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Supplementary Figure 5 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 4 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 2 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 7 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 3 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 6 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Supplementary Figure 1 from Tumor-Specific CD8+ T Cells Expressing Interleukin-12 Eradicate Established Cancers in Lymphodepleted Hosts

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future., Graphical abstract, Schaser and colleagues found that lentiviral vectors encoding B cell-specific CARs display the respective CAR on the particle surface. The LV-displayed CARs retained their antigen-binding potential and specifically promoted transduction of malignant B cells. These findings provide a potential explanation for the emergence of CAR-expressing malignant cells in B-ALL patients.

The potential of adoptive cell therapy can be extended when combined with genome editing. However, variation in the quality of the starting material and the different manufacturing steps are associated with production failure and product contamination. Here, we present an automated T cell engineering process to produce off-the-shelf chimeric antigen receptor (CAR) T cells on an extended CliniMACS Prodigy platform containing an in-line electroporation unit. This setup was used to combine lentiviral delivery of a CD19-targeting CAR with transfer of mRNA encoding a TRAC locus-targeting transcription activator-like effector nuclease (TALEN). In three runs at clinical scale, the T cell receptor (TCR) alpha chain encoding TRAC locus was disrupted in >35% of cells with high cell viability (>90%) and no detectable off-target activity. A final negative selection step allowed the generation of TCRα/β-free CAR T cells with >99.5% purity. These CAR T cells proliferated well, maintained a T cell memory phenotype, eliminated CD19-positive tumor cells, and released the expected cytokines when exposed to B cell leukemia cells. In conclusion, we established an automated, good manufacturing practice (GMP)-compliant process that integrates lentiviral transduction with electroporation of TALEN mRNA to produce functional TCRα/β-free CAR19 T cells at clinical scale., Graphical Abstract, The potential of CAR T cell immunotherapy can likely be extended when combined with genome editing. Cathomen and colleagues developed a GMP-compliant, automated process that integrates lentiviral CAR delivery with TRAC editing at clinical scale. Such protocols will facilitate the production of off-the-shelf products and their successful clinical application.

Simple Summary Chimeric antigen receptor (CAR) T cell therapy can be associated with substantial side effects primarily due to intense immune activation following treatment, or target antigen recognition on off-tumor tissue. Consequently, temporal and tunable control of CAR T cell activity is of major importance for the clinical translation of innovative CAR designs. This work demonstrates the transcriptional regulation of an anti-CD20 CAR in primary T cells using a drug inducible zinc finger-based transcription factor. The switch system enables titratable induction of CAR expression and CAR T cell effector function with the clinically relevant inducer drug tamoxifen and its metabolites both in vitro and in vivo, whereby CAR activity is strictly dependent on the presence of the inducer drug. The results obtained can readily be transferred to other CARs for which an improved control of expression is required. Abstract Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Purpose The aim of this study was to perform, from the patient’s point of view, a nationwide assessment of nuclear medicine practices regarding diabetic management before 18F-fluorodeoxyglucose (FDG) PET/CT for oncologic indications. Methods This prospective observational study was exempt from institutional review board oversight. Sixty-five nuclear medicine scheduling lines (33 academic, 32 private practice, 12-17 in each of the five US regions) were called using a prewritten script under the guise of a nonexpert patient’s family member about scheduling a patient with diabetes with “cancer” for FDG PET/CT. Each center was called three times on three different days. The following data were collected: (1) blood glucose threshold for rescheduling an examination, (2) when or if to stop various medications, (3) fasting requirements, and (4) time-of-day scheduling preferences. Withheld information was not specifically requested. Descriptive statistics were calculated. Results There were 195 phone calls (mean duration, 2.9 min; range, 2-6 min). Relevant information was often withheld; withholding rates were as follows: blood glucose threshold, 71% (138 of 195); short-acting insulin instructions, 30% (59 of 195); long-acting insulin instructions, 99% (193 of 195); metformin instructions, 88% (179 of 195); fasting duration, 37% (72 of 195); and time-of-day scheduling preference, 91% (177 of 195). Mean provided data were as follows: blood glucose threshold, 195 mg/dL (range, 150-210 mg/dL); short-acting insulin withholding, 4.9 hours (range, 4-8 hours); long-acting insulin withholding, 12 hours (range, 12-24 hours); fasting duration, 5 hours (range, 4-8 hours); and preferred examination time, 91% (177 of 195). When specified (n = 18), morning scheduling was preferred (8% [15 of 195] versus 2% [3 of 195]). Conclusions Diabetes-specific information is commonly withheld by nuclear medicine call centers throughout the United States when discussing oncologic FDG PET/CT despite local and national policies indicating its importance.

