Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.

One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs.

Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Vaccine-induced effector-memory CD8+ T cell responses predict therapeutic efficacy against tumors.

CD8(+) T cells have the potential to attack and eradicate cancer cells. The efficacy of therapeutic vaccines against cancer, however, lacks defined immune correlates of tumor eradication after (therapeutic) vaccination based on features of Ag-specific T cell responses. In this study, we examined CD8(+) T cell responses elicited by various peptide and TLR agonist-based vaccine formulations in nontumor settings and show that the formation of CD62L(-)KLRG1(+) effector-memory CD8(+) T cells producing the effector cytokines IFN-γ and TNF predicts the degree of therapeutic efficacy of these vaccines against established s.c. tumors. Thus, characteristics of vaccine-induced CD8(+) T cell responses instill a predictive determinant for the efficacy of vaccines during tumor therapy.

Hypomethylating drugs convert HA-1-negative solid tumors into targets for stem cell-based immunotherapy.

Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1(neg)) and cannot be targeted by HA-1-specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2'-deoxycytidine induced HA-1 expression only in HA-1(neg) tumor cells and sensitized them for recognition by HA-1-specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1(neg) tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SCT.

Adult and cord blood T cells can acquire HA-1 specificity through HA-1 T-cell receptor gene transfer.

BACKGROUND AND OBJECTIVES: Minor histocompatibility antigen (mHag)-specific graft-versus-leukemia reactivities are observed following unselected donor lymphocyte infusion for the treatment of relapse after HLA-matched mHag-mismatched stem cell transplantation (SCT). Adoptive transfer of donor-derived ex vivo-generated HA-1-specific oligoclonal T cells or HA-1 peptide patient vaccination are currently being explored as curative tools for stem cell based immunotherapy of hematologic malignancies. Another treatment modality to eradicate residual leukemic cells after SCT is the transfer of the HA-1 hematopoietic-specific T-cell receptor (TCR) into cells from the stem cell donor. This strategy would be particularly useful in case of relapse after cord blood transplantation (CBT) and is explored in this study. DESIGN AND METHODS: HLA-A2(neg) adult peripheral blood and cord blood mononuclear cells were transduced with the genes encoding the HA-1 alpha and beta TCR chains derived from established HA-1 specific cytotoxic T lymphocyte clones. RESULTS: The T cells transduced with HA-1 TCR alpha beta showed consistent marker gene expression, but low staining with HLA-A2/HA-1 tetrameric complexes. They did, however, show hematopoietic-restricted cytolytic activity against HLA-A2(pos)/HA-1(pos) target cells, including leukemic cells. INTERPRETATION AND CONCLUSIONS: The low level of HA-1-specific tetramer staining of HA-1 TCR alpha beta transduced T cells may be caused by hybrid TCR formation of the transferred TCRalpha and beta chains with endogenous TCR alpha and beta chains. This may cause unwanted alloreactivity and requires attention. The HA-1 TCR alpha beta transduced T cells show that the HA-1 TCR can be functionally transferred into donor mononuclear cells, which can be exploited in immunotherapeutic settings of SCT and CBT for hematologic malignancies.

Detection of microchimerism by minor histocompatibility antigen HA-1 allele-specific nested polymerase chain reaction.

Minor histocompatibility antigens (mHags) can induce T-cell reactivities with important consequences for the graft-versus-leukemia effect and the development of graft-versus-host disease in HLA-matched stem cell transplantation settings. Recently, mHag-specific T cells were also demonstrated in multiparous woman and in solid organ transplant recipients. Microchimeric cells have been detected in the latter settings. To study whether microchimerism is instrumental in the induction and/or maintenance of mHag T cells, we developed an HA-1 allele-specific nested polymerase chain reaction. To optimize and validate the reliability of this method at different levels of microchimerism, serial dilutions of HA-1(H) cells titrated into HA-1(R) cells were tested. We demonstrated that the HA-1(H) allele can be reliably and consistently detected at concentrations as low as 1:10(5) without losing specificity. The developed HA-1-specific nested polymerase chain reaction is an important tool that facilitates the detection of HA-1 microchimerism in various clinical specimens and that promotes investigation of the effects of microchimerism on induction of mHag-specific T cells in the various settings of immunization.

Postgenomic approach to identify novel Mycobacterium leprae antigens with potential to improve immunodiagnosis of infection.

Early detection of Mycobacterium leprae infection is considered an important component of strategies aiming at reducing transmission of infection, but currently available diagnostic tools often lack sufficient sensitivity and specificity to reach this goal. Recent comparative genomics have revealed the presence of 165 M. leprae genes with no homologue in M. tuberculosis. We selected 17 of these genes for further study. All 17 genes were found to be expressed at the mRNA level in M. leprae from infected mice and from a multibacillary leprosy patient. Additional comparative genomic analyses of all currently available mycobacterial genome databases confirmed 12 candidate genes to be unique to M. leprae, whereas 5 genes had homologues in mycobacteria other than M. tuberculosis. Evaluation of the immunogenicity of all 17 recombinant proteins in PBMC from 127 Brazilians showed that five antigens (ML0576, ML1989, ML1990, ML2283, and ML2567) induced significant gamma interferon levels in paucibacillary leprosy patients, reactional leprosy patients, and exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. Importantly, among exposed healthy controls 71% had no detectable immunoglobulin M antibodies to the M. leprae-specific PGL-I but responded to one or more M. leprae antigen(s). Collectively, the M. leprae proteins identified are expressed at the transcriptome level and can efficiently activate T cells of M. leprae-exposed individuals. These proteins may provide new tools to develop tests for specific diagnosis of M. leprae infection and may enhance our understanding of leprosy and its transmission.

Minor histocompatibility antigens--big in tumour therapy.

Technical advances combined with the deciphering of the human genome have facilitated the identification of the molecular nature of human minor histocompatibility (H) antigens. To date, it is believed that minor H antigens result from just any polymorphic protein, regardless of their functional properties. A closer look at the first series of autosomally encoded human minor H proteins reveals a striking functional relationship. Here, we propose that T cells generated after HLA-identical stem cell transplantation (SCT) for malignancies are likely to be directed towards peptides derived from minor H proteins involved in tumourigenesis. This novel insight has important consequences in the search for, and the use of, minor H antigens as immunotherapeutics in stem-cell-based immunotherapy of haematological malignancies and solid tumours.

Identification and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis.

In this paper we describe identification and characterization of Mycobacterium leprae ESAT-6 (L-ESAT-6), the homologue of M. tuberculosis ESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed to M. tuberculosis and M. leprae. Moreover, leprosy patients who were not responsive to M. leprae failed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potent M. leprae antigen that stimulates T-cell-dependent gamma interferon production in a large proportion of individuals exposed to M. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic.