scholarly journals Recent advances in T-cell engineering for use in immunotherapy

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2344 ◽  
Author(s):  
Preeti Sharma ◽  
David M. Kranz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Complex Molecule ◽  
Major Histocompatibility Complex Molecule ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Chain ◽  
Cell Therapies ◽  
Recent Advances

Adoptive T-cell therapies have shown exceptional promise in the treatment of cancer, especially B-cell malignancies. Two distinct strategies have been used to redirect the activity of ex vivo engineered T cells. In one case, the well-known ability of the T-cell receptor (TCR) to recognize a specific peptide bound to a major histocompatibility complex molecule has been exploited by introducing a TCR against a cancer-associated peptide/human leukocyte antigen complex. In the other strategy, synthetic constructs called chimeric antigen receptors (CARs) that contain antibody variable domains (single-chain fragments variable) and signaling domains have been introduced into T cells. Whereas many reviews have described these two approaches, this review focuses on a few recent advances of significant interest. The early success of CARs has been followed by questions about optimal configurations of these synthetic constructs, especially for efficacy against solid tumors. Among the many features that are important, the dimensions and stoichiometries of CAR/antigen complexes at the synapse have recently begun to be appreciated. In TCR-mediated approaches, recent evidence that mutated peptides (neoantigens) serve as targets for endogenous T-cell responses suggests that these neoantigens may also provide new opportunities for adoptive T-cell therapies with TCRs.

scholarly journals In a small multideterminant peptide, each determinant is recognized by a different V beta gene segment.

1992 ◽  
Vol 176 (1) ◽  
pp. 297-302 ◽  
Author(s):  
N K Nanda ◽  
K K Arzoo ◽  
E E Sercarz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Segment ◽  
Complex Molecule ◽  
Major Histocompatibility Complex Molecule ◽  
Sperm Whale ◽  
Cell Receptor ◽  
T Cell Response ◽  
Absolute Requirement ◽  
Beta Gene

Given the vast potential for diversification of the T cell receptor (TCR) repertoire and the fact that V(a) beta mice exist in the wild, it would have been predicted that in spite of the absence of 10 TCR V beta gene segments, V(a) beta mice would still have been able to produce an antigen-specific T cell response to all determinants. We have recently shown that Vb beta mice, with a wild-type TCR V beta repertoire, respond to peptide 110-121 of sperm whale myoglobin, with a majority of T cells expressing TCR V beta 8.2 and restricted to a hybrid I-A(d)/I-E(d) major histocompatibility complex molecule, and a smaller number of T cells expressing TCR V beta 8.1 and restricted to the I-A(d) molecule. However, V(a) beta mice, lacking members of the TCR V beta 8 gene family, responded only with I-A(d)-restricted T cells. Thus, it appeared that the I-A(d)-restricted response was less constrained, or more plastic. We now show that the two separate panels of I-A(d)-restricted T cell hybrids derived from V(a) beta or Vb beta mice in fact recognize distinct determinants within the same peptide 110-121. The determinant recognized by V(a) beta T cells is NH2 terminal (core: 110-118) with an absolute requirement for the residue Ala-110 for a successful interaction with TCRs. On the other hand, Vb beta T cells recognize the COOH-terminal region (core: 112-118) on the same peptide with an absolute requirement for COOH-terminal residue 118. In the dominance hierarchy displayed by the three distinct determinants of peptide 110-121, V(a) beta mice cannot recognize the two most dominant: the hybrid I-A(d)/I-E(d)-restricted determinant and the COOH-terminal, I-A(d)-restricted determinant. They instead respond with T cells specific for a third, distinctly NH2-terminal determinant. Our results show a strict association between recognition of a particular specificity and TCR V beta usage. This evidence suggests that even when a small peptide induces a heterogenous group of TCR V beta S, this need not be considered evidence for plasticity. Rather, at the level of individual determinants within the peptide, the results can point in the opposite direction, towards serious constraints in recognition at the level of V beta expression.