BackgroundThere is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition.MethodsWe used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR.ResultsWe defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns.ConclusionsWe report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules., There is an unmet clinical need to identify therapeutic options for the treatment of pancreatic cancer (PDAC). Here the authors present a systematic screening approach for the identification of potential PDAC cell surface target candidates for CAR-T cell based immunotherapy, followed by their functional validation in preclinical models.

Simple Summary Metastatic disease remains one of the biggest challenges for tumor therapy. The aim of our study was the preclinical evaluation of adapter chimeric antigen receptor (AdCAR)-engineered NK-92 cell efficacy as a possible treatment strategy for various types of bone metastatic cancers. We confirmed that AdCAR NK-92 cells successfully induces tumor cell lysis in bone metastasis cell lines derived from mammary, renal cell and colorectal carcinoma as well as melanoma in a specific and controllable manner, thus, establishing a potent cellular product with universal applicability and quick clinical translation potential for the treatment of solid tumors, including metastases. Abstract Background: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed. Methods: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models. Conclusions: AdCAR NK-92 cells may provide an interesting and promising “off-the-shelf” cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1–dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

Background Adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has emerged as a promising treatment modality for a broad range of cancers highlighted by the approval of Kymriah™ and Yescarta™ for the treatment of B cell malignancies. However, lack of control of CAR T cell function and consequent excessive inflammation in patients can result in severe side effects especially when targeting tumor-associated rather than tumor-specific antigens. Thus, temporal and tunable control of CAR activity is of major importance for the clinical translation of innovative CAR designs. While the activation of suicide switches results in the apoptotic elimination of the transferred cells, other strategies, e.g. anti-tag CARs or small molecule-gated CARs, enable the reversible control of CAR-mediated function at the protein level but are restricted to a particular CAR design. Focusing on the control of expression rather than CAR signaling, transcriptional regulators represent a versatile tool facilitating a wide range of CAR T cell applications. Materials and Methods To maintain control over the infused CAR T cell product and mitigate risks for the patient, we describe here the development of an inducible switch system for the transcriptional regulation of transgene expression in primary, human T cells. Chemically regulated synthetic transcription factors composed of a zinc finger DNA-binding domain, an inducible control domain and a transcription activation domain were designed, screened for functionality, and evaluated in T cells regarding their potential to control CAR expression both in vitro and in vivo. Results By screening, we identified a synthetic transcription factor, which shows high transcriptional output in T cells in the presence of a clinically relevant inducer drug and absence of background activity in the non-induced state. Using this system we were able to control the expression of a CAR recognizing the CD20 antigen present on B cells and B cell leukemic blasts. The addition of the inducer drug resulted in rapid expression of the anti-CD20 CAR on the T cell surface. Moreover, inducible anti-CD20 CAR T cells executed cytolytic activity against CD20 positive target cells and secreted cytokines upon stimulation in vitro. Effectivity in co-cultures was thereby comparable to T cells expressing the anti-CD20 CAR under a conventional constitutive promoter. Furthermore, we could fine-tune CAR activity by titrating the inducer concentration. By defining the time-point of induction, modulation of the onset of therapy was achieved. Upon inducer drug discontinuation, inducible CD20 CAR T cells lost CAR expression and concurrently all CAR-related functions, indicating that the ‘on’ and ‘off’ status can be tightly controlled by the administration of the drug. After pausing of CAR T cell-mediated activity, we could re-induce CAR expression suggesting complete reversibility of effector function. Finally, we were able to show that inducible CD20 CAR T cells mediate a significant, strictly inducer-dependent antitumor activity in a well-established mouse model of B cell lymphoma. Conclusions The zinc-finger-based transcriptional control system investigated in this study provides small molecule-inducible control over a therapeutically relevant anti-CD20 CAR in primary T cells in a time- and dose-dependent manner. The tight regulation of CAR expression will pave the way for safer cellular therapies. Disclosure Information B. Kotter: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. N. Werchau: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. W. Krueger: A. Employment (full or part-time); Significant; Lentigen Technology Inc. A. Roy: A. Employment (full or part-time); Significant; Lentigen Technology Inc. J. Mittelstaet: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG. A. Kaiser: A. Employment (full or part-time); Significant; Miltenyi Biotec B.V. & Co. KG.

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) has shown remarkable clinical efficacy against advanced B cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+ and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B and carcinoma cells. Our results indicate that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only interact at first with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively infiltrate the center of tumor cell regions. The analysis of this two-step entry process shows that activated CAR T cells trigger the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The blockade of ICAM-1/LFA-1 interactions prevents CAR T cell recruitment into tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T cell entry into tumor islets is of significance for improving CAR T strategy in solid tumors.

