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7023 Background: Results of a phase I trial of donor-derived CD7 chimeric antigen receptor (CAR) T cells for relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) (Pan et al. J Clin Oncol 2021;39:3340-3351) have been reported previously, and are updated now. Methods: The target dose is 1 × 106 (±30%) CAR T cells per kg of body weight. Patients with prior stem cell transplantation (SCT) received CAR T cells from prior SCT donors, while patients without SCT history received CAR T cells from new donors who also provided stem cells for transplantation post CAR T therapy. The primary endpoint was safety with efficacy secondary. Survival status were continuously followed up while severe adverse events (SAEs) were recorded until receiving other anti-leukemia therapy. Results: Nineteen (95%) of twenty enrolled patients responded and were followed up with a median time of 15.8 months (range 13-18.3) until Feb, 14th, 2022. Short-term adverse events included grade 3 or higher cytokine release syndrome (10%) and grade 1-2 graft-versus-host disease (GVHD, 60%), which were all reversible. Six late-onset ( > 30 days post-infusion) severe adverse events (SAEs) occurred in 5 responders. Two had been reported previously. Four SAEs were newly observed, including a grade 4 intestinal GVHD at month 11 and a grade 5 pneumonia at month 12.3 in one patient, a grade 5 Pseudomonas Aeruginosa pneumonia at month 8.7 in one patient, and a grade 3 cytomegalovirus (CMV) encephalitis at month 11 which recovered at month 13.3 in another patient. All severe infections occurred in patients with no further therapy, and the total T cells in them reached a median count of 300.03/μL (range 121.46-512.83), which were substantially lower than normal levels despite steadily increasing. The objective response and complete remission rate was 95% and 85% at day 30 post-infusion. Of 19 responders that were followed up, two (11%) withdrew for other therapy at day 55 and 271 respectively. Of 10 (53%) patients who received no further therapy, all had continuously detectable CAR T cells until the last visit, three remained in remission, three had a relapse (one CD7+, and two CD7-), and four died of infection; Seven (37%) patients proceeded to SCT and no CAR T cells were detectable after SCT, and among them two remained in remission, four had a relapse (three CD7+, and one CD7-), and one died of transplant-related mortality. Patients relapsed at a median time of 6 (range 4-10.9) months. The one-year progressive-free survival (PFS) and overall survival (OS) rates were 51.6% (95% CI, 24.7-78.4%) and 72.5% (95% CI, 51.9-93.0%). Conclusions: Donor-derived CD7 CAR T cell therapy showed encouraging activity in treating r/r T-ALL. Relapse emerges as major issues impeding long-term outcomes. CD7-negative relapse was commonly observed under CAR T cell surveillance. Late onset GVHD and infections may occur and should be carefully managed. Clinical trial information: ChiCTR2000034762.

BackgroundIn clinical applications of CAR T-cell therapy, life-threatening adverse events including cytokine release syndrome and neurotoxicity can lead to treatment failure. Outcomes of patients treated with anti-CD30 CAR T- cell have been disappointing in relapsing/refractory (r/r) classical Hodgkin’s Lymphoma (cHL).MethodsIn order to understand the applicable population of multiple CAR T-cell therapy, we examined the expression of CD19, CD20, and CD30 by immunohistochemistry (IHC) in 38 paraffin-embedded specimens of cHL. In the past two years, we found only one patient with cHL who is eligible for combined anti-CD19 and CD30 CAR T-cell treatment. This patient’s baseline characteristics were prone to severe adverse events. We treated this patient with low doses and multiple infusions of anti-CD19 and CD30 CAR T-cell.ResultsThe positive expression of CD19+ + CD30+ in Reed-Sternberg (RS) cells is approximately 5.2% (2/38). The patient we treated with combined anti-CD19 and CD30 CAR T-cell did not experience severe adverse events related to CAR T-cell therapy and received long term progression-free survival (PFS).ConclusionFor high risk r/r cHL patients, low doses of CAR T-cell used over different days at different times might be safe and effective. More clinical trials are warranted for CD19 and CD30 CAR T-cell combination therapy.