Transgenic mice to study T-cell receptor gene regulation and repertoire formation

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 652-655 ◽  
Author(s):  
Michael Steinmetz ◽  
Horst Blüthmann ◽  
Stefan Ryser ◽  
Yasushi Uematsu
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Transgenic Mice ◽  
T Cell Receptor ◽  
Complex Molecule ◽  
Major Histocompatibility Complex Molecule ◽  
Cell Receptor ◽  
Target Cells ◽  
Allelic Exclusion

Transgenic mice have been obtained with genes coding for an αβ T-cell receptor that recognizes the male-specific antigen H-Y in association with the Db class I major histocompatibility complex molecule. Most if not all of the T-cells express the β chain encoded by the transgene and show allelic exclusion of endogenous β genes. In contrast, the expression of the α transgene does not completely block rearrangement and formation of functional endogenous α genes. In H-2b transgenic female mice the transgenic T-cell receptor is functionally expressed on at least 30% of CD8+ peripheral T-lymphocytes as indicated by their ability to lyse male target cells. Also in transgenic H-2b male mice a large proportion of peripheral T-cells appear to express the transgenic receptor. However, these cells do not react with male target cells because they show only low level or no expression of CD8 cell interaction molecules. Tolerance is established in the male transgenic thymus through deletion of CD4+CD8+ immature thymocytes.Key words: transgenic mice, immune system, T-lymphocytes, T-cell receptor, tolerance, CD8 surface antigen, enhancer, gene rearrangement, allelic exclusion.

scholarly journals Identification of NY-ESO-1157–165 Specific Murine T Cell Receptors With Distinct Recognition Pattern for Tumor Immunotherapy

2021 ◽  
Vol 12 ◽  
Author(s):  
Helin Zhang ◽  
Meng Sun ◽  
Jie Wang ◽  
Bin Zeng ◽  
Xiaoqing Cao ◽  
...  
Keyword(s):  
New York ◽  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Target Cells ◽  
Single Chain ◽  
Recognition Pattern ◽  
Clinical Benefits

New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a promising target for T-cell receptor-engineered T cell (TCR-T) therapy, and targeting the human leukocyte antigen (HLA)-A2 restricted NY-ESO-1157−165 epitope has yielded remarkable clinical benefits in the treatment of multiple advanced malignancies. Herein, we report the identification of two NY-ESO-1157−165 epitope-specific murine TCRs obtained from HLA-A*0201 transgenic mice. NY-ESO-1157−165 specific TCRs were isolated after vaccinating HLA-A2 transgenic mice with epitope peptides. HZ6 and HZ8 TCRs could specifically bind to NY-ESO-1157−165/HLA-A2 and were capable of cytokine secretion with engineered Jurkat T cells and primary T cells upon recognition with K562 target cells expressing the single-chain trimer (SCT) of NY-ESO-1157−165/HLA-A2. The reactivity profiles of the HZ6 and HZ8 TCRs were found to be distinct from one another when co-cultured with K562 target cells carrying alanine-substituted NY-ESO-1157−165 SCTs. The binding characterization revealed that the recognition pattern of the HZ6 TCR to NY-ESO-1157−165/HLA-A2 was substantially different from the widely used 1G4 TCR. These findings would broaden the understanding of immunogenicity of the NY-ESO-1157−165, and the two identified TCRs may serve as promising candidates for the future development of TCR-T therapy for tumors.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

scholarly journals 192 Pooled T cell receptor screening (POTS) provides unbiased, high-throughput method for TCR discovery

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A204-A204
Author(s):  
Jack Reid ◽  
Shihong Zhang ◽  
Ariunaa Munkhbat ◽  
Matyas Ecsedi ◽  
Megan McAfee ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Receptor ◽  
High Throughput ◽  
Ex Vivo ◽  
Cell Receptor ◽  
High Avidity ◽  
Reporter System ◽  
High Throughput Method