The purpose of this study was to determine whether routine pelvic imaging is necessary during postoperative surveillance of pathologic T2-T4 renal cell carcinoma after nephrectomy for curative intent.A retrospective single-institution cohort study with 603 subjects undergoing partial or radical nephrectomy of T2-T4 renal cell carcinoma with curative intent was conducted from January 1, 2000, through December 31, 2015. Clinical and imaging (CT or MRI) follow-up findings were evaluated in a prospectively maintained registry to determine the timing and location of recurrent and metastatic disease. The primary outcome was the proportion of subjects with positive or equivocal findings in the pelvis and negative findings in the chest and abdomen. Binomial CIs were calculated and compared with a prespecified minimum detection threshold of 5%.The T category distribution was as follows: T2 (28.9% [174/603]), T3 (70.3% [424/603]), and T4 (0.8% [5/603]). Most (81.8% [493/603]) of the patients underwent radical nephrectomy, and 27.0% (163/603) had recurrence or metastasis (mean time to first recurrence, 600 ± 695 days). Pelvic imaging findings were negative in 97.0% (585/603) of cases. Four subjects (0.7% [95% CI, 0.2-1.7%]) had isolated positive findings in the pelvis (p0.0001 vs the 5% threshold). Two (0.3% overall [95% CI, 0.04-1.1%]) of these positive findings were in subjects who did not have symptoms.Routine pelvic imaging of patients undergoing surveillance for asymptomatic T2-T4 renal cell carcinoma after nephrectomy performed with curative intent has minimal value and probably should not be performed.

Relapsed/refractory B-precursor acute lymphoblastic leukemia (pre-B ALL) remains a major therapeutic challenge. Chimeric antigen receptor (CAR) T cells are promising treatment options. Central memory T cells (Tcm) and stem cell-like memory T cells (Tscm) are known to promote sustained proliferation and persistence after T-cell therapy, constituting essential preconditions for treatment efficacy. Therefore, we set up a protocol for anti-CD19 CAR T-cell generation aiming at high Tcm/Tscm numbers. 100 ml peripheral blood from pediatric pre-B ALL patients was processed including CD4+/CD8+-separation, T-cell activation with modified anti-CD3/-CD28 reagents and transduction with a 4-1BB-based second generation CAR lentiviral vector. The process was performed on a closed, automated device requiring additional manual/open steps under clean room conditions. The clinical situation of these critically ill and refractory patients with leukemia leads to inconsistent cellular compositions at start of the procedure including high blast counts and low T-cell numbers with exhausted phenotype. Nevertheless, a robust T-cell product was achieved (mean CD4+ = 50%, CD8+ = 39%, transduction = 27%, Tcm = 50%, Tscm = 46%). Strong proliferative potential (up to > 100-fold), specific cytotoxicity and low expression of co-inhibitory molecules were documented. CAR T cells significantly released TH1 cytokines IFN-γ, TNF-α and IL-2 upon target-recognition. In conclusion, partly automated GMP-generation of CAR T cells from critically small blood samples was feasible with a new stimulation protocol that leads to high functionality and expansion potential, balanced CD4/CD8 ratios and a conversion to a Tcm/Tscm phenotype.

International audience; Cell and gene therapies are finally becoming viable patient treatment options, with both T cell- and hematopoietic stem cell (HSC)-based therapies being approved to market in Europe. However, these therapies, which involve the use of viral vector to modify the target cells, are expensive and there is an urgent need to reduce manufacturing costs. One major cost factor is the viral vector production itself, therefore improving the gene modification efficiency could significantly reduce the amount of vector required per patient. This study describes the use of a transduction enhancing peptide, Vectofusin-1((R)), to improve the transduction efficiency of primary target cells using lentiviral and gammaretroviral vectors (LV and RV) pseudotyped with a variety of envelope proteins. Using Vectofusin-1 in combination with LV pseudotyped with viral glycoproteins derived from baboon endogenous retrovirus, feline endogenous virus (RD114), and measles virus (MV), a strongly improved transduction of HSCs, B cells and T cells, even when cultivated under low stimulation conditions, could be observed. The formation of Vectofusin-1 complexes with MV-LV retargeted to CD20 did not alter the selectivity in mixed cell culture populations, emphasizing the precision of this targeting technology. Functional, ErbB2-specific chimeric antigen receptor-expressing T cells could be generated using a gibbon ape leukemia virus (GALV)-pseudotyped RV. Using a variety of viral vectors and target cells, Vectofusin-1 performed in a comparable manner to the traditionally used surface-bound recombinant fibronectin. As Vectofusin-1 is a soluble peptide, it was possible to easily transfer the T cell transduction method to an automated closed manufacturing platform, where proof of concept studies demonstrated efficient genetic modification of T cells with GALV-RV and RD114-RV and the subsequent expansion of mainly central memory T cells to a clinically relevant dose.

