[Show abstract][Hide abstract] ABSTRACT: Survivin is a tumor-associated antigen (TAA) that inhibits apoptosis and is widely overexpressed in cancer cells; therefore, survivin has potential as a target for cancer immunotherapy. Application of HLA-A2-restricted survivin-specific T cell receptors (TCRs) isolated from allogeneic HLA-mismatched TCR repertoires has, however, been impeded by the inability of these TCRs to distinguish healthy cells expressing low levels of survivin from cancer cells with high survivin expression levels. Here, we identified an HLA-A2-restricted survivin-specific TCR isolated from autologous TCR repertoires that targets tumor cells in vitro and in vivo but does not cause fratricidal toxicity. Molecular modeling of the TCR-peptide-HLA ternary complexes and alanine scanning revealed that the autologously derived TCRs had tighter interactions with the survivin peptide than did fratricidal TCRs. Similar recognition patterns were observed among 7 additional TAA-specific TCRs isolated from allogeneic versus autologous repertoires. Together, the results from this study indicate that maximal peptide recognition is key for TCR selectivity and likely critical for reducing unwanted off-target toxicities. Moreover, isolating TCRs from autologous repertoires to maximize TCR selectivity has potential as a useful strategy to identify and select other shared tumor- and self-antigen-specific TCRs and ensure selective antitumor activity.
Journal of Clinical Investigation 11/2014; · 13.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 12-year-old boy with refractory acute lymphoblastic leukemia received a haploidentical transplant from his mother. As prophylaxis for EBV, CMV and adenovirus, he received ex-vivo expanded virus-specific donor T cells 3.5 months after transplant. Four weeks later leukemic blasts bearing the E2A deletion, identified by FISH, appeared transiently in the blood followed by a FISH negative hematological remission, which was sustained until a testicular relapse 3.5 months later. Clearance of the circulating leukemic cells coincided with a marked increase in circulating virus specific T cells. The virus-specific cytotoxic T-cell (CTL) line showed strong polyfunctional reactivity with the patient's leukemic cells but not PHA blasts, suggesting that virus-specific CTL lines may have clinically significant anti-leukemia activity.Molecular Therapy (2014); doi:10.1038/mt.2014.192.
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr virus (EBV) reactivation can cause significant morbidity and mortality after allogeneic hematopoietic stem cell transplant. Delays in reconstitution of EBV-specific T lymphocyte activity can lead to life-threatening EBV lymphoproliferative disease (EBV-PTLD). This review highlights recent advances in the understanding of pathophysiology, risk factors, diagnosis, and management of EBV viremia and PTLD.
Current Opinion in Hematology 08/2014; · 4.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It remains difficult to treat the multiplicity of distinct viral infections that afflict immunocompromised patients. Adoptive transfer of virus-specific T cells (VSTs) can be safe and effective, but such cells have been complex to prepare and limited in antiviral range. We now demonstrate the feasibility and clinical utility of rapidly generated single-culture VSTs that recognize 12 immunogenic antigens from five viruses (Epstein-Barr virus, adenovirus, cytomegalovirus, BK virus, and human herpesvirus 6) that frequently cause disease in immunocompromised patients. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term.
Science translational medicine 06/2014; 6(242):242ra83. · 14.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Broader implementation of cell-based therapies has been hindered by the logistics associated with the expansion of clinically relevant cell numbers ex vivo. To overcome this limitation, Wilson Wolf Manufacturing developed the G-Rex, a cell culture flask with a gas-permeable membrane at the base that supports large media volumes without compromising gas exchange. Although this culture platform has recently gained traction with the scientific community due to its superior performance when compared with traditional culture systems, the limits of this technology have yet to be explored. In this study, we investigated multiple variables including optimal seeding density and media volume, as well as maximum cell output per unit of surface area. Additionally, we have identified a novel means of estimating culture growth kinetics. All of these parameters were subsequently integrated into a novel G-Rex “M” series, which can accommodate these optimal conditions. A multicenter study confirmed that this fully optimized cell culture system can reliably produce a 100-fold cell expansion in only 10 days using 1L of medium. The G-Rex M series is linearly scalable and adaptable as a closed system, allowing an easy translation of preclinical protocols into the good manufacturing practice.
