Combined CD28 and 4-1BB Costimulation Potentiates Affinity-tuned Chimeric Antigen Receptor-engineered T Cells

Purpose: Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CART) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of lowaffinity CAR-T cells. Experimental Design: We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. Results: We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (K d < 1.9 × 10 -6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. Conclusions: A combinatorial costimulatory design allows the use of very low-affinity binding domains (K d < 1 mmol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs

High-resolution CT phenotypes in pulmonary sarcoidosis: a multinational Delphi consensus study

One view of sarcoidosis is that the term covers many different diseases. However, no classification framework exists for the future exploration of pathogenetic pathways, genetic or trigger predilections, patterns of lung function impairment, or treatment separations, or for the development of diagnostic algorithms or relevant outcome measures. We aimed to establish agreement on high-resolution CT (HRCT) phenotypic separations in sarcoidosis to anchor future CT research through a multinational two-round Delphi consensus process. Delphi participants included members of the Fleischner Society and the World Association of Sarcoidosis and other Granulomatous Disorders, as well as members' nominees. 146 individuals (98 chest physicians, 48 thoracic radiologists) from 28 countries took part, 144 of whom completed both Delphi rounds. After rating of 35 Delphi statements on a five-point Likert scale, consensus was achieved for 22 (63%) statements. There was 97% agreement on the existence of distinct HRCT phenotypes, with seven HRCT phenotypes that were categorised by participants as non-fibrotic or likely to be fibrotic. The international consensus reached in this Delphi exercise justifies the formulation of a CT classification as a basis for the possible definition of separate diseases. Further refinement of phenotypes with rapidly achievable CT studies is now needed to underpin the development of a formal classification of sarcoidosis

Towards safe and effective CD38-CAR T cell therapy for myeloma

Immunotherapy is a promising field within cancer therapy. The recent progresses resulted in 'Immunotherapy for the treatment of cancer' as break-through of the year in 2013. This was partly due to the great successes with Chimeric Antigen Receptor (CAR) T cell therapy. With CAR T cells, recognition takes place via a cleverly synthesized receptor made in the laboratory on the basis of an antibody, which has been made part of the T cell receptor via genetic manipulation. Clinical studies with CAR T cells have shown how strong this anti-tumor weapon is. In order to make CAR T cell therapy accessible to more patients, we focus on different targets, such as CD38 for multiple myeloma. In the past 30 years, the treatment of multiple myeloma has greatly improved, with improved survival rates. Very recent research has shown that antibodies against CD38 have proven clinical benefit. CD38 is a molecule that is present in large numbers (high expression) on multiple myeloma cells. In this thesis we study the possibility to target CARs against CD38 and we studied ways to improve the therapy and reduce toxicity. These important aspects of the development of CAR T cells have been studied and presented in this thesis: towards a safe and applicable CAR T cell treatment for multiple myeloma

Three-Dimensional Modeling of Solid Tumors and Their Microenvironment to Evaluate T Cell Therapy Efficacy In Vitro

Immunotherapy development for solid tumors remains challenging, partially due to a lack of reproducible, cost-effective in vitro three-dimensional (3D) models to mimic the heterogeneous and complex tumor microenvironment. Here, we investigate the cellular anti-tumor reactivity of ab T cells engineered to express a defined gd TCR (TEG A3). For that purpose, we developed a 3D cytotoxicity assay targeting cell linederived spheroids or patient-derived tumor organoids formed in serum-free media. Tumor cell lysis by TEG A3 was monitored using the Incucyte S3 live-cell imaging system with the apoptosis marker caspase 3/7 green and endpoint readouts of IFN-g secretion in the supernatant. The 3D cytotoxicity assay model system was able to adequately demonstrate TEG A3 reactivity toward targets expressing an isoform of CD277 (CD277J). To obtain a more complex heterogeneous tumor microenvironment, patient-derived organoids were mixed with unmatched patient-derived fibroblasts or matched cancer-associated fibroblasts. In all assays, we demonstrated the tumor target specificity of TEG A3, lysing tumor cells within 48 h. Our study demonstrates the utility of complex 3D cytotoxicity assay model systems incorporating the tumor microenvironment in the functional evaluation of T cellbased adoptive immunotherapy, providing a useful platform for early-stage preclinical development of immunotherapies

Preclinical evaluation of invariant natural killer t cells modified with cd38 or bcma chimeric antigen receptors for multiple myeloma

Due to the CD1d restricted recognition of altered glycolipids, Vα24-invariant natural killer T (iNKT) cells are excellent tools for cancer immunotherapy with a significantly reduced risk for graft-versus-host disease when applied as off-the shelf-therapeutics across Human Leukocyte Antigen (HLA) barriers. To maximally harness their therapeutic potential for multiple myeloma (MM) treatment, we here armed iNKT cells with chimeric antigen receptors (CAR) directed against the MM-associated antigen CD38 and the plasma cell specific B cell maturation antigen (BCMA). We demonstrate that both CD38-and BCMA-CAR iNKT cells effectively eliminated MM cells in a CAR-dependent manner, without losing their T cell receptor (TCR)-mediated cytotoxic activity. Importantly, iNKT cells expressing either BCMA-CARs or affinity-optimized CD38-CARs spared nor-mal hematopoietic cells and displayed a Th1-like cytokine profile, indicating their therapeutic util-ity. While the costimulatory domain of CD38-CARs had no influence on the cytotoxic functions of iNKT cells, CARs containing the 4-1BB domain showed a better expansion capacity. Interestingly, when stimulated only via CD1d+ dendritic cells (DCs) loaded with α-galactosylceramide (α-GalCer), both CD38-and BCMA-CAR iNKT cells expanded well, without losing their CAR-or TCR-dependent cytotoxic activities. This suggests the possibility of developing an off-the-shelf therapy with CAR iNKT cells, which might even be boostable in vivo by administration α-GalCer pulsed DCs

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Off-tumor effect of inducible CD38-CAR T cells.