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

Gadolinium has been widely used as the contrast agent of choice for magnetic resonance imaging (MRI). However, gadolinium administration is not always desired due to its inherent enhancement properties and potential side effects (nephrogenic systemic fibrosis). This article reviews gadolinium alternatives, iron-, and manganese- based agents, and their current clinical usage for contrast-enhanced MRI examinations.

In recent years, CAR T cell mediated immunotherapy has shown clinical efficacy in the fight against cancer. Increased research efforts to address several challenges associated with CAR T cell therapy for solid tumor treatment for instance, such as poor efficacy and unwanted side effects, are required to progress current approaches. Fast, efficient and flexible CAR T workflows can offer the opportunity to progress such research. At Miltenyi Biotec we have established an optimized CAR T cell research workflow, combining cell isolation, culture and analysis tailored for CAR T cell generation that can be transferred into clinical setting. Using our StraightFrom® MicroBeads, the fundamental step of isolating high quality T cells as starting material to generate functional CAR T cells, can be performed directly from whole blood, buffy coat and blood products without density gradient centrifugation. Thereby, experimental procedure is accelerated and allows for automation on platforms such as the MultiMACS™ Cell24 Separator Plus and the MultiMACS™ X, whilst maintaining the quality, functionality and downstream compatibility of the isolated T cells, resulting in an ideal starting population for the generation of fully functional CAR T cells. In conclusion, our innovative tools can reduce experimental time and increase the reproducibility and quality of data obtained in your CAR T cell research efforts in immuno-oncology and immunotherapy.

Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor in adults. The current standard-of-care treatment, which includes surgical resection, radiation, and chemotherapy, extends the survival of patients to merely 14.6 months. GBM is virtually incurable due to its heterogeneity, which is represented by tumor cells with different patterns of gene expression (proneural, neural, classical, and mesenchymal GBM), the presence of GBM cancer stem (like) cells (CSC), and immune evasion. Thus, new therapeutic strategies directed at eliminating GBM CSC in particular are urgently needed. Utilizing fresh patient material from surgical resections, GBM CSC were successfully enriched and cultivated in serum-free medium and subsequently differentiated by switching to medium containing FBS. The gene expression pattern of both, GBM CSC and differentiated cells, was characterized. Furthermore, a screening platform was tailored for flow-cytometric characterization of patient-specific antigen expression of all newly established glioblastoma cell lines. In this work we present a promising immunotherapeutic approach towards eradication of GBM CSC by combining the universal targeting and controllability of our recently developed adapter chimeric antigen receptor (AdCAR) system with the “off-the-shelf” properties of the continuously expandable and well-characterized NK cell line NK-92. The AdCAR system is based on the unique properties of a novel scFv targeting a “neo”-epitope-like structure derived from the endogenous vitamin biotin, which can be linked to monoclonal antibodies (mAb). Utilizing these biotinylated mAb as adapter molecules, the system allows precise quantitative (on-/off switch) as well as qualitative (change and combination of target antigen) regulation of immune cell function. AdCAR-transduced NK-92 cells demonstrated significant target cell lysis, which, importantly, is largely independent from activation of endogenously expressed NK receptors and the presence of their ligands on target cells but mainly mediated by AdCAR activation through binding to the respective tumor antigen. In the presence of the respective antigen, first and foremost biotinylated antibodies directed at CD9, CD146, CD276, and EGFR were capable of inducing significant AdCAR NK-92-mediated lysis of GBM cells after 2 hours. Interestingly, due to overexpression of various target antigens on GBM cancer stem cells, CSC lysis was noticeably higher as compared to AdCAR-mediated lysis of differentiated glioblastoma cells. In conclusion, we have successfully generated a CAR-modified NK cell line, AdCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens, allowing universal and tunable targeting of glioblastoma cells. Most importantly, AdCAR NK-92 cells provide a promising strategy to eradicate GBM cancer stem (like) cells. Citation Format: Stefan Grote, Chun-Ho Chan, Caroline Baden, Stephan M. Huber, Franziska Eckert, Joerg Mittelstaet, Andrew Kaiser, Christian Seitz, Patrick Schlegel, Rupert Handgretinger, Sabine Schleicher. Universal adapter CAR-engineered NK-92 cells target patient-derived glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B70.

