Search

Although Chimeric Antigen Receptor (CAR) T-cells have shown high efficacy in hematologic malignancies, they can cause severe to life-threatening side effects. To address these safety concerns, we have developed adaptor CAR platforms, like the UniCAR system. The redirection of UniCAR T-cells to target cells relies on a Target Module (TM), containing the E5B9 epitope and a tumor-specific binding moiety. Appropriate UniCAR-T activation thus involves two interactions: between the TM and the CAR T-cell, and the TM and the target cell. Here, we investigate if and how alterations of the amino acid sequence of the E5B9 UniCAR epitope impact the interaction between TMs and the UniCAR.We identify the new epitope E5B9L, for which the monoclonal antibody 5B9 has the greatest affinity. We then integrate the E5B9L peptide in previously established TMs directed to Fibroblast Activation Protein (FAP) and assess if such changes in the UniCAR epitope of the TMs affect UniCAR T-cell potency. Binding properties of the newly generated anti-FAP-E5B9L TMs to UniCAR and their ability to redirect UniCAR T-cells were compared side-by-side with the ones of anti-FAP-E5B9 TMs. Despite a substantial variation in the affinity of the different TMs to the UniCAR, no significant differences were observed in the cytotoxic and cytokine-release profiles of the redirected T-cells. Overall, our work indicates that increasing affinity of the UniCAR to the TM does not play a crucial role in such adaptor CAR system, as it does not significantly impact the potency of the UniCAR T-cells.

Discussed are two picolinate appended bispidine ligands (3,7-diazabicyclo[3.3.1]nonane derivatives) in comparison with an earlier described bis-pyridine derivative, which are all known to strongly bind CuII. The radiopharmacological characterization of the two isomeric bispidine complexes includes quantitative labeling with 64CuII at ambient conditions with high radiochemical purities and yields (molar activities > 200 MBq/nmol). Challenge experiments in presence of EDTA, cyclam, human serum and SOD demonstrate high stability and inertness of the 64Cu-bispidine complexes. Biodistribution studies performed in Wistar rats indicate a rapid renal elimination for both 64Cu-labeled chelates. The bispidine ligand with the picolinate group in N7 position was selected for further biological experiments, and its backbone was therefore substituted with a benzyl-NCS group at C9. Two tumor target modules (TMs), targeting prostate stem cell antigen (PSCA), overexpressed in prostate cancer, and the fibroblast activation protein (FAP) in fibrosarcoma, were selected for thiourea coupling with the NCS-functionalized ligand and lysine residues of TMs. Small animal PET experiments on tumor-bearing mice showed very good and specific accumulation of the 64Cu-labeled TMs in tumors.

Immunotherapies are already successfully used for cancer treatment. In our lab two modular platform technologies were developed which demonstrated high success against various types of malignant cells: the universal chimeric antigen receptor (UniCAR) system, and the bispecific antibody (bsAb) based UniMAB system. Both approaches have the aim to recruit T cells to kill the malignant cells carrying a tumor associated antigen (TAA) on their surface. In the first case, UniCAR modified T cells are linked via a TAA specific target module (TM) to cancer cells. In the case of the UniMAB system, natural T cells are recruited to cancer cells via a TAA specific TM and a bsAb based effector module (EM) which binds to CD3 of T cells and to an epitope tag of the TM. In both cases, an immune complex is formed leading to T cell activation and tumor cell elimination. Most importantly, the T cell activation is dependent on the cross-linkage of T cells and tumor cells via the TM or TM/EM complex. Thus, T cell activation is steerable which increases the safety of these approaches. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to millions of deaths worldwide, we aimed to apply the UniCAR and UniMAB technology against SARS-CoV-2 and virus infected cells as novel immunotherapeutic treatment approach. For this purpose, we designed two TMs binding to the receptor binding domain (RBD) of SARS-CoV-2. The first TM is based on a single chain fragment variable (scFv) with specificity for RBD, whereas the second one contains the extracellular domain of the human ACE2 receptor which is the entry receptor of SARS-CoV-2. Both TMs were able to efficiently recruit either UniCAR T cells or, in combination with the EM of the UniMAB system, natural T cells to efficiently kill human cells having the RBD of SARS-CoV-2 on their surface. Remarkably, the ACE2-Mb-TM is additionally able to block RBD/ACE2 interaction and potently neutralizes pseudo- as well as live S

Immunotherapies are already successfully used for cancer treatment. In our lab two modular platform technologies were developed which demonstrated high success against various types of malignant cells: the universal chimeric antigen receptor (UniCAR) system, and the bispecific antibody (bsAb) based UniMAB system. Both approaches have the aim to recruit T cells to kill the malignant cells carrying a tumor associated antigen (TAA) on their surface. In the first case, UniCAR modified T cells are linked via a TAA specific target module (TM) to cancer cells. In the case of the UniMAB system, natural T cells are recruited to cancer cells via a TAA specific TM and a bsAb based effector module (EM) which binds to CD3 of T cells and to an epitope tag of the TM. In both cases, an immune complex is formed leading to T cell activation and tumor cell elimination. Most importantly, the T cell activation is dependent on the cross-linkage of T cells and tumor cells via the TM or TM/EM complex. Thus, T cell activation is steerable which increases the safety of these approaches. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leading to millions of deaths worldwide, we aimed to apply the UniCAR and UniMAB technology against SARS-CoV-2 and virus infected cells as novel immunotherapeutic treatment approach. For this purpose, we designed two TMs binding to the receptor binding domain (RBD) of SARS-CoV-2. The first TM is based on a single chain fragment variable (scFv) with specificity for RBD, whereas the second one contains the extracellular domain of the human ACE2 receptor which is the entry receptor of SARS-CoV-2. Both TMs were able to efficiently recruit either UniCAR T cells or, in combination with the EM of the UniMAB system, natural T cells to efficiently kill human cells having the RBD of SARS-CoV-2 on their surface. Remarkably, the ACE2-Mb-TM is additionally able to block RBD/ACE2 interaction and potently neutralizes pseudo- as well as live S

Chimeric antigen receptors (CARs) are used for redirection of T cells against tumor cells and have demonstrated remarkable therapeutic effects against some hematological cancers. The modular system termed RevCAR can overcome the dangerous side effects associated with conventional CAR T cell therapy, such as cytokine release syndrome. It is composed of RevCAR T cells and a bispecific molecule target module, termed RevTM. This system is highly safe since RevCAR T cell activity can be controlled by RevTM and immediately switched off if side effects are detected. It is also versatile, since the same RevCAR T cell can be directed to different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, to improve the effectiveness in the treatment of solid tumors, the immunosuppressive tumor microenvironment (TME) still needs to be addressed. For this purpose, we have developed novel RevTMs targeting immune checkpoint molecules such as PD-L1, which are often upregulated by cancer cells to escape the immune cells. We have demonstrated that our novel RevTMs can redirect RevCAR T cells to specifically kill cell lines expressing immune checkpoint molecules. In addition to this strategy, the combinatorial targeting of a TAA and an immune checkpoint is very promising strategy to overcome the immunosuppressive microenvironment of solid tumors and thus to improve the treatment outcome.