Chimeric antigen receptor (CAR)-T cell therapy emerges as a new treatment for refractory or relapsed (r/r) B-cell non-Hodgkin lymphoma (B-NHL); however, the overall response rate (ORR) of which in the B-NHL patients is much lower compared to the patients with r/r B acute lymphoblastic leukemia (B-ALL). We previously confirmed that sequential infusions of CD20 and CD22 CAR-T cells significantly improved the prognosis of the B-NHL patients, while some advanced patients still progressed to death during these CAR-T cell treatments. In this study, we showed that timely sequential administration of the second CAR-T cells could enhance expansion of prior CAR-T cells with stronger tumor-killing capacity in vitro and in vivo. We further conducted compassionate treatments on two advanced B-NHL patients with short-interval sequential infusions of CD19/22/20 CAR-T cells. Disease progression was observed in both patients after primary CAR-T cell infusion but robust re-expansion of prior CAR-T cells and anti-tumor effects was induced by infusion of a secondary CAR-T cells. These results indicate sequential infusions of CAR-T cells with a short interval may improve therapeutic efficacy in the B-NHL patients by promoting expansion of prior CAR-T cells.

Chimeric antigen receptor (CAR) T-cell therapies that target either CD19 or CD22 alone have potent antilymphoma effects. However, antigen escape–mediated relapse often occurs. CAR T cells targeting both CD19 and CD22 may overcome this limitation. In this study, we developed bispecific CAR T cells simultaneously recognizing CD19- and CD22-expressing targets and assessed their safety and efficacy profiles in patients with relapsed/refractory aggressive B-cell lymphoma. Twenty-four patients were screened, and 16 were found eligible for the study. CAR T-cell–associated toxicities were recorded. Responses, overall survival (OS), and progression-free survival (PFS) were assessed. Of the 16 eligible patients, 14 (87.5%) achieved objective response and 10 (62.5%) achieved complete response (CR). The 2-year OS and PFS rates were 77.3% and 40.2%, respectively. Achieving CR (P = 0.046) and the number of prior chemotherapy lines (n = 2; P = 0.047) were independent prognostic factors associated with favorable PFS. The 2-year OS and PFS among patients who achieved CR were higher than among those who did not (P = 0.015 and P < 0.001, respectively). The 2-year PFS among patients who received two prior lines of chemotherapy was higher than that among patients who received more than two lines of chemotherapy (P = 0.049); OS did not differ between the groups. Severe grade 4 cytokine-release syndrome (CRS) was observed in 1 patient; 4 and 11 patients had grades 1 and 2 CRS, respectively. No patients developed neurotoxicity. CD19/CD22 dual-targeted CAR T cells may be a safe, potent antilymphoma cell-based targeted immunotherapy.

Although recent clinical trials have demonstrated the efficacy of CD19-directed chimeric antigen receptor (CAR) T-cell therapy for refractory or relapsed B acute lymphoblastic leukemia (r/r B-ALL), most trials exclude patients with high-burden CNS leukemia (CNSL) to avoid the risk of severe neurotoxicity. There were only sparse cases describing the effect of CAR T cells on low-burden CNSL, and the safety and effectiveness of CAR T cells in high-burden CNSL remains unknown.Here, we retrospectively analyzed the results of CD19 CAR T-cell therapy in 12 pediatric patients that had low (Blasts 20/μL in CSF) or high-burdens (Blasts or intracranial solid mass) of CNS B-ALL, that are enrolled in three clinical trials and one pilot study at Beijing Boren Hospital RESULTS: Eleven patients (91.7%) achieved complete remission (CR) on day 30, and one patient got CR on day 90 after infusion. Most patient experienced mild cytokine-release syndrome. However, of the five patients who retained 5/μL blasts in CSF or a solid mass before CAR T-cell expansion, four developed severe (grade 3-4) neurotoxicity featured by persistent cerebral edema and seizure, and they fully recovered after intensive managements. Sustained remission was achieved in 9 of the 12 patients, resulted in a 6-month leukemia-free survival rate of 81.8% (95% CI 59.0-100). Only one patient has CNS relapse again.Our study demonstrates that CAR T cells are effective in clearing both low- and high-burden CNSL, but a high CNSL burden before CAR T-cell expansion may cause severe neurotoxicity requiring intense intervention.

Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is overexpressed in more than 90% of non-small cell lung cancer (NSCLC) but not significantly in normal lung tissue. It is therefore an important tumor antigen target for chimeric antigen receptors (CAR)-T-based therapy in NSCLC. Here, we developed a specific CAR targeted to EphA2, and the anti-tumor effects of this CAR were investigated. A second generation CAR with co-stimulatory receptor 4-1BB targeted to EphA2 was developed. The functionality of EphA2-specific T cells in vitro was tested with flow cytometry and real-time cell electronic sensing system assays. The effect in vivo was evaluated in xenograft SCID Beige mouse model of EphA2 positive NSCLC. These EphA2-specifc T cells can cause tumor cell lysis by producing the cytokines IFN-γ when cocultured with EphA2-positive targets, and the cytotoxicity effects was specific in vitro. In vivo, the tumor signals of mice treated with EphA2-specifc T cells presented the tendency of decrease, and was much lower than the mice treated with non-transduced T cells. The anti-tumor effects of this CAR-T technology in vivo and vitro had been confirmed. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive NSCLC.

Refractory or relapsed B lymphoblastic leukemia (B-ALL) patients have a dismal outcome with current therapy. We treated 42 primary refractory/hematological relapsed (R/R) and 9 refractory minimal residual disease by flow cytometry (FCM-MRD+) B-ALL patients with optimized second generation CD19-directed CAR-T cells. The CAR-T-cell infusion dosages were initially ranged from 0.05 to 14 × 105/kg and were eventually settled at 1 × 105/kg for the most recent 20 cases. 36/40 (90%) evaluated R/R patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 9/9 (100%) FCM-MRD+ patients achieved MRD-. All of the most recent 20 patients achieved CR/CRi. Most cases only experienced mild to moderate CRS. 8/51 cases had seizures that were relieved by early intervention. Twenty three of twenty seven CR/CRi patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HCT) remained in MRD- with a median follow-up time of 206 (45-427) days, whereas 9 of 18 CR/CRi patients without allo-HCT relapsed. Our results indicate that a low CAR-T-cell dosage of 1 × 105/kg, is effective and safe for treating refractory or relapsed B-ALL, and subsequent allo-HCT could further reduce the relapse rate.

Mantle cell lymphoma (MCL) is a distinct histological type of B-cell lymphoma with a poor prognosis. Several agents, such as proteasome inhibitors, immunomodulatory drugs, and inhibitors of B cell lymphoma-2 and Bruton's tyrosine kinase have shown efficacy for relapsed or refractory (r/r) MCL but often have short-term responses. Chimeric antigen receptor (CAR) T-cell therapy has emerged as a novel treatment modality for r/r non-Hodgkin's lymphoma. However, long-term safety and tolerability associated with CAR T-cell therapy are not defined well, especially in MCL. In this report, we described a 70-year-old patient with r/r MCL with 48-month duration of follow-up who achieved long-term remission after CAR T-cell therapy. CAR T-cell-related toxicities were also mild and tolerated well even in this elderly patient. This report suggested that CAR T-cell therapy is a promising treatment modality for patients with MCL, who are generally elderly and have comorbid conditions.

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2-99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

Neovasculature imaging is a promising approach for tumor diagnosis. We constructed tumor neovasculature targeted paramagnetic nanoliposomes with RGD10, F56, and K237 peptides, which can bind to Integrin αvβ3 and VEGFR-1, VEGFR-2, respectively, and compared their potential value as MRI contrast agents for detecting small tumors in animal models.Peptide-Ahx-palmitic acid conjugate was synthesized using Fmoc solid-phase synthesis chemistry. Targeted paramagnetic nanoliposomes were prepared by the thin film dispersion-sonication method. The tumor signal enhancements of liposome particles were evaluated by MRI in a xenograft mice model.The apparent affinity constants of RGD10, K237, and F56 peptides binding to their cell receptors were 9.15 × 10(7), 6.01 × 10(7), and 3.85 × 10(7) mol/L, respectively. RGD10 and K237 targeted paramagnetic nanoliposomes have shown much greater tumor-specific MRI signal enhancement in xenograft of the nude mice compared to F56 targeted paramagnetic nanoliposome. Tumor signal enhancement rate (SER %) increased 2.21 ± 0.09 and 1.82 ± 0.05 fold, respectively, for RGD10 and K237 compared to non-targeted control in T1 weighted MR image.RGD10 and K237 targeted paramagnetic nanoliposomes can be developed as potential tumor-specific MRI contrast agents and are helpful for tumor detection.