BackgroundT Cell Receptor (TCR)-T cell therapies have shown some promising results in cancer clinical trials, however the efficacy of treatment remains suboptimal. Outcomes could potentially be improved by utilizing highly functional TCRs for future trials. Current TCR discovery methods are relatively low throughput and rely on synthesis and screening of individual TCRs based on tetramer binding and peptide specificity, which is costly and labor intensive. We have developed and validated a pooled approach relying on directly cloned TCRs transduced into a fluorescent Jurkat reporter system (figure 1). This approach provides an unbiased, high-throughput method for TCR discovery.MethodsAs a model for POTS, T cells specific for a peptide derived adenovirus structural protein were sorted on tetramer and subjected to 10x single cell VDJ analysis. Pools of randomly paired TCR alpha and beta chains were cloned from the 10x cDNA into a lentiviral vector and transduced into a Jurkat reporter cells. Consecutive stimulations with cognate antigen followed by cell sorts were performed to enrich for functional TCRs. Full length TCRab pools were sequenced by Oxford Nanopore Technologies (ONT) and compared to a 10x dataset to find naturally paired TCRs.ResultsComparison between the ex vivo single cell VDJ sequencing and ONT sequencing of the transduced antigen specific TCRs showed more than 99% of the TCR pairs found in reporter positive Jurkat cells were naturally paired TCRs. The functionality of 8 TCR clonotypes discovered using POTS were compared and clone #2 showed the strongest response. Of the selected clonotypes, clone #2 showed a low frequency of 0.9% in the ex vivo single cell VDJ sequencing. After the first round of stimulation and sequencing, clone #2 takes up of 5% of all reporter-positive clones. The abundance of clone #2 further increased to 17% after another round of stimulation, sorting and sequencing, suggesting this method can retrieve and enrich for highly functional antigen specific TCRs.Abstract 192 Figure 1Outline of the POTS workflow.ConclusionsPOTS provides a high-throughput method for discovery of naturally paired, high-avidity T cell receptors. This method mitigates bias introduced by T cell differentiation state by screening TCRs in a clonal reporter system. Additionally, POTS allows for screening of low abundance clones when compared with traditional TCR discovery techniques. Pooled TCRs could also be screened in vivo with primary T cells in a mouse model to screen for the most functional and physiologically fit TCR for cancer treatment.

scholarly journals CD1: From Molecules to Diseases

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 1909 ◽  
Author(s):  
D. Branch Moody ◽  
Sara Suliman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Cell Receptor ◽  
High Genetic Diversity ◽  
Disease States ◽  
New Research ◽  
Antigen Presenting ◽  
Cluster Of Differentiation

The human cluster of differentiation (CD)1 system for antigen display is comprised of four types of antigen-presenting molecules, each with a distinct functional niche: CD1a, CD1b, CD1c, and CD1d. Whereas CD1 proteins were thought solely to influence T-cell responses through display of amphipathic lipids, recent studies emphasize the role of direct contacts between the T-cell receptor and CD1 itself. Moving from molecules to diseases, new research approaches emphasize human CD1-transgenic mouse models and the study of human polyclonal T cells in vivo or ex vivo in disease states. Whereas the high genetic diversity of major histocompatibility complex (MHC)-encoded antigen-presenting molecules provides a major hurdle for designing antigens that activate T cells in all humans, the simple population genetics of the CD1 system offers the prospect of discovering or designing broadly acting immunomodulatory agents.

scholarly journals Activation-induced Modification in the CD3 Complex of the γδ T Cell Receptor

2002 ◽  
Vol 196 (10) ◽  
pp. 1355-1361 ◽  
Author(s):  
Sandra M. Hayes ◽  
Karen Laky ◽  
Dalal El-Khoury ◽  
Dietmar J. Kappes ◽  
B.J. Fowlkes ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Cell Receptor ◽  
Intraepithelial Lymphocytes ◽  
Subunit Composition ◽  
Γδ T Cell ◽  
Receptor Complexes ◽  
Functional Consequences

The T cell antigen receptor complexes expressed on αβ and γδ T cells differ not only in their respective clonotypic heterodimers but also in the subunit composition of their CD3 complexes. The γδ T cell receptors (TCRs) expressed on ex vivo γδ T cells lack CD3δ, whereas αβ TCRs contain CD3δ. While this result correlates with the phenotype of CD3δ−/− mice, in which γδ T cell development is unaffected, it is inconsistent with the results of previous studies reporting that CD3δ is a component of the γδ TCR. Since earlier studies examined the subunit composition of γδ TCRs expressed on activated and expanded peripheral γδ T cells or γδ TCR+ intestinal intraepithelial lymphocytes, we hypothesized that activation and expansion may lead to changes in the CD3 subunit composition of the γδ TCR. Here, we report that activation and expansion do in fact result in the inclusion of a protein, comparable in mass and mobility to CD3δ, in the γδ TCR. Further analyses revealed that this protein is not CD3δ, but instead is a differentially glycosylated form of CD3γ. These results provide further evidence for a major difference in the subunit composition of αβ- and γδ TCR complexes and raise the possibility that modification of CD3γ may have important functional consequences in activated γδ T cells.

