We previously demonstrated the benefit of using a test-dose of IV Busulfan to predict systemic exposure in patients with hematologic malignancies undergoing allogeneic stem cell transplant (Walko et al, J Clin Oncol, ASCO, 24, : 16502, 2006). We now report on this approach in 30 pts receiving a 90-hr CI of Bu in a dose-escalation study. Methods: Pts received a 0.8 mg/kg test dose over 2 hours and PK sampling at 0, 2.5, 4, 5, and 6 hours one week prior to initiation of full-dose treatment. Conditioning consisted of 30 mg/m2 Flu qd x 5 and targeted Bu to achieve an AUC of 4800 (9 pts), 5760 (6 pts), 6912 (8 pts), 7603 (3 pts) or 8363 (2 pts) umol∗min/hr/d on days -7 to -3. PK sampling occurred at hours 0, 12, 16, 18, 48, 60, 72, and 89.5. All pts received tacrolimus and IV alemtuzumab in doses of 30mg for matched related donors (MRD) or 60mg for matched unrelated donors (MUD) for prevention of GVHD. Results: 30 pts (14 MRD, 16 MUD) ages 18–55 (median 37) with high-risk AML (11), ALL (7), MDS (4), or other (8) were enrolled. All engrafted with a median of 13 days to ANC 〉 500 and 14 days to platelet count 〉 20K with no late graft failures. There were 9 treatment related deaths. Seven were from infection: pneumonia (1) and biliary sepsis (1) prior to day 100, and aspergillus (2), nocardia (1), biliary sepsis (1), and hemorrhagic cystitis (1) after day 100. One death was from late VOD and liver failure (d 201), and one from leukoencephalopathy (d 196). Grade 4 toxicities included 3 cases of mucositis (2 at dose level 5, AUC 8363, and 1 at level 2), and 1 liver failure at level 2. Grade 3 toxicities included mucositis (8), hepatitis (3) pneumonia (2) and seizures (1). 11 pts developed grade 2, two grade 3, and one grade 4 aGVHD. Two pts developed extensive and two developed limited cGVHD. 11 pts (37%) relapsed at a median of 117 days (range 17–652) post-transplant. PFS at 18 months was 36%. The MTD was level 4 with a target AUC of 7603 umol∗min/hr per day x 4 days. Dose limiting toxicity was grade 4 mucositis in 2/2 pts at dose level 5 with an actual mean AUC of 8600 umol∗min/hr per day x 4 days. Conclusion: This approach permits accurate delivery of a 55% increase in target systemic exposure to IV Bu compared to the standard AUC of 4800 umol∗min/hr per day x 4 days, is well-tolerated, and will be studied in a Phase II trial with ATG and MTX as GVH prophylaxis in an attempt to reduce the rate of deaths from late infections and relapse.
<p>The document contains 4 supplemental tables: 1. RT-PCR Primers for TCRbeta sequencing 2. HPLC-MS identified peptide sequences 3. The amino acid sequences of recurrent TCRbeta clonotypes 4. The distribution of the CDR3 amino acid length in the analyzed patient. There are 2 supplementary figures: 1. Tetramer flow cytometry showing the generation of an oligoclonal T-cell population with a UNC-CDK4-1 high affinity T-cell population. 2. More detailed information regarding the tetramer gating strategy employed.</p>
There remains little consensus about the best means for mobilizing stem cells prior to ASCT. Cytokine alone, usually G-CSF, provides easy scheduling while the combination approaches generally provide higher cell yields and additional cytoreduction. Because of its value as a cytoreductive agent, and its lack of stem cell damaging properties or other toxicities, we investigated the use of mid-dose VP-16 plus G-CSF as a mobilizing regimen.
36 A novel approach to gene therapy for insulin dependent diabetes mellitus (IDDM) is to transplant genetically modified human liver cells in the patients. This therapy requires the development of human liver cells to replace beta cells that can synthesize and process active insulin (mature insulin). Human liver cells cannot simply process proinsulin cDNA to make mature insulin because they lack the endoprotease to cleave the B-chain-C-peptide junction and C-peptide-A-chain junction. To achieve this link we used the human hepatoma cell line (HepG2) as a model to genetically engineer the human insulin gene. Methods: PCR-based site-directed mutagenesis was performed to create two mutation sites on insulin cDNA between the B-chain and C-peptide and between the C-peptide and A-chain, which were recognized by furin endoprotease in HepG2 cells. The mutated and wild-type insulin cDNA were cloned into mammalian expression vectors driven by the CMV promoter, respectively. In the permanent transfection of HepG2 with the mutated insulin cDNA construct, the mature insulin was secreted into the media and radioimmunoassay (RIA), (Linco Research Inc, St. Louis MO), was used to detect the mature insulin. Results: The concentration of mature insulin that accumulated in the culture media increased and reached a peak on day 5 at 38uU/ml without changing media (3.5 fold above background). No active insulin was detected in transfected cells with wild-type cDNA and untransfected cells. Production of mature insulin was confirmed by 35S-Cysteine labeling of these transfected cells in vivo, followed by immuno-precipitation, SDS-PAGE, and phosphor imaging. A4-Kd mature insulin band was observed from the media of HepG2 cells transfected with mutated insulin cDNA. However, in the media of HepG2 cells transfected with wild-type insulin cDNA only a 9-Kd proinsulin band was found. This is evidence that HepG2 cells process mutated insulin cDNA to be mature insulin efficiently. Application of conditioned media containing mature insulin to stimulate normal HepG2 cells, which express the insulin receptors, confers a biological response. The beta unit (cytosolic domain) of insulin receptor was immuno-precipated and subsequently subjected to Western blot to examine phosphotyrosine protein. It was shown that there was a specific 100-Kd band comparable to control cells treated with media alone. Conclusions: We demonstrated that mutated human insulin cDNA can be produced in human HepG2 cells correctly and efficiently. This genetically modified insulin is biologically functional. These techniques will be applied to cultured normal human hepatocytes with a replication stimulus, in future experiments.
Acute graft-versus-host disease (aGvHD) is a major limitation to the use of allogeneic stem cell transplantation for the treatment of patients with relapsed malignant disease. Previous work using animals lacking secondary lymphoid tissue (SLT) suggested that activation of donor T cells in SLT is critically important for the pathogenesis of aGvHD. However, these studies did not determine if impaired migration into, and more importantly, out of SLT, would ameliorate aGvHD. Here, we show that T cells from mice lacking Coronin 1A (Coro 1A(-/-)), an actin-associated protein shown to be important for thymocyte egress, do not mediate acute GvHD. The attenuation of aGvHD was associated with decreased expression of the critical trafficking proteins C-C chemokines receptor type 7 (CCR7) and sphingosine 1 phosphate receptor on donor T cells. This was mediated in part by impaired activation of the canonical NF-κB pathway in the absence of Coro 1A. As a result of these alterations, donor T cells from Coro 1A(-/-) mice were not able to initially traffic to SLT or exit SLT after BM transplantation. However, this alteration did not abrogate the graft-versus-leukemia response. Our data suggest that blocking T-cell migration into and out of SLT is a valid approach to prevent aGvHD.