The La-protein is a multifunctional abundantly expressed protein in the nucleus of all human cells, where it for example functions as a chaperone for newly synthesized RNA-polymerase III transcripts. Recently, it has been shown that La is overexpressed in malignant cells and can be translocated to the cell surface under certain conditions, e. g. radio- or chemotherapy-induced necrosis, where it becomes a promising, inducible target for so-called “Death Targeting” approaches. The aim of this study was to investigate whether the La-protein could also be used as an inducible target for CAR T cell therapy. Therefore, three different CAR T cells directed against the La-protein (La-CARs) were developed and tested regarding their potential for “Death Targeting” of tumor cells. For construction of the La-CARs, we selected three different anti-La mAbs (5B9, 7B6 and 312B), which recognize distinct epitopes within the La-protein in a redox-dependent manner. This allowed us to determine, if the redox-state influences the ability of the La-CARs to target cell surface-bound La-protein. We first examined if La-CARs could eliminate tumor cells that were artificially labeled with wildtype La-protein or its oxidation-resistant triple-cysteine-mutant (TCM). By performing luciferase-based killing assays, we showed, that all La-CARs were specifically activated for lysis of La- or TCM-decorated tumor cells, which also resulted in significant secretion of pro-inflammatory cytokines. Redox-dependent differences of the La-CARs could be observed, whereby the non-redox-dependent 5B9-CAR was activated by La-wt- and TCM-labeled cells, the 7B6-CAR preferably by La-wt-labeled and the 312B-CAR preferably by TCM-labeled tumor cells. We could further prove that La-release from tumor cells could be induced by treatment with the cytotoxic drug cisplatin and La-CARs could be redirected against remaining tumor-cells after cisplatin treatment. Thereby, the 5B9-CAR, which recognizes La-protein independent

Immunotherapy based on chimeric antigen receptor (CAR) T-cells has demonstrated remarkable therapeutic effects, particularly against some hematological cancers. A versatile adaptor CAR system called RevCAR, consisting of RevCAR T-cells and a bispecific target module (RevTM) has been developed to overcome severe side effects associated with conventional CAR T-cell therapy. As the activity of RevCAR T-cells can be steered based on the availability of RevTM, working as an on/off switch, the system can be immediately turned off if side effects occur. Furthermore, the RevCAR system is highly flexible, since the same RevCAR T-cell can be directed towards different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, the effectiveness of CAR T-cells against solid tumors remains limited particularly due to their immunosuppressive tumor microenvironment (TME). To overcome these hurdles, we have established a novel RevCAR system targeting immune checkpoint molecules such as PD-L1, which are frequently overexpressed by cancer cells to suppress immune responses. We have constructed novel RevTMs that can redirect RevCAR T-cells to kill tumor cells that express such immune checkpoint molecules. Furthermore, true AND gate tumor targeting was achieved by targeting a TAA in addition to PD-L1 in a combinatorial manner using our Dual-RevCAR system. In this way, targeting PD-L1 not only results in Dual-RevCAR T-cell activation but simultaneously blocks the immunosuppressive PD-L1/PD-1 axis. Altogether, we have turned an immunosuppressive marker into an immune-activating signal that might modulate the TME in a beneficial manner, showing promise for the development of an effective immunotherapy against solid tumors.

This presentation was given in Lisbon for the first OncoProTools Summer School.

Chimeric antigen receptor (CAR)-engineered immune effector cells constitute a promising approach for adoptive cancer immunotherapy. Nevertheless, on-target/off-tumor toxicity and immune escape due to antigen loss represent considerable challenges. These may be overcome by adaptor CARs that are selectively triggered by bispecific molecules that crosslink the CAR with a tumor-associated surface antigen. Here, we generated NK cells carrying a first- or second-generation universal CAR (UniCAR) and redirected them to tumor cells with so-called target modules (TMs) which harbor an ErbB2 (HER2)-specific antibody domain for target cell binding and the E5B9 peptide recognized by the UniCAR. To investigate differential effects of the protein design on activity, we developed homodimeric TMs with one, two or three E5B9 peptides per monomer, and binding domains either directly linked or separated by an IgG4 Fc domain. The adaptor molecules were expressed as secreted proteins in Expi293F cells, purified from culture supernatants and their bispecific binding to UniCAR and ErbB2 was confirmed by flow cytometry. In cell killing experiments, all tested TMs redirected NK cell cytotoxicity selectively to ErbB2-positive tumor cells. Nevertheless, we found considerable differences in the extent of specific cell killing depending on TM design and CAR composition, with adaptor proteins carrying two or three E5B9 epitopes being more effective when combined with NK cells expressing the first-generation UniCAR, while the second-generation UniCAR was more active in the presence of TMs with one E5B9 sequence. These results may have important implications for the further development of optimized UniCAR and target module combinations for cancer immunotherapy.

Next-generation Radioimmunotherapy using CAR T Cells Combined with Photon vs. Proton vs. Carbon Irradiation

The COVID-19 pandemic caused by SARS-CoV-2 led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options, which can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2 infected animal models, but also for (ii) clearance of virus infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Despite the success of chimeric antigen receptor (CAR) T-cells especially for treating hematological malignancies, critical drawbacks, such as “on-target, off-tumor” toxicities, need to be addressed to improve safety in translating to clinical application. This is especially true, when targeting tumor-associated antigens (TAAs) that are not exclusively expressed by solid tumors but also on healthy tissues. To improve the safety profile, we developed switchable adaptor CAR systems including the RevCAR system. RevCAR T-cells are activated by cross-linking of bifunctional adaptor molecules termed target modules (RevTM). In a further development, we established a Dual-RevCAR system for an AND-gated combinatorial targeting by splitting the stimulatory and co-stimulatory signals of the RevCAR T-cells on two individual CARs. Examples of common markers for colorectal cancer (CRC) are the carcinoembryonic antigen (CEA) and the epithelial cell adhesion molecule (EpCAM), while these antigens are also expressed by healthy cells. Here we describe four novel structurally different RevTMs for targeting of CEA and EpCAM. All anti-CEA and anti-EpCAM RevTMs were validated and the simultaneous targeting of CEA+ and EpCAM+ cancer cells redirected specific in vitro and in vivo killing by Dual-RevCAR T-cells. In summary, we describe the development of CEA and EpCAM specific adaptor RevTMs for monospecific and AND-gated targeting of CRC cells via the RevCAR platform as an improved approach to increase tumor specificity and safety of CAR T-cell therapies.

Adaptor chimeric antigen receptor (CAR) T-cell therapy offers solutions for improved safety and antigen escape which represent main obstacles for the clinical translation of CAR T-cell therapy in myeloid malignancies. The adaptor CAR T-cell platform “UniCAR” is currently under early clinical investigation. Recently, first proof-of-concept of a well-tolerated, rapidly switchable, CD123-directed UniCAR T-cell product treating patients with acute myeloid leukaemia (AML) was reported. Relapsed and refractory AML is prone to a high plasticity under therapy pressure targeting one single tumour antigen. Thus, targeting of multiple tumour antigens seems to be required to achieve durable anti-tumour responses, underlining the need to further design alternative AML-specific target modules (TM) for the UniCAR platform. We here present the preclinical development of a novel FMS-like tyrosine kinase 3 (FLT3)-directed UniCAR T-cell therapy, which is highly effective for in vitro killing of both AML cell lines and primary AML samples. Furthermore, we show in vivo functionality in a murine xenograft model. PET analyses further demonstrate a short serum half-life of FLT3 TMs, which will enable a rapid on/off-switch of UniCAR T-cells. Overall, the presented preclinical data encourage the further development and clinical translation of FLT3-specific UniCAR T-cells for the therapy of AML.

Background: Chimeric antigen receptor (CAR) T-cells are a promising approach in cancer immunotherapy, particularly for treating hematologic malignancies. Yet, their effectiveness is limited when tackling solid tumors, where immune cell infiltration and immunosuppressive tumor microenvironments (TME) are major hurdles. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) and various tumor cells, playing an important role in tumor growth and immunosuppression. Aiming to modulate the TME with increased clinical safety and effectiveness, we developed novel small and size-extended immunotheranostic UniCAR target modules (TMs) targeting FAP. Methods: The specific binding and functionality of the anti-FAP-scFv TM and the size extended anti-FAP-IgG4 TM were assessed using 2D and 3D in vitro models as well as in vivo. Their specific tumor accumulation and diagnostic potential was evaluated using PET studies after functionalization with a chelator and suitable radionuclide. Results: The anti-FAP-scFv and -IgG4 TMs effectively and specifically redirected UniCAR T-cells using 2D, 3D, and in vivo models. Moreover, a remarkably high and specific accumulation of radiolabeled FAP-targeting TMs at the tumor site of xenograft mouse models was observed. Conclusions: These findings demonstrate that the novel anti-FAP TMs are promising immunotheranostic tools to foster cancer imaging and treatment, paving the way for a more convenient, individualized, and safer treatment of cancer patients.