Repertoire Analysis of Ex Vivo Polyclonally Expanded Regulatory T Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3222-3222
Author(s):  
Jenny Zilberberg ◽  
Kira Goldgirsh ◽  
Robert Korngold ◽  
Thea M. Friedman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Cell Receptor ◽  
Clinical Situation ◽  
Post Transplantation ◽  
Cell Repertoire ◽  
Hematopoietic Stem ◽  
Pcr Products

Abstract CD4+CD25+ regulatory T cells (Treg) are essential for the maintenance of self-tolerance and have also been implicated in the control of alloreactive immune responses. Several studies using murine models of graft-vs.-host disease (GVHD) have shown that addition of equivalent numbers of Treg to the donor T cell inoculum at time of hematopoietic stem cell transplantation can significantly reduce the incidence of GVHD. In addition, in an MHC-matched, minor histocompatibility disparate model, the infusion of Treg ten days post-transplantation was shown to ameliorate the progression of GVHD while permitting a graft-versus-leukemia effect. However, because Treg constitute <5% of peripheral CD4+ T cells in humans, the use of freshly isolated Treg to treat and/or prevent GVHD, as well as other diseases in the clinical situation, is limited. Therefore, much effort is now under way to expand Treg in order to have sufficient numbers for therapeutic use. There is little available information regarding the repertoire complexity of ex vivo, polyclonally expanded regulatory T cells. We hypothesize that like their CD4+CD25− T cell counterparts, the diversity of the Treg T cell receptor (TCR) repertoire will also be complex. To this end, CD4+CD25− and CD4+CD25+ T cells from B10.BR mice were purified using fluorescence activated cell sorting; both populations were polyclonally expanded using CD3/CD28 paramagnetic microbeads in combination with high levels (100 IU/ml) of hrIL-2. After achieving a greater than 50 fold expansion, RNA from 1–1.5×107 cells was isolated for RT-PCR. The complexity of the T cell repertoire of expanded CD4+CD25− and CD4+CD25+ was determined using TCR Vb CDR3-size spectratype analysis. The PCR products were run on a sequencing gel and analyzed by the GeneMapper Software from Applied Biosystems. This comparison revealed that the number of resolvable Vb families is more heterogeneous in the CD25− populations. Whether this reflected a lack of complexity in the regulatory repertoire warrants further investigation. However, for the resolvable Vb families there were no significant differences in the complexity indexes between these two groups.

Safe and Effective Adoptive T-Cell Receptor Transfer with a High Affinity Single Chain p53(264–272)-Specific TCR

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4226-4226
Author(s):  
Hakim Echchannaoui ◽  
Jutta Petschenka ◽  
Edite Antunes ◽  
Matthias Theobald
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Cell Transfer ◽  
Single Chain ◽  
Adoptive T Cell Transfer ◽  
High Affinity ◽  
T Cell Transfer

Abstract Abstract 4226 Several studies have demonstrated the clinical efficacy of adoptive T cell therapy for targeting cancer. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264–272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. A safety concern of TCR gene transfer is the pairing of endogenous and introduced TCR chains resulting in the potential generation of self-reactive T cells (off-target autoimmunity). Several strategies to favor matched TCR chains pairing and thus enhancing TCR cell surface expression, including optimization of TCR encoding nucleotide sequences, introduction of an additional inter-chain disulfide bond between the TCR α and β chain constant domains, coexpression of both TCR α and β encoding-genes using self-cleaving 2A virus peptide-based retroviral vectors have been applied. However, adoptive transfer of mouse T cells transduced with modified p53-specific TCRs into p53-deficient humanized (A2Kb) mice was inducing lethal autoimmunity due to the formation of self-reactive TCRs infiltrating vital organs, such as spleen, liver and bone marrow. Therefore, an optimized single chain (sc) p53-specific TCR was engineered to avoid the formation of mismatched TCR heterodimers. The safety and therapeutic efficiency of this approach were evaluated in humanized mouse models of adoptive T cell transfer and successfully demonstrated that optimized p53-specific scTCR-redirected T cells (i) do not induce OFF-target autoimmunity and (ii) mediate antitumor reactivity. Importantly, because the expression of p53 antigen on normal tissues raises the concern of potential on-target toxicity, we performed adoptive T cell transfer experiments in humanized mice expressing the Human p53 protein (Hupki mice) and did not observe any sign of TCR gene transfer-mediated GvHD in this model. In conclusion, these mouse studies suggest that the optimized p53(264–272)-specific scTCR could represent a safe and efficient approach for TCR-based gene therapy. Disclosures: No relevant conflicts of interest to declare.

