A minority of T cells recognizing tumor-associated antigens presented in self-HLA can provoke antitumor reactivity.

Tumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient avidity to recognize naturally overexpressed self-antigens in the context of self-HLA can be found in the T-cell repertoire of healthy donors. Minor histocompatibility antigen (MiHA)-specific T cells were used as a model, as the influence of thymic selection on the T-cell repertoire directed against MiHA can be studied in both self (MiHApos donors) and non-self (MiHAneg donors) backgrounds. T-cell clones directed against the HLA*02:01-restricted MiHA HA-1H were isolated from HA-1Hneg/HLA-A*02:01pos and HA-1Hpos/HLA-A*02:01pos donors. Of the 16 unique HA-1H-specific T-cell clones, five T-cell clones derived from HA-1Hneg/HLA-A*02:01pos donors and one T-cell clone derived from an HA-1Hpos/HLA-A*02:01pos donor showed reactivity against HA-1Hpos target cells. In addition, in total, 663 T-cell clones (containing at least 91 unique clones expressing different T-cell receptors) directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, proteinase-3-VLQ, PRAME-VLD, and NY-eso-1-SLL were isolated from HLA-A*02:01pos donors. Only 3 PRAME-VLD-specific and one NY-eso-1-SLL-specific T-cell clone provoked interferon-γ production and/or cytolysis upon stimulation with HLA-A*02:01pos malignant cell lines (but not primary malignant samples) naturally overexpressing the TAA. These results show that self-HLA-restricted T cells specific for self-antigens such as MiHA in MiHApos donors and TAAs are present in peripheral blood of healthy individuals. However, clinical efficacy would require highly effective in vivo priming by peptide vaccination in the presence of proper adjuvants or in vitro expansion of the low numbers of self-antigen-specific T cells of sufficient avidity to recognize endogenously processed antigen.

The simultaneous isolation of multiple high and low frequent T-cell populations from donor peripheral blood mononuclear cells using the major histocompatibility complex I-Streptamer isolation technology.

BACKGROUND: Adoptive transfer of donor-derived T cells can be applied to improve immune reconstitution in immune-compromised patients after allogeneic stem cell transplantation. The separation of beneficial T cells from potentially harmful T cells can be achieved by using the major histocompatibility complex (MHC) I-Streptamer isolation technology, which has proven its feasibility for the fast and pure isolation of T-cell populations with a single specificity. We have analyzed the feasibility of the simultaneous isolation of multiple antigen-specific T-cell populations in one procedure by combining different MHC I-Streptamers. METHODS: First, the effect of combining different amounts of MHC I-Streptamers used in the isolation procedure on the isolation efficacy of target antigen-specific T cells and on the number of off-target co-isolated contaminating cells was assessed. The feasibility of this approach was demonstrated in large-scale validation procedures targeting both high and low frequent T-cell populations using the Good Manufacturing Practice (GMP)-compliant CliniMACS Plus device. RESULTS: T-cell products targeting up to 24 different T-cell populations could be isolated in one, simultaneous MHC I-Streptamer procedure, by adjusting the amount of MHC I- Streptamers per target antigen-specific T-cell population. Concurrently, the co-isolation of potentially harmful contaminating T cells remained below our safety limit. This technology allows the reproducible isolation of high and low frequent T-cell populations. However, the expected therapeutic relevance of direct clinical application without in vitro expansion of these low frequent T-cell populations is questionable. DISCUSSION: This study provides a feasible, fast and safe method for the generation of highly personalized MHC I-Streptamer isolated T-cell products for adoptive immunotherapy.

Antigen-specific TIL therapy for melanoma: A flexible platform for personalized cancer immunotherapy.

Tumor infiltrating lymphocyte (TIL) therapy has shown objective clinical response rates of 50% in stage IV melanoma patients in a number of clinical trials. Nevertheless, the majority of patients progress either directly upon therapy or after an initial period of tumor control. Recent data have shown that most TIL products that are used for therapy contain only low frequencies of T cells reactive against known melanoma-associated epitopes. Because of this, the development of a technology to create T-cell products that are enriched for reactivity against defined melanoma-associated antigens would seem valuable, both to evaluate the tumoricidal potential of T cells directed against different antigen classes and to potentially increase response rates. Here, we developed and validated a conditional MHC streptamer-based platform for the creation of TIL products with defined antigen reactivities. We have used this platform to successfully enrich both high-frequency (≥1%) and low-frequency (<1%) tumor-specific CD8(+) T-cell populations, and thereby created T-cell products with enhanced tumor recognition potential. Collectively, these data demonstrate that selection of antigen-specific T-cell populations can be used to create defined T-cell products for clinical use. This strategy thus forms a highly flexible platform for the development of antigen-specific cell products for personalized cancer immunotherapy.

