Emma Song

Interim analysis of toxicity and response in phase 1 trial of systemic targeted alpha therapy for metastatic melanoma

Purpose. The aim is to assess toxicity and response of systemic alpha therapy for metastatic melanoma. Experimental design. This is an open-labelled Phase 1 dose escalation study to establish the effective dose of the alpha-immunoconjugate 213Bi-cDTPA-9.2.27 mAb (AIC). Tools used to investigate the effects were physical examination; imaging of tumours; pathology; GFR; CT and changes in tumour marker. Responses were assessed using RECIST criteria. Results and Discussion. 22 patients with stage IV melanoma/ in-transit metastasis were treated with activities of 55-947 MBq. Using RECIST criteria 50% showed stable disease and 14% showed partial response. One patient (6%) showed near complete response and was retreated because of an excellent response to the initial treatment. Another patient showed response in his tumour on mandible and reduction in lung lesions. Overall 30% showed progressive disease. The tumour marker melanoma inhibitory activity protein (MIA) showed reductions over 8 weeks in most of the patients. The disparity of dose with responders is discussed. No toxicity was observed over the range of administered activities. Conclusion. Observation of responses without any toxicity indicates that targeted alpha therapy has the potential to be a safe and effective therapeutic approach for metastatic melanoma.

Glycogen Synthase Kinase-3β Inhibition Preserves Hematopoietic Stem Cell Activity and Inhibits Leukemic Cell Growth

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase‐3β (GSK‐3β) regulates the degradation of β‐catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK‐3β inhibition activates β‐catenin in cord blood CD34+ cells and upregulates β‐catenin transcriptional targets c‐myc and HoxB4, both known to regulate HSC self‐renewal. GSK‐3β inhibition resulted in delayed ex vivo expansion of CD34+ cells, yet enhanced the preservation of stem cell activity as tested in long‐term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long‐term cultures treated with GSK‐3β inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK‐3β inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK‐3β inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK‐3β may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK‐3β inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK‐3β may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.

Targeted alpha therapy for cancer

Targeted alpha therapy (TAT) offers the potential to inhibit the growth of micrometastases by selectively killing isolated and preangiogenic clusters of cancer cells. The practicality and efficacy of TAT is tested by in vitro and in vivo studies in melanoma, leukaemia, colorectal, breast and prostate cancers, and by a phase 1 trial of intralesional TAT for melanoma. The alpha-emitting radioisotope used is Bi-213, which is eluted from the Ac-225 generator and chelated to a cancer specific monoclonal antibody (mab) or protein (e.g. plasminogen activator inhibitor-2 PAI2) to form the alpha-conjugate (AC). Stable alpha-ACs have been produced which have been tested for specificity and cytotoxicity in vitro against melanoma (9.2.27 mab), leukaemia (WM60), colorectal (C30.6), breast (PAI2, herceptin), ovarian (PAI2, herceptin, C595), prostate (PAI2, J591) and pancreatic (PAI2, C595) cancers. Subcutaneous inoculation of 1-1.5 million human cancer cells into the flanks of nude mice causes tumours to grow in all mice. Tumour growth is compared for untreated controls, nonspecific AC and specific AC, for local (subcutaneous) and systemic (tail vein or intraperitoneal) injection models. The 213Bi-9.2.27 AC is injected into secondary skin melanomas in stage 4 patients in a dose escalation study to determine the effective tolerance dose, and to measure kinematics to obtain the equivalent dose to organs. In vitro studies show that TAT is one to two orders of magnitude more cytotoxic to targeted cells than non-specific ACs, specific beta emitting conjugates or free isotopes. In vivo local TAT at 2 days post-inoculation completely prevents tumour formation for all cancers tested so far. Intra-lesional TAT can completely regress advanced sc melanoma but is less successful for breast and prostate cancers. Systemic TAT inhibits the growth of sc melanoma xenografts and gives almost complete control of breast and prostate cancer tumour growth. Intralesional doses up to 450 microCi in human patients are effective in regressing melanomas, with no concomitant complications. These results point to the application of local and systemic TAT in the management of secondary cancer. Results of the phase 1 clinical trial of TAT of subcutaneous, secondary melanoma indicate proof of the principle that TAT can make tumours in patients regress.

