Successful Generation of CD19 Chimeric Antigen Receptor T Cells from Patients with Advanced Systemic Lupus Erythematosus.

Although it has been shown that the production of functional chimeric antigen receptor T cells is feasible in patients with B-cell malignancies, it is currently unclear whether sufficient amounts of functional autologous CAR T cells can be generated from patients with autoimmune diseases. Intrinsic T-cell abnormalities and T-cell-targeted immune suppression in patients with autoimmunity may hamper the retrieval of sufficient T cells and their transduction and expansion into CAR T cells. Patients with active systemic lupus erythematosus (SLE) underwent leukapheresis after tapering glucocorticoids and stopping T-cell-suppressive drugs. This material was used as source for manufacturing anti-CD19 CAR T-cell products (CAR) in clinical scale. Cells were transduced with a lentiviral anti-CD19 CAR vector and expanded under good manufacturing practice (GMP) conditions using a closed, semi-automatic system. Functionality of these CAR T cells derived from autoimmune patient cells was tested in vitro. Six SLE patients were analyzed. Leukapheresis could be successfully performed in all patients yielding sufficient T-cell numbers for clinical scale CAR T-cell production. In addition, CAR T cells showed high expansion rates and viability, leading to CAR T cells in sufficient doses and quality for clinical use. CAR T cells from all patients showed specific cytotoxicity against CD19+ cell lines in vitro. GMP grade generation of CD19 CAR T-cell products suitable for clinical use is feasible in patients with autoimmune disease.

Oxidative DNA damage in reconstituting T cells is associated with relapse and inferior survival after allo-SCT.

Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only curative treatment option for a number of hematologic malignancies. Its therapeutic potential relies on the potency of donor T cells to eliminate residual malignant cells, the so-called graft-versus-leukemia (GVL) effect. Disease relapse remains the most frequent treatment failure and is associated with poor outcome. Therefore, it is inevitable to decipher mechanisms that weaken GVL. In recent years, studies of tumor biology have revealed that metabolic remodeling of the micromilieu can critically regulate immune responses. Accumulation of reactive oxygen species leads to a metabolic condition known as oxidative stress, which can severely hamper T cells. Currently, only a few studies, mainly using preclinical models, have demonstrated the occurrence of oxidative stress after allo-SCTs. Therefore, we sought to investigate oxidative stress in a well-characterized group of patients who underwent allo-SCT and its impact on reconstituting T cells. We identified high concentrations of serum 8-hydroxydeoxyguanosine (8-OHdG) as an established biomarker for oxidative stress. 8-OHdG is one of the major products of DNA oxidation, which is normally rapidly removed. After allo-SCT, T cells accumulated oxidative DNA damage. High cellular 8-OHdG content (8-OHdGhi) was associated not only with signs of enhanced T-cell activation but also premature exhaustion. The inability of 8-OHdGhi T cells to efficiently target malignant cells or produce cytotoxic granzyme B and interferon gamma was associated with a significantly increased relapse risk and a shorter overall survival. Taken together, our novel findings could give reason to focus on bolstering DNA repair in reconstituting T cells as a means to improve GVL efficacy.

Reduced cytotoxicity by mutation of lysine 590 of Pseudomonas exotoxin can be restored in an optimized, lysine-free immunotoxin.

Immunotoxins, which are fusion proteins of an antibody fragment and a fragment of a bacterial or a plant toxin, induce apoptosis in target cells by inhibition of protein synthesis. ADP-ribosylating toxins often have few lysine residues in their catalytic domain. As they are the target for ubiquitination, the low number of lysines possibly prevents ubiquitin-dependent degradation of the toxin in the cytosol. To reduce this potential degradation, we aimed to generate a lysine-free (noK), Pseudomonas exotoxin (PE)-based immunotoxin. The new generation 24 kDa PE, which lacks all but the furin-cleavage site of domain II, was mutated at lysine 590 (K590) and at K606 in a CD22-targeting immunotoxin and activity was determined against various B cell malignancies in vitro and in vivo. On average, K590 mutated to arginine (R) reduced cytotoxicity by 1.3-fold and K606R enhanced cytotoxicity by 1.3-fold compared to wild type (wt). Mutating K590 to histidine or deleting K590 did not prevent this loss in cytotoxicity. Neither stability nor internalization rate of K590R could explain reduced cytotoxicity. These results highlight the relevance of lysine 590 for PE intoxication. In line with in vitro results, the K606R mutant was more than 1.8-fold more active than the other variants in vivo suggesting that this single mutation may be beneficial when targeting CD22-positive malignancies. Finally, reduced cytotoxicity by K590R was compensated for by K606R and the resulting lysine-free variant achieved wt-like activity in vitro and in vivo. Thus, PE24-noK may represent a promising candidate for down-stream applications that would interfere with lysines.

