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Purpose: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated.Experimental Design: Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-γ were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-γ were examined. The in vitro effect of sunitinib on patient MDSC was evaluated.Results: Metastatic RCC patients had elevated levels of CD33+HLA-DR− and CD15+CD14− MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3+CD4+CD25hiFoxp3+ Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-γ. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at ≥1.0 μg/mL. Sunitinib did not induce MDSC maturation in vitro.Conclusions: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.

Supplementary Tables S1-S2 from Sunitinib Mediates Reversal of Myeloid-Derived Suppressor Cell Accumulation in Renal Cell Carcinoma Patients

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

A patient with adenosine deaminase–deficient severe combined immune deficiency (ADA-SCID) was enrolled in a study of retroviral-mediated ADA gene transfer to bone marrow hematopoietic stem cells. After the discontinuation of ADA enzyme replacement, busulfan (75 mg/m2) was administered for bone marrow cytoreduction, followed by infusion of autologous, gene-modified CD34+ cells. The expected myelosuppression developed after busulfan but then persisted, necessitating the administration of untransduced autologous bone marrow back-up at day 40. Because of sustained pancytopenia and negligible gene marking, diagnostic bone marrow biopsy and aspirate were performed at day 88. Analyses revealed hypocellular marrow and, unexpectedly, evidence of trisomy 8 in 21.6% of cells. Trisomy 8 mosaicism (T8M) was subsequently diagnosed by retrospective analysis of a pretreatment marrow sample that might have caused the lack of hematopoietic reconstitution. The confounding effects of this preexisting marrow cytogenetic abnormality on the response to gene transfer highlights another challenge of gene therapy with the use of autologous hematopoietic stem cells.

Adenosine deaminase (ADA) deficiency is a form of severe combined immunodeficiency (SCID) that has long been considered a good candidate for gene therapy (GTx). In 2001–2002, we treated 4 ADA-SCID patients in a clinical trial evaluating the efficacy of 2 different retroviral vectors while continuing enzyme replacement with pegylated bovine ADA (PEG-ADA). No chemotherapy was used. All patients have been monitored for 6 years. No treatment-related serious adverse events occurred. A mild transient elevation in absolute lymphocyte count (ALC) was seen in 2 patients early post-treatment, however, no durable immunologic changes were observed. Low levels (0.1–0.7%) of vector-marked peripheral blood mononuclear cells (PBMCs) persist in 2 patients treated at the age of 4–5 years. All patients remain on PEG-ADA, prophylactic antibiotics and intravenous immunoglobulins. In 2004, we revised the protocol in order to facilitate engraftment and selective advantage of gene-corrected cells by withdrawing PEG-ADA and giving busulfan (75 mg/m^2) before GTx. In November 2005, a first patient was treated who developed unexpected prolonged bone marrow (BM) aplasia. Cytogenetics revealed trisomy 8 aberrations that were found to be present on a BM specimen obtained pre-GTx (Blood2007; 109:503). Our second patient enrolled in January 2007. He received 5x10^6 CD34+ cells/kg that showed 40–200 units (U) of ADA activity (normal range 58–128). Over 6 months, this patient showed a slow increase in ALC (up to 750/mcL) and lymphocyte function. PBMC ADA activity has been up to 50U. The deoxyadenosine metabolite (dAXP) level has decreased to 500/mcL were 36 and 45 days, respectively. In >200 combined days of observation, there has been only one temperature above 38 °C, which resolved with acetaminophen. These data are consistent with the positive results of GTx for ADA-SCID obtained in Milan and London and show that PEG-ADA withdrawal and reduced conditioning improve the outcome of GTx for this disease. Longer follow-up should allow us to study engraftment of cells containing vector-specific sequences and conclude if either vector contributes more to recovery of lymphoid immunity.

Current studies involving the clinical application of genetically modified hematopoietic stem cells (HSC) require an ex vivo culture period during which the cells must maintain their ability to self- renew, repopulate the bone marrow, and produce the genetically corrected progeny cells. Recent advances in the formulation of optimized serum-free HSC tissue culture media led us to compare several recently commercially available culture media with the medium used in our current clinical gene therapy protocols for the transduction of HSC. We compared expansion, transduction efficiency, and differentiation of both cord blood (CB) and bone marrow (BM) derived CD34+ cells using X-Vivo15 (BioWhittaker, supplemented with 1% HSA), Cellgro|[reg]| SCGM (Cellgenix), StemSpan|[trade]| H300 (Stemline Technologies), Stemline|[trade]| I (Sigma- Aldrich), and Stemline|[trade]| II (Sigma-Aldrich). We observed comparable cell viability among the various media when using BM and CB derived CD34+ cells after 4 days of culture. Both BM and CB CD34+ cells cultured in X-Vivo 15, Cellgro|[reg]| SCGM, and Stemline|[trade]| II expanded to a greater extent than cells cultured in StemSpan|[trade]| H3000 or Stemline|[trade]|. Higher transduction efficiency using a retrovirus containing the eGFP gene was also observed in X- Vivo 15, Cellgro|[reg]| SCGM, and Stemline|[trade]| II media. A direct comparison between BM and CB CD34+ cells cultured in X-Vivo 15 or Stemline|[trade]| II resulted in 23% (BM) and 27% (CB) more transduced cells in the Stemline|[trade]| II medium. We then examined the clonogenicity of the transduced BM CD34+ cells in a Colony Forming Unit (CFU) assay. BM CD34+ cells cultured in Stemline|[trade]| II medium displayed a two-fold higher (16%) plating efficiency in the CFU assay compared to BM CD34+ cells cultured in X-Vivo 15 (8-9.5%). The number of colonies that were transduced (eGFP+) was higher in the BM CD34+ cells cultured in Stemline|[trade]| II medium compared to those cultured in X-Vivo 15 (29.9% and 10.3% respectively). From these results it is evident that BM CD34+ cells cultured in Stemline|[trade]| II medium have both a higher percentage of transduced cells and a higher percentage of these transduced cells remain capable of producing colonies in a CFU assay when compared with X-Vivo 15. From these data, Stemline|[trade]| II appears to be a more suitable medium for the ex vivo culturing and transduction of HSC for use in clinical therapies.