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-g 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.

Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hor-mone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor pro-gression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide 177Lu. The re-sulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images re-vealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.

Glioblastoma (GBM) is still an incurable tumor that is associated with high recurrence rate and poor survival despite the current treatment regimes. With the urgent need for novel therapeutic strategies, immunotherapies, especially chimeric antigen receptor (CAR)-expressing T cells, represent a promising approach for specific and effective targeting of GBM. However, CAR T cells can be associated with serious side effects. To overcome such limitation, we applied our switchable RevCAR system to target both the epidermal growth factor receptor (EGFR) and the disialoganglioside GD2, which are expressed in GBM. The RevCAR system is a modular platform that enables controllability, improves safety, specificity and flexibility. Briefly, it consists of RevCAR T cells having a peptide epitope as extracellular domain, and a bispecific target module (RevTM). The RevTM acts as a switch key that recognizes the RevCAR epitope and the tumor-associated antigen, and thereby activating the RevCAR T cells to kill the tumor cells. However, in the absence of the RevTM, the RevCAR T cells are switched off. In this study, we show that the novel EGFR/GD2-specific RevTMs can selectively activate RevCAR T cells to kill GBM cells. Moreover, we show that gated targeting of GBM is possible with our Dual-RevCAR T cells, which have their internal activation and co-stimulatory domains separated into two receptors. Therefore, a full activation of Dual-RevCAR T cells can only be achieved when both receptors recognize EGFR and GD2 simultaneously via RevTMs, leading to a significant killing of GBM cells both in vitro and in vivo.

Immunotherapy based on chimeric antigen receptor (CAR) T-cells has demonstrated remarkable therapeutic effects, particularly against some hematological cancers. A versatile adaptor CAR system called RevCAR, consisting of RevCAR T-cells and a bispecific target module (RevTM) has been developed to overcome severe side effects associated with conventional CAR T-cell therapy. As the activity of RevCAR T-cells can be steered based on the availability of RevTM, working as an on/off switch, the system can be immediately turned off if side effects occur. Furthermore, the RevCAR system is highly flexible, since the same RevCAR T-cell can be directed towards different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, the effectiveness of CAR T-cells against solid tumors remains limited particularly due to their immunosuppressive tumor microenvironment (TME). To overcome these hurdles, we have established a novel RevCAR system targeting immune checkpoint molecules such as PD-L1, which are frequently overexpressed by cancer cells to suppress immune responses. We have constructed novel RevTMs that can redirect RevCAR T-cells to kill tumor cells that express such immune checkpoint molecules. Furthermore, true AND gate tumor targeting was achieved by targeting a TAA in addition to PD-L1 in a combinatorial manner using our Dual-RevCAR system. In this way, targeting PD-L1 not only results in Dual-RevCAR T-cell activation but simultaneously blocks the immunosuppressive PD-L1/PD-1 axis. Altogether, we have turned an immunosuppressive marker into an immune-activating signal that might modulate the TME in a beneficial manner, showing promise for the development of an effective immunotherapy against solid tumors.

Chimeric antigen receptors (CARs) are used for redirection of T cells against tumor cells and have demonstrated remarkable therapeutic effects against some hematological cancers. The modular system termed RevCAR can overcome the dangerous side effects associated with conventional CAR T cell therapy, such as cytokine release syndrome. It is composed of RevCAR T cells and a bispecific molecule target module, termed RevTM. This system is highly safe since RevCAR T cell activity can be controlled by RevTM and immediately switched off if side effects are detected. It is also versatile, since the same RevCAR T cell can be directed to different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, to improve the effectiveness in the treatment of solid tumors, the immunosuppressive tumor microenvironment (TME) still needs to be addressed. For this purpose, we have developed novel RevTMs targeting immune checkpoint molecules such as PD-L1, which are often upregulated by cancer cells to escape the immune cells. We have demonstrated that our novel RevTMs can redirect RevCAR T cells to specifically kill cell lines expressing immune checkpoint molecules. In addition to this strategy, the combinatorial targeting of a TAA and an immune checkpoint is very promising strategy to overcome the immunosuppressive microenvironment of solid tumors and thus to improve the treatment outcome.

The La-protein is a multifunctional abundantly expressed protein in the nucleus of all human cells, where it for example functions as a chaperone for newly synthesized RNA-polymerase III transcripts. Recently, it has been shown that La is overexpressed in malignant cells and can be translocated to the cell surface under certain conditions, e. g. radio- or chemotherapy-induced necrosis, where it becomes a promising, inducible target for so-called “Death Targeting” approaches. The aim of this study was to investigate whether the La-protein could also be used as an inducible target for CAR T cell therapy. Therefore, three different CAR T cells directed against the La-protein (La-CARs) were developed and tested regarding their potential for “Death Targeting” of tumor cells. For construction of the La-CARs, we selected three different anti-La mAbs (5B9, 7B6 and 312B), which recognize distinct epitopes within the La-protein in a redox-dependent manner. This allowed us to determine, if the redox-state influences the ability of the La-CARs to target cell surface-bound La-protein. We first examined if La-CARs could eliminate tumor cells that were artificially labeled with wildtype La-protein or its oxidation-resistant triple-cysteine-mutant (TCM). By performing luciferase-based killing assays, we showed, that all La-CARs were specifically activated for lysis of La- or TCM-decorated tumor cells, which also resulted in significant secretion of pro-inflammatory cytokines. Redox-dependent differences of the La-CARs could be observed, whereby the non-redox-dependent 5B9-CAR was activated by La-wt- and TCM-labeled cells, the 7B6-CAR preferably by La-wt-labeled and the 312B-CAR preferably by TCM-labeled tumor cells. We could further prove that La-release from tumor cells could be induced by treatment with the cytotoxic drug cisplatin and La-CARs could be redirected against remaining tumor-cells after cisplatin treatment. Thereby, the 5B9-CAR, which recognizes La-protein independent

