Hiroki Torikai

A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR.

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR(+) T cells to eliminate expression of the endogenous αβ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR(+)TCR(neg) T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

A Novel HLA-A*3303-Restricted Minor Histocompatibility Antigen Encoded by an Unconventional Open Reading Frame of Human TMSB4Y Gene

Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5′-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8+ CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8+ T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.

Sleeping beauty system to redirect T-cell specificity for human applications

The Sleeping Beauty (SB) transposon/transposase DNA plasmid system is used to genetically modify cells for long-term transgene expression. We adapted the SB system for human application and generated T cells expressing a chimeric antigen receptor (CAR) specific for CD19. Electrotransfer of CD19-specific SB DNA plasmids in peripheral blood mononuclear cells and propagation on CD19+ artificial antigen presenting cells was used to numerically expand CD3+ T cells expressing CAR. By day 28 of coculture, >90% of expanded CD3+ T cells expressed CAR. CAR+ T cells specifically killed CD19+ target cells and consisted of subsets expressing biomarkers consistent with central memory, effector memory, and effector phenotypes. CAR+ T cells contracted numerically in the absence of the CD19 antigen, did not express SB11 transposase, and maintained a polyclonal TCR V&agr; and TCR V&bgr; repertoire. Quantitative fluorescence in situ hybridization revealed that CAR+ T cells preserved the telomere length. Quantitative polymerase chain reaction and fluorescence in situ hybridization showed CAR transposon integrated on average once per T-cell genome. CAR+ T cells in peripheral blood can be detected by quantitative polymerase chain reaction at a sensitivity of 0.01%. These findings lay the groundwork as the basis of our first-in-human clinical trials of the nonviral SB system for the investigational treatment of CD19+ B-cell malignancies (currently under 3 INDs: 14193, 14577, and 14739).

HapMap scanning of novel human minor histocompatibility antigens

Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Identification of human minor histocompatibility antigens based on genetic association with highly parallel genotyping of pooled DNA

Minor histocompatibility (H) antigens are the molecular targets of allo-immunity responsible both for the development of antitumor effects and for graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, despite their potential clinical use, our knowledge of human minor H antigens is largely limited by the lack of efficient methods of their characterization. Here we report a robust and efficient method of minor H gene discovery that combines whole genome association scans (WGASs) with cytotoxic T-lymphocyte (CTL) assays, in which the genetic loci of minor H genes recognized by the CTL clones are precisely identified using pooled-DNA analysis of immortalized lymphoblastoid cell lines with/without susceptibility to those CTLs. Using this method, we have successfully mapped 2 loci: one previously characterized (HMSD encoding ACC-6), and one novel. The novel minor H antigen encoded by BCL2A1 was identified within a 26 kb linkage disequilibrium block on chromosome 15q25, which had been directly mapped by WGAS. The pool size required to identify these regions was no more than 100 individuals. Thus, once CTL clones are generated, this method should substantially facilitate discovery of minor H antigens applicable to targeted allo-immune therapies and also contribute to our understanding of human allo-immunity.

CD62L+ NKT cells have prolonged persistence and antitumor activity in vivo

Vα24-invariant natural killer T cells (NKTs) localize to tumors and have inherent antitumor properties, making them attractive chimeric antigen receptor (CAR) carriers for redirected cancer immunotherapy. However, clinical application of CAR-NKTs has been impeded, as mechanisms responsible for NKT expansion and the in vivo persistence of these cells are unknown. Here, we demonstrated that antigen-induced expansion of primary NKTs in vitro associates with the accumulation of a CD62L+ subset and exhaustion of CD62L- cells. Only CD62L+ NKTs survived and proliferated in response to secondary stimulation. When transferred to immune-deficient NSG mice, CD62L+ NKTs persisted 5 times longer than CD62L- NKTs. Moreover, CD62L+ cells transduced with a CD19-specific CAR achieved sustained tumor regression in a B cell lymphoma model. Proliferating CD62L+ cells downregulated or maintained CD62L expression when activated via T cell receptor alone or in combination with costimulatory receptors. We generated HLAnull K562 cell clones that were engineered to express CD1d and costimulatory ligands. Clone B-8-2 (HLAnullCD1dmedCD86high4-1BBLmedOX40Lhigh) induced the highest rates of NKT expansion and CD62L expression. B-8-2-expanded CAR-NKTs exhibited prolonged in vivo persistence and superior therapeutic activities in models of lymphoma and neuroblastoma. Therefore, we have identified CD62L as a marker of a distinct NKT subset endowed with high proliferative potential and have developed artificial antigen-presenting cells that generate CD62L-enriched NKTs for effective cancer immunotherapy.

