Purpose A first-in-human pilot safety and feasibility trial evaluating chimeric antigen receptor (CAR) engineered, autologous primary human CD8+ cytolytic T lymphocytes (CTLs) targeting IL13Rα2 for the treatment of recurrent glioblastoma (GBM). Experimental Design Three patients with recurrent GBM were treated with IL13(E13Y)-zetakine CD8+ CTL targeting IL13Rα2. Patients received up to twelve local infusions at a maximum dose of 108 CAR-engineered T cells via a catheter/reservoir system. Results We demonstrate the feasibility of manufacturing sufficient numbers of autologous CTL clones expressing an IL13(E13Y)-zetakine CAR for redirected HLA-independent IL13Rα2-specific effector function for a cohort of patients diagnosed with GBM. Intracranial delivery of the IL13-zetakine+ CTL clones into the resection cavity of three patients with recurrent disease was well-tolerated, with manageable temporary CNS inflammation. Following infusion of IL13-zetakine+ CTLs, evidence for transient anti-glioma responses was observed in two of the patients. Analysis of tumor tissue from one patient before and after T cell therapy suggested reduced overall IL13Rα2 expression within the tumor following treatment. MRI analysis of another patient indicated an increase in tumor necrotic volume at the site of IL13-zetakine+ T cell administration. Conclusion These findings provide promising first-in-human clinical experience for intracranial administration of IL13Rα2-specific CAR T cells for the treatment of GBM, establishing a foundation on which future refinements of adoptive CAR T cell therapies can be applied.
Metastatic neuroblastoma is a poor-prognosis malignancy arising during childhood that overexpresses the L1-cell adhesion molecule (CD171). We have previously described a tumor L1-cell adhesion molecule-specific, single chain antibody-derived, chimeric antigen receptor designated CE7R for re-directing the antigen-specific effector functioning of cytolytic T lymphocytes. Here, we report on the feasibility of isolating, and the safety of infusing, autologous CD8(+) cytolytic T lymphocyte clones co-expressing CE7R and the selection-suicide expression enzyme HyTK in children with recurrent/refractory neuroblastoma. The cytolytic T lymphocyte products were derived from peripheral blood mononuclear cells that were subjected to polyclonal activation, plasmid vector electrotransfer, limiting dilution hygromycin selection, and expansion to numbers sufficient for adoptive transfer. In total, 12 infusions (nine at 10(8) cells/m(2), three at 10(9) cells/m(2)) were administered to six patients. No overt toxicities to tissues known to express L1-cell adhesion molecule (e.g., central nervous system, adrenal medulla, and sympathetic ganglia) were observed. The persistence of cytolytic T lymphocyte clones in the circulation, measured by vector-specific quantitative polymerase chain reaction, was short (1-7 days) in patients with bulky disease, but significantly longer (42 days) in a patient with a limited disease burden. This first-in-humans pilot study sets the stage for clinical trials employing adoptive transfer in the context of minimal residual disease.
Key Points CD123 CAR T cells specifically target CD123+ AML cells. AML patient-derived T cells can be genetically modified to lyse autologous tumor cells.
Monoclonal antibodies and T cells modi- IntroductionB-cell chronic lymphocytic leukemia (B-CLL) and mantle cell lymphoma (MCL) are common B-cell malignancies that respond to chemotherapy but are rarely cured. Allogeneic hematopoietic stem cell transplantation (HCT) enables a T cell-mediated graft-versus-leukemia (GVL) effect and induces durable remissions in a subset of patients with chemotherapy-refractory B-CLL and MCL, demonstrating that these malignancies are susceptible to recognition and elimination by T cells. 1,2 In a previous study, we identified tumor-reactive CD8 ϩ T cells directed against minor histocompatibility (H) and tumor-associated antigens (TAA) expressed by B-CLL in patients with sustained tumor regression after allogeneic HCT. 3 These results have encouraged the development of T cell-adoptive immunotherapy to augment the GVL effect after HCT. However, major challenges for therapy with ␣ ⌻-cell receptor (TCR)-bearing T cells include the need to identify antigens with restricted expression on malignant cells to avoid graft-versus-host disease, and the population distribution and requirement for human leukocyte antigen (HLA)-restriction for both minor H antigens and TAA. 4 An approach that could overcome these challenges and also enable T-cell therapy for B-CLL and MCL in the nontransplant setting is to genetically modify T cells to express a chimeric antigen receptor (CAR) that is specific for a cell surface protein expressed by malignant cells. CARs consists of a single-chain antibody fragment (scFv) that is derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb) linked to the TCR CD3 chain that mediates T-cell activation and cytotoxicity. 5 Costimulatory signals can also be provided through the CAR by fusing the costimulatory domain of CD28 or 4-1BB to the CD3 chain. 5,6 CARs are specific for cell surface molecules independent from HLA, thus overcoming the limitations of TCR-recognition including HLA-restriction and low levels of HLA-expression on tumor cells. B-cell lineage differentiation molecules such as CD19 and CD20 are retained on most B-cell tumors, and T cells modified with CD19-and CD20-specific CARs are currently being evaluated in clinical trials. 7,8 However, targeting B-cell lineage-specific antigens with immunotherapy has the disadvantage of eliminating normal mature B cells, which can increase the risk of infection. 9,10 Here, we evaluate a strategy to selectively eliminate malignant B cells without damaging the mature normal B-cell compartment by targeting the receptor tyrosine kinase-like orphan receptor 1 (ROR1). ROR1 was identified as a highly expressed gene in B-CLL by expression profiling and it has been shown that ROR1-protein is uniformly expressed on the cell surface of B-CLL. 11-14 The ROR1-gene encodes a 105-kDa protein with a Submitted May 3, 2010; accepted August 4, 2010. Prepublished online as Blood First Edition paper, August 11, 2010; DOI 10.1182 DOI 10. /blood-2010 An Inside Blood analysis of this article appears at...
Key Points TCM-derived CD19 CAR T–cell therapy is safe for treatment of poor-risk NHL patients undergoing autologous HSCT. Addition of a CD28 costimulatory domain to the CAR, plus changes to T-cell product manufacturing, resulted in improved T-cell expansion.
The success of adoptive therapy using chimeric antigen receptor (CAR)-expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (FcγRs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble FcγRs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid FcγR binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2Δ). These mutations reduced binding to soluble FcγRs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2Δ T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular FcγR interactions to improve T cell persistence and antitumor responses.
A key determinant of the therapeutic potency of adoptive T cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR) redirected anti-tumor CD8+ effector T cells derived from CD45RA−CD62L+ central memory (TCM) precursors engraft long-term and reconstitute functional memory following adoptive transfer. Here, we describe a clinical-scale, closed system, immunomagnetic selection method to isolate CD8+ TCM from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS™ device to first deplete CD14+, CD45RA+ and CD4+ cells from PBMC, and then to positively select CD62L+ cells. The average purity and yield of CD8+CD45RA−CD62L+ TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8+ TCM are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in IL-2/IL-15 over 3–6 weeks. The resulting CD8+ TCM-derived effectors (TE(CM)) are polyclonal, retain expression of CD62L and CD28, exhibit CAR redirected anti-tumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NSG mice. Adoptive atherapy using purified TCM cells are now the subject of an FDA authorized clinical trial for the treatment of CD19+ B-cell malignancies, and three clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.
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