Modular Medical Imaging Agents Based on Azide-Alkyne Huisgen Cycloadditions: Synthesis and Pre-Clinical Evaluation of 18 F-Labeled PSMA-Tracers for Prostate Cancer Imaging.

Since the seminal contribution of Rolf Huisgen to develop the [3+2] cycloaddition of 1,3-dipolar compounds, its azide-alkyne variant has established itself as the key step in numerous organic syntheses and bioorthogonal processes in materials science and chemical biology. In the present study, the copper(I)-catalyzed azide-alkyne cycloaddition was applied for the development of a modular molecular platform for medical imaging of the prostate-specific membrane antigen (PSMA), using positron emission tomography. This process is shown from molecular design, through synthesis automation and in vitro studies, all the way to pre-clinical in vivo evaluation of fluorine-18- labeled PSMA-targeting 'F-PSMA-MIC' radiotracers (t1/2 =109.7 min). Pre-clinical data indicate that the modular PSMA-scaffold has similar binding affinity and imaging properties to the clinically used [68 Ga]PSMA-11. Furthermore, we demonstrated that targeting the arene-binding in PSMA, facilitated through the [3+2]cycloaddition, can improve binding affinity, which was rationalized by molecular modeling. The here presented PSMA-binding scaffold potentially facilitates easy coupling to other medical imaging moieties, enabling future developments of new modular imaging agents.

Photochemical internalization (PCI)-mediated enhancement of bleomycin cytotoxicity by liposomal mTHPC formulations in human head and neck cancer cells.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) with photosensitizers that locate in endocytic vesicles of cancer cells can be exploited to promote the intracellular release of anticancer drugs entrapped in endolysosomal vesicles. This new approach is commonly referred to as Photochemical Internalization (PCI). Here we report on the PCI effects of three different formulations (Foscan, Foslip, and Fospeg) of the clinically approved photosensitizer, meta-tetrahydroxyphenyl chlorin (mTHPC) on the anticancer drug bleomycin (BLM) in the head and neck cancer cell lines. MATERIALS AND METHODS: Uptake and localization of Foscan, Foslip, and Fospeg in head and neck cancer cells were evaluated by fluorescence spectrophotometry and fluorescence microscopy. Photodynamic efficacy of Foscan, Foslip, and Fospeg were compared with cell proliferation assay. Moreover, PCI effects of Foscan, Foslip, and Fospeg on BLM were compared using protocols in which PDT was applied after or before BLM treatment. RESULTS: Cellular uptake of Foscan, Foslip, and Fospeg increased in a dose-dependent fashion with consistent higher uptakes of Foslip and Fospeg than Foscan. Fluorescence microscopy showed diffuse intracellular localization pattern for Foscan, Foslip, and Fospeg similar to that of a specific ER probe. However, the subcellular localization pattern of the Rhodamine-labelled same type of pegylated liposomes as Fospeg was similar to that of a specific endolysosomal probe, suggesting that Fospeg uptake appeared to initially proceed via endolysosomal trafficking. Foscan, Foslip, and Fospeg showed no apparent PCI-mediated cytotoxicity when PDT was performed after BLM treatment. However, significantly increased cytotoxicity of BLM (P < 0.05) was observed for both Foslip and Fospeg when PDT was performed before BLM treatment. The observed differences of PCI-mediated cytotoxicity between these two timing protocols appears to be related to the differential intracellular trafficking and localization of Foscan, Foslip, and Fospeg. CONCLUSION: Liposomal formulations of mTHPC may be candidates for developing mTHPC-based PCI protocols to enhance the efficacy of anticancer drugs entrapped in endolysosomal vesicles.

Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein.

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity. In this study, we report visible light control over the cytolytic activity of a protein. A water-soluble visible-light-operated tetra-ortho-fluoro-azobenzene photoswitch was synthesized by utilizing the nucleophilic aromatic substitution reaction for installing a solubilizing sulfonate group onto the electron-poor photoswitch structure. The azobenzene was attached to two cysteine mutants of the pore-forming protein fragaceatoxin C (FraC), and their respective activities were evaluated on red blood cells. For both mutants, the green-light-irradiated sample, containing predominantly the cis-azobenzene isomer, was more active compared to the blue-light-irradiated sample. Ultimately, the same modulation of the cytolytic activity pattern was observed toward a hypopharyngeal squamous cell carcinoma. These results constitute the first case of using low energy visible light to control the biological activity of a toxic protein.

Novel Fab-peptide-HLA-I fusion proteins for redirecting pre-existing anti-CMV T cell immunity to selective eliminate carcinoma cells.

Typically, anticancer CD8pos T cells occur at low frequencies and become increasingly impaired in the tumor micro environment. In contrast, antiviral CD8pos T cells display a much higher polyclonality, frequency, and functionality. In particular, cytomegalovirus (CMV) infection induces high numbers of 'inflationary' CD8pos T cells that remain lifelong abundantly present in CMV-seropositive subjects. Importantly, these so-called inflationary anti-CMV T cells increase with age, maintain a ready-to-go state, populate tumors, and do not become exhausted or senescent. Given these favorable attributes, we devised a novel series of recombinant Fab-peptide-HLA-I fusion proteins and coined them 'ReTARGs'. A ReTARG fusion protein consists of a high-affinity Fab antibody fragment directed to carcinoma-associated cell surface antigen EpCAM (or EGFR), fused in tandem with soluble HLA-I molecule/ß2-microglobulin, genetically equipped with an immunodominant peptide derived from CMV proteins pp65 (or IE-1). Decoration with EpCAM-ReTARGpp65 rendered EpCAM-expressing primary patient-derived carcinoma cells highly sensitive to selective elimination by cognate anti-CMV CD8pos T cells. Importantly, this treatment did not induce excessive levels of proinflammatory T cell-secreted IFNγ. In contrast, analogous treatment with equimolar amounts of EpCAM/CD3-directed bispecific T-cell engager solitomab resulted in a massive release of IFNγ, a feature commonly associated with adverse cytokine-release syndrome. Combinatorial treatment with EpCAM-ReTARGpp65 and EGFR-ReTARGIE-1 strongly potentiated selective cancer cell elimination owing to the concerted action of the corresponding cognate anti-CMV CD8pos T cell clones. In conclusion, ReTARG fusion proteins may be useful as an alternative or complementary form of targeted cancer immunotherapy for 'cold' solid cancers.

A proof-of-concept study on the use of a fluorescein-based 18F-tracer for pretargeted PET.

BACKGROUND: Pretargeted immuno-PET tumor imaging has emerged as a valuable diagnostic strategy that combines the high specificity of antibody-antigen interaction with the high signal and image resolution offered by short-lived PET isotopes, while reducing the irradiation dose caused by traditional 89Zr-labelled antibodies. In this work, we demonstrate proof of concept of a novel 'two-step' immuno-PET pretargeting approach, based on bispecific antibodies (bsAbs) engineered to feature dual high-affinity binding activity for a fluorescein-based 18F-PET tracer and tumor markers. RESULTS: A copper(I)-catalysed click reaction-based radiolabeling protocol was developed for the synthesis of fluorescein-derived molecule [18F]TPF. Binding of [18F]TPF on FITC-bearing bsAbs was confirmed. An in vitro autoradiography assay demonstrated that [18F]TPF could be used for selective imaging of EpCAM-expressing OVCAR3 cells, when pretargeted with EpCAMxFITC bsAb. The versatility of the pretargeting approach was showcased in vitro using a series of fluorescein-binding bsAbs directed at various established cancer-associated targets, including the pan-carcinoma cell surface marker EpCAM, EGFR, melanoma marker MCSP (aka CSPG4), and immune checkpoint PD-L1, offering a range of potential future applications for this pretargeting platform. CONCLUSION: A versatile pretargeting platform for PET imaging, which combines bispecific antibodies and a fluorescein-based 18F-tracer, is presented. It is shown to selectively target EpCAM-expressing cells in vitro and its further evaluation with different bispecific antibodies demonstrates the versatility of the approach.