The human Cathepsin H gene encodes two novel minor histocompatibility antigen epitopes restricted by HLA-A*3101 and -A*3303

Minor histocompatibility antigens (mHags) play crucial roles in the induction of graft versus host disease (GVHD) and/or graft versus leukaemia (GVL) effects following human leucocyte antigen (HLA)‐identical haematopoietic stem cell transplantation (HSCT). Using HLA‐A*3101‐ and ‐A*3303‐restricted cytotoxic T lymphocyte (CTL) clones generated from different post‐HSCT recipients, we identified two novel mHag epitopes encoded by the leader sequence of cathepsin H (CTSH) isoform a. The nonameric sequence ATLPLLCAR was defined as an HLA‐A*3101‐restricted epitope (CTSHR/A31), while a decameric peptide featuring a one N‐terminal amino acid extension, WATLPLLCAR, was presented by HLA‐A*3303 (CTSHR/A33). The immunogenicity of both epitopes was totally dependent on the polymorphic C‐terminal arginine residue and substitution with glycine completely abolished binding to the corresponding HLA molecules. Thus, the immunogenicity of this mHag is exerted by differential HLA binding capacity. CTSH is relatively ubiquitously expressed at protein levels, thus it may be involved in GVHD and anti‐leukaemic/tumour responses. Interestingly, however, CTL clones predominantly lysed targets of haematopoietic cell origin, which could not be explained in terms of the immunoproteasome system. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that CTSH‐encoded mHags could be targets for GVL effects.

Identification of an HLA-A24-restricted cytotoxic T lymphocyte epitope from human papillomavirus type-16 E6: The combined effects of bortezomib and interferon-γ on the presentation of a cryptic epitope

About 50% of cervical cancers are associated with human papillomavirus type 16 (HPV‐16), and since the HPV‐16 E6 and E7 oncoproteins are constitutively expressed in the tumor cells, they are attractive targets for cytotoxic T lymphocyte (CTL)‐mediated immunotherapy. Nevertheless, only a limited number of HPV‐16 E6 epitopes have been identified to date. Using reverse immunological methods, we have generated a CTL clone against the HPV‐16 E649–57 epitope restricted by HLA‐A*2402, which is the most common allele in Japan and relatively frequent worldwide, capable of lysing 293T cells transduced with HLA‐A*2402 and HPV‐16 E6. Although it was unable to recognize the SiHa cervical cancer cell line positive for HPV‐16 and HLA‐A*2402, the cells became susceptible to lysis when transduced with E6‐E7 genes, which was unexpectedly offset by pretreatment with interferon (IFN)‐γ alone. Interestingly, however, combined pretreatment with a proteasome inhibitor, bortezomib and IFN‐γ fully restored CTL‐mediated lysis of the original SiHa cells. Furthermore, such intervention of 2 of 4 other cervical cancer cell lines expressing HPV‐16 E6 and HLA‐A*2402 was found to induce IFN‐γ production by specific CTLs. Tetramer analysis further revealed that induction of E649–57‐specific T cells was possible in 5 of 7 patients with HPV‐16‐positive high grade cervical intraepithelial neoplasia or cervical cancer by in vitro stimulation with E649–57 peptide. Thus, these findings together indicate that E649–57 is a candidate epitope for immunotherapy and immunological monitoring of such patients.

The HLA-A*0201-restricted minor histocompatibility antigen HA-1H peptide can also be presented by another HLA-A2 subtype, A*0206.

HA-1H is one of the most attractive minor histocompatibility antigens (mHA) as a target for immunotherapy of hematopoietic malignancies, but HLA-A*0201 and HLA-B60 molecules capable of presenting HA-1H-derived peptides are less common in eastern Asian populations when compared with Caucasian populations. Therefore, an attempt was made to search for novel epitopes presented by HLA alleles other than those previously reported by generating CTL lines from patients undergoing HLA-identical, HA-1 disparate hematopoietic stem cell transplantation (hematopoietic SCT) by stimulation with a 29-mer HA-1H peptide spanning a central polymorphic histidine (His). Two CTL clones established were found to be restricted by HLA-A*0206, which is the second or third most common HLA-A2 subtype worldwide. Epitope mapping revealed that the clones recognized the same nonameric peptide as A*0201-restricted HA-1H, VLHDDLLEA. This epitope was unexpected, since it does not contain any preferred anchor motifs for HLA-A*0206. However, an HLA peptide binding assay revealed stronger binding of this peptide to A*0206 than to A*0201. Interestingly, HLA-A*0206-restricted CTL clones could lyse both HLA-A*0206+ and HLA-A*0201+ targets (including leukemic blasts) that express HA-1H peptide endogenously, whereas an HLA-A*0201-restricted, HA-1H-specific CTL clone failed to lyse HLA-A*0206+ targets. This finding will expand the patient population who can benefit from HA-1H-based immunotherapy.

Redirecting Specificity of T cells Using the Sleeping Beauty System to Express Chimeric Antigen Receptors by Mix-and-Matching of VL and VH Domains Targeting CD123+ Tumors

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR’s in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.