Introduction: The source of chimeric antigen receptor (CAR) T cells can be autologous or allogeneic, depending on the history of allogeneic hematopoietic stem cell transplantation (allo-HSCT). For patients with a history of allo-HSCT, CAR T cells are allogeneic, which can be manufactured from donors' peripheral blood mononuclear cells (PBMC) (donor-derived allogeneic CAR T cells, DD-alloCAR) or patients' own PBMC (recipient-derived allogeneic CAR T cells, RD-alloCAR). Unlike autoCAR T cells, which are activated by CAR only, alloCAR T cells receive activation signals from both T-cell receptor and CAR. The difference could affect CAR T-cell biology, resulting in different clinical outcomes. Methods: We retrospectively reviewed 31 consecutive patients who received CAR T-cell therapy in our center. Patients were divided into three groups, as autoCAR group, DD-alloCAR group and RD-alloCAR group. We compared the efficacy and safety profiles among the three groups. We also performed a subgroup analysis of patients received alloCAR. Results: Altogether there were 17 patients in the autoCAR, 11 in the RD-alloCAR and 3 in the DD-alloCAR groups. After a median follow-up of 9 months, the CR rate was 88.2% (95% CI 63.6-98.5%) in the autoCAR group and 100% (95% CI 71.5-100%) in the RD-alloCAR group, which had no significant difference (p=0.73). CR rate in DD-alloCAR was 66.7% (95% CI 9.43-99.1%). The median peak expansion of CAR T cells in the autoCAR was significantly higher than the RD-alloCAR group (p=0.007, Figure A). However, the median OS and EFS did not differ significantly among the groups. As far as adverse effects, RD-alloCAR group had significantly less patients with severe CRS (defined as Grade ≥ 3) than the autoCAR group (p=0.049), with significantly lower peak IL-6 level (p=0.021, Figure B). Neurotoxicity and the degree of cytopenia did not have significant difference among the groups. Acute GVHD occurred in 2 (18.2%) RD-alloCAR patients and 1 (33.3%) DD-alloCAR patients following CAR T-cell infusion. Univariate subgroup analysis of the patients who received alloCAR T-cell therapy showed the presence of cGVHD at the time of PBMC collection was significantly associated with less 6-month relapses (p=0.022); the median RFS in the no-cGVHD group was 2.00 (95% CI 0.72 to 3.28) months, compared to 12.0 (95% CI 2.40 to 21.6) months in the cGVHD group (p=0.084, Figure C); the median OS had no significant difference (Figure D). RD-alloCAR patients with or without cGVHD at PBMC collection did not differ in terms of the peak CAR T-cell expansion, CRS grades or OS. Conclusions: Compared with autoCAR, RD-alloCAR had similar efficacy but less severe CRS. For RD-alloCAR T-cell therapy, the presence of cGVHD at the time of PBMC collection was associated with fewer 6-month relapses. DD-alloCAR and RD-alloCAR T-cell therapies were effective and safe without causing significant GVHD. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Background: CD19-targeting chimeric antigen receptor (CAR) T-cell therapy has shown great efficacy in patients with refractory/relapsed non-Hodgkin lymphoma (NHL), but was associated with serious adverse effects such as cytokine release syndrome (CRS). It has been speculated that NHL baseline disease burden might affect clinical outcome and CRS, but such assumption has not been explored in detail in previous studies. Metabolic tumor volume (MTV) and total lesion glycolysis (TLG), calculated using FDG PET-CT, are quantitative indicators of baseline tumor burden. Methods: Utilizing FDG PET-CT, we calculated MTV and TLG at baseline and post CAR T-cell therapy in 15 patients with NHL. Results: Among all the patients, the median MTV was 72 (range 0.02-3024.9) cm3 and the median TLG was 610.1 (range 0.011-13156.3). After a median follow-up of 6 months, the overall response rate (ORR) was 66.7% (95% CI 38.4-88.2%). The baseline MTV and TLG did not have significant difference in patients with or without response (p=0.271 and 0.95, respectively). Cox-regression analysis did not find lower baseline MTV and TLG significantly associated with better overall survival (p=0.67 and 0.45, respectively). Patients with mild and moderate CRS (defined as Grade 0-2) had significantly lower MTV and TLG than those with severe CRS (Grade 3, 4) (median MTV: 49.3 v.s. 1137.7 cm3, p=0.012; median TLG: 379.1 v.s. 9384, p=0.012, Figure A, B). The median MTV in patients received tocilizumab, an IL-6 antagonist for the treatment of severe CRS, was 963.4 cm3, which was significantly higher than the patients not receiving tocilizumab (58.1 cm3, p=0.037). The median TLG in patients received tocilizumab was 9187.1, compared with 610.1 in patients not receiving tocilizumab (p=0.053). Using FDG PET-CT, we also demonstrated that CAR T-cell therapy in NHL patients could associate with severe local complications such as local compression and local inflammation. Conclusions: Low NHL baseline disease burden is not associated with better response rate or long-term outcome. Patients with higher baseline disease burden have more severe CRS Figure. Figure. Disclosures No relevant conflicts of interest to declare.

The in vivo regulation of T lymphocyte activity by the activation of a suicide mechanism is an essential paradigm for the safety of adoptive cell therapies. In light of reports showing that gamma-retroviral vector-encoded herpes simplex virus thymidine kinase (hsvtk) undergoes recombination, we undertook a thorough investigation of the genomic stability of SFG-based vectors using two variants of the wild-type hsvtk gene. In a large panel of independent clones, we demonstrate that both hsvtk genes undergo recombination with molecular signatures indicative of template switching in GC-rich regions displaying homology at the deletion junctions or RNA splicing. In the absence of ganciclovir selection, the frequency of recombination is 3% per retroviral replication cycle. Our results underscore the importance of the five nucleotide difference between the two hsvtk genes that account for the presence of recombinogenic hot spots in one variant and not the other, indicating that the probability of RNA splicing is influenced by minute nucleotide changes in sequences adjacent to the splice donor and acceptor sites. Furthermore, our mutational analysis in an unbiased panel of human lymphoid cells (that is, without immune or ganciclovir-mediated selective pressure) provides a robust in vitro assay to predict and quantify clinically relevant mutations in hsvtk suicide genes, which can be applied to studying and improving the stability of any transgene expressed in gamma-retroviral or lentiviral vectors.

The ability of specific virally encoded proteins to down-regulate MHC class I molecules may enable infected cells to elude killing by CTL. In the case of HIV-1, Nef appears to be responsible for this effect. Thus, interfering with Nef-induced MHC class I down-regulation would be a strategy for increasing HIV-1-specific CTL activity, particularly towards long-lived T cell populations suchas memory T cells that harbor replication-competent virus. Here, using two Nef-expressing human cell model systems, we show that a dominant-negative mutant derived from the Hck protein-tyrosine kinase, composed of the Hck N-terminal region, as well as the SH3 and SH2 domains, was able to inhibit Nef-induced MHC class I molecule down-regulation. This effect was SH3 domain dependent as it was not evident when the cells were transfected with DN-Hck-W93F, an SH3 domain mutant. The inhibitory effect of dominant-negative-Hck (DN-Hck) on Nef-induced class I down-regulation suggests that this Nef-mediated effect requires an interaction between the Nef polyproline site and an SH3-containing cellular protein that is involved in MHC class I molecule turnover. Interfering with the function of the Nef SH3 binding site in this way represents a strategy for assisting the host CTL response to clear HIV-1-infected cells.