The aim of this study was to perform, from the patient's point of view, a nationwide assessment of nuclear medicine practices regarding diabetic management beforeThis prospective observational study was exempt from institutional review board oversight. Sixty-five nuclear medicine scheduling lines (33 academic, 32 private practice, 12-17 in each of the five US regions) were called using a prewritten script under the guise of a nonexpert patient's family member about scheduling a patient with diabetes with "cancer" for FDG PET/CT. Each center was called three times on three different days. The following data were collected: (1) blood glucose threshold for rescheduling an examination, (2) when or if to stop various medications, (3) fasting requirements, and (4) time-of-day scheduling preferences. Withheld information was not specifically requested. Descriptive statistics were calculated.There were 195 phone calls (mean duration, 2.9 min; range, 2-6 min). Relevant information was often withheld; withholding rates were as follows: blood glucose threshold, 71% (138 of 195); short-acting insulin instructions, 30% (59 of 195); long-acting insulin instructions, 99% (193 of 195); metformin instructions, 88% (179 of 195); fasting duration, 37% (72 of 195); and time-of-day scheduling preference, 91% (177 of 195). Mean provided data were as follows: blood glucose threshold, 195 mg/dL (range, 150-210 mg/dL); short-acting insulin withholding, 4.9 hours (range, 4-8 hours); long-acting insulin withholding, 12 hours (range, 12-24 hours); fasting duration, 5 hours (range, 4-8 hours); and preferred examination time, 91% (177 of 195). When specified (n = 18), morning scheduling was preferred (8% [15 of 195] versus 2% [3 of 195]).Diabetes-specific information is commonly withheld by nuclear medicine call centers throughout the United States when discussing oncologic FDG PET/CT despite local and national policies indicating its importance.

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process.

Targeted therapy with sunitinib, pazopanib or everolimus has improved treatment outcome for patients with metastatic renal cell carcinoma patients (RCC). However, despite considerable efforts in sequential or combined modalities, durable remissions are rare. Immunotherapy like cytokine therapy with interleukin-2, T cell checkpoint blockade or adoptive T cell therapies can achieve long-term benefit and even cure. This raises the question of whether combining targeted therapy with immunotherapy could also be an effective treatment option for RCC patients. Sunitinib, one of the most frequently administered therapeutics in RCC patients has been implicated in impairing T cell activation and proliferation in vitro. In this work, we addressed whether this notion holds true for expansion of tumor-infiltrating lymphocytes (TILs) in sunitinib-treated patients. We compared resected primary RCC tumor material of patients pretreated with sunitinib with resection specimen from sunitinib-naïve patients. We found improved TIL expansion from sunitinib-pretreated tumor digests. These TIL products contained more PD-1 expressing TIL, while the regulatory T cell infiltration was not altered. The improved TIL expansion was associated with reduced intratumoral myeloid-derived suppressor cell (MDSC) content. Depletion of MDSCs from sunitinib-naïve RCC tissue-digest improved TIL expansion, proving the functional relevance of the MDSC alteration by sunitinib. Our in vivo results do not support previous in vitro observations of sunitinib inhibiting T cell function, but do provide a possible rationale for the combination of sunitinib with immunotherapy.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Background Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated. Methods Antigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice. Results Tandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs. Conclusion Tandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0246-1) contains supplementary material, which is available to authorized users.

Adoptive immunotherapy for solid tumors relies on infusing large numbers of T cells to mediate successful antitumor responses in patients. While long-term rapid-expansion protocols (REPs) produce sufficient numbers of CD8+ T cells for treatment, they also cause decline in the cell's therapeutic fitness. In contrast, we discovered that IL-17-producing CD4+ T cells (Th17 cells) do not require REPs to expand 5,000-fold over 3 weeks. Also, unlike Th1 cells, Th17 cells do not exhibit hallmarks of senescence or apoptosis, retaining robust antitumor efficacy in vivo. Three-week-expanded Th17 cells eliminated melanoma as effectively as Th17 cells expanded for 1 week when infused in equal numbers into mice. However, treating mice with large recalcitrant tumors required the infusion of all cells generated after 2 or 3 weeks of expansion, while the cell yield obtained after 1-week expansion was insufficient. Long-term-expanded Th17 cells also protected mice from tumor rechallenge including lung metastasis. Importantly, 2-week-expanded human chimeric antigen receptor-positive (CAR+) Th17 cells also retained their ability to regress human mesothelioma, while CAR+ Th1 cells did not. Our results indicate that tumor-reactive Th17 cells are an effective cell therapy for cancer, remaining uncompromised when expanded for a long duration owing to their resistance to senescence.