[Show abstract][Hide abstract] ABSTRACT: Antigen-specific T cells provide a therapy for cancer that is highly specific, self-replicating, and potentially devoid of toxicity. Ideally, tumor-specific T cells should recognize multiple epitopes on multiple antigens to prevent tumor immune escape. However the large-scale expansion of such broad-spectrum T cells has been limited by the availability of potent autologous antigen-presenting cells that can present antigens on the polymorphic array of each patient's HLA allotype. We evaluated a novel antigen-presenting complex (KATpx) in which antigens in the form of peptide libraries can be presented by autologous activated T cells, whereas costimulation is complemented in trans by an HLA-negative K562 cell line genetically modified to express CD80, CD83, CD86, and 4-1BBL (K562cs). The additional costimulation provided by K562cs significantly enhanced T-cell expansion in culture over autologous activated T cells alone while maintaining antigen specificity. We validated this antigen-presenting system by generating Epstein-Barr virus (EBV) antigen-specific T cells from healthy donors and from patients with EBV-positive malignancies including nasopharyngeal carcinoma and multiply relapsed EBV-positive lymphoma. These T cells were specific for EBNA1, LMP1, and LMP2, the viral antigens expressed in these type 2 latency EBV-associated malignancies. The KATpx system consistently activated and expanded antigen-specific T cells both from healthy donors and from 5 of 6 patients with lymphoma and 6 of 6 with nasopharyngeal carcinoma, while simplifying the process for generating APCs by eliminating the need for live virus (EBV) or viral vectors to force expression of transgenic EBV antigens. Hence, KATpx provides a robust, reliable, and scalable process to expand tumor-directed T cells for the treatment of virus-associated cancers.
[Show abstract][Hide abstract] ABSTRACT: Adoptive transfer of T lymphocytes expressing a CD19-specific chimeric antigen receptor (CAR.CD19) induces complete tumor regression in patients with lymphoid malignancies. While in vivo persistence of CAR-T cells correlates with clinical responses, it remains unknown whether specific cell subsets within the CAR-T cell product correlate with their subsequent in vivo expansion and persistence. We analyzed 14 patients with B-cell malignancies infused with autologous CAR.CD19-redirected T cells expanded ex vivo using IL-2, and found that their in vivo expansion only correlated with the frequency within the infused product of a CD8(+)CD45RA(+)CCR7(+) subset, whose phenotype is closest to "T-memory stem cells". Preclinical models showed that increasing the frequency of CD8(+)CD45RA(+)CCR7(+) CAR-T cells in the infused line by culturing the cells with IL-7 and IL-15 produced greater antitumor activity of CAR-T cells mediated by increased resistance to cell death, following repetitive encounters with the antigen, whilst preserving their migration to secondary lymphoid organs. Studies are registered at clinicaltrials.gov, identifiers: NCT00586391 and NCT00709033.
[Show abstract][Hide abstract] ABSTRACT: Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk of uncontrolled graft versus host disease (GvHD). Thus patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase-9 (iC9) had their disease effectively controlled by a single administration of a small molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality, and a robust immunologic benefit, mediated initially by the infused cells themselves, and subsequently by an apparently accelerated reconstitution of endogenous naïve T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens including cytomegalovirus, adenovirus, BK virus and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study is registered at www.clinicaltrials.gov as NCT00710892.
[Show abstract][Hide abstract] ABSTRACT: This review is intended to reflect upon the current status and perspectives of cell-based immunotherapy at a time when the promise of extensive pre-clinical research has been translated into encouraging clinical responses. However, some of these have also been complicated by significant adverse reactions. As the field moves towards definitive late stage trials, with a growing interest from pharmaceutical companies, we realize that novel cell therapy strategies pose questions that are familiar to traditional drug development, along with new considerations due to the potential of T cells to persist long term and to expand after adoptive transfer. These questions address the safety of the product, the efficacy, the mode of action, and the anticipation of risks. From different perspectives, we intend to address exciting opportunities and safety concerns in current concepts of cellular immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: SIRS is a rare systemic inflammatory response associated with fever, tachycardia, profound hypotension and respiratory distress that has been reported in cancer patients receiving T cells genetically modified with chimeric antigen receptors to retarget their specificity to tumor associated antigens. The syndrome usually occurs following significant in vivo expansion of the infused cells and has been associated with tumor destruction/lysis. Analysis of patient plasma has shown elevated cytokine levels, and resolution of symptoms has been reported after administration of steroids and/or antibodies such as anti-TNF and anti-IL6 receptor that interfere with cytokine responses.To date SIRS has not been reported in subjects receiving genetically unmodified T-cells with native receptors directed to tumor antigens, in which more physiological control of T cell activation and expansion may occur. Here, however, we report a patient with bulky refractory EBV-associated lymphoma who developed this syndrome two weeks after receiving T cells directed to EBV antigens through their native receptors. She was treated with steroids and Etanercept with rapid resolution of symptoms. SIRS may therefore occur even when T cells recognize antigens physiologically through their "wild-type" native receptors, and should be acknowledged as a potential complication of this therapyMolecular Therapy (2014); doi:10.1038/mt.2014.48.