Flow cytometry-based TCR-ligand Koff -rate assay for fast avidity screening of even very small antigen-specific T cell populations ex vivo.

High epitope-specific sensitivity of CD8(+) T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8(+) T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR re-expression experiments. Based on reversible MHCI multimer staining and koff -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8(+) T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8(+) T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand koff -rate values for poly- or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. © 2016 International Society for Advancement of Cytometry.

Lowest numbers of primary CD8(+) T cells can reconstitute protective immunity upon adoptive immunotherapy.

Patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT) are threatened by potentially lethal viral manifestations like cytomegalovirus (CMV) reactivation. Because the success of today's virostatic treatment is limited by side effects and resistance development, adoptive transfer of virus-specific memory T cells derived from the stem cell donor has been proposed as an alternative therapeutic strategy. In this context, dose minimization of adoptively transferred T cells might be warranted for the avoidance of graft-versus-host disease (GVHD), in particular in prophylactic settings after T-cell-depleting allo-HSCT protocols. To establish a lower limit for successful adoptive T-cell therapy, we conducted low-dose CD8(+) T-cell transfers in the well-established murine Listeria monocytogenes (L.m.) infection model. Major histocompatibility complex-Streptamer-enriched antigen-specific CD62L(hi) but not CD62L(lo) CD8(+) memory T cells proliferated, differentiated, and protected against L.m. infections after prophylactic application. Even progenies derived from a single CD62L(hi) L.m.-specific CD8(+) T cell could be protective against bacterial challenge. In analogy, low-dose transfers of Streptamer-enriched human CMV-specific CD8(+) T cells into allo-HSCT recipients led to strong pathogen-specific T-cell expansion in a compassionate-use setting. In summary, low-dose adoptive T-cell transfer (ACT) could be a promising strategy, particularly for prophylactic treatment of infectious complications after allo-HSCT.

Selection of adenovirus-specific and Epstein-Barr virus-specific T cells with major histocompatibility class I streptamers under Good Manufacturing Practice (GMP)-compliant conditions.

BACKGROUND AIMS: Despite antiviral drug therapies, human adenovirus (HAdV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections still contribute substantially to transplant-related death of patients after hematopoietic stem cell transplantation. Earlier clinical studies demonstrated successful adoptive transfer of magnetically selected CMV-specific T cells via removable, and thus Good Manufacturing Practice-compliant, major histocompatibility class I streptamers. Thus, the primary focus of the present study was the selection of HAdV-streptamer+ T cells, although in three experiments, EBV-streptamer+ T cells were also selected. METHODS: Cells from leukaphereses of healthy donors were prepared in large (1-6 × 10(9)) and small (25 × 10(6)) cell batches. Whereas the larger batch was directly labeled with streptamers to select HAdV- and/or EBV-specific T cells (large-scale), the smaller batch was used to generate in vitro virus-specific T-cell lines before streptamer labeling for streptamer selection (small-scale). Isolation of HAdV- and/or EBV-specific T cells was performed with the use of the CliniMACS device. RESULTS: The purity of HAdV- and EBV-streptamer+ T cells among CD3+ cells, obtained from large-scale selection, was up to 6.7% and 44%, respectively. If HAdV- and EBV-streptamers were applied simultaneously, the purity of antigen-specific T cells reached up to 50.7%. A further increase in purity reaching up to 98% was achieved by small-scale selection of HAdV-specific T cells. All final products fulfilled the microbiological and chemical release criteria. Interferon-γ-response indicating functional activity was seen in 6 of 9 HAdV and 2 of 3 EBV large-scale selections and in 2 of 3 HAdV small-scale selections. CONCLUSIONS: HAdV-streptamers were shown to be clinically feasible for few patients after the large-scale approach but for larger patient numbers if combined with EBV-streptamers or after the small-scale approach.