Immunotherapy based on chimeric antigen receptor (CAR) T-cells has demonstrated remarkable therapeutic effects, particularly against some hematological cancers. A versatile adaptor CAR system called RevCAR, consisting of RevCAR T-cells and a bispecific target module (RevTM) has been developed to overcome severe side effects associated with conventional CAR T-cell therapy. As the activity of RevCAR T-cells can be steered based on the availability of RevTM, working as an on/off switch, the system can be immediately turned off if side effects occur. Furthermore, the RevCAR system is highly flexible, since the same RevCAR T-cell can be directed towards different tumor-associated antigens (TAA) simply by adding RevTMs with different specificities. However, the effectiveness of CAR T-cells against solid tumors remains limited particularly due to their immunosuppressive tumor microenvironment (TME). To overcome these hurdles, we have established a novel RevCAR system targeting immune checkpoint molecules such as PD-L1, which are frequently overexpressed by cancer cells to suppress immune responses. We have constructed novel RevTMs that can redirect RevCAR T-cells to kill tumor cells that express such immune checkpoint molecules. Furthermore, true AND gate tumor targeting was achieved by targeting a TAA in addition to PD-L1 in a combinatorial manner using our Dual-RevCAR system. In this way, targeting PD-L1 not only results in Dual-RevCAR T-cell activation but simultaneously blocks the immunosuppressive PD-L1/PD-1 axis. Altogether, we have turned an immunosuppressive marker into an immune-activating signal that might modulate the TME in a beneficial manner, showing promise for the development of an effective immunotherapy against solid tumors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a pandemic with millions of infections and deaths worldwide and devastating impact on global economy. Up to now, vaccines and monoclonal antibody (mAb) therapies lack to provide a long-lasting protection against rapidly evolving new emerging SARS-CoV-2 variants. Thus, novel therapeutic options are pressingly needed especially for immunocompromised patients and/or patients with high risk for developing a severe coronavirus disease 2019 (COVID-19). In that regard, we developed a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2). This ACE2 decoy potently binds to the SARS-CoV-2 receptor binding domain (RBD), neutralizes SARS-CoV-2 as well as the Delta and Omicron variant and protects hamsters from a SARS-CoV-2 infection. To additionally use this ACE2 decoy for elimination of virus infected cells, we equipped it with an epitope tag. Thus, it can be applied as adapter molecule in the modular platform technologies UniMAB and UniCAR, which already demonstrated great success in the setting of malignant diseases. As adapter molecule the ACE2 decoy is able to efficiently recruit either universal chimeric antigen receptor (UniCAR) modified T cells or, in combination with an anti-peptide epitope-anti-CD3 bispecific Ab of the UniMAB system, unmodified T cells to efficiently kill SARS-CoV-2 RBD expressing human cells. Taken together, the ACE2 decoy represents a very promising immunotherapeutic drug for both SARS-CoV-2 variant neutralization and infected cell killing via the UniMAB and UniCAR system and might, therefore, clearly improve the treatment of COVID-19 patients.

Seagrass meadows have disappeared on many coastal sections due to anthropogenic disturbances, diseases, and/or eutrophication. To facilitate informed seagrass restoration, we i) quantified the hydrodynamic dislodgement thresholds for newly transplanted Z. marina shoots, and ii) tested the effect of artificial seagrass (ASG) as a hydrodynamic protection measure. Experiments were carried out by planting Z. marina rhizomes with living shoots into a sediment bed and exposing them to a range of wave and current conditions in a flume. The use of ASG significantly reduced wave height, as well as current velocity. The applied waves led to the development of ripples whereas currents led to erosion of the sediment bed. The number of shoots that were uprooted and dislodged increased with increasing bed shear stress and erosion. By reducing bed shear stress, the ASG raised the input current velocity threshold, which the transplanted shoots were able to withstand. The present study offers insight into the effect of artificial seagrass (ASG) on wave and current attenuation, as well as sediment erosion and shoot dislodgement. Our results help to inform the setting of hydrodynamic thresholds for the early establishment of Z. marina and to define the improvement of hydrodynamic conditions by ASG.

Seagrass meadows have disappeared on many coastal sections due to anthropogenic disturbances, diseases, and/or eutrophication. To facilitate informed seagrass restoration, we i) quantified the hydrodynamic dislodgement thresholds for newly transplanted Z. marina shoots, and ii) tested the effect of artificial seagrass (ASG) as hydrodynamicprotection measure. Experiments were carried out by planting Z. marina rhizomes with living shoots into a sediment bed and exposing them to a range of wave and current conditions in a flume. The use of ASG significantly reduced wave height, as well as current velocity. The applied waves led to the development of ripples whereas currents led to erosion of the sediment bed. The number of shoots that were uprooted and dislodged increased with increasing bed shear stress and erosion. By reducing bed shear stress, the ASG raised the input current velocity threshold, which the transplanted shoots were able to withstand. The present study offers insight into the effect of artificial seagrass (ASG) on wave and current attenuation, as well as sediment erosion and shoot dislodgement. Our results help to inform on hydrodynamic thresholds for the early establishment of Z. marina and to define the improvement of hydrodynamic conditions by ASG.

In Africa, cattle are often fed low quality tropical roughages resulting in low-yielding animals with high methane (CH4) emission intensity (EI, g CH4/per unit of product). Supplementation with protein is known to improve the nutritive value of the otherwise low-quality diets. However, animal nutrition studies in East Africa that are accompanied by CH4 emission measurements are lacking. Thus, an animal experiment was conducted to quantify the effect of supplementing cattle fed mainly on low-quality Urochloa brizantha hay (control diet; CON; crude protein (CP) = 7.4%) or supplemented with either a tannin-rich leguminous fodder, Desmodium intortum hay (DES) or a commercial dairy concentrate (CUBES) on voluntary dry matter intake (DMI), nutrient apparent total tract digestibility, nitrogen (N) retention, enteric CH4 production and animal performance (milk and average daily gain). Twelve mid-lactating crossbred (Friesian × Boran) cows (initial liveweight = 335 kg) were used in a 3×3 (Period × Diet) Latin square design with each period running for four weeks. Compared to CON, DES decreased nutrient (DM, OM, CP) intake, apparent total tract digestibility and daily milk yield. In contrast, CUBES increased nutrient intake and animal performance compared to CON, while nutrients’ apparent total tract digestibility was not different, except for CP digestibility that increased. Compared to CON, DES and CUBES improved overall N retention by the animals as a proportion of N intake. The DES diet compared with CON and CUBES, shifted the proportion of N excretion via urine to the fecal route, likely because of its tannin content. Both DES and CUBES, compared to CON, reduced methane yield (MY, g CH4/kg DMI) by 15% and 9%, respectively. The DES diet reduced absolute enteric CH4 emissions by 26% while CUBES increased emissions by 11% compared to CON. Based on the present findings, high supplementation levels (>50%) of Desmodium intortum hay is not recommended especially when the

Livestock are an important source of livelihoods in agricultural systems in sub-Saharan Africa (SSA), while also being the largest source of national greenhouse gas (GHG) emissions in most African countries. As a consequence, there is a critical need for data on livestock GHG sources and sinks to develop national inventories, as well as conduct baseline measurements and intervention testing to mitigate GHG emissions and meet ambitious national climate goals. Our objective was to review studies on GHG emissions from livestock systems in SSA, as well as soil carbon storage in livestock-dominated systems (i.e., grasslands and rangelands), to evaluate best current data and suggest future research priorities. To this end, we compiled studies from SSA that determined emission factors (EFs) for enteric methane and manure emissions, along with studies on soil organic carbon (SOC) stocks in SSA. We found that there has been limited research on livestock GHG emissions and SOC relative to national ambitions for climate change mitigation in SSA. Enteric methane emission factors (EFs) in low productivity cattle systems may be lower than IPCC Tier 1 default EFs, whereas small ruminants (i.e. sheep and goats) had higher EFs compared to IPCC Tier 1 EFs. Manure EFs were equal to or lower than IPCC Tier 1 EFs for deposited manure (while grazing), manure applied as fertilizer, and manure management. SOC stocks for grasslands and rangelands in SSA show broad agreement with IPCC estimates, but there was a strong geographic bias and many studies did not report soil type, bulk density, or SOC stocks at >30 cm depth. In general, the largest data gaps included information for manure (quantity, quality, management), small ruminants, agropastoral/pastoralist systems, and in general from West Africa. Future research should focus on filling major data gaps on locally appropriate mitigation interventions and improving livestock activity data for developing Tier 2 GHG inventories in SSA. A

Seagrass meadows have disappeared on many coastal sections due to anthropogenic disturbances, diseases, and/or eutrophication. To facilitate informed seagrass restoration, we i) quantified the hydrodynamic dislodgement thresholds for newly transplanted Z. marina shoots, and ii) tested the effect of artificial seagrass (ASG) as hydrodynamicprotection measure. Experiments were carried out by planting Z. marina rhizomes with living shoots into a sediment bed and exposing them to a range of wave and current conditions in a flume. The use of ASG significantly reduced wave height, as well as current velocity. The applied waves led to the development of ripples whereas currents led to erosion of the sediment bed. The number of shoots that were uprooted and dislodged increased with increasing bed shear stress and erosion. By reducing bed shear stress, the ASG raised the input current velocity threshold, which the transplanted shoots were able to withstand. The present study offers insight into the effect of artificial seagrass (ASG) on wave and current attenuation, as well as sediment erosion and shoot dislodgement. Our results help to inform on hydrodynamic thresholds for the early establishment of Z. marina and to define the improvement of hydrodynamic conditions by ASG.