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) has shown remarkable results in patients with metastatic melanoma. However, only a small fraction within the TIL population reacts against the tumor. Therefore, the pre-enrichment of tumor-specific T-cells and subsequent ex vivo expansion may improve the efficiency of ACT therapies. In addition, tumor-reactive T lymphocytes circulating in the blood (TRLs) have been found in low frequencies, which represents a challenge for their isolation, but also an advantage over TIL therapy since blood is a more reliable and accessible source than tumor excisions. Another impediment to the widespread application of ACT is the conventional rapid expansion protocol (REP) that constitutes a laborious and extensive process with frequent culture manipulations, and thus requires specialized personell and equipment. Our aim is to develop a fully automated large scale ex vivo T-cell isolation and expansion procedure in the CliniMACS Prodigy in order to simplify the manufacturing of tumor-reactive T-cells for ACT. The CliniMACS Prodigy instrument is a controlled system that integrates a series of cell processes, from magnetic cell separation and cell culture to final product formulation, under GMP conditions in a closed system. This process focuses on the optimization of the REP procedure on the CliniMACS Prodigy for TILs and TRLs. As a proof of concept, we used both cryopreserved outgrown TILs and magnetically isolated virus-specific T-cells from healthy donor leukapheresis via CD137 upregulation upon in vitro antigen stimulation. The first results show expansions ranging from 3,000-15,000 fold, both for TILs and CD137-expressing T-cells. From low cell numbers (2x10e5 - 1x10e6 cells) and after 14 days of cell culture in TexMACS medium and in the presence of high amounts of IL-2 and irradiated feeder cells, we were able to obtain around 3-4x10e9 cells. We also compared two different stimulation reagents, anti-CD3 antibody (OKT3) and TransAct (a soluble polymeric nanomatrix conjugated to humanized CD3 and CD28 agonist), which resulted in comparable expansion rates. Furthermore, small-scale experiments showed no differences between TexMACS and the conventional TIL culture medium (50% RPMI/50% AIM-V medium). The phenotype and reactivity of the expanded T-cells were also assessed by flow cytometry. Currently, higher starting cell numbers up to 1x10e7 cells are being assessed and first results are promising when shaking and media exchange are commenced earlier in the process. In summary, these data provide proof of concept for the expansion of low numbers of TILs and virus-specific T-cells from peripheral blood in a closed, automated manner in the CliniMACS Prodigy. In the future, this expansion process will be combined with a tumor-reactive T-cell enrichment process (e.g., via CD137-conjugated magnetic beads) to achieve the desired efficiency, simplicity and automated production of ACT therapies against cancer. Citation Format: Bianca Heemskerk, Christina Maeder, Elvira Criado-Moronati, Lisa Boettcher, Andrew Kaiser, Mario Assenmacher, Andrzej Dzionek. Automated ex vivo expansion of low numbers of tumor-reactive T-cells on the CliniMACS Prodigy® [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B016.