CD137 (4-1BB) stimulation leads to metabolic and functional reprogramming of human monocytes/macrophages enhancing their tumoricidal activity.

Immunotherapies have heralded a new era in the cancer treatment. In addition to checkpoint inhibitors, agonistic antibodies against co-stimulatory immune receptors hold the potential to invoke efficient antitumor immunity. Targeting CD137 has gained momentum based on its ability to drive NK- and T-cell-based responses. CD137-engaging mAbs have already entered clinical trials for different types of tumors showing promising results. Despite the efforts to translate CD137-mediated immunotherapy into clinical practice, little remains known regarding the role of CD137 in human monocytes/macrophages.We found CD137 being expressed on monocytes of healthy controls and at even higher levels in patients with multiple myeloma or CLL. CD137HI(GH) monocytes displayed a distinct phenotypic, transcriptomic, and metabolic profile. They possessed an increased phagocytic capacity enabling superior antibody-dependent phagocytosis (ADPC) of multiple myeloma and lymphoma cells that were treated with anti-CD38 or anti-CD20 mAbs. Triggering CD137 promoted both metabolic and tumoricidal activity in an extracellular signal-regulated kinase (ERK)-dependent fashion. In addition, we observed a phenotypic, transcriptomic, and functional skewing towards a M1-like phenotype.Overall, we introduce CD137 as a positive immune checkpoint on human monocytes/macrophages, which can have therapeutic implications especially in view of synergistic effects when combining CD137 agonists with tumor-targeting antibodies.

Inflammation-induced glycolytic switch controls suppressivity of mesenchymal stem cells via STAT1 glycosylation.

Mesenchymal stem cells (MSCs) represent key contributors to tissue homeostasis and promising therapeutics for hyperinflammatory conditions including graft-versus-host disease. Their immunomodulatory effects are controlled by microenvironmental signals. The MSCs' functional response towards inflammatory cues is known as MSC-"licensing" and includes indoleamine 2,3-dioxygenase (IDO) upregulation. MSCs use tryptophan-depleting IDO to suppress T-cells. Increasing evidence suggests that several functions are (co-)determined by the cells' metabolic commitment. MSCs are capable of both, high levels of glycolysis and of oxidative phosphorylation. Although several studies have addressed alterations of the immune regulatory phenotype elicited by inflammatory priming metabolic mechanisms controlling this process remain unknown. We demonstrate that inflammatory MSC-licensing causes metabolic shifts including enhanced glycolysis and increased fatty acid oxidation. Yet, only interfering with glycolysis impacts IDO upregulation and impedes T-cell-suppressivity. We identified the Janus kinase (JAK)/signal transducer and activator of transcription (STAT)1 pathway as a regulator of both glycolysis and IDO, and show that enhanced glucose turnover is linked to abundant STAT1 glycosylation. Inhibiting the responsible O-acetylglucosamine (O-GlcNAc) transferase abolishes STAT1 activity together with IDO upregulation. Our data suggest that STAT1-O-GlcNAcylation increases its stability towards degradation thus sustaining downstream effects. This pathway could represent a target for interventions aiming to enhance the MSCs' immunoregulatory potency.

[TILGen study-immunological targets in patients with breast cancer : Influence of tumor-infiltrating lymphocytes]. / TILGen-Studie ­ Immunologische Angriffspunkte bei Patientinnen mit Brustkrebs : Einfluss der tumorinfiltrierenden Lymphozyten.

BACKGROUND: The interaction of our immune system with breast cancer (BC) cells prompted the investigation of tumor-infiltrating lymphocytes (TILs) and targeted, tumor antigen-specific immunotherapy. OBJECTIVES: Correlation between TILs and pathological complete response (pCR) after neoadjuvant systemic therapy (NACT). Tumor-specific antigens (TSAs) in HER2+ and triple negative BC and establishment of TSA-specific therapies within the interdisciplinary TILGen study. METHODS: Illustration of the TILGen study design. Assessment of TILs and correlation with pCR within this BC study. RESULTS: pCR was achieved in 38.4% (56/146) and associated with estrogen receptor/progesterone receptor negative (ER-/PR-) and HER2+ tumors. Lymphocytic predominant BC (LPBC) was found in 16.4% (24/146), particularly in ER-/PR- (ER-: 27.3% vs. ER+: 9.9%, PR-: 22.3% vs. PR+: 8.2%), large, and poorly differentiated BC. TILs were significantly correlated with pCR in multivariate analysis. In LPBC, pCR was achieved in 66.7%, whereas it was 32.8% in non-LPBC. CONCLUSIONS: First results confirm the influence of the human immune system on the response to NACT in HER2+ and triple negative BC. TSA-specific immunotherapy might improve the outcome in BC patients but there is an urgent need for comprehensive studies to further investigate this issue.

[Recent development of hypertension and acute renal failure in a 59-year-old woman]. / Neu aufgetretene Hypertonie und akutes Nierenversagen bei einer 59-jährigen Patientin.

We report on a 59-year-old woman who presented with nausea, fatigue, arterial hypertension, and acute renal failure. Clinical examination, laboratory findings of blood and urine and abdominal sonography were inconclusive. Renal biopsy revealed infiltration by a diffuse large B­cell lymphoma. In fluorodeoxyglucose positron emission tomography/computed tomography tracer enrichment was demonstrated in both kidneys, skeletal system and retroperitoneal lymph nodes. Renal function improved already after the first cycle of R­CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). After chemotherapy, a complete remission, normalization of blood pressure and renal function were achieved.

The PD-1/PD-L1 axis contributes to immune metabolic dysfunctions of monocytes in chronic lymphocytic leukemia.

Immune dysfunctions in chronic lymphocytic leukemia (CLL) contribute to tumor immune escape and attenuate immune-based therapies. Monocytes/macrophages represent key components of cancer immune surveillance and effectors for antibody-mediated antitumor effects. Monocytes display an altered subset composition in CLL. Moreover, we find a changed metabolic phenotype: glucose uptake, glucose transporters and expression of glycolytic molecules are reduced. Our data establish a link between glycolytic competence and monocyte-mediated phagocytosis of tumor cells. Furthermore, we report that CLL monocytes express Bruton's tyrosine kinase (BTK). Our observations suggest that using BTK inhibitors in CLL might further aggravate the observed immune metabolic defects in monocytes. Triggering the programmed cell death-1 (PD-1) checkpoint on monocytes hampers glycolysis, phagocytosis and BTK signaling. Conversely, disrupting PD-1/PD-L1 signaling reverses these immune metabolic dysfunctions. Taken together, our findings imply a novel metabolic interplay between CLL cells and monocytes and that blocking PD-1/PD-L1 might restore metabolic together with antitumor activity of CLL monocytes/macrophages.

Nilotinib combined with interleukin-2 mediates antitumor and immunological effects in a B16 melanoma model.

