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.

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.

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.

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.

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.

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.

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.

Engrafting human regulatory T cells with a flexible modular chimeric antigen receptor technology.

As regulatory T cells (Tregs) play a fundamental role in immune homeostasis their adoptive transfer emerged as a promising treatment strategy for inflammation-related diseases. Preclinical animal models underline the superiority of antigen-specific Tregs compared to polyclonal cells. Here, we applied a modular chimeric antigen receptor (CAR) technology called UniCAR for generation of antigen-specific human Tregs. In contrast to conventional CARs, UniCAR-endowed Tregs are indirectly linked to their target cells via a separate targeting module (TM). Thus, transduced Tregs can be applied universally as their antigen-specificity is easily adjusted by TM exchange. Activation of UniCAR-engrafted Tregs occurred in strict dependence on the TM, facilitating a precise control over Treg activity. In order to augment efficacy and safety, different intracellular signaling domains were tested. Both 4-1BB (CD137) and CD28 costimulation induced strong suppressive function of genetically modified Tregs. However, in light of safety issues, UniCARs comprising a CD137-CD3ζ signaling domain emerged as constructs of choice for a clinical application of redirected Tregs. In that regard, Tregs isolated from patients suffering from autoimmune or inflammatory diseases were, for the first time, successfully engineered with UniCAR 137/ζ and efficiently suppressed patient-derived effector cells. Overall, the UniCAR platform represents a promising approach to improve Treg-based immunotherapies for tolerance induction.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

BACKGROUND: Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. METHODS: In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. RESULTS: Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. CONCLUSIONS: Overall, the novel modular system represents a promising tool for multiple tumor targeting.

Redirection of CD4+ and CD8+ T lymphocytes via a novel antibody-based modular targeting system triggers efficient killing of PSCA+ prostate tumor cells.

BACKGROUND: There is still a need for new therapeutic options against prostate cancer. Conventional single-chain bispecific antibodies (bsAbs), that directly cross-link T cells and tumor cells, hold great potential for efficient tumor treatment. However, rapid development of novel bsAbs is hampered by laborious optimization to improve their efficacy and reduce potential side effects. To accelerate the development of a novel antibody tool for the redirection of T cells to different tumor-associated antigens, we recently introduced a modular targeting system. METHODS: We here describe a novel modular system for treatment of prostate cancer by retargeting of T cells to the prostate stem cell antigen (PSCA). Functionality of the novel PSCA-specific modular system was investigated in vitro by T cell activation and chromium release assays as well as in immunodeficient mice. RESULTS: Similar to a conventional bsAb CD3-PSCA, the novel PSCA-specific modular system induces activation of both CD4+ and CD8+ T cells leading to secretion of pro-inflammatory cytokines and highly efficient target-specific tumor cell lysis. The novel TM was ready-to-use from the time point of construction and functional at low E:T ratios and picomolar concentrations without further optimization. In addition, the PSCA-specific modular system delays outgrowth of s.c. tumors in mice comparable to bsAb CD3-PSCA. CONCLUSIONS: We have developed a novel PSCA-specific modular system which triggers an efficient T cell-mediated killing of PSCA+ tumor cells in vitro and in vivo. The new Ab-based targeting strategy can functionally replace conventional bsAbs and allows a flexible redirection of T cells to different tumor-associated antigens.

Retargeting of human regulatory T cells by single-chain bispecific antibodies.

