Palbociclib impairs the proliferative capacity of activated T cells while retaining their cytotoxic efficacy.

The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib is an emerging cancer therapeutic that just recently gained Food and Drug Administration approval for treatment of estrogen receptor (ER)-positive, human epidermal growth factor receptor (Her)2-negative breast cancer in combination with the ER degrader fulvestrant. However, CDK4/6 inhibitors are not cancer-specific and may affect also other proliferating cells. Given the importance of T cells in antitumor defense, we studied the influence of palbociclib/fulvestrant on human CD3+ T cells and novel emerging T cell-based cancer immunotherapies. Palbociclib considerably inhibited the proliferation of activated T cells by mediating G0/G1 cell cycle arrest. However, after stopping the drug supply this suppression was fully reversible. In light of combination approaches, we further investigated the effect of palbociclib/fulvestrant on T cell-based immunotherapies by using a CD3-PSCA bispecific antibody or universal chimeric antigen receptor (UniCAR) T cells. Thereby, we observed that palbociclib clearly impaired T cell expansion. This effect resulted in a lower total concentration of interferon-γ and tumor necrosis factor, while palbociclib did not inhibit the average cytokine release per cell. In addition, the cytotoxic potential of the redirected T cells was unaffected by palbociclib and fulvestrant. Overall, these novel findings may have implications for the design of treatment modalities combining CDK4/6 inhibition and T cell-based cancer immunotherapeutic strategies.

Combining Radiation- with Immunotherapy in Prostate Cancer: Influence of Radiation on T Cells.

Radiation of tumor cells can lead to the selection and outgrowth of tumor escape variants. As radioresistant tumor cells are still sensitive to retargeting of T cells, it appears promising to combine radio- with immunotherapy keeping in mind that the radiation of tumors favors the local conditions for immunotherapy. However, radiation of solid tumors will not only hit the tumor cells but also the infiltrated immune cells. Therefore, we wanted to learn how radiation influences the functionality of T cells with respect to retargeting to tumor cells via a conventional bispecific T cell engager (BiTE) and our previously described modular BiTE format UNImAb. T cells were irradiated between 2 and 50 Gy. Low dose radiation of T cells up to about 20 Gy caused an increased release of the cytokines IL-2, TNF and interferon-γ and an improved capability to kill target cells. Although radiation with 50 Gy strongly reduced the function of the T cells, it did not completely abrogate the functionality of the T cells.

Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy.

Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

T Cell Mediated Conversion of a Non-Anti-La Reactive B Cell to an Autoreactive Anti-La B Cell by Somatic Hypermutation.

Since the first description of nuclear autoantigens in the late 1960s and early 1970s, researchers, including ourselves, have found it difficult to establish monoclonal antibodies (mabs) against nuclear antigens, including the La/SS-B (Sjögrens' syndrome associated antigen B) autoantigen. To date, only a few anti-La mabs have been derived by conventional hybridoma technology; however, those anti-La mabs were not bona fide autoantibodies as they recognize either human La specific, cryptic, or post-translationally modified epitopes which are not accessible on native mouse La protein. Herein, we present a series of novel murine anti-La mabs including truly autoreactive ones. These mabs were elicited from a human La transgenic animal through adoptive transfer of T cells from non-transgenic mice immunized with human La antigen. Detailed epitope and paratope analyses experimentally confirm the hypothesis that somatic hypermutations that occur during T cell dependent maturation can lead to autoreactivity to the nuclear La/SS-B autoantigen.

Versatile chimeric antigen receptor platform for controllable and combinatorial T cell therapy.

Chimeric antigen receptor (CAR) T cells show remarkable therapeutic effects in some hematological malignancies. However, CAR T cells can also cause life-threatening side effects. In order to minimize off-target and on-target/off-tumor reactions, improve safety, enable controllability, provide high flexibility, and increase tumor specificity, we established a novel humanized artificial receptor platform termed RevCARs. RevCAR genes encode for small surface receptors lacking any antigen-binding moiety. Steering of RevCAR T cells occurs via bispecific targeting molecules (TMs). The small size of RevCAR-encoding genes allows the construction of polycistronic vectors. Here, we demonstrate that RevCAR T cells efficiently kill tumor cells, can be steered by TMs, flexibly redirected against multiple targets, and used for combinatorial targeting following the "OR" and "AND" gate logic.