Despite the fact that chimeric antigen receptor (CAR) engineered T cells have shown encouraging therapeutic effects in hematological setups, the targeting of solid tumor-related antigens still represents a challenge. One main issue is that the expression of this type of antigens is not restricted to cancer cells, but normal tissues express them as well to some degree. In order to avoid strong side-effects caused by on-target/off-tumor effects, more controllable and specific CAR T cell-derived technologies need to be developed. In this line of thought, we developed the switchable, flexible and programmable Reverse (Rev) CAR platform. This system is based on engineered T cells expressing RevCAR molecules, which have extracellular short peptide epitopes incapable of recognizing surface antigens. Thus, the RevCAR T cells are per se inert and will only recognize the target cell through the interaction with an antigen-specific target module, named RevTM. RevTMs are bispecific antibodies designed to bind simultaneously to the target antigen and the short epitopes on the RevCAR engineered T cells. This interaction triggers a specific activation and cytotoxic activity of the RevCAR T cells redirecting them to eradicate the cancer cells. Here, we adapted our RevCAR technology to target the carcinoembryonic antigen (CEA), which is a significant tumor marker for colorectal cancers and other carcinomas. Moreover, we developed two RevTMs with different structures: scFv- and IgG4-based. The first one is built by linking a scFv against CEA to a scFv that recognizes the RevCAR epitope through glycine and serine residues, resulting in a small sized molecule (<60 kDa) with two binding sites. The IgG4-based RevTM connects the same scFv-structures but in this case through the hinge and constant Fc regions (CH2 and CH3) of an IgG4 antibody. The formation of disulfide bridges on the hinge portion causes the formation of homodimers, resulting in a molecule with increased molecular weight (

Chimeric antigen receptor (CAR) T-cells are unquestionably considered one of the most promising approaches in cancer immunotherapy. Nonetheless, mild to severe toxicities are associated with this approach which include e.g. on-target/off-tumour toxicities and cytokine release syndrome. Aiming for increased clinical safety, a modular universal CAR (UniCAR) platform was developed by our group in which UniCAR T-cells are exclusively activated in the presence of a target module (TM) that specifically establishes the cross-link between target cells and UniCAR T-cells. Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts (CAFs) present in the tumour stroma and also found to be overexpressed in tumour cells. This protein plays an important role in promoting tumour growth, metastasis, and immunosuppression and has therefore been studied as a target for cancer diagnosis and treatment. Given this and the demonstrated efficacy, flexibility and switchability of the UniCAR system, currently demonstrated in phase I clinical trials, we hereby aimed to develop TMs targeting FAP that can be used for both immunotherapeutic and theranostic approaches. For that, the single-chain variable fragment (scFv) of an anti-human FAP mAb was fused to the peptide epitope E5B9 that is recognized by the UniCAR T-cells, creating low molecular weight TMs that are rapidly eliminated allowing a specific and recurrent on/off switch of UniCAR T-cell activity via TM dosing. Additionally, extended half-life anti-FAP TMs based on the human IgG4 Fc-domain, including a mutated version, were created intending to strengthen anti-tumour responses and to ease the clinical TM administration at later stages of tumour therapy. All TMs were tested in vitro based on naturally and artificially overexpressing 2D and 3D models, and proven to specifically redirect UniCAR T-cells to FAP-expressing target cells. Positron emission tomography (PET) using 64Cu radiolabelled anti-FAP IgG4 TMs

As next generation for CAR T cells, adaptor CAR platforms have been developed, which are designed to improve safety, but at the same time maintain the high efficiency of the CAR T cell approach. In our lab, we developed the UniCAR system, which consists of universal (Uni)CAR T cells and tumor-specific target modules (TMs), which work as bridging molecules between the UniCAR T cells and the target cells. Until now, type 1 and type 2 UniCARs were developed. Both UniCAR types consist of an extracellular binding domain derived from a monoclonal antibody (mAb) directed to the nuclear La/SS-B protein. Type 1 UniCARs are derived from the anti-La mAb 5B9. Type 2 UniCARs from the anti-La mAb 7B6. As both anti-La mAbs are not able to precipitate native La protein, both anti-La mAbs are directed to a specific cryptic epitope which is not accessible on the cell surface. Thus, the UniCAR T cell is per se inert. To activate the UniCAR T cell for tumor cell killing a TM is needed as a second component. Typically, a TM is composed of a tumor-specific binding domain and the respective La epitope. Consequently, the affinity of the TM towards the target antigen but also towards the UniCAR T cell via the E5B9-tag plays an important role for functionality of the respective UniCAR system. In this study, we representatively aimed to elucidate if and how the affinity of the type 1 UniCAR domain to the E5B9 epitope impacts the functionality of the UniCAR system. To alter the interaction of UniCAR and TM, we designed different mutated E5B9 peptides (M1-M3) carrying one or two amino acid (aa) changes. In detail, aspartic acid and/or glutamic acid were mutated to glycine residues as they most probably are involved in epitope/paratope interactions. We subsequently fused these mutated peptides to an scFv domain, resulting in three different mutated TM versions. By conducting ELISA and flow-cytometry based binding studies, we showed that a single aa exchange (D3>G3) in M1 did not alter the affini

Chimeric antigen receptor (CAR) T-cells are undoubtedly a promising approach in cancer immunotherapy. Nevertheless, mild to severe toxicities are associated with this approach including on-target/off-tumor effects and cytokine release syndrome. Aiming for increased clinical safety, adaptor CAR technologies were developed which include the modular universal CAR (UniCAR) platform developed by our group. UniCAR T-cells are exclusively activated in the presence of a target module (TM), which establishes the cross-link between cancer cells and UniCAR T-cells. These TMs are highly versatile molecules that can be constructed not only by using antibody fragments but also e.g. peptides specifically targeting a receptor or molecule on the cell´s surface. Somatostatin receptor (SSTR) subtype 2 is highly expressed in a variety of malignancies and has therefore been studied as a marker and target for cancer diagnosis and treatment. Currently, SSTR2 agonists and antagonists, such as Tyr3-octreotate (TATE) and BASS or JR11, respectively, are particularly well established and clinically implemented mostly used for diagnostic nuclear medicine. Given this and the proven flexibility and efficacy of the UniCAR system, we hereby aimed to develop small peptide-derived TMs targeting SSTR2 that can be used for both immunotherapeutic and diagnostic approaches. For that, the abovementioned SSTR2 agonist and antagonists were chemically linked to the E5B9 peptide and equipped with the radiometal chelator NODAGA. These TMs were tested in vitro and in vivo, in which they have proven to specifically redirect UniCAR T-cells to human neuroendocrine and breast SSTR2-expressing cancer cells. Furthermore, the enrichment of these anti-SSTR2 peptide TMs at the tumor site was confirmed by positron emission tomography (PET) studies. We hereby designed novel small peptide-derived TMs that can be used for redirection of UniCAR T-cells to SSTR2-expressing cancer cells as well as for PET imaging, proving to b