The immune system contributes to tumor cell killing which can be enhanced by cancer chemotherapeutics and immune modulatory pharmaceuticals such as tyrosine kinase inhibitors (TKIs). Recently, the beneficial effect of natural killer (NK) cells was demonstrated when combining interleukin-2 (IL-2) with the TKI imatinib. The aim of the present study was to address the antitumor and immunological effects of recently approved TKIs. Therefore, we focused on the comparison of the efficacy between imatinib and nilotinib in combination with IL-2 in a murine B16F10 melanoma model. Both TKIs possessed antitumor activity in vivo. However, the combination of nilotinib and IL-2 showed a superior outcome. Importantly, both the use of immunodeficient Rag2γc-/- mice, which lack T-lymphocytes, B-lymphocytes and NK cells, as well as NK cell-depletion in C57Bl/6 mice reduced the therapeutic effect of nilotinib. Flow cytometry revealed a significant increase in the IFN-γ-producing CD27+ NK cell subpopulation following treatment with nilotinib and IL-2. Furthermore, the therapeutic antitumor effect of nilotinib/IL-2 was completely lost in IFN-γ-/- mice. In summary, we suggest that nilotinib combined with IL-2 confers high antitumor activity involving the subset of IFN-γ-producing CD27+ NK cells. These new insights are of high importance for the understanding and development of immunotherapeutic protocols using TKIs.

T lymphocytes can be effectively recruited for ex vivo and in vivo lysis of AML blasts by a novel CD33/CD3-bispecific BiTE antibody construct.

Patients with acute myelogenous leukemia (AML) are in high need of novel targeted therapies. Here we explored the ex vivo activity of AMG330, a novel T-cell-engaging BiTE (bi-specific T-cell engagers) antibody (Ab) construct, that is bispecific for the myeloid differentiation antigen, CD33 and CD3, in primary samples from AML patients (N=23) and AML cell lines. KG-1 and U937 cells were lysed in co-culture with healthy donor T-cells at AMG330 concentrations as low as 0.1 ng/ml (1.8 pM). T-cells derived from AML patient samples were found to be as active in redirected lysis by AMG330 as T-cells from healthy donors. In an autologous setting, AMG330 could activate and expand T-cells in primary AML patient samples, and effectively mediated the redirected lysis of AML blasts and normal myeloid cells. A deficiency in target-cell lysis was only observed in samples with very low initial effector-to-target (E:T) ratio. However, this could be overcome if previously stimulated autologous T-cells were tested in patient samples at a higher E:T ratio. In vivo experiments in immunodeficient mice demonstrated significant inhibition of tumor growth by AMG330 and an inducible infiltration of human T-cells into subcutaneous HL60 tumors. The activities of the CD33/CD3-bispecific BiTE Ab construct AMG330 warrant further development for the treatment of AML.

[Fundus leucaemicus as first manifestation of chronic myeloid leukemia. Diagnosis and monitoring with OCT under treatment with imatinib and interferon-alpha]. / Fundus leucaemicus als Erstmanifestation einer chronischen myeloischen Leukämie. Diagnosestellung und Verlaufskontrolle mit OCT unter Therapie mit Imatinib und Interferon-alpha.

Fundus leucaemicus with reduction of visual acuity can be one of the first signs of chronic myeloid leukemia (CML). The ocular manifestations are unspecific, but characteristic for severe systemic diseases. Optical coherence tomography (OCT) is helpful for documentation and quantification of the pathological retinal changes. However, peripheral blood counts and differential haemograms are seminal for the diagnosis of CML and can be important for survival. First line therapy for CML is the application of the tyrosine kinase inhibitor imatinib (Glivec). Ophthalmological adverse effects of this therapy, such as periorbital edema, are possible. Therefore regular ophthalmic monitoring should be performed.

In situ analysis of the antigen-processing machinery in acute myeloid leukaemic blasts by tissue microarray.

Altered expression of major histocompatibility complex (MHC) class I molecules can be caused by defects in genes of the antigen-processing machinery (APM), and is often correlated to progression in solid tumours. However, little is known about expression of the APM components in blasts from patients with acute myeloid leukaemia (AML). In this study, we investigated the expression of the APM components large multifunctional peptidases (LMP) 2 and 7, transporter-associated with antigen processing (TAP) 1 and 2, beta-2-microglobulin (beta2m) and MHC class I heavy chain in situ by tissue microarray from bone marrow biopsies of 30 AML patients. APM components were heterogeneously expressed in all AML samples tested, but no significant correlation with the AML subtype according to the French-American-British classification was found. Depending on the APM component tested, up to 90% of the trephines showed no or weak expression, whereas the LMP7 protein was detected in 66% of all samples. By following disease progression in individual AML patients, we found severe downregulation of APM components in two out of four patients from initial diagnosis to relapse. We conclude that downregulation of APM components may play a role in the failure of immuno-surveillance and may therefore contribute to relapse in acute leukaemia.