Nanocytometer for smart analysis of peripheral blood and acute myeloid leukemia: a pilot study.

We realize an ultracompact nanocytometer for real-time impedimetric detection and classification of subpopulations of living cells. Nanoscopic nanowires in a microfluidic channel act as nanocapacitors and measure in real time the change of the amplitude and phase of the output voltage and, thus, the electrical properties of living cells. We perform the cell classification in the human peripheral blood (PBMC) and demonstrate for the first time the possibility to discriminate monocytes and subpopulations of lymphocytes in a label-free format. Further, we demonstrate that the PBMC of acute myeloid leukemia and healthy samples grant the label free identification of the disease. Using the algorithm based on machine learning, we generated specific data patterns to discriminate healthy donors and leukemia patients. Such a solution has the potential to improve the traditional diagnostics approaches with respect to the overall cost and time effort, in a label-free format, and restrictions of the complex data analysis.

Highly Efficient Targeting of EGFR-Expressing Tumor Cells with UniCAR T Cells via Target Modules Based on Cetuximab®.

INTRODUCTION: Since epithelial growth factor receptor (EGFR) overexpression is linked to a variety of malignancies, it is an attractive target for immune therapy including chimeric antigen receptor (CAR)-engineered T cells. Unfortunately, CAR T cell therapy harbors the risk of severe, even life-threatening side effects. Adaptor CAR T cell platforms such as the previously described UniCAR system might be able to overcome these problems. In contrast to conventional CARs, UniCAR T cells are per se inert. Their redirection towards target cells occurs only in the presence of a tumor-specific target molecule (TM). TMs are bifunctional molecules being able to recognize a tumor-associated antigen and to cross-link the CAR T cell via a peptide epitope recognized by the UniCAR domain. MATERIALS AND METHODS: Here, we compare αEGFR TMs: a nanobody (nb)-based αEGFR TM derived from the camelid αEGFR antibody 7C12 with a murine and humanized single-chain fragment variable (scFv) based on the clinically used antibody Cetuximab®. RESULTS: In principle, both the nb- and scFv-based TM formats are able to redirect UniCAR T cells to eliminate EGFR-expressing tumor cells in an antigen-specific and TM-dependent manner. However, the scFv-based αEGFR TM was significantly superior to the nb-based TM especially with respect to lysis of tumor cells. DISCUSSION: Improved efficiency of the scFv-based TM allowed the redirection of UniCAR T cells towards tumor cells expressing high as well as low EGFR levels in comparison to nb-based αEGFR TMs.

Extended half-life target module for sustainable UniCAR T-cell treatment of STn-expressing cancers.

BACKGROUND: Adapter chimeric antigen receptor (CAR) approaches have emerged has promising strategies to increase clinical safety of CAR T-cell therapy. In the UniCAR system, the safety switch is controlled via a target module (TM) which is characterized by a small-size and short half-life. The rapid clearance of these TMs from the blood allows a quick steering and self-limiting safety switch of UniCAR T-cells by TM dosing. This is mainly important during onset of therapy when tumor burden and the risk for severe side effects are high. For long-term UniCAR therapy, the continuous infusion of TMs may not be an optimal setting for the patients. Thus, in later stages of treatment, single infusions of TMs with an increased half-life might play an important role in long-term surveillance and eradication of residual tumor cells. Given this, we aimed to develop and characterize a novel TM with extended half-life targeting the tumor-associated carbohydrate sialyl-Tn (STn). METHODS: The extended half-life TM is composed of the STn-specific single-chain variable fragment (scFv) and the UniCAR epitope, fused to the hinge region and Fc domain of a human immunoglobulin 4 (IgG4) antibody. Specific binding and functionality of the αSTn-IgG4 TM as well as pharmacokinetic features were assessed using in vitro and in vivo assays and compared to the already established small-sized αSTn TM. RESULTS: The novel αSTn-IgG4 TM efficiently activates and redirects UniCAR T-cells to STn-expressing tumors in a target-specific and TM-dependent manner, thereby promoting the secretion of proinflammatory cytokines and tumor cell lysis in vitro and in experimental mice. Moreover, PET-imaging results demonstrate the specific enrichment of the αSTn-IgG4 TM at the tumor site, while presenting a prolonged serum half-life compared to the short-lived αSTn TM. CONCLUSION: In a clinical setting, the combination of TMs with different formats and pharmacokinetics may represent a promising strategy for retargeting of UniCAR T-cells in a flexible, individualized and safe manner at particular stages of therapy. Furthermore, as these molecules can be used for in vivo imaging, they pose as attractive candidates for theranostic approaches.

UniCAR T cell immunotherapy enables efficient elimination of radioresistant cancer cells.

Induction or selection of radioresistant cancer (stem) cells following standard radiotherapy is presumably one of the major causes for recurrence of metastatic disease. One possibility to prevent tumor relapse is the application of targeted immunotherapies including, e.g., chimeric antigen receptor (CAR) T cells. In light of long-term remissions, it is highly relevant to clarify whether radioresistant cancer cells are susceptible to CAR T cell-mediated killing. To answer this question, we evaluated the anti-tumor activity of the switchable universal chimeric antigen receptor (UniCAR) system against highly radioresistant head and neck squamous cell carcinoma cells both in vitro and in vivo. Following specific UniCAR T cell engagement via EGFR or CD98 target modules, T cell effector mechanisms were induced including secretion of pro-inflammatory cytokines, up-regulation of granzyme B and perforin, as well as T cell proliferation. CD98- or EGFR-redirected UniCAR T cells further possess the capability to efficiently lyse radioresistant tumor cells. Observed anti-tumor effects were comparable to those against the radiosensitive parental cell lines. Finally, redirected UniCAR T cells significantly inhibited the growth of radioresistant cancer cells in immunodeficient mice. Taken together, our obtained data underline that the UniCAR system is able to overcome radioresistance. Thus, it represents an attractive technology for the development of combined radioimmunotherapeutic approaches that might improve the outcome of patients with metastatic radioresistant tumor diseases.

Adaptor CAR Platforms-Next Generation of T Cell-Based Cancer Immunotherapy.

The success of conventional chimeric antigen receptor (CAR) therapy in the treatment of refractory hematologic malignancies has triggered the development of novel exciting experimental CAR technologies. Among them, adaptor CAR platforms have received much attention. They combine the flexibility and controllability of recombinant antibodies with the power of CARs. Due to their modular design, adaptor CAR systems propose answers to the central problems of conventional CAR therapy, such as safety and antigen escape. This review provides an overview on the different adaptor CAR platforms available, discusses the possibilities and challenges of adaptor CAR therapy, and summarizes the first clinical experiences.

"UniCAR"-modified off-the-shelf NK-92 cells for targeting of GD2-expressing tumour cells.

Antigen-specific redirection of immune effector cells with chimeric antigen receptors (CARs) demonstrated high therapeutic potential for targeting cancers of different origins. Beside CAR-T cells, natural killer (NK) cells represent promising alternative effectors that can be combined with CAR technology. Unlike T cells, primary NK cells and the NK cell line NK-92 can be applied as allogeneic off-the-shelf products with a reduced risk of toxicities. We previously established a modular universal CAR (UniCAR) platform which consists of UniCAR-expressing immune cells that cannot recognize target antigens directly but are redirected by a tumour-specific target module (TM). The TM contains an antigen-binding moiety fused to a peptide epitope which is recognized by the UniCAR molecule, thereby allowing an on/off switch of CAR activity, and facilitating flexible targeting of various tumour antigens depending on the presence and specificity of the TM. Here, we provide proof of concept that it is feasible to generate a universal off-the-shelf cellular therapeutic based on UniCAR NK-92 cells targeted to tumours expressing the disialoganglioside GD2 by GD2-specific TMs that are either based on an antibody-derived single-chain fragment variable (scFv) or an IgG4 backbone. Redirected UniCAR NK-92 cells induced specific killing of GD2-expressing cells in vitro and in vivo, associated with enhanced production of interferon-γ. Analysis of radiolabelled proteins demonstrated that the IgG4-based format increased the in vivo half-life of the TM markedly in comparison to the scFv-based molecule. In summary, UniCAR NK-92 cells represent a universal off-the-shelf platform that is highly effective and flexible, allowing the use of different TM formats for specific tumour targeting.

A theranostic PSMA ligand for PET imaging and retargeting of T cells expressing the universal chimeric antigen receptor UniCAR.

Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics.

Conventional CARs versus modular CARs.

The clinical application of immune effector cells genetically modified to express chimeric antigen receptors (CARs) has shown impressive results including complete remissions of certain malignant hematological diseases. However, their application can also cause severe side effects such as cytokine release syndrome (CRS) or tumor lysis syndrome (TLS). One limitation of currently applied CAR T cells is their lack of regulation. Especially, an emergency shutdown of CAR T cells in case of life-threatening side effects is missing. Moreover, targeting of tumor-associated antigens (TAAs) that are not only expressed on tumor cells but also on vital tissues requires the possibility of a switch allowing to repeatedly turn the activity of CAR T cells on and off. Here we summarize the development of a modular CAR variant termed universal CAR (UniCAR) system that promises to overcome these limitations of conventional CARs.

Anti-CAR-engineered T cells for epitope-based elimination of autologous CAR T cells.

Although CAR T-cell therapy has demonstrated tremendous clinical efficacy especially in hematological malignancies, severe treatment-associated toxicities still compromise the widespread application of this innovative technology. Therefore, developing novel approaches to abrogate CAR T-cell-mediated side effects is of great relevance. Several promising strategies pursue the selective antibody-based depletion of adoptively transferred T cells via elimination markers. However, given the limited half-life and tissue penetration, dependence on the patients' immune system and on-target/off-side effects of proposed monoclonal antibodies, we sought to exploit αCAR-engineered T cells to efficiently eliminate CAR T cells. For comprehensive and specific recognition, a small peptide epitope (E-tag) was incorporated into the extracellular spacer region of CAR constructs. We provide first proof-of-concept for targeting this epitope by αE-tag CAR T cells, allowing an effective killing of autologous E-tagged CAR T cells both in vitro and in vivo whilst sparing cells lacking the E-tag. In addition to CAR T-cell cytotoxicity, the αE-tag-specific T cells can be empowered with cancer-fighting ability in case of relapse, hence, have versatile utility. Our proposed methodology can most probably be implemented in CAR T-cell therapies regardless of the targeted tumor antigen aiding in improving overall safety and survival control of highly potent gene-modified cells.

T cells engrafted with a UniCAR 28/z outperform UniCAR BB/z-transduced T cells in the face of regulatory T cell-mediated immunosuppression.

Adoptive transfer of chimeric antigen receptor (CAR)-equipped T cells have demonstrated astonishing clinical efficacy in hematological malignancies recently culminating in the approval of two CAR T cell products. Despite this tremendous success, CAR T cell approaches have still achieved only moderate efficacy against solid tumors. As a major obstacle, engineered conventional T cells (Tconvs) face an anti-inflammatory, hostile tumor microenvironment often infiltrated by highly suppressive regulatory T cells (Tregs). Thus, potent CAR T cell treatment of solid tumors requires efficient activation of Tconvs via their engrafted CAR to overcome Treg-mediated immunosuppression. In that regard, selecting an optimal intracellular signaling domain might represent a crucial step to achieve best clinical efficiency. To shed light on this issue and to investigate responsiveness to Treg inhibition, we engrafted Tconvs with switchable universal CARs (UniCARs) harboring intracellularly the CD3ζ domain alone or in combination with costimulatory CD28 or 4-1BB. Our studies reveal that UniCAR ζ-, and UniCAR BB/ζ-engineered Tconvs are strongly impaired by activated Tregs, whereas UniCARs providing CD28 costimulation overcome Treg-mediated suppression both in vitro and in vivo. Compared to UniCAR ζ- and UniCAR BB/ζ-modified cells, UniCAR 28/ζ-armed Tconvs secrete significantly higher amounts of Th1-related cytokines and, furthermore, levels of these cytokines are elevated even upon exposure to Tregs. Thus, in contrast to 4-1BB costimulation, CD28 signaling in UniCAR-transduced Tconvs seems to foster a pro-inflammatory milieu, which contributes to enhanced resistance to Treg suppression. Overall, our results may have significant implications for CAR T cell-based immunotherapies of solid tumors strongly invaded by Tregs.

An oligo-His-tag of a targeting module does not influence its biodistribution and the retargeting capabilities of UniCAR T cells.

Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised. In order to facilitate their purification all these TMs are expressed as recombinant proteins equipped with an oligo-His-tag. The aim of the here presented manuscript was to learn whether or not the oligo-His-tag of the TM influences the UniCAR system. For this purpose, we constructed TMs against PSCA equipped with or lacking an oligo-His-tag. Both TMs were compared side by side including for functionality and biodistribution. According to our data, an oligo-His-tag of a UniCAR TM has only little if any effect on its binding affinity, in vitro and in vivo killing capability and in vivo biodistribution.

Midostaurin abrogates CD33-directed UniCAR and CD33-CD3 bispecific antibody therapy in acute myeloid leukaemia.

Combinatory therapeutic approaches of different targeted therapies in acute myeloid leukaemia are currently under preclinical/early clinical investigation. To enhance anti-tumour effects, we combined the tyrosine kinase inhibitor (TKI) midostaurin and T-cell mediated immunotherapy directed against CD33. Clinically relevant concentrations of midostaurin abrogated T-cell mediated cytotoxicity both after activation with bispecific antibodies and chimeric antigen receptor T cells. This information is of relevance for clinicians exploring T-cell mediated immunotherapy in early clinical trials. Given the profound inhibition of T-cell functionality and anti-tumour activity, we recommend specific FLT3 TKIs for further clinical testing of combinatory approaches with T-cell based immunotherapy.

Theranostic CAR T cell targeting: A brief review.

More than hundred years ago, Paul Ehrlich postulated that our immune system should be able to recognize tumor cells. Just recently, the development of check point inhibitors, bispecific antibodies, and T cells genetically modified to express chimeric antigen receptors (CARs) underlines the true power of our immune system. T cells genetically modified with CARs can lead to complete remission of malignant hematologic diseases. However, they can also cause life-threatening side effects. In case of cytokine release syndrome, tumor lysis syndrome, or deadly side effects on the central nervous system, an emergency shut down of CAR T cells is needed. Targeting of tumor-associated antigens that are also expressed on vital tissues require a possibility to repeatedly switch the activity of CAR T cells on and off on demand and to follow the treatment by imaging. Theranostic, modular CARs such as the UniCAR system may help to overcome these problems.

Tonic Signaling and Its Effects on Lymphopoiesis of CAR-Armed Hematopoietic Stem and Progenitor Cells.

Long-term survival of adoptively transferred chimeric Ag receptor (CAR) T cells is often limited. Transplantation of hematopoietic stem cells (HSCs) transduced to express CARs could help to overcome this problem as CAR-armed HSCs can continuously deliver CAR+ multicell lineages (e.g., T cells, NK cells). In dependence on the CAR construct, a variable extent of tonic signaling in CAR T cells was reported; thus, effects of CAR-mediated tonic signaling on the hematopoiesis of CAR-armed HSCs is unclear. To assess the effects of tonic signaling, two CAR constructs were established and analyzed 1) a signaling CAR inducing a solid Ag-independent tonic signaling termed CAR-28/ζ and 2) a nonstimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow cells from immunocompetent mice were isolated, purified for HSC-containing Lin-cKit+ cells or the Lin-cKit+ Sca-1+ subpopulation (Lin-Sca-1+cKit+), and transduced with both CAR constructs. Subsequently, modified bone marrow cells were transferred into irradiated mice, in which they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ζ led to elimination of mature CAR+ T and B cells, suggesting that the CAR-mediated tonic signaling mimics autorecognition via the newly recombined immune receptors in the developing lymphocytes.

Native Polyacrylamide Gels.

Proteins can easily be separated by polyacrylamide gel electrophoresis (PAGE) in the presence of a detergent and under (heat-) denaturing and (non- or) reducing conditions. The most commonly used detergent is sodium dodecyl sulfate (SDS). The major function of SDS is to shield the respective charge of the proteins present in the mixture to be analyzed and to provide all proteins with a negative charge. As a consequence, the proteins will be separated according to their molecular weight. Electrophoresis of proteins can also be performed in the absence of SDS. Using such "native" conditions, the charge of each of the proteins, which will depend on the primary amino acid sequence of the protein (isoelectric point) and the pH during electrophoresis, will mainly influence the mobility of the respective protein during electrophoresis. Here we describe a starting protocol for "native" PAGE.