CAR T-cell therapy achieved unparalleled clinical success rates for treatment of patients with hematological malignancies. However, progress and clinical translation towards solid tumors is slow and hampered by many factors e.g. increased complexity, high heterogeneity and an immunosuppressive tumor microenvironment. Thus, CAR T-cell therapy alone might not result in durable antitumor responses. Combinatorial approaches are promising strategies to improve CAR T-cell efficacy in solid tumor treatment. In this regard, we here aim to combine conventional cancer theranostics with CAR T-cell immunotherapy in one single approach. By using the well-established UniCAR system, a novel, multifunctional tool termed PSCA-IgG4 target module (TM) was developed for dual prostate cancer theranostics. It comprises a human PSCA-specific binding domain, the hinge and Fc-domain of human IgG4 molecules as well as the UniCAR epitope E5B9. As shown by in vitro assays with PSCA-positive and PSCA-negative prostate cancer cells, the novel TM redirected UniCAR T cells for efficient tumor cell lysis in a strictly antigen- and TM-dependent manner. After radiolabeling with copper-64 or actinium-225, the novel PSCA-IgG4 TM was successfully applied for diagnostic imaging and targeted radioimmunotherapy. The 64Cu-labeled PSCA-IgG4 TM showed maximal tumor accumulation with optimal tumor-to-background ratios after 1.5 days. Furthermore, targeted alpha-therapy with the 225Aclabeled TM significantly delayed the outgrowth of established tumors in mice. In summary, the here presented, novel PSCA-IgG4 TM is a promising candidate for dual theranostics of prostate cancer that may help to overcome present hurdles in solid tumor therapy. After radiolabeling it facilitates not only targeted alpha-therapy and diagnostic imaging of PSCA, but can be also repurposed as a TM for UniCAR T-cell immunotherapy.

Modifying of immune effector cells with chimeric antigen receptors (CARs) has revealed a promising therapeutic potential for targeting cancer, especially with CAR-modified T cells. However, the use of other immune cells like primary NK cells or NK cell lines appeared as another advantageous approach that can be combined with CAR technology. Unlike T cells, established NK cell lines can be used allogenically as an off-the-shelf product with reduced risk of toxicities. We have established previously a modular Universal CAR platform (UniCAR) which can be switched on/off and allows the flexible targeting of various tumor antigens. This system consists of two parts, the UniCAR-expressing immune effector cells and a target module (TM). The UniCAR-immune cells cannot recognize surface antigens but are only redirected with the TM which contains an antigen-binding moiety on one hand and an epitope recognized by the UniCAR molecules on the other hand. Here, we provide a proof of concept for using the UniCAR system in combination with the NK-92 cell line to target disialoganglioside GD2-expressing tumors. The UniCAR NK-92 cells induced increase in lysis of neuroblastoma and melanoma cell lines in the presence of scFv- or human IgG4-based TMs, associated with specific release of pro-inflammatory cytokines. Moreover, UniCAR NK-92 cells were shown to be functional in eradicating GD2-expressing tumors in experimental mice. In order to investigate the in vivo half-life of the scFv- and IgG4-based TMs, they were radiolabeled with 64Cu and detected using PET imaging. Dynamic PET scanning has shown that the IgG4 format increased the half-life of the TM to around 24 folds in comparison to the scFv-based TMs. In summary, UniCAR NK-92 provides an off-the-shelf universal platform that can be combined with various antibody formats, and can be easily expanded for therapeutic use.

Background: Glioblastoma (GBM) is a very aggressive brain tumor, associated with poor prognosis and survival. So far, the efficiency of available therapies is limited. After proving their effectiveness in targeting hematological malignancies, chimeric antigen receptor (CAR) T cells might provide a promising therapeutic approach for GBM. Here, we present our switchable Reverse CAR technology (RevCAR). Unlike conventional CAR T cells, RevCAR T cells contain an epitope in their extracellular receptor domain, and can only be activated via bispecific target modules (RevTM) which recognize the RevCAR T cells on one side and tumor cells on the other side. Once these TMs are eliminated, RevCARs are switched off. In addition, we have developed dual-targeting RevCARs allowing the control of T cells according to the AND gate logic of Boolean algebra. Methods: RevTMs directed against GMB were expressed in eukaryotic cells and purified with affinity chromatography. The ability of these RevTMs to bind GBM cells and RevCAR T cells was analyzed using flow cytometry. Moreover, the capability of the mono-specific and the dual-targeting RevCAR T cells to kill GBM cells was analyzed using luminescence-based cytotoxicity assay in the absence or presence of a range of RevTM concentrations. Secreted pro-inflammatory cytokines were also evaluated by ELISA. In addition to the in vitro assays, a proof of concept co-injection experiment was performed in vivo. Results: In this study, we show that GBM-specific RevTMs can bind both the RevCAR T cells and the GBM cells. Importantly, the RevCAR T cells can be activated to secrete pro-inflammatory cytokines and to efficiently kill GBM cells via the RevTMs. Moreover, we were able to prove that dual-targeting RevCAR T cells can be activated only upon recognition of two different GBM targets, thereby allowing a highly specific and selective killing of GBM both in vitro and in vivo. Conclusion: In conclusion, the switchable RevCAR platform is a novel t

As next generation for CAR T cells, adaptor CAR platforms have been developed, which are designed to improve safety, but at the same time maintain the high efficiency of the CAR T cell approach. In our lab, we developed the UniCAR system, which consists of universal (Uni)CAR T cells and tumor-specific target modules (TMs), which work as bridging molecules between the UniCAR T cells and the target cells. Until now, type 1 and type 2 UniCARs were developed. Both UniCAR types consist of an extracellular binding domain derived from a monoclonal antibody (mAb) directed to the nuclear La/SS-B protein. Type 1 UniCARs are derived from the anti-La mAb 5B9. Type 2 UniCARs from the anti-La mAb 7B6. As both anti-La mAbs are not able to precipitate native La protein, both anti-La mAbs are directed to a specific cryptic epitope which is not accessible on the cell surface. Thus, the UniCAR T cell is per se inert. To activate the UniCAR T cell for tumor cell killing a TM is needed as a second component. Typically, a TM is composed of a tumor-specific binding domain and the respective La epitope. Consequently, the affinity of the TM towards the target antigen but also towards the UniCAR T cell via the E5B9-tag plays an important role for functionality of the respective UniCAR system. In this study, we representatively aimed to elucidate if and how the affinity of the type 1 UniCAR domain to the E5B9 epitope impacts the functionality of the UniCAR system. To alter the interaction of UniCAR and TM, we designed different mutated E5B9 peptides (M1-M3) carrying one or two amino acid (aa) changes. In detail, aspartic acid and/or glutamic acid were mutated to glycine residues as they most probably are involved in epitope/paratope interactions. We subsequently fused these mutated peptides to an scFv domain, resulting in three different mutated TM versions. By conducting ELISA and flow-cytometry based binding studies, we showed that a single aa exchange (D3>G3) in M1 did not alter the affini

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic PET imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+ the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

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 IL2, TNF and interferon-g 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.

Most patients with head and neck squamous cell carcinomas (HNSCC) are diagnosed at a locally advanced stage and show heterogeneous treatment responses. Low SLC3A2 (solute carrier family 3 member 2) mRNA and protein (CD98hc) expression levels are associated with higher locoregional control in HNSCC patients treated with primary radiochemotherapy or postoperative radiochemotherapy, suggesting that CD98hc could be a target for HNSCC radiosensitization. One of the targeted strategies for tumor radiosensitization is precision immunotherapy, e.g., the use of chimeric antigen receptor (CAR) T cells. This study aimed to define the potential clinical value of new treatment approaches combining conventional radiotherapy with CD98hc-targeted immunotherapy. To address this question, we analyzed the antitumor activity of the combination of fractionated irradiation and switchable universal CAR (UniCAR) system against radioresistant HNSCC cells in 3D culture. CD98hc-redirected UniCAR T cells showed the ability to destroy radioresistant HNSCC spheroids. Also, the infiltration rate of the UniCAR T cells was enhanced in the presence of the CD98hc target module. Furthermore, sequential treatment with fractionated irradiation followed by CD98hc-redirected UniCAR T treatment showed a synergistic effect. Taken together, our obtained data underline the improved antitumor effect of the combination of radiotherapy with CD98hc-targeted immunotherapy. Such a combination presents an attractive approach for the treatment of high-risk HNSCC patients.