Clinical-scale isolation of 'minimally manipulated' cytomegalovirus-specific donor lymphocytes for the treatment of refractory cytomegalovirus disease.

BACKGROUND AIMS: Reactivation of cytomegalovirus (CMV) after hematopoietic stem cell transplantation remains a major cause of morbidity despite improved antiviral drug therapies. Selective restoration of CMV immunity by adoptive transfer of CMV-specific T cells is the only alternative approach that has been shown to be effective and non-toxic. We describe the results of clinical-scale isolations of CMV-specific donor lymphocytes with the use of a major histocompatibility (MHC) class I peptide streptamer-based isolation method that yields minimally manipulated cytotoxic T cells of high purity. METHODS: Enrichment of CMV-specific cytotoxic T lymphocytes (CTLs) was performed by labeling 1 × 10(10) leukocytes from a non-mobilized mononuclear cell (MNC) apheresis with MHC class I streptamers and magnetic beads. Thereafter, positively labeled CMV-specific CTLs were isolated through the use of CliniMACS (magnetic-activated cell sorting), and MHC streptamers were released through the use of d-biotin. The purity of enriched CMV-specific CTLs was determined on the basis of MHC streptamer staining and fluorescence-activated cell sorting. RESULTS: A total of 22 processes were performed with the use of five different MHC class I streptamers. The median frequency of CMV-specific CTLs in the starting apheresis product was 0.41% among CD3+ T cells. The isolation process yielded a total of 7.77 × 10(6) CMV-specific CTLs, with a median purity of 90.2%. Selection reagents were effectively removed from the final cell product; the CMV-specific CTLs displayed excellent viability and cytotoxicity and were stable for at least 72 h at 4°C after MNC collection. CONCLUSIONS: Clinical-scale isolation of "minimally manipulated" CMV-specific donor CTLs through the use of MHC class I streptamers is feasible and yields functional CTLs at clinically relevant dosages.

Miniaturized CAR knocked onto CD3ε extends TCR function with CAR specificity under control of endogenous TCR signaling cascade.

Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.

Activation-inducible CAR expression enables precise control over engineered CAR T cell function.

CAR T cell therapy is a rapidly growing area of oncological treatments having a potential of becoming standard care for multiple indications. Coincidently, CRISPR/Cas gene-editing technology is entering next-generation CAR T cell product manufacturing with the promise of more precise and more controllable cell modification methodology. The intersection of these medical and molecular advancements creates an opportunity for completely new ways of designing engineered cells to help overcome current limitations of cell therapy. In this manuscript we present proof-of-concept data for an engineered feedback loop. We manufactured activation-inducible CAR T cells with the help of CRISPR-mediated targeted integration. This new type of engineered T cells expresses the CAR gene dependent on their activation status. This artifice opens new possibilities to regulate CAR T cell function both in vitro and in vivo. We believe that such a physiological control system can be a powerful addition to the currently available toolbox of next-generation CAR constructs.

Next generation automated traceless cell chromatography platform for GMP-compliant cell isolation and activation.

Large-scale target cell isolation from patient blood preparations is one of the critical operations during drug product manufacturing for personalized cell therapy in immuno-oncology. Use of high-affinity murine antibody coated magnetic nanoparticles that remain on isolated cells is the current standard applied for this purpose. Here, we present the transformation of previously described technology - non-magnetic immunoaffinity column chromatography-based cell selection with reversible reagents into a new clinical-grade cell isolation platform called Automated Traceless Cell affinity chromatography (ATC). ATC is a fully closed and GMP-compliant cell selection and manufacturing system. Reversibility of reagents enables (sequential) positive cell selection, optionally in combination with depletion columns, enabling capture of highly specific cell subsets. Moreover, synergy with other Streptamer-based technologies allows novel uses beyond cell isolation including integrated and automated on-column target cell activation. In conclusion, ATC technology is an innovative as well as versatile platform to select, stimulate and modify cells for clinical manufacturing and downstream therapies.

Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal. STUDY DESIGN AND METHODS: Here, the novel streptamer technology was used at good manufacturing practice (GMP) level for adoptive transfer of CMV-specific T cells into acute leukemia patients with recurrent high CMV antigenemia after allogeneic stem cell transplantation. RESULTS: After a single transfusion, the frequency of CMV-specific CD8+CD45RA+CCR7- effector T cells increased dramatically from 0.0% to a maximum of 27.1% of all T cells. These T cells were clearly donor derived and did not stem from intrinsic reconstitution, as demonstrated by analysis of 1) donor chimerism through single-tandem repeats, 2) T-cell receptor excision circles, and 3) Vß-chain typing by polymerase chain reaction. Clinically, the specific T-cell transfer resulted in a persistent clearance of the CMV antigenemia, which allowed the patients to discontinue toxic antiviral drug therapy without further high-level reactivation of CMV, demonstrating the power of the streptamer technology. CONCLUSION: Taken together, the streptamer technology offers the advantage of selecting virus-specific CD8+ T cells at GMP level for adoptive T-cell transfer, thus inducing long-lasting specific CD8+ T-cell responses without increasing the risk for graft-versus-host disease.

Expamers: a new technology to control T cell activation.

T cell activation is a cornerstone in manufacturing of T cell-based therapies, and precise control over T cell activation is important in the development of the next generation T-cell based therapeutics. This need cannot be fulfilled by currently available methods for T cell stimulation, in particular not in a time dependent manner. Here, we describe a modular activation reagent called Expamers, which addresses these limitations. Expamers are versatile stimuli that are intended for research and clinical use. They are readily soluble and can be rapidly bound and removed from the cell surface, allowing nearly instantaneous initiation and termination of activation signal, respectively. Hence, Expamers enable precise regulation of T cell stimulation duration and provide promise of control over T cell profiles in future products. Expamers can be easily adopted to different T cell production formats and have the potential to increase efficacy of T cell immunotherapeutics.

Generation and infusion of multi-antigen-specific T cells to prevent complications early after T-cell depleted allogeneic stem cell transplantation-a phase I/II study.

Prophylactic infusion of selected donor T cells can be an effective method to restore specific immunity after T-cell-depleted allogeneic stem cell transplantation (TCD-alloSCT). In this phase I/II study, we aimed to reduce the risk of viral complications and disease relapses by administrating donor-derived CD8pos T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus antigens, tumor-associated antigens (TAA) and minor histocompatibility antigens (MiHA). Twenty-seven of thirty-six screened HLA-A*02:01pos patients and their CMVpos and/or EBVpos donors were included. Using MHC-I-Streptamers, 27 T-cell products were generated containing a median of 5.2 × 106 cells. Twenty-four products were administered without infusion-related complications at a median of 58 days post alloSCT. No patients developed graft-versus-host disease during follow-up. Five patients showed disease progression without coinciding expansion of TAA/MiHA-specific T cells. Eight patients experienced CMV- and/or EBV-reactivations. Four of these reactivations were clinically relevant requiring antiviral treatment, of which two progressed to viral disease. All resolved ultimately. In 2/4 patients with EBV-reactivations and 6/8 patients with CMV-reactivations, viral loads were followed by the expansion of donor-derived virus target-antigen-specific T cells. In conclusion, generation of multi-antigen-specific T-cell products was feasible, infusions were well tolerated and expansion of target-antigen-specific T cells coinciding viral reactivations was illustrated in the majority of patients.

Multimer staining of cytomegalovirus phosphoprotein 65-specific T cells for diagnosis and therapeutic purposes: a comparative study.

BACKGROUND: Cytomegalovirus (CMV) disease represents a serious complication after allogeneic peripheral blood stem cell (PBSC) transplantation. If possible, stem cell donors for transplantation are selected on the basis of their CMV serostatus. However, the cytomegalovirus-specific immune status can be further characterized by measuring CMV phosphoprotein 65-specific CD8(+) T cell frequencies using tetramers, pentamers, and streptamers. We therefore investigated the specificity and sensitivity of all 3 methods and compared the results to patient serostatus. METHODS: Twenty-three samples from CMV-seropositive healthy volunteers and 15 samples from CMV-seropositive patients before and after allogeneic PBSC transplantation were stained with tetramers, pentamers, or streptamers and analyzed by flow cytometry. RESULTS: Similar frequencies of CD8(+) and multimer(+) T cells could be measured by all 3 multimer technologies. The lowest background signals (< or =0.02%) were obtained using tetramer technology. Frequencies of 0.19%-2.48% of CMV phosphoprotein 65 495-503-specific CD8(+) T cells were detected in healthy volunteers. Antigen-specific T cells were detected in only 11 (48%) of 23 seropositive healthy volunteers. CMV antigenemia before day 100 after allogeneic PBSC transplantation occurred in 2 of 3 patients without any specific T cells. CONCLUSION: These findings demonstrate the power of multimer staining and a certain limitation of serologic testing to define appropriate donors for transplantation. Therefore, whenever possible, CMV-seropositive donors of transplants to seropositive recipients should be screened for their CD8(+) T cell frequency. All 3 multimer technologies can be used, yielding similar results. The streptamer technology additionally offers the advantage of selecting CMV phosphoprotein 65-specific CD8(+) T cells at the good manufacturing practice level for adoptive T cell transfer.