[Show abstract][Hide abstract] ABSTRACT: The success of adoptively transferred tumor-directed T cells requires them to survive and expand in vivo. Most tumors, however, employ immune evasion mechanisms, including the production of inhibitory cytokines that limit in vivo T-cell persistence and effector function. To protect tumor-directed T cells from such negative influences, we generated a chimeric cytokine receptor in which the interleukin (IL) 4 receptor exodomain was fused to the IL7 receptor endodomain. We thereby inverted the effects of tumor-derived IL4 so that the proliferation and activation of tumor directed cytotoxic T cells was enhanced rather than inhibited in the tumor microenvironment, resulting in superior antitumor activity. These transgenic T cells were only activated in the tumor environment since triggering required exposure to both tumor antigen (signal 1) and tumor-derived IL4 (signal 2). This selectivity supports future clinical adaptation.Molecular Therapy (2014); doi:10.1038/mt.2014.47.
[Show abstract][Hide abstract] ABSTRACT: Serious viral infections are a common cause of morbidity and mortality after allogeneic stem cell transplantation. They occur in the majority of allograft recipients and are fatal in 17-20%. These severe infections may be prolonged or recurrent and add substantially to the cost, both human and financial, of the procedure. Many features of allogeneic stem cell transplantation contribute to this high rate of viral disease. The cytotoxic and immunosuppressive drugs administered pretransplant to eliminate the host hematopoietic/immune system and any associated malignancy, the delay in recapitulating immune ontogeny post-transplant, the immunosuppressive drugs given to prevent graft versus host disease (GvHD), and the effects of GvHD itself, all serve to make stem cell transplant recipients vulnerable to disease from endogenous (latent) and exogenous (community) viruses, and to be incapable of controlling them as quickly and effectively as most normal individuals.
[Show abstract][Hide abstract] ABSTRACT: Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with low numbers of circulating CD4+CD25+FoxP3+ regulatory T-cells(Tregs). Because Tregs express high levels of the IL-2 receptor, they may selectively expand in vivo in response to doses of IL-2 insufficient to stimulate T-effector T-cell populations, thereby preventing GvHD.
We prospectively evaluated the effects of ultra low-dose (ULD) IL-2 injections on Treg recovery in pediatric patients after alloSCT and compared this recovery with Treg reconstitution post alloSCT in patients without IL-2. Sixteen recipients of related(n=12) or unrelated(n=4) donor grafts received ULD-IL-2 post HSCT (100,000-200,000 IU/m2 3xweekly), starting <day30 and continuing for 6-12weeks.
No grade 3/4 toxicities were associated with ULD-IL-2. CD4+CD25+FoxP3+ Tregs increased from a mean of 4.8%(range, 0-11.0%) pre IL-2 to 11.1%(range,1.2-31.1%) following therapy, with the greatest change occurring in the recipients of MRD transplants. No IL-2 patients developed grade II-IV aGvHD, compared to 4/33(12%) of the comparator group who did not receive IL-2. IL-2 recipients retained T-cells reactive to viral and leukemia antigens, and in the MRD recipients, only 2/13(15%) of the IL-2 patients developed viral infections versus 63% of the comparator group (p=0.022).
Hence, ULD-IL-2 is well-tolerated, expands a Treg population in vivo, and may be associated with a lower incidence of viral infections and GvHD.
Clinical Cancer Research 02/2014; · 8.19 Impact Factor