo donor-specific hyporesponsiveness was demonstrated on day 0 before TBI in eight dogs that received CTLA4-Ig combined with donor PBMC infusions. Four of five dogs treated with increased doses of CTLA4-Ig achieved initial engraftment but eventually rejected, with a duration of mixed chimerism ranging from 12 to 22 weeks. CTLA4-Ig did not show any effect on host natural killer (NK) cell function in vitro or in vivo. No graft-versus-host disease (GvHD) was observed in dogs receiving CTLA4-Ig treatment. CONCLUSIONS. Non-myeloablative conditioning with 200 cGy TBI and CTLA4-Ig combined with donor PBMC infusion was able to overcome the T-cell barrier to achieve initial engraftment without GvHD in dogs receiving DLA haplo-identical grafts. However, rejection eventually occurred; we hypothesize because of the inability of CTLA4-Ig to abate natural killer cell function.

Evaluation of posttransplant methotrexate to facilitate engraftment in the canine major histocompatibility complex-haploidentical nonmyeloablative transplant model.

BACKGROUND: Posttransplant cyclophosphamide has been shown to control graft-versus-host disease and facilitate engraftment in the major histocompatibility complex-haploidentical transplant setting. Here, we hypothesized that methotrexate (MTX) could be used in a similar fashion. In patients with genetic diseases, the use of MTX rather than an alkylating agent such as cyclophosphamide would be preferable due to its reduced risk of promoting secondary malignancies. METHOD: Using our standard conditioning regimen consisting of a specific anti-CD44 mAb (S5) and 200 cGy total body irradiation followed by postgrafting immunosuppression with cyclosporine and mycophenolate mofetil as a control group, we compared outcomes with experimental animals receiving the same regimen with the addition of a single, large dose of posttransplant MTX on day +3 (50-400 mg/m2). RESULTS: Adding MTX at all dose levels did not abrogate initial engraftment and controlled graft-versus-host disease in most cases. Dogs receiving MTX at the first dose level (50 mg/m2) improved time to rejection compared with controls (P=0.03) but did not decrease overall rates of rejection (P=0.56). However, increasing the dose of MTX beyond 50 mg/m2 seemed to have detrimental effects in both average (P=0.04) and peak (P=0.002) donor chimerism. Increasing the dose of MTX also promoted more profound lymphopenia. Finally, delaying cyclosporine and mycophenolate mofetil until after MTX administration did not seem to significantly improve engraftment kinetics. CONCLUSION: Adding high-dose MTX seemed to benefit the duration of donor chimerism at the lowest dose studied, but there was no benefit when escalating MTX doses to toxicity.

Pilot study of a (213)bismuth-labeled anti-CD45 mAb as a novel nonmyeloablative conditioning for DLA-haploidentical littermate hematopoietic transplantation.

BACKGROUND: A pilot study was conducted to determine whether conditioning using selective targeting of hematopoietic cells with an alpha-particle emitter, bismuth-213 ((213)Bi)-labeled anti-CD45 monoclonal antibody (mAb) is sufficient to overcome the major histocompatibility barrier in a canine model of dog leukocyte antigen-haploidentical hematopoietic cell transplantation (HCT). METHODS: Six dogs were administered 0.5 mg/kg (213)Bi-labeled anti-CD45 mAb (dose (213)Bi=2.26-4.9 mCi/kg) in six to eight injections. For postgrafting immunosuppression, all dogs received cyclosporine and mycophenolate mofetil. RESULTS: All dogs had initial donor engraftment, with three of six dogs having sustained engraftment to last point of follow-up. Two dogs receiving 2.26 and 3.25 mCi/kg of (213)Bi rejected their grafts at day +127 and +125, respectively, whereas dogs receiving (213)Bi doses of 3.3 mCi/kg or greater achieved high level donor chimerism. CONCLUSION: The results suggest that nonmyeloablative conditioning with (213)Bi-labeled anti-CD45 mAb could be applicable to major histocompatibility haploidentical HCT without excessive nonhematologic regimen-related toxicity.