Bispecific Abs hold great potential for immunotherapy of malignant diseases. Because the first components of this new drug class are now entering clinical trials, all aspects of their mode of action should be well understood. Several studies proved that CD8(+) and CD4(+) effector T cells can be successfully redirected and activated against tumor cells by bispecific Abs both in vitro and in vivo. To our knowledge, this study provides the first evidence that bispecific Abs can also redirect and activate regulatory T cells against a surface Ag, independently of their TCR specificity. After cross-linking, via a bispecific Ab, redirected regulatory T cells upregulate the activation markers CD69 and CD25, as well as regulatory T cell-associated markers, like CTLA-4 and FOXP3. The activated regulatory T cells secrete the immunosuppressive cytokine IL-10, but, in contrast to CD8(+) and CD4(+) effector T cells, almost no inflammatory cytokines. In addition, the redirected regulatory T cells are able to suppress effector functions of activated autologous CD4(+) T cells both in vitro and in vivo. Therefore, the potential risk for activation of regulatory T cells should be taken into consideration when bispecific Abs are applied for the treatment of malignant diseases. In contrast, an Ag/tissue-specific redirection of regulatory T cells with bispecific Abs holds great potential for the treatment of autoimmune diseases and graft rejection.

Novel humanized and highly efficient bispecific antibodies mediate killing of prostate stem cell antigen-expressing tumor cells by CD8+ and CD4+ T cells.

Prostate cancer is the most common noncutaneous malignancy in men. The prostate stem cell Ag (PSCA) is a promising target for immunotherapy of advanced disease. Based on a novel mAb directed to PSCA, we established and compared a series of murine and humanized anti-CD3-anti-PSCA single-chain bispecific Abs. Their capability to redirect T cells for killing of tumor cells was analyzed. During these studies, we identified a novel bispecific humanized Ab that efficiently retargets T cells to tumor cells in a strictly Ag-dependent manner and at femtomolar concentrations. T cell activation, cytokine release, and lysis of target cells depend on a cross-linkage of redirected T cells with tumor cells, whereas binding of the anti-CD3 domain alone does not lead to an activation or cytokine release. Interestingly, both CD8+ and CD4+ T cells are activated in parallel and can efficiently mediate the lysis of tumor cells. However, the onset of killing via CD4+ T cells is delayed. Furthermore, redirecting T cells via the novel humanized bispecific Abs results in a delay of tumor growth in xenografted nude mice.

Retargeting of regulatory T cells to surface-inducible autoantigen La/SS-B.

The nuclear autoantigen La can be detected on the surface of dying cells. Here we present an assay which enables us to show that La protein is not limited to the surface of dying cells but will be released upon stress-induced cell death. As released La protein tightly binds to the surface of neighboring intact cells we asked the question whether or not La protein could serve as a stress-inducible target e.g. for redirecting of regulatory T cells (Tregs) into damaged tissues to downregulate an immune response. In order to provide first proof of concept we developed a novel fully humanized single-chain bispecific antibody (bsAb) which on the one hand is directed to the La antigen and on the other hand to the CD3 complex of T cells. A cross-linkage of Tregs with La-decorated target cells mediated by this bsAb resulted indeed in the activation of the Tregs in a target-dependent manner. Moreover, such bsAb activated Tregs displayed a potent suppressive capacity and negatively influenced proliferation, expansion and cytokine production of autologous CD4(+) and CD8(+) Teff cells.

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.

Generation of single-chain bispecific green fluorescent protein fusion antibodies for imaging of antibody-induced T cell synapses.

There is growing interest in the development of novel single-chain bispecific antibodies for retargeting of immune effector T cells to tumor cells. Until today, functional fusion constructs consisting of a single-chain bispecific antibody and a fluorescent protein were not reported. Such molecules could be useful for an in vivo visualization of this retargeting process. Recently, we established two novel single-chain bispecific antibodies. One is capable of retargeting T cells to CD33, and the other is capable of retargeting T cells to the prostate stem cell antigen (PSCA). CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). The PSCA is a potential target on prostate cancer cells. Flanking the reading frame encoding the green fluorescent protein (GFP) with a recently described novel helical linker element allowed us to establish novel single-chain bispecific fusion antibodies. These fluorescent fusion antibodies were useful to efficiently retarget T cells to the respective tumor cells and visualize the formation of immune synapses between effector and target 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.

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.

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.

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.