Immunotherapy using CAR-T cells is a new paradigm technology for cancer treatment. To avoid severe side effects and tumor escape variants observed for conventional CAR-T cells approach, adaptor CAR technologies are under development, where intermediate target modules redirect immune cells against cancer cells. In this work, silicon nanowire field effect transistors are used to assist in the development of target modules for an optimized CAR-T cell operation. Focusing on a library of seven variants of E5B9 peptide that is used as CAR peptide epitope, we performed multiplexed binding tests in serum using nanosensor chips. Peptides have been immobilized onto the sensor to compare the signals of transistor upon titration with anti-E5B9 antibodies. Correlation analysis of binding affinities and sensitivities enabled a selection of best candidates for the interaction between CAR and target modules. Finally, cytotoxic functionality of CAR-T cells in combination with the selected target modules were successfully proven. Our results open the perspective for the nanobiosensorics to go beyond the early diagnostics in the field of clinical cancer research, and paves the way towards personalization and efficient monitoring of the immunotherapeutic treatment, where the quantitative analysis with the standard techniques is not an option.

Chimeric antigen receptor (CAR)-expressing T-cells are without a doubt a breakthrough therapy for hematological malignancies. Despite their success, clinical experience has revealed several challenges, which include relapse after targeting single antigens such as CD19 in the case of B-cell acute lymphoblastic leukemia (B-ALL), and the occurrence of side effects that could be severe in some cases. Therefore, it became clear that improved safety approaches, and targeting multiple antigens, should be considered to further improve CAR T-cell therapy for B-ALL. In this paper, we address both issues by investigating the use of CD10 as a therapeutic target for B-ALL with our switchable UniCAR system. The UniCAR platform is a modular platform that depends on the presence of two elements to function. These include UniCAR T-cells and the target modules (TMs), which cross-link the T-cells to their respective targets on tumor cells. The TMs function as keys that control the switchability of UniCAR T-cells. Here, we demonstrate that UniCAR T-cells, armed with anti-CD10 TM, can efficiently kill B-ALL cell lines, as well as patient-derived B-ALL blasts, thereby highlighting the exciting possibility for using CD10 as an emerging therapeutic target for B-cell malignancies.

CAR T-cell therapy achieved unparalleled clinical success rates for treatment of patients with hematological malignancies. However, progress and clinical translation towards solid tumors is slow and hampered by many factors e.g. increased complexity, high heterogeneity and an immunosuppressive tumor microenvironment. Thus, CAR T-cell therapy alone might not result in durable antitumor responses. Combinatorial approaches are promising strategies to improve CAR T-cell efficacy in solid tumor treatment. In this regard, we here aim to combine conventional cancer theranostics with CAR T-cell immunotherapy in one single approach. By using the well-established UniCAR system, a novel, multifunctional tool termed PSCA-IgG4 target module (TM) was developed for dual prostate cancer theranostics. It comprises a human PSCA-specific binding domain, the hinge and Fc-domain of human IgG4 molecules as well as the UniCAR epitope E5B9. As shown by in vitro assays with PSCA-positive and PSCA-negative prostate cancer cells, the novel TM redirected UniCAR T cells for efficient tumor cell lysis in a strictly antigen- and TM-dependent manner. After radiolabeling with copper-64 or actinium-225, the novel PSCA-IgG4 TM was successfully applied for diagnostic imaging and targeted radioimmunotherapy. The 64Cu-labeled PSCA-IgG4 TM showed maximal tumor accumulation with optimal tumor-to-background ratios after 1.5 days. Furthermore, targeted alpha-therapy with the 225Aclabeled TM significantly delayed the outgrowth of established tumors in mice. In summary, the here presented, novel PSCA-IgG4 TM is a promising candidate for dual theranostics of prostate cancer that may help to overcome present hurdles in solid tumor therapy. After radiolabeling it facilitates not only targeted alpha-therapy and diagnostic imaging of PSCA, but can be also repurposed as a TM for UniCAR T-cell immunotherapy.

Background: Glioblastoma (GBM) is a very aggressive brain tumor, associated with poor prognosis and survival. So far, the efficiency of available therapies is limited. After proving their effectiveness in targeting hematological malignancies, chimeric antigen receptor (CAR) T cells might provide a promising therapeutic approach for GBM. Here, we present our switchable Reverse CAR technology (RevCAR). Unlike conventional CAR T cells, RevCAR T cells contain an epitope in their extracellular receptor domain, and can only be activated via bispecific target modules (RevTM) which recognize the RevCAR T cells on one side and tumor cells on the other side. Once these TMs are eliminated, RevCARs are switched off. In addition, we have developed dual-targeting RevCARs allowing the control of T cells according to the AND gate logic of Boolean algebra. Methods: RevTMs directed against GMB were expressed in eukaryotic cells and purified with affinity chromatography. The ability of these RevTMs to bind GBM cells and RevCAR T cells was analyzed using flow cytometry. Moreover, the capability of the mono-specific and the dual-targeting RevCAR T cells to kill GBM cells was analyzed using luminescence-based cytotoxicity assay in the absence or presence of a range of RevTM concentrations. Secreted pro-inflammatory cytokines were also evaluated by ELISA. In addition to the in vitro assays, a proof of concept co-injection experiment was performed in vivo. Results: In this study, we show that GBM-specific RevTMs can bind both the RevCAR T cells and the GBM cells. Importantly, the RevCAR T cells can be activated to secrete pro-inflammatory cytokines and to efficiently kill GBM cells via the RevTMs. Moreover, we were able to prove that dual-targeting RevCAR T cells can be activated only upon recognition of two different GBM targets, thereby allowing a highly specific and selective killing of GBM both in vitro and in vivo. Conclusion: In conclusion, the switchable RevCAR platform is a novel t

Despite the fact that chimeric antigen receptor (CAR) engineered T cells have shown encouraging therapeutic effects in hematological setups, the targeting of solid tumor-related antigens still represents a challenge. One main issue is that the expression of this type of antigens is not restricted to cancer cells, but normal tissues express them as well to some degree. In order to avoid strong side-effects caused by on-target/off-tumor effects, more controllable and specific CAR T cell-derived technologies need to be developed. In this line of thought, we developed the switchable, flexible and programmable Reverse (Rev) CAR platform. This system is based on engineered T cells expressing RevCAR molecules, which have extracellular short peptide epitopes incapable of recognizing surface antigens. Thus, the RevCAR T cells are per se inert and will only recognize the target cell through the interaction with an antigen-specific target module, named RevTM. RevTMs are bispecific antibodies designed to bind simultaneously to the target antigen and the short epitopes on the RevCAR engineered T cells. This interaction triggers a specific activation and cytotoxic activity of the RevCAR T cells redirecting them to eradicate the cancer cells. Here, we adapted our RevCAR technology to target the carcinoembryonic antigen (CEA), which is a significant tumor marker for colorectal cancers and other carcinomas. Moreover, we developed two RevTMs with different structures: scFv- and IgG4-based. The first one is built by linking a scFv against CEA to a scFv that recognizes the RevCAR epitope through glycine and serine residues, resulting in a small sized molecule (<60 kDa) with two binding sites. The IgG4-based RevTM connects the same scFv-structures but in this case through the hinge and constant Fc regions (CH2 and CH3) of an IgG4 antibody. The formation of disulfide bridges on the hinge portion causes the formation of homodimers, resulting in a molecule with increased molecular weight (