Abstract Introduction: Patients (pts) with relapsed refractory B-ALL are mostly incurable by chemotherapy. The disease free survival (DFS) is low even treatment with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We explored treatment of 47 cases of relapsed refractory B-ALL with low dose CD19 CAR-T cells and assessed the clinical safety and efficacy. Patients and Methods: (6-8)X107 pts' peripheral blood mononuclear cells(PBMCs) were activated with CD3 and CD28 antibodies for 24h, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR (Image1/Picture1) and cultured for 5-6 days in serum-free media containing IL2,IL7,IL15,IL21. All pts except one who had persistent cytopenia received cyclophosphamide 250 mg/m2/d X 3d, and fludarabine 30 mg/m2/d X 3d, then CAR-T cell infusion. Between Jul.31 2015 and Jul. 15 2016, a total of 47 cases were treated with CAR-T cells (Chart1). 37/47 cases had frank hematologically relapsed refractory B-ALL, who could not achieve complete remission (CR) after more than 1 cycles of chemotherapy. The median prior chemotherapy duration was 18 months. The median pre-treatment bone marrow blast percentage was 67% (6.5-98.5%). The most recently treated 15 cases had their PB blasts Chart 1: Characteristics of Patients Pre-CAR-T Image 1/Picture 1: Results: The pts received a median of 10 (0.5-140) X104cells/kg CAR-T cells, and the most recently treated 15 cases all received 10 X 104cells/kg. The median observation period was 201 days (20-368 days). On day 16-20 after CAR-T infusion, 31/35 (88.6%) relapsed hematological refractory cases achieved CR or incomplete CR(Cri), and 29/35 cases (82.9%) achieved negative MRD by FCM(CMR: complete molecular remission). No extramedullary leukemia was detected in any cases with residual disease. The most recently treated 15 consecutive cases all achieved CR with only mild ( Conclusion: Our anti-CD19 CAR-T cell therapy can result in a high CR/CRi /CMR rate in pts with refractory B-ALL and could overcome pre-existing risk factors for poor outcomes, including complex chromosome abnormalities, poor gene mutations, inherited predisposing gene mutation, extramedullary leukemia etc. Pts could be safely bridged into allo-HSCT for potential cure. The dose of infused CAR-T cells in our patients was far lower than previously reported in the literature and the culture period was only 6-7 days, which could dramatically reduce the cost of the CAR-T therapy. 10X104cells/kg of CAR-T cells was a safe and effective number for treating B-ALL. The major complication was CRS and the severity of CRS was directly correlated with the number of malignant B cells in the PB. The efficacy of CAR-T therapy was correlated to the infused number of CAR-T cells. The most recently treated 15 consecutive cases all achieved CR without severe CRS suggesting that the optimal number of CAR-T cells and patient selection are important for the efficacy and safety. Table. Table. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Systemically distributed proteins such as clotting factors have been traditionally expressed from muscle or liver to achieve therapeutic, long-term expression. As long-lived cell capable of generating an abundant progeny, hematopoietic stem cells (HSCs) also merit consideration for this purpose. To be clinically relevant, this approach would require that hematopoietic cells be capable of expressing high levels of functional, secreted proteins, that the risk of insertional oncogenesis be minimized, and that sufficient stem cell engraftment be achieved following minimally invasive conditioning. Recent reports demonstrate the feasibility of expressing either factor IX (FIX) or factor VIII (FVIII) in erythroblasts and platelets using lineage-restricted vectors, resulting in effective treatments in mouse models of hemophilia. The erythroid system is especially powerful in providing high protein output, yielding FIX levels approaching 1 micro g/ml per vector copy in the plasma of long-term hematopoietic chimeras, a secretion level that vastly outperforms any other current mammalian constitutive or long-terminal repeat (LTR)-driven vector system. These early but promising studies raise the prospect of further developing these strategies for clinical investigation.