[Chronic myeloproliferative diseases. Current therapeutic standards and new developments]. / Chronische myeloproliferative Erkrankungen. Aktuelle Therapiestandards und neue Entwicklungen.

Chronic myeloproliferative diseases (CMPD) are haematopoetic neoplasias with indolent course and preserved cellular function of the maturing malignant cells. In Philadelphia-positive chronic myeloid leukaemia the discovery of molecular disease mechanisms led to the successful introduction of targeted therapy with imatinib. Ph-negative CMPD are conventionally treated by cytoreduction and low dose ASS in order to minimise the risk of vascular complications. For all CMPD, lifelong surveillance and therapy are necessary.

The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays.

The interpretation of the results obtained from immunomonitoring of clinical trials is a difficult task due to the variety of methods and protocols available to detect vaccine-specific T-cell responses. This heterogeneity as well as the lack of standards has led to significant scepticism towards published results. In February 2005, a working group was therefore founded under the aegis of the Association for Immunotherapy of Cancer ("CIMT") in order to compare techniques and protocols applied for the enumeration of antigen-specific T-cell responses. Here we present the results from two consecutive phases of an international inter-laboratory testing project referred to as the "CIMT monitoring panel". A total of 13 centers from six European countries participated in the study in which pre-tested PBMC samples, synthetic peptides and PE-conjugated HLA-tetramers were prepared centrally and distributed to participants. All were asked to determine the number of antigen-specific T-cells in each sample using tetramer staining and one functional assay. The results of the first testing round revealed that the total number of cells analyzed was the most important determinant for the sensitive detection of antigen-specific CD8(+) T-cells by tetramer staining. Analysis by ELISPOT was influenced by a combination of cell number and a resting phase after thawing of peripheral blood mononuclear cells. Therefore, the experiments were repeated in a second phase but now the participants were asked to change their protocols according to the new guidelines distilled from the results of the first phase. The recommendations improved the number of antigen-specific T-cell responses that were detected and decreased the variability between the laboratories. We conclude that a two-step approach in inter-laboratory testing allows the identification of distinct variables that influence the sensitivity of different T-cell assays and to formally show that a defined correction to the protocols successfully increases the sensitivity and reduces the inter-center variability. Such "two-step" inter-laboratory projects could define rational bases for accepted international guidelines and thereby lead to the harmonization of the techniques used for immune monitoring.

Dendritic cells for specific cancer immunotherapy.

The characterization of tumor-associated antigens recognized by human T lymphocytes in a major histocompatibility complex (MHC)-restricted fashion has opened new possibilities for immunotherapeutic approaches to the treatment of human cancers. Dendritic cells (DC) are professional antigen presenting cells that are well suited to activate T cells toward various antigens, such as tumor-associated antigens, due to their potent costimulatory activity. The availability of large numbers of DC, generated either from hematopoietic progenitor cells or monocytes in vitro or isolated from peripheral blood, has profoundly changed pre-clinical research as well as the clinical evaluation of these cells. Accordingly, appropriately pulsed or transfected DC may be used for vaccination in the field of infectious diseases or tumor immunotherapy to induce antigen-specific T cell responses. These observations led to pilot clinical trials of DC vaccination for patients with cancer in order to investigate the feasibility, safety, as well as the immunologic and clinical effects of this approach. Initial clinical studies of human DC vaccines are generating encouraging preliminary results demonstrating induction of tumor-specific immune responses and tumor regression. Nevertheless, much work is still needed to address several variables that are critical for optimizing this approach and to determine the role of DC-based vaccines in tumor immunotherapy.