Reagents for astatination of biomolecules. 4. Comparison of maleimido-closo-decaborate(2-) and meta-[(211)At]astatobenzoate conjugates for labeling anti-CD45 antibodies with [(211)At]astatine.

An investigation was conducted to compare the in vivo tissue distribution of a rat antimurine CD45 monoclonal antibody (30F11) and an irrelevant mAbs (CA12.10C12) labeled with (211)At using two different labeling methods. In the investigation, the mAbs were also labeled with (125)I to assess the in vivo stability of the labeling methods toward deastatination. One labeling method employed N-hydroxysuccinimidyl meta-[(211)At]astatobenzoate, [(211)At]1c, and N-hydroxysuccinimidyl meta-[(125)I]iodobenzoate, [(125)I]1b, in conjugation reactions to obtain the radiolabeled mAbs. The other labeling method involved conjugation of a maleimido-closo-decaborate(2-) derivative, 2, with sulfhydryl groups on the mAbs, followed by labeling of the mAb-2 conjugates using Na[(211)At]At or Na[(125)I]I and chloramine-T. Concentrations of the (211)At/(125)I pair of radiolabeled mAbs in selected tissues were examined in BALB/c mice at 1, 4, and 24 h post injection (pi). The co-injected anti-CD45 mAb, 30F11, labeled with [(125)I]1b and [(211)At]1c targeted the CD45-bearing cells in the spleen with the percent injected dose (%ID) of (125)I in that tissue being 13.31 ± 0.78; 17.43 ± 2.56; 5.23 ± 0.50; and (211)At being 6.56 ± 0.40; 10.14 ± 1.49; 7.52 ± 0.79 at 1, 4, and 24 h pi (respectively). However, better targeting (or retention) of the (125)I and (211)At was obtained for 30F11 conjugated with the closo-decaborate(2-), 2. The %ID in the spleen of (125)I (i.e., [(125)I]30F11-2) being 21.15 ± 1.33; 22.22 ± 1.95; 12.41 ± 0.75; and (211)At (i.e., [(211)At]30F11-2) being 22.78 ± 1.29; 25.05 ± 2.35; 17.30 ± 1.20 at 1, 4, and 24 h pi (respectively). In contrast, the irrelevant mAb, CA12.10C12, labeled with (125)I or (211)At by either method had less than 0.8% ID in the spleen at any time point, except for [(211)At]CA12.10C12-1c, which had 1.62 ± 0.14%ID and 1.21 ± 0.08%ID at 1 and 4 h pi. The higher spleen concentrations in that conjugate appear to be due to in vivo deastatination. Differences in (125)I and (211)At concentrations in lung, neck, and stomach indicate that the meta-[(211)At]benzoyl conjugates underwent deastatination, whereas the (211)At-labeled closo-decaborate(2-) conjugates were very stable to in vivo deastatination. In summary, using the closo-decaborate(2-) (211)At labeling approach resulted in higher concentrations of (211)At in target tissue (spleen) and higher stability to in vivo deastatination in this model. These findings, along with the simpler and higher-yielding (211)At-labeling method, provide the basis for using the closo-decaborate(2-) labeling reagent, 2, in our continued studies of the application of (211)At-labeled mAbs for conditioning in hematopoietic cell transplantation.

Transmission and expansion of HOXB4-induced leukemia in two immunosuppressed dogs: implications for a new canine leukemia model.