We have developed a lentiviral vector system for human factor IX (hFIX) gene transfer in hematopoietic stem cells (HSCs) that provides erythroid cell–derived systemic protein delivery following nonmyeloablative conditioning and in vivo methylguanine methyltransferase (MGMT) drug selection. After bone marrow transplantation under moderate Busulfan conditioning, the initial hFIX expression in the chimeras was minimally detectable. However, the hFIX levels rose sharply following in vivo MGMT-drug selection and eventually reached a level that is considered curative in hemophilia B therapy (>500 ng/ml). The rise of hFIX levels was proportional to the increase in vector copy (VC) number in peripheral blood cells. High levels of hFIX expression were maintained in serially engrafted mice chimeras for 18 months. Importantly, high-level hFIX expression by erythroid cells did not result in anemia or adversely affect red blood cell counts. The prospect of combining reduced intensity conditioning, a presumably lowered risk of insertional mutagenesis due to low VC number requirement and erythroid-restricted transgene expression, as well as long-term protein expression at high level, strongly supports the potential applicability of adult stem cell–based gene therapy in nonlethal blood or metabolic disorders, as demonstrated here for hemophilia.

To reject tumors, T cells must overcome poor tumor immunogenicity and an adverse tumor microenvironment. Providing agonistic costimulatory signals to tumor-infiltrating T cells to augment T cell function remains a challenge for the implementation of safe and effective immunotherapy. We hypothesized that T cells overexpressing selected costimulatory ligands could serve as cellular vehicles mediating powerful, yet constrained, anatomically targeted costimulation. Here, we show that primary human T cells expressing CD80 and 4-1BB ligand (4-1BBL) vigorously respond to tumor cells lacking costimulatory ligands and provoke potent rejection of large, systemic tumors in immunodeficient mice. In addition to showing costimulation of bystander T cells (transcostimulation), we show the effect of CD80 and 4-1BBL binding to their respective receptors in the immunological synapse of isolated single cells (autocostimulation). This new strategy of endowing T cells with constitutively expressed costimulatory ligands could be extended to other ligand-receptor pairs and used to enhance any targeted adoptive transfer therapy.

Recent studies on the integration patterns of different categories of retroviral vectors, the genotoxicity of long-terminal repeats (LTRs) and other genetic elements, the rise of lentiviral technology and the emergence of regulated vector systems providing tissue-restricted transgene expression and RNA interference, are profoundly changing the landscape of stem cell-based therapies. New developments in vector design and an increasing understanding of the mechanisms underlying insertional oncogenesis are ushering in a new phase in hematopoietic stem cell (HSC) engineering, thus bringing the hitherto exclusive reliance on LTR-driven, gamma-retroviral vectors to an end. Based on their ability to transduce non-dividing cells and their genomic stability, lentiviral vectors offer new prospects for the manipulation of HSCs. Tissue-specific vectors, as exemplified by globin vectors, not only provide therapeutic efficacy, but may also enhance safety, insofar that they restrict transgene expression in stem cells, progenitor cells and blood cells in all but the transcriptionally targeted lineage. This review provides a survey of these advances as well as several remaining challenges, focusing in particular on the importance of achieving adequate levels of protein expression from a limited number of vector copies per cell-ideally one to two.

We demonstrate here the capacity of erythroid cells to mediate long-term, systemic and therapeutic protein delivery in vivo. By targeting human factor IX (hFIX) expression to late-stage erythropoiesis, we achieve long-term hFIX secretion at levels significantly higher (>tenfold) than those obtained with an archetypal ubiquitous promoter in a mouse model of hemophilia B. Erythroid cell-derived hFIX is biologically active, resulting in phenotypic correction of the bleeding disorder. In addition to achieving high expression levels and resistance to transcriptional silencing, red cell-mediated protein delivery offers multiple advantages including immune tolerance induction, reduction of the risk of insertional oncogenesis and relative ease of application by either engrafting transduced hematopoietic stem cells or transfusing ex vivo-generated, stem cell-derived erythroid cells.