Despite tremendous clinical success of chimeric antigen receptor (CAR) expressing T cell (CAR-T) therapies, targeting B-phenotypic antigens in ALL, CLL, NHL or multiple myeloma, there are still major limitations for broader clinical application. CAR-Ts are capable to generate a specific immune response against defined surface-expressed antigens leading to sustained depletion of target antigen expressing tissues e.g. B cells. While B cell function can be substituted by repetitive IgG infusions, prolonged depletion of vitally essential tissues is not compatible with life. In AML for instance, most promising target antigens are expressed along myeloid lineage differentiation, limiting the therapeutic applicability. Therefore, CAR-Ts targeting essential shared antigens must allow tight regulation of CAR-T function and/or be able to differentiate between cancerous and healthy tissue. To address these issues, we have developed the adapter CAR-T cell (aCAR-T) system. By splitting antigen recognition and CAR-T activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combine target antigens) regulation of CAR-T function. aCAR-Ts are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated to novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. We were able to demonstrate that aCAR-Ts allow simultaneous targeting of various antigens ("OR"-gate) , preventing antigen evasion by selection of antigen or epitope-loss variants. In the present study we intended to investigate whether aCAR-Ts are capable to identify and differentiate target cells due to versatile antigen expression profiles ("AND"-gate). In theory, AMs against different target antigens can be assembled on the surface of a target cell , leading to aCAR-T activation independent of the targeted antigens (surface painting) , by binding to the presented LLE-tags. Therefore, combinatorial AMs treatment might allow to translate complex and multiple antigen-dependent target cell identification into an aCAR-T activation. To test this hypothesis, we have generated LLE-AMs against ALL/NHL - (CD10, CD19, CD20, CD22, CD37, CD138, ROR1) and AML - (CD32, CD33, CD38, CD123, CD135, CD305, CLL1) associated antigens. Individual threshold concentrations for aCAR-T activation by different AMs, targeti ng the model cell lines Nalm 6 (ALL), JeKo1 (NHL), HL-60 , U973 and Molm13 ( all AML), have been analyzed. Cut offs were found to be between 10 and 100 pg/ml, dependent on target expression and target cell line. Importantly, combinations of 2, 3 or 5 AMs, targeting different antigens expressed on the same target cell, cause target- cell lysis at concentrations below the activation threshold for single AMs (exemplified for HL-60 in Figure A) . Our results clearly demonstrate an additive effect in combining different AMs to hurdle the activation threshold. Moreover, in a JeKo 1 CD19 and/or CD20 knock out (KO) antigen-loss model, combinations of AMs targeting CD19, CD20 and ROR1 can differentiate between wild type and antigen -1 (CD19 or CD20 KO) or antigen -2 (CD19 and CD20 KO) variants in medi ating target- cell lysis, even though at least one target antigen is expressed. Finally, we found that combinations of CD10, CD19, CD22 and CD138 sufficiently eliminate Nalm-6 BCP-ALL cells, while sparing healthy B cells in co - culture experiments. Similar results were obtained in co - culture experiments of HL-60 AML cells with monocytes, neutrophils as well as CD34- enriched hematopoietic progenitor cells, applying combinations of CD32, CD33, CD38, CD123, CD305 and CLL1. Co - culturing experiments using autologous blasts, monocytes, neutrophils and aCAR-Ts are ongoing. Together, our results indicate that aCAR-Ts in combination with selected AM combinations might have the ability to identify and specifically eliminate cancer cells based on complex antigen expression profiles. This would have major implications for clinical translation, enabling combinatorial therapy, essential to avoid antigen evasion, and the possibility to spare vitally essential tissue from elimination. Figure. Figure. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Lock:Miltenyi Biotec: Employment. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding. Lang:Miltenyi Biotec: Patents & Royalties, Research Funding. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding.

Chimeric antigen receptor (CAR) expressing T cells (CAR-Ts) have demonstrated tremendous clinical success, especially when targeted against the B-phenotypic antigens CD19 and CD22 in ALL, CLL as well as NHL. Despite the recent success, production of CAR-Ts still requires an extensive and time consuming manufacturing process. Moreover, CAR-Ts have to be prepared individually for each patient. Especially in heavily pretreated and rapidly progressing patients, which often lack sufficient numbers of healthy T cells for CAR-T production, alternatives are a significant clinical need. NK cells might represent a promising alternative effector cell source. The continuously expandable and well established NK cell line NK-92 can provide a safe and consistent way to produce NK effector cells in a GMP-compliant and cost-effective way. Irradiated NK-92 CARs as an "off-the-shelf on-demand" cell therapeutic are currently tested in pre-clinical and early-phase clinical trials. Furthermore, NK-92 can be redirected by CARs to mediate direct antigen specific lysis. We have recently developed a universal adapter CAR (aCAR) system. By splitting antigen recognition and CAR-immune cell activation, introducing adapter molecules (AMs), the system allows precise quantitative (on-/off-switch) as well as qualitative (change and combination of target antigens) regulation of immune cell function. aCARs are based on the unique properties of a novel scFv targeting a "neo"-epitope-like structure consisting of the endogenous vitamin biotin in the context of a specific linker, referred to as linker-label-epitope (LLE). LLEs can be easily conjugated on novel or preexisting AM formats like monoclonal antibodies (mAbs) or mAb fragments in a GMP-compliant manner. In the present study, we intended to combine the universal and flexible targeting as well as controllability of the aCAR with the "off-the-shelf" properties of NK-92 cells. NK-92 was obtained from ATCC and transduced with aCARs containing either CD28 or 4-1BB co-stimulatory plus CD3-ζ signaling domains. Importantly, only CD28 containing aCARs sufficiently mediated specific target cell lysis in the presence of biotinylated antibodies (LLE-AMs). Using single cell sorting, aCAR NK-92 clones with the highest CAR expression were selected and demonstrated significantly improved target cell lysis, in a LLE-AMs dependent manner. Both, LLE-AMs against CD19 and CD20, were capable of inducing significant NK-92-mediated lysis against the NHL cell lines Raji, Daudi and JeKo-1. Cytotoxicity experiments using aCAR NK-92 cells and primary lymphoma cells are ongoing. Specificity of the LLE-AM directed effector cell elimination was further proven using a JeKo-1 CD19 and/or CD20 knock out (KO) antigen-loss model. aCAR NK-92 mediated antigen specific lysis only in the presence of the target antigen and the specific LLE-AM. Moreover, combinations of anti-CD19 and anti-CD20 LLE-AMs are capable of avoiding antigen evasion. To test the universal applicability of aCAR NK-92, specific target cell lysis against a variety of different tumor entities was demonstrated using LLE-conjugated therapeutic antibodies. Importantly, irradiation of effector cells, as required in all active clinical trials using NK-92, prior to testing had no observable effect on target cell lysis. Finally, the investigation of potential on-target off-tumor reactivity against healthy B cells showed no cytotoxic effects of aCAR NK-92 cells in combination with LLE-AMs against CD19 or CD20. In conclusion, we have generated an NK cell line, aCAR NK-92, whose effector function can be tightly regulated and redirected against one or multiple antigens allowing tunable and universal targeting. Moreover, aCAR NK-92 cells can be manufactured as an "off-the-shelf on-demand" standardized product improving the practicality of NK CAR therapy combined with the possibility of tailoring a specific LLE-AM platform for patient-individualized treatment. Disclosures Seitz: Miltenyi Biotec: Patents & Royalties, Research Funding. Mittelstaet:Miltenyi Biotec: Employment, Patents & Royalties. Kaiser:Miltenyi Biotec: Employment, Patents & Royalties. Schlegel:Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.