GSK‐3β Inhibition Promotes Engraftment of Ex Vivo‐Expanded Hematopoietic Stem Cells and Modulates Gene Expression

Glycogen synthase kinase‐3β (GSK‐3β) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK‐3β, 6‐bromoindirubin 3′‐oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non‐obese diabetic/severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo‐expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine‐induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK‐3β provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation. STEM CELLS 2011;29:108–118

Bismuth-213 radioimmunotherapy with C595 anti–MUC1 monoclonal antibody in an ovarian cancer ascites model

Purpose: Control of ovarian cancer (OC) ascites remains a major objective in post-surgical treatment. The aim of this study was to investigate the effect of targeted alpha therapy (TAT) for the control of ascites in an OC ascites mouse model; the biodistribution of 213Bi-C595 and its long term toxicity. Methods: The expression of tumor-associated antigen mucin-1 (MUC-1) in OVCAR3 ascites cells in mice and OC cancer tissues in patients was detected by indirect immmunostaining. The monoclonal antibody (MAb) C595 was labeled with 213Bi using the chelator cDTPA to form the alpha-immunoconjugate (AIC). Mice were injected with different concentrations of AIC by i.p administration. Changes in tumor progression were assessed by measurement of the circumference of the abdomen. Results: MUC-1 is strongly expressed in 73% of OC tissues. At 9 days post-cell inoculation in mice, a single injection of 355 MBq/kg of 213Bi-C595 can prolong survival by 25 days. A high tumor: blood ratio (5.8) was found in biodistribution study. The maximum tolerance dose (MTD) was more than 1180 MBq/kg up to 21 weeks. Conclusions: C595 is a specific targeting vector for ovarian cancer cells, which show a high percentage of expression of MUC1. 213Bi-C595 can effectively target and kill ovarian cancer cells in vitro and in vivo. 213Bi-C595 is the recommended alpha conjugate for a Phase I clinical trial for ovarian cancer.

Using Small Molecule GSK3β Inhibitors to Treat Inflammation

Glycogen Synthase Kinase 3 beta (GSK3β) is a serine-threonine kinase originally identified for its role in the conversion of glucose to glycogen. Pharmacological inhibition can be achieved by drug binding to ATP or magnesium binding sites on the enzyme. Pharmaceutical companies have developed several small molecule GSK3β inhibitors for diabetes research. Additionally, GSK3β inhibitors are being clinically tested as therapeutics for neurological diseases, however, the mechanisms of involvement are unclear. Several studies have shown that the therapeutic effect of GSK3β inhibition is associated with the inhibition of inflammation. Similarly, the mechanisms underlying the anti-inflammatory function of GSK3β inhibition are not well understood. GSK3β inhibition attenuates activation of the pro-inflammatory transcription factor NFκB, and activates the immuno-modulatory transcription factor β-catenin. GSK3β inhibition has also been shown to induce secretion of the anti-inflammatory cytokine IL-10. In addition, pharmacological inhibition of GSK3β suppressed alloreactive T-cell responses. The combined anti-proliferative and anti-inflammatory properties of small molecule inhibitors of GSK3β make them an attractive treatment modality towards the control of inflammation.

Glycogen synthase kinase−3β inhibitors suppress leukemia cell growth

OBJECTIVE The objective of this study was to investigate the effect of small molecule inhibitors of glycogen synthase kinase-3β (GSK-3β) on leukemia cell growth and survival. MATERIALS AND METHODS Analysis of cytotoxicity and cell proliferation was conducted using the MTS assay, cell-cycle analysis, and division tracking. Apoptosis was investigated by Annexin-V/7-aminoactinomycin D and caspase-3 expression. The effect of GSK-3β inhibitors was also tested in vivo in an animal model of leukemia. Gene expression analysis was performed to identify the genes modulated by GSK-3β inhibition in leukemia cells. RESULTS GSK-3β inhibitors suppress cell growth and induce apoptosis in seven leukemia cell lines of diverse origin, four acute myeloid leukemia, one myelodysplastic syndrome, and one acute lymphoblastic leukemia samples. GSK-3β inhibitors are cytotoxic for rapidly dividing clonogenic leukemia blasts, and higher doses of the inhibitors are needed to eliminate primitive leukemia progenitor/stem cells. Slow cell-division rate, low drug uptake, and interaction with bone marrow stroma make leukemia cells more resistant to apoptosis induced by GSK-3β inhibitors. Global gene expression analysis combined with functional approaches identified multiple genes and specific signaling pathways modulated by GSK-3β inhibition. An important role for Bcl2 in the regulation of apoptosis induced by GSK-3β inhibitors was defined by expression analysis and confirmed by using pharmacological inhibitors of the protein. In vivo administration of GSK-3β inhibitors delayed tumor formation in a mouse leukemia model. GSK-3β inhibitors did not affect hematopoietic recovery following irradiation. CONCLUSIONS Our data support further evaluation of GSK-3β inhibitors as promising novel agents for therapeutic intervention in leukemia and warrant clinical investigation in leukemia patients.