Top of pageAbstract Various cell types have been targeted for expression of clotting factors in experimental models of hemophilia, with muscle and hepatocyte-directed gene transfer advancing to clinical trials. Hematopoietic stem cells (HSCs) are attractive alternative targets because of their self-renewing properties, their ability to generate blood cells over extended periods of time, and their ease of manipulation ex vivo. However, several hurdles must be overcome to make this approach safe and practical. These include the need to express sufficient amount of therapeutic gene products and minimize the risk of insertional mutagenesis and the toxicity of host conditioning to facilitate HSC engraftment. We have previously developed an erythoid cell-specific platform for long-term and systemic therapeutic protein delivery in vivo using an erythroid- specific lentiviral vector. We demonstrated in an immune competent C57BL/6 hemophilia B mouse model, that therapeutic levels of human factor IX (hFIX) can be expressed at low average vector copy (VC) number (250-350 ng/mL with under 0.5 VC per cell). In this study, we examined the level of erythroid-specific protein delivery obtained after engraftment following minimal conditioning, eventually in the presence of in vivo methylguanine methyltransferase (MGMT)-mediated drug resistance. Low to moderate levels of hFIX expression were achieved in control, lethally irradiated mice (average 100-200 ng/ml), 4-weeks post-transplantation. In non-myeloablated recipients conditioned with 400 cGy or Busulfan (20 mg/kg, given twice at 48 h and 24 h before engraftment), the levels of hFIX expression were ranged from negligible to low (0-20 ng/ml and 0 |[ndash]| 100 ng/ml, respectively). Four weeks after the first round of BG/BCNU (30 mg/kg and 5 mg/kg, respectively), a wide range of hFIX expression levels were achieved, reaching in some mice levels equivalent to 20% of physiological levels in humans. We are currently monitoring long-term hFIX expression, as well as the average VC number in blood cells and CFUs. Further rounds of drug selection are also in progress. In conclusion, our data suggest that it is possible to express high levels of a secreted protein from HSC-derived erythroid cells engrafted under minimal conditioning. The combined features of reduced intensity conditioning, reduced insertional mutagenesis due to low vector copy number requirement and erythroid-specific transgene expression, as well as long-term protein expression at therapeutic levels, increase the potential applicability of adult stem cell-based gene therapy in non-lethal disorders such as hemophilia.

The light and heavy chain variable regions of three mouse hybridoma cell lines (AG11, AE6 and EH1) that produce monoclonal antibodies against an overlapping epitope at the C-terminus of Nef were cloned. Sequence analysis of the light and heavy chain variable regions indicated that clones AG11 and AE6, but not EH1, were highly related. Single-chain antibodies were constructed from the cDNA clones of AG11 and EH1, and subcloned into an eukaryotic expressing vector with the green fluorescent protein as marker for expression. Such intracellular antibodies may provide a way in which to inhibit the function of Nef during HIV-1 infection of cells.

Recombinant lentiviral vectors derived from human immunodeficiency virus-1 are promising vectors for gene therapy applications. This is due to their ability to transduce nondividing cells, their genomic stability, their relatively large genomic capacity, as well as their ability to stably maintain long term transgene expression. Recent studies have uncovered that the central polypurine tract (cPPT) plays an important role in increasing lentivirus-mediated gene transfer efficiency. The cPPT participates in lentiviral reverse transcription by initiating the synthesis of a downstream plus strand cDNA. Together with the upstream plus strand initiated at the 3' polypurine tract, it forms a central DNA flap that bears in its center a short plus strand overlap. A possible role in nuclear import enhancement of the viral preintegration complex has been suggested (Zennou, Cell, 2000), but the mechanism of cPPT function is still not fully elucidated. It has also been reported that the incorporation of cPPT in lentiviral vectors can increase particle infectivity (Follenzi, Nature Genetics, 2000). However, a systematic evaluation of the cPPT in complex lentiviral vectors has not been reported to date.

Current attempts to treat hemophilia with stem cell-based gene therapy make use of oncoretroviral or lentiviral vectors with ubiquitous promoters to express human FVIII or FIX. However, studies in mouse models of hemophilia have resulted in low transgene expression (Evans & Morgan, 1998; Kootstra & Verma, 2003; Tiede & Scherr, 2003), only achieving therapeutic expression levels following high copy gene transfer (>5 copies/cell, Bigger and Thermis, 2004). A recent study by Moayeri et al. (Mol. Ther., 2004) demonstrated long-term therapeutic levels of factor VIII expression in immunocompromised hemophilic mice engrafted with pre-selected, oncoretroviral vector transduced hematopoietic stem cells (HSCs). However, non-specific promoters may pose a greater risk of inducing insertional oncogenesis than vectors whose expression is restricted to terminally differentiated progeny, such as maturing erythroblasts.