<p>(A) Representative flow cytometry density plots of MM-BM with CD38⁺/CD138⁺ cells (MM) after treatment with inducible mock (+/- 1000 ng/ml DOX for 48 hours) and inducible high affinity (028) CD38-CAR T cells (- DOX or + 1000 ng/ml DOX for 48 hours and 0, 24 or 120 hours after DOX removal). (B) Pooled data obtained from the analysis of five MM patient bone marrow samples (patient 1–5, see for their phenotype data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>) with ~20% MM cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>) were co-incubated with inducible high affinity (028) CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g003" target="_blank">Fig 3A</a>. Shown are the mean CAR-dependent % lysis of MM (CD138⁺/CD38⁺ ; open squares) and % lysis of healthy non-MM cells (CD138^-/CD56^-/CD38^+/-; grey diamonds) by inducible CD38-CAR T cells. Incubated with DOX for 24 hours 1000 ng/ml (upper left), 48 hours 1000 ng/ml (upper right), 24 hours 10 ng/ml (lower left), 48 hours 10 ng/ml (lower right). Presented is the pooled data from 5 independent experiments. Error bars indicate the mean +/- SEM. (Pt 1–5, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>).</p

Off-tumor effect of inducible low affinity CD38-CAR T cells.

<p>MM patient bone marrow samples (n = 4) with ~20% MM cells were co-incubated with inducible low affinity (B1) CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g003" target="_blank">Fig 3A</a>. Depicted are the average CAR-dependent lysis of MM cells (CD138⁺/CD38⁺ ; open squares) and lysis of healthy non-MM cells (CD138^-/CD56^-/CD38^+/- ; grey diamonds) by inducible CD38-CAR T cells. Incubated with DOX for 24 hours 1000 ng/ml (upper left), 48 hours 1000 ng/ml (upper right), 24 hours 10 ng/ml (lower left), 48 hours 10 ng/ml (lower right). Presented is the pooled data from 4 independent experiments. Error bars indicate the mean +/- SEM. (Pt 2–5, same pts as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.g004" target="_blank">Fig 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197349#pone.0197349.s004" target="_blank">S4 Fig</a>).</p

DOX dose-dependent induction of CD38-CAR expression and anti-MM cytotoxicity.

<p>(A) Lysis of luciferase-transduced CD38⁺ MM cell lines UM9 and RPMI8226 after co-incubation with Mock and inducible, high affinity (028) CD38-CAR, after treatment with no or 1000 ng/ml DOX for 48 hours. Grey lines indicate the lysis by constitutively expressed high affinity (028) CD38-CAR T cells The BLI signal from surviving MM cells was measured after 16 hours using a luminometer and the percentage lysis was calculated as indicated in the material & methods. Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD (B) Mean fluorescent intensity (MFI) of the CAR measured by staining with soluble CD38-his after 48 hours incubation with 0, 1, 10, 100 or 1000 ng/ml DOX, Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD. (C) The cytotoxic activity of untreated or DOX treated inducible CD38-CAR T cells against luc+ MM cell line UM9 after 16 hours. Presented is the pooled data from 2 independent experiments. Error bars indicate the mean +/- SD. In all panels * indicates p value <0.05 and ** <0.01 using one-way analysis of variance and subsequent multiple comparison.</p

Induction and decay kinetics of CD38-CAR expression.

<p>(A) schematic overview of CAR induction and decay assay. Black bars indicate the DOX incubation times, gray bars indicate the period of decay after the removal of DOX. (B and C) Representative results of five independent experiments of mean fluorescent intensity (MFI) of the CAR measured by staining with soluble his-tagged CD38 after 6, 24 or 48 hours incubation with (B) 10 or 1000 ng/ml DOX or 6, 24, 48 or 120 hours after washing of DOX (C) (an MFI of 600, observed by Mock cells was considered background expression). (D) A MM patient bone marrow sample with 20% MM cells was co-incubated with inducible, high affinity (028), CD38-CAR T cells (E:T ratio 3:1) treated with DOX according to the schedule depicted in Fig 3A. are the CAR-dependent % lysis of CD138+/CD38+ MM cells (% lysed by CAR—% lysed by Mock). Presented is the representative data of n = 5. (E) Significant Pearson correlation of MFI of CAR expression as detected with soluble CD38 (sCD38) with % lysis of MM cells. High dose DOX R² = 0.60 and p = 0.012, low dose DOX R² = 0.61 and p = 0.015.</p