Modifying of immune effector cells with chimeric antigen receptors (CARs) has revealed a promising therapeutic potential for targeting cancer, especially with CAR-modified T cells. However, the use of other immune cells like primary NK cells or NK cell lines appeared as another advantageous approach that can be combined with CAR technology. Unlike T cells, established NK cell lines can be used allogenically as an off-the-shelf product with reduced risk of toxicities. We have established previously a modular Universal CAR platform (UniCAR) which can be switched on/off and allows the flexible targeting of various tumor antigens. This system consists of two parts, the UniCAR-expressing immune effector cells and a target module (TM). The UniCAR-immune cells cannot recognize surface antigens but are only redirected with the TM which contains an antigen-binding moiety on one hand and an epitope recognized by the UniCAR molecules on the other hand. Here, we provide a proof of concept for using the UniCAR system in combination with the NK-92 cell line to target disialoganglioside GD2-expressing tumors. The UniCAR NK-92 cells induced increase in lysis of neuroblastoma and melanoma cell lines in the presence of scFv- or human IgG4-based TMs, associated with specific release of pro-inflammatory cytokines. Moreover, UniCAR NK-92 cells were shown to be functional in eradicating GD2-expressing tumors in experimental mice. In order to investigate the in vivo half-life of the scFv- and IgG4-based TMs, they were radiolabeled with 64Cu and detected using PET imaging. Dynamic PET scanning has shown that the IgG4 format increased the half-life of the TM to around 24 folds in comparison to the scFv-based TMs. In summary, UniCAR NK-92 provides an off-the-shelf universal platform that can be combined with various antibody formats, and can be easily expanded for therapeutic use.

Modification of autologous T cells with tumor-specific chimeric antigen receptors (CARs) has been shown to be an effective tool for treatment of hematologic malignancies. However, clinical translation towards solid tumors is still limited as demonstrated by the suboptimal performance of CAR T cells in first in-human studies. Combining advances in CAR-T cell therapy and cancer theranostics may provide a promising multimodal approach to increase therapeutic efficacy and achieve durable responses in cancer patients. Here we present the development and functional characterization of different peptide- or antibody constructs targeting tumor-associated antigens or different structures of the tumor microenvironment. Equipment of these molecules with the E5B9 peptide epitope, enabled their use in the well-established UniCAR system. Accordingly, the constructs were able to specifically activate UniCAR T cells for efficient killing of both hematological and solid tumors in vitro and in vivo. Upon conjugation of chelators like DOTAGA and NODAGA, the peptide- or antibody derivates were further successfully applied for cancer theranostics, using e.g. 68Ga, 64Cu as diagnostic radionuclides and e.g. 67Cu, 225Ac as therapeutic radionuclides. PET imaging in xenotransplanted mice has demonstrated high contrast tumor accumulation. Consistent with the stable tumor accumulation, 225Ac-labeled molecules demonstrated therapeutic efficacy in a xenograft mouse model. In summary, E5B9-tagged peptide- or antibody constructs open a new era of cancer immunotheranostics as they can be used multimodally for (i) UniCAR-T cell therapy, (ii) non-invasive diagnostic imaging and (iii) targeted radioimmunotherapy. Prospectively, they could thus bridge the gap between the fields of CAR-T cells and cancer theranostics.

Chimeric antigen receptor (CAR) T-cells show remarkable therapeutic effects especially in B-cell derived leukemias and lymphomas. However, clinical translation of such an innovative immunotherapeutic approach in highly heterogeneous hematological malignancies like acute myeloid leukemia (AML) or solid tumors is still challenging due to life-threatening side effects, immune escape and disease relapse. To overcome such major hurdles and to address the unmet need for further improvements in CAR therapy, we have established flexible, switchable and programmable adaptor CAR platform technologies named UniCAR and RevCAR. These modular strategies consist of T-cells engineered with adaptor CARs which are primarily inactive as they are incapable to recognize surface antigens. Universal adaptor CAR T-cells can be flexibly redirected to any tumor antigen and controlled by targeting modules (TMs) cross-linking adaptor CAR T- and tumor cells resulting in tumor cell lysis. As an advancement of UniCARs, RevCARs lack the extracellular antigen-binding moiety reducing the receptor size and facilitating the genetical modification of T-cells with several RevCARs possessing different specificity and functionality. Thus, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. So far, we have successfully shown preclinical applicability of the UniCAR and RevCAR approaches to target hematological malignancies as well as solid tumors in a flexible and specific manner using tumor cell lines and patient-derived materials. Remarkably, efficiency and switchability of UniCAR T-cells were even proven for the first time in patients in a clinical phase I study. Furthermore, by targeting of two different tumor antigens, combinatorial OR and AND gate logic targeting according to the rules of Boolean algebra was accomplished using the RevCAR platform. These achievements have a high potential for an improved and personalized tumor immunotherapy.

Adoptive therapy using CAR-T cells as "living drugs" is an emerging field in cancer immunotherapy. Our group particularly focusses on the development and clinical translation of novel adaptor CAR-T platforms (UniCAR & RevCAR), in which CAR T-cell activity is controlled by tumor-specific adaptor molecules. Apart from their immunotherapeutic potential demonstrated by numerous preclinical and an initial clinical proof-of-concept study, our adaptor CAR technologies represent also an ideal starting point for the development of cancer theranostics. Preclinical studies have shown that adaptor molecules can be easily radiolabeled for tumor therapy and diagnostics. Conversely, clinically used PET tracers have been successfully converted into highly efficient adaptor molecules for CAR T-cell immunotherapy. Overall, adaptor molecules are versatile tools for (i) CAR-T cell therapy, (ii) noninvasive diagnostic imaging, and (iii) targeted radioimmunotherapy, underscoring that combinatorial theranostic adaptor CAR-T approaches may open new avenues for effective cancer therapy.

Fibroblast activation protein (FAP), mainly expressed by cancer-associated fibroblasts (CAFs) in the tumour stroma, promotes tumour growth, metastasis, and immunosuppression and, therefore, has been studied as a target for cancer diagnosis and treatment. With regard to immunotherapy, the innovative modular universal CAR (UniCAR) platform developed by our group is one of the most promising approach due to the reduced risk for e.g. on-target/off-tumour toxicities and cytokine release syndrome. Thereby, chimeric antigen receptor (CAR) T-cells (UniCAR T cells) are exclusively activated in the presence of a target module (TM) that specifically establishes the crosslinking between target cells and UniCAR T-cells. FAP specific TMs are hypothesized to be not only immunotherapeutics with increased safety but in addition to be suitable as radionuclide-based theranostic agents. For that, low molecular weight TMs that are rapidly eliminated allowing a specific and recurrent on/off switch of UniCAR T-cell activity via TM dosing were developed by fusion of the single-chain variable fragment (scFv) of an anti-human FAP mAb to the peptide epitope E5B9 that is recognized by the UniCAR T-cells. To ease the clinical TM administration at later stages of tumour therapy and for targeted radionuclide therapy, however, TMs with extended half-life may be advantageous. Therefore, anti-FAP TMs based on the human IgG4 Fc-domain, including a mutated version, were created. All TMs were tested (i) in vitro based on naturally and artificially overexpressing 2D and 3D models and (ii) in vivo by positron emission tomography (PET) and single-photon emission tomography (SPECT) in NMRI nude mice bearing both mock transfected and FAP overexpressing HT1080 tumor xenografts. In vitro, all TMs were proven to specifically redirect UniCAR T-cells to FAP-expressing target cells. Moreover, FAP specific TMs could be conjugated to different chelators, e.g. Bispidines, NODAGA, and CHX-A-DTPA and, afterwards, radi