Tumour anti-vascular alpha therapy: a mechanism for the regression of solid tumours in metastatic cancer

Targeted alpha therapy (TAT) is an emerging therapeutic modality, thought to be best suited to cancer micrometastases, leukaemia and lymphoma. TAT has not been indicated for solid tumours. However, several melanoma patients in a phase 1 clinical trial of systemic targeted alpha therapy for melanoma experienced marked regression of subcutaneous and internal tumours. This response cannot be ascribed to killing of all cancer cells in the tumours by targeted alpha therapy. These new observations support the original hypothesis that tumours can be regressed by a mechanism called tumour anti-vascular alpha therapy. This effect depends on the expression of targeted receptors by capillary pericytes and contiguous cancer cells, and on the short-range and high-energy transfer of alpha radiation.

The cytokinesis-block micronucleus assay as a biological dosimeter for targeted alpha therapy.

Ionizing radiation causes structural chromosomal aberrations, a proportion of which give rise to chromosome fragments without spindle attachment organelles. When a cell divides, some of these fragments are excluded from the main daughter nuclei and form small nuclei within the cytoplasm. The cytokinesis-block micronucleus assay allows these micronuclei (MN) to be counted, providing an in situ biological dosimeter. In this study, we evaluated the micronucleus frequency in peripheral blood lymphocytes after in vitro incubation with the alpha conjugates (213)BiI(3) and (213)Bi-9.2.27 (AIC). Lymphocytes were inoculated in vitro AIC for 3 h. Further, we report the first MN measurements in melanoma patients after targeted alpha therapy (TAT) with (213)Bi-9.2.27. Patients were injected with 260-360 MBq of AIC, and blood samples taken at 3 h, 2 weeks and 4 weeks post-treatment. Absorbed dose (MIRD) and effective total body dose (PED) were calculated. The MN frequency in lymphocytes was similar for equal in vitro incubation activities of (213)BiI(3) and (213)Bi-9.2.27 (P=0.5), indicating that there is no selective targeting of lymphocytes by the alpha conjugates. After inoculation with 10-1200 kBq mL-1 of AIC, there was a substantial activity-related increase in MN. The number of MN in the blood of treated patients peaked at 3 h post-TAT, slowly returning to baseline levels by 4 weeks. The mean photon equivalent dose (PED) is 0.43 Gy (SD 0.15) and the mean MIRD calculated absorbed dose is 0.11 Gy (SD 0.03), giving an RBE=4+/-0.4 for this study.

Preparation and testing of bevacizumab radioimmunoconjugates with Bismuth-213 and Bismuth-205/Bismuth-206.

Bevacizumab, a humanized anti-VEGF monoclonal antibody has shown promise in various clinical trials. We report the development and testing of Bi-213 (an alpha-emitting radionuclide) labeled bevacizumab for in vitro and in vivo studies using two different chelators viz cDTPA and CHX-A″. The developed labeling method showed high labeling yields of 93.6% and 89.7% for cDTPA and CHX-A″ respectively and the results were reproducible. The in vitro and in vivo stability tests were carried out using Bi-213 and long half-life Bismuth isotope (Bi-205 / Bi-206) for pharmacokinetics. The in vitro results showed remarkable stability of the radiolabeled bevacizumab regardless of the chelator. The in vivo pharmacokinetics studies however, showed that the uptake and retention of cDTPA- bevacizumab was significantly higher in kidneys (p-value 0.02) and lower in liver and spleen (p-value