T-cell–based immunotherapies can be effective in the treatment of large vascularized tumors, but they rely on adoptive transfer of substantial numbers (∼20 million) of tumor-specific T cells administered together with vaccination and high-dose interleukin (IL)-2. In this study, we report that ∼10,000 T cells gene-engineered to express a single-chain IL-12 molecule can be therapeutically effective against established tumors in the absence of exogenous IL-2 and vaccine. Although IL-12–engineered cells did not perist long-term in hosts, they exhibited enhanced functionality and were detected in higher numbers intratumorally along with increased numbers of endogenous natural killer and CD8+ T cells just before regression. Importantly, transferred T cells isolated from tumors stably overproduced supraphysiologic amounts of IL-12, and the therapeutic effect of IL-12 produced within the tumor microenvironment could not be mimicked with high doses of exogenously provided IL-12. Furthermore, antitumor effects could be recapitulated by engineering wild-type open-repertoire splenocytes to express both the single-chain IL-12 and a recombinant tumor-specific T-cell receptor (TCR), but only when individual cells expressed both the TCR and IL-12, indicating that arrested migration of T cells at the tumor site was required for their activities. Successful tumor eradication was dependent on a lymphodepleting preconditioning regimen that reduced the number of intratumoral CD4+ Foxp3+ T regulatory cells. Our findings reveal an approach to genetically modify T cells to reduce the cell number needed, eliminate the need for vaccines or systemic IL-2, and improve immunotherapy efficacy based on adoptive transfer of gene-engineered T cells. Cancer Res; 70(17); 6725–34. ©2010 AACR.

Adoptive cell transfer (ACT), either using rapidly expanded tumor infiltrating lymphocytes or T-cell receptor transduced peripheral blood lymphocytes, can be considered one of the most promising approaches in cancer immunotherapy. ACT results in the repopulation of the host with high frequencies of tumor-specific T cells; however, optimal function of these cells within the tumor micro-environment is required to reach long-term tumor clearance. We and others have shown that ongoing anti-tumor immune responses can be impaired by the expression of ligands, such as PD-L1 (B7-H1) on tumor cells. Such inhibitory molecules can affect T cells at the effector phase via their receptor PD-1. PD-L1/PD-1 interaction has indeed been shown crucial in inducing T-cell anergy and maintaining peripheral tolerance. In order to maximize anti-tumor responses, antibodies that target the PD-1/PD-L1 axis are currently in phase I/II trials. Alternatively, a more refined approach could be the selective targeting of PD-1 in tumor-specific T cells to obtain long-term resistance against PD-1-mediated inhibition. We addressed whether this goal could be achieved by means of retroviral siRNA delivery. Effective siRNA sequences resulting in the reduction of surface PD-1 expression led to improved murine as well as human T-cell immune functions in response to PD-L1 expressing melanoma cells. These data suggest that blockade of PD-1-mediated T-cell inhibition through siRNA forms a promising approach to achieve long-lasting enhancement of tumor-specific T-cell function in adoptive T-cell therapy protocols.