Reversible HLA multimers (Streptamers) for the isolation of human cytotoxic T lymphocytes functionally active against tumor- and virus-derived antigens.

The development of MHC/peptide multimers has facilitated the visualization and purification of antigen-specific T cells. However, the persistence of multimers leads to prolonged T cell receptor signaling and subsequently to altered T-cell function. We have recently developed a new type of MHC/peptide multimers, which can be dissociated from the T cell. Herein, we have generated and tested for the first time reversible HLA/peptide multimers, termed Streptamers, for the isolation of human T cells. The Streptamer technique demonstrates the specificity and sensitivity of conventional HLA/peptide tetramers with regards to the sorting of human T lymphocytes. This is shown for T cells directed against immunogenic peptides derived from viral and tumor-associated antigens. We show that antigen-specific cytotoxic T cells remain functionally active following Streptamer dissociation, whereas lytic function and proliferation of the T cells is impaired in the presence of conventional tetramers. These novel HLA/peptide Streptamer reagents allow the isolation of antigen-specific T cells with preserved function and, therefore, facilitate the development of adoptive T cell transfer regimens for the treatment of patients with cancer or infectious diseases.

Longitudinal analyses of leukemia-associated antigen-specific CD8+ T cells in patients after allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment approach for patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). Graft versus leukemia (GVL) effects, which are exerted by donor T cells directed against leukemic-associated antigens (LAAs), are considered to play a crucial role in disease eradication. Although the expansion of cytotoxic T lymphocytes (CTLs) specific for cytomegalovirus (CMV) in response to an infection has been shown in multiple studies, data on CTLs mediating GVL effects are limited. To evaluate a potential increase or decrease of T lymphocytes specific for LAAs in the setting of allogeneic HSCT, we monitored leukemia-specific CD8+ T cells throughout the first year after HSCT in 18 patients using streptamer technology. A broad panel of promising LAAs was selected: Wilms tumor protein, proteinase 3, receptor for hyaluronan acid-mediated motility, apoptosis regulator Bcl-2, survivin, nucleophosmin, and fibromodulin. T cells specifically directed against AML- or CLL-associated antigens were found at very low frequencies in peripheral blood. Substantial frequencies of LAA-specific T cells could not be measured at any time point by flow cytometry. In contrast, abundant CMV-pp65-specific T cells were detected in CMV-seropositive patient-recipient pairs and an increase prompted by CMV infection could be demonstrated. In conclusion, T lymphocytes with specificities for the aforementioned LAAs can only be detected in minimal quantities in the early phase after allogeneic HSCT.

Potent inhibition of angiogenesis by D,L-peptides derived from vascular endothelial growth factor receptor 2.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and plays a central role in angiogenesis and vasculogenesis. Therefore, VEGF and its receptors VEGFR-1 and VEGFR-2 are prime targets for anti-angiogenic intervention which is thought to be one of the most promising approaches in cancer therapy. Recently, we have discovered a VEGFR-2-derived peptide ((247)RTELNVGIDFNWEYP(261)) representing a potential binding site to VEGF. Using the spot synthesis technique, systematic D-amino acid substitutional analyses of this peptide were conducted and the resulting D,L-peptides inhibit VEGF binding to VEGFR-2 at half maximal concentration of 30 nM. The serum-stable D,L-peptides further inhibited autophosphorylation of the VEGFR-2 at nanomolar concentrations. Testing of the peptides in a spheroid-based angiogenesis assay demonstrated a potent anti-angiogenic effect in vitro. The rational design of potent and stable anti-angiogenic peptide inhibitors from their parent receptors provides a feasible route to develop novel leads for anti-angiogenic medicines.

Novel serial positive enrichment technology enables clinical multiparameter cell sorting.

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.