OBJECTIVE: There are currently no large animal models to study the biology of leukemia and development of novel antileukemia therapies. We have previously shown that dogs transplanted with homeobox B4 (HOXB4)-transduced autologous CD34(+) cells developed myeloid leukemia associated with HOXB4 overexpression. Here we describe the transmission, engraftment, and expansion of these canine leukemia cells into two genetically unrelated, immunosuppressed dogs. MATERIALS AND METHODS: Two dogs immunosuppressed after major histocompatibility complex-haploidentical hematopoietic cell transplantation and exhibiting mixed donor-host chimerism were accidentally infused trace amounts of HOXB4-overexpressing leukemia cells from a third-party dog. RESULTS: Six weeks after infusion of HOXB4-overexpressing leukemia cells, these two dogs rapidly developed myeloid leukemia consisting of marrow and organ infiltration, circulating blasts, and, in one dog, chloromatous masses. Despite neither of these dogs sharing any dog leukocyte antigen haplotypes with the sentinel case, the HOXB4-transduced clones engrafted and proliferated without difficulty in the presence of immunosuppression. Chimerism studies in both dogs confirmed that donor and, in one case, host hematopoietic cell engraftment was lost and replaced by third-party HOXB4 cells. CONCLUSIONS: The engraftment and expansion of these leukemia cells in dogs will allow studies into the biology of leukemia and development and evaluation of novel antileukemia therapies in a clinically relevant large animal model.

Biodistributions, myelosuppression, and toxicities in mice treated with an anti-CD45 antibody labeled with the alpha-emitting radionuclides bismuth-213 or astatine-211.

We previously investigated the potential of targeted radiotherapy using a bismuth-213 ((213)Bi)-labeled anti-CD45 antibody to replace total body irradiation as conditioning for hematopoietic cell transplantation in a canine model. Although this approach allowed sustained marrow engraftment, limited availability, high cost, and short half-life of (213)Bi induced us to investigate an alternative alpha-emitting radionuclide, astatine-211 ((211)At), for the same application. Biodistribution and toxicity studies were conducted with conjugates of the anti-murine CD45 antibody 30F11 with either (213)Bi or (211)At. Mice were injected with 2 to 50 muCi on 10 microg or 20 muCi on 2 or 40 microg of 30F11 conjugate. Biodistribution studies showed that the spleen contained the highest concentration of radioactivity, ranging from 167 +/- 23% to 417 +/- 109% injected dose/gram (% ID/g) after injection of the (211)At conjugate and 45 +/- 9% to 166 +/- 11% ID/g after injection of the (213)Bi conjugate. The higher concentrations observed for (211)At-labeled 30F11 were due to its longer half-life, which permitted better localization of isotope to the spleen before decay. (211)At was more effective at producing myelosuppression for the same quantity of injected radioactivity. All mice injected with 20 or 50 muCi (211)At, but none with the same quantities of (213)Bi, had lethal myeloablation. Severe reversible acute hepatic toxicity occurred with 50 muCi (213)Bi, but not with lower doses of (213)Bi or with any dose of (211)At. No renal toxicity occurred with either radionuclide. The data suggest that smaller quantities of (211)At-labeled anti-CD45 antibody are sufficient to achieve myelosuppression and myeloablation with less nonhematologic toxicity compared with (213)Bi-labeled antibody.

Dog leukocyte antigen-haploidentical stem cell allografts after anti-CD44 therapy and nonmyeloablative conditioning in a preclinical canine model.

BACKGROUND: We previously described a reduced-intensity hematopoietic cell transplantation (HCT) regimen in dog leukocyte antigen (DLA)-haploidentical littermate recipients consisting of 450 cGy total body irradiation (TBI) and anti-CD44 monoclonal antibody (mAb) S5 before and mycophenolate mofetil (MMF)/cyclosporine (CSP) after HCT. METHODS: We tested a nonmyeloablative regimen of mAb S5 and 200 cGy TBI with postgrafting MMF/CSP in 44 DLA-haploidentical recipients using eight different regimens. Ten dogs also received escalating doses of donor lymphocyte infusion (DLI) alone or with pentostatin to convert to complete donor chimerism. RESULTS: All dogs achieved initial engraftment between one to two weeks after HCT with peripheral blood mononuclear cell (PBMC) donor chimerism ranging from 2% to 98% (median 37%) on day +35. Twenty-five (57%) dogs rejected their donor grafts at a median of seven (range; 1-19) weeks after HCT. Low levels of PBMC donor chimerism at three weeks predicted graft rejection. DLI neither facilitated conversion to full donor chimerism after HCT nor prevented rejection. Higher total nucleated cells, CD4+, CD8+, and CD14+ cell subset numbers in the PBMC graft were associated with stable full donor engraftment. Dogs given higher cell subset doses of infused PBMC achieved longer duration of mixed chimerism. CONCLUSIONS: Nonmyeloablative conditioning with 200 cGy TBI and anti-CD44 mAb was sufficient for initial uniform engraftment across DLA haplotype-mismatched barriers. However, sustained donor engraftment was seen in only 43% of recipients. Graft composition and donor-dominant chimerism early after HCT may be the most important factors for sustained donor engraftment.

Prolonged allogeneic marrow engraftment following nonmyeloablative conditioning using 100 cGy total body irradiation and pentostatin before and pharmacological immunosuppression after transplantation.

In a canine model of dog leukocyte antigen (DLA)-identical nonmyeloablative marrow transplantation including postgrafting immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF), engraftment was only transient with 100 cGy total body irradiation (TBI) conditioning, indicating suboptimal pretransplant immunosuppression. In contrast, grafts after 200 cGy TBI were durable in 11/12 recipients. We hypothesized that addition of pentostatin before transplantation could, in part, substitute for 100 cGy TBI. Pharmacokinetic studies showed pentostatin significantly inhibited adenosine deaminase in canine lymphocytes. Eight dogs were conditioned with 6x4 mg/m pentostatin and 100 cGy TBI, whereas two dogs received 3x4 mg/m pentostatin plus 100 cGy TBI. All were given MMF/CSP posttransplant. All showed initial engraftment; four remained stable mixed chimeras for >32 weeks. The median duration of engraftment was 13 (range 9 to >39) weeks, which was significantly longer than in six historical controls conditioned with 100 cGy TBI alone (median 10, range 3-12 weeks) (P=0.01).

Radioimmunotherapy with bismuth-213 as conditioning for nonmyeloablative allogeneic hematopoietic cell transplantation in dogs: a dose deescalation study.

BACKGROUND: Using a canine model of nonmyeloablative hematopoietic cell transplantation (HCT), the authors demonstrated that pretransplant radioimmunotherapy with the alpha-emitter bismuth-213 (Bi) coupled to anti-CD45 or anti-T-cell receptor alphabeta (TCRalphabeta) monoclonal antibodies (mAb), together with postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine A (CsA), achieved stable engraftment of dog leukocyte antigen (DLA)-identical marrow. Engraftment was achieved with doses of 3.6 to 8.8 mCi/kg Bi, but signs of liver toxicity were noted in all dogs. To find a safe and effective dose for further trials, the authors performed a dose deescalation study in 15 dogs with 2.7 to 0.8 mCi/kg Bi. METHODS: Bi was linked to the mAb using the metal-binding chelate CHX-A"-DTPA. All dogs received three to six injections of Bi linked to anti-CD45 or anti-TCRalphabeta mAb followed by marrow grafts from DLA-identical littermates and postgrafting MMF and CsA. RESULTS: During follow-up of greater than 30 weeks, engraftment remained stable in all evaluable dogs conditioned with 1.4 to 2.1 mCi/kg Bi-anti-CD45 or 2.0 to 2.7 mCi/kg Bi-anti-TCRalphabeta. Only one dog conditioned with 1.5 mCi/kg Bi-anti-TCRalphabeta had stable engraftment, whereas two rejected their grafts. In both groups, all dogs conditioned with less than 1.3 mCi/kg Bi rejected their grafts. No signs of graft-versus-host disease or other toxicities were noted. Only mild and transient elevation of liver function tests occurred in 4 of 15 dogs. CONCLUSIONS: This study demonstrates that dose deescalation of radioimmunotherapy with Bi labeled to anti-CD45 or anti-TCRalphabeta as conditioning for nonmyeloablative HCT minimizes toxicity without compromising engraftment. With a dose of 2 mCi/kg Bi, further trials using radioimmunotherapy with Bi for nonmyeloablative HCT seem feasible.