Evaluation Of CD33 Expression and Functional Analysis Of The CD33/CD3 Bispecific BiTE® Antibody AMG 330 In Primary AML Samples

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 239-239 ◽  
Author(s):  
Christina Krupka ◽  
Peter Kufer ◽  
Roman Kischel ◽  
Gerhard Zugmaier ◽  
Jan Boegeholz ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Ex Vivo ◽  
Natural Setting ◽  
Single Chain ◽  
Activation Markers ◽  
Expression Levels ◽  
Equity Ownership

Abstract Antibody-based immunotherapy represents a promising strategy to specifically target and eliminate chemoresistant leukemic cells in acute myeloid leukemia (AML). We evaluated a single-chain bispecific CD33/CD3 BiTE® antibody (AMG 330) for its suitability as immunotherapy in AML. A prerequisite for successful immunotherapeutic approaches using this molecule is the expression of CD33 on AML blasts including leukemic stem cells (LSCs). Therefore, we quantified CD33 expression on AML blasts and LSCs by flow cytometry (mean fluorescence intensity, MFI) and correlated expression intensity with cytogenetic and molecular disease characteristics in order to identify patient cohorts possibly most suited for CD33-targeted therapies. CD33 expression was detected in >99% of patient samples (n=621, MFI ratio ≥ 1.5) although highly variable. A strong correlation between high CD33 expression levels and NPM1 mutations (p<0.001) was found. In contrast, low CD33 expression levels were significantly associated with complex karyotypes and t(8,21) translocations (p<0.001). Furthermore, LSCs within the CD34+/CD38- compartment displayed CD33 at higher levels than healthy donor stem cells (p=0.047). Importantly, colony formation of CD34+/Lin-cells from healthy donors was not affected after pre-incubation with AMG 330 and T-cells. A major hurdle for measuring cytotoxic effects on AML blasts has long been that primary AML patient samples show progressive cell death within a few days ex-vivo. To simulate the natural setting of target and T-cells in AML patients, we developed a long-term culture system for AML blasts that allowed us to observe these co-cultures for up to 5 weeks. Thus, we were able to show effective elimination of AML blasts within primary samples by AMG 330-activated and expanded residual CD3+/CD45RA-/CCR7+ memory T-lymphocytes. While the functional relevance of CD33 expression levels was shown by faster lysis kinetics of CD33BRIGHT vs. CD33DIM AML cell lines in an in-vitro cytotoxicity assay potent anti-leukemic activity on primary AML blasts was observed irrespective of CD33 expression level. At low effector to target ratios (up to 1:79), the recruited T-cells lysed autologous blasts completely in the majority of samples. Further T-cell analysis showed that naive T-cells (CD45RA+/CCR7+) were not expanded by AMG 330; neither were terminally differentiated T-cells (CD45RA+/CCR7-), probably due to their poor proliferative capacity. We did not observe an increase in percentage of CD3+/CD4+/CD25+/FoxP3+regulatory T-cells in the presence of AMG 330, suggesting that these cells may not have impacted AMG 330-mediated T-cell activity in our experiments. Compared to control cultures, T-cells were shown to up-regulate the activation markers CD25, PD-1, TIM3 and LAG3 upon response to AMG 330, which was partially reversible after complete target cell elimination. However, PD-1 up-regulation did not correlate with an up-regulation of PD-L1 on AML blasts despite substantial INFγ secretion by activated T-cells. This study provides the first correlation of CD33 expression levels to a comprehensive genotype analysis in adult AML patients. While CD33 expression may vary by AML biologic subgroup, AMG 330 exposure led to lysis of AML blasts even in samples with low levels of expression. Targeting CD33 using AMG 330 in primary AML samples led to efficient T-cell activation and expansion as expected from the mechanism of action of BiTE® antibodies. The remarkable ex-vivo activity of AMG 330 supports further development of AMG 330 as an immunotherapy for patients with AML. Disclosures: Kufer: AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Kischel:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Baeuerle:AMGEN Research (Munich) GmbH: Employment; AMGEN Inc.: Equity Ownership. Riethmüller:AMGEN Inc.: Equity Ownership.

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