Dog leukocyte antigen nonidentical unrelated canine marrow grafts: enhancement of engraftment by CD4 and CD8 T cells.

BACKGROUND: Previous studies have demonstrated that most marrow grafts from dog leukocyte antigen (DLA)-mismatched unrelated donors were rejected after 9.2 Gy total body irradiation (TBI), and that graft resistance could be overcome by infusing viable peripheral blood mononuclear cells (PBMCs) in addition to marrow. METHODS: To investigate the donor cell populations that facilitate engraftment, we determined the minimal dose of PBMCs required to ensure stable engraftment. Nineteen dogs underwent transplantation with DLA-mismatched unrelated marrow and PBMCs in a dose de-escalation study. In subsequent studies, 12 dogs were given selected CD4 or CD8 cells in addition to marrow. RESULTS: When 3 x 108 PBMC/kg were given in addition to a median of 4 x 108 marrow cells/kg, five of six animals engrafted. At a dose of 1 x 108 PBMC/kg, four of eight animals engrafted, and none of five dogs engrafted at a dose of 3 x 107 PBMC/kg. Accordingly, 12 dogs were given 9.2 Gy TBI, marrow grafts from DLA-mismatched unrelated dogs, and a median of 5.2 x 107 selected CD8 cells/kg or 10.4 x 107 selected CD4 cells/kg corresponding to the number of CD8 or CD4 cells contained in 3 x 108 PBMC/kg. Five of six dogs given CD8 cells and five of six dogs given CD4 cells engrafted. CONCLUSION: Results indicate that at least 3 x 108 unmodified PBMC/kg are needed for stable engraftment of DLA-mismatched unrelated marrow, and that both CD4 and CD8 cell subpopulations are capable of facilitating engraftment.

Selective T-cell ablation with bismuth-213-labeled anti-TCRalphabeta as nonmyeloablative conditioning for allogeneic canine marrow transplantation.

Two major immunologic barriers, the host-versus-graft (HVG) and graft-versus-host (GVH) reactions, have to be overcome for successful allogeneic hematopoietic cell transplantation. T cells were shown to be primarily involved in these barriers in the major histocompatibility complex identical setting. We hypothesized that selective ablation of T cells using radioimmunotherapy together with postgrafting immunosuppression would suffice to ensure stable allogeneic engraftment. We had described a canine model of nonmyeloablative marrow transplantation in which host immune reactions were impaired by a single dose of 200 cGy total body irradiation (TBI), and both GVH and residual HVG reactions were controlled by postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP). Here, we substituted the alpha-emitter bismuth-213 (213Bi) linked to a monoclonal antibody (mAb) against T-cell receptor (TCR) alphabeta, using the metal-binding chelate diethylenetriaminepentaacetic acid (DTPA) derivative cyclohexyl-(CHX)-A", for 200 cGy TBI. Biodistribution studies using a gamma-emitting indium-111-labeled anti-TCRalphabeta mAb showed uptake primarily in blood, marrow, lymph nodes, spleen, and liver. Four dogs were treated with 0.13 to 0.46 mg/kg TCRalphabeta mAb labeled with 3.7 to 5.6 mCi/kg (137-207 MBq/kg) 213Bi. The treatment was administered in 6 injections on days -3 and -2 followed by transplantation of dog leukocyte antigen-identical marrow on day 0 and postgrafting immunosuppression with MMF/CSP. The therapy was well tolerated except for elevations of transaminases that were transient in all but one of the dogs. No other organ toxicities or signs of graft-versus-host disease were noted. The dogs had prompt allogeneic hematopoietic engraftment and achieved stable mixed donor-host hematopoietic chimerism with donor contributions ranging from 5% to 55% after more than 30 weeks of follow up.