Cancer cells under immune attack acquire CD47-mediated adaptive immune resistance independent of the myeloid CD47-SIRPα axis.

Cancer cells exploit CD47 overexpression to inhibit phagocytic elimination and neoantigen processing via the myeloid CD47-SIRPα axis and thereby indirectly evade adaptive T cell immunity. Here, we report on a hitherto unrecognized direct immunoinhibitory feature of cancer cell-expressed CD47. We uncovered that in response to IFNγ released during cognate T cell immune attack, cancer cells dynamically enhance CD47 cell surface expression, which coincides with acquiring adaptive immune resistance toward pro-apoptotic effector T cell mechanisms. Indeed, CRISPR/Cas9-mediated CD47-knockout rendered cancer cells more sensitive to cognate T cell immune attack. Subsequently, we developed a cancer-directed strategy to selectively overcome CD47-mediated adaptive immune resistance using bispecific antibody (bsAb) CD47xEGFR-IgG2s that was engineered to induce rapid and prolonged cancer cell surface displacement of CD47 by internalization. Treatment of CD47pos cancer cells with bsAb CD47xEGFR-IgG2s potently enhanced susceptibility to cognate CD8pos T cells. Targeting CD47-mediated adaptive immune resistance may open up new avenues in cancer immunotherapy.

Synthesis and Evaluation of 18F-Enzalutamide, a New Radioligand for PET Imaging of Androgen Receptors: A Comparison with 16ß-18F-Fluoro-5α-Dihydrotestosterone.

16ß-18F-fluoro-5α-dihydrotestosterone (18F-FDHT) is a radiopharmaceutical that has been investigated as a diagnostic agent for the assessment of androgen receptor (AR) density in prostate cancer using PET. However, 18F-FDHT is rapidly metabolized in humans and excreted via the kidneys into the urine, potentially compromising the detection of tumor lesions close to the prostate. Enzalutamide is an AR signaling inhibitor currently used in different stages of prostate cancer. Enzalutamide and its primary metabolite N-desmethylenzalutamide have an AR affinity comparable to that of FDHT but are excreted mainly via the hepatic route. Radiolabeled enzalutamide could thus be a suitable candidate PET tracer for AR imaging. Here, we describe the radiolabeling of enzalutamide with 18F. Moreover, the in vitro and in vivo behavior of 18F-enzalutamide was evaluated and compared with the current standard, 18F-FDHT. Methods:18F-enzalutamide was obtained by fluorination of the nitro precursor. In vitro cellular uptake studies with 18F-enzalutamide and 18F-FDHT were performed in LNCaP (AR-positive) and HEK293 (AR-negative) cells. Competition assays with both tracers were conducted on the LNCaP (AR-positive) cell line. In vivo PET imaging, ex vivo biodistribution, and metabolite studies with 18F-enzalutamide and 18F-FDHT were conducted on athymic nude male mice bearing an LNCaP xenograft in the shoulder. Results:18F-enzalutamide was obtained in 1.4% ± 0.9% radiochemical yield with an apparent molar activity of 6.2 ± 10.3 GBq/µmol. 18F-FDHT was obtained in 1.5% ± 0.8% yield with a molar activity of more than 25 GBq/µmol. Coincubation with an excess of 5α-dihydrotestosterone or enzalutamide significantly reduced the cellular uptake of 18F-enzalutamide and 18F-FDHT to about 50% in AR-positive LNCaP cells but not in AR-negative HEK293 cells. PET and biodistribution studies on male mice bearing a LnCaP xenograft showed about 3 times higher tumor uptake for 18F-enzalutamide than for 18F-FDHT. Sixty minutes after tracer injection, 93% of 18F-enzalutamide in plasma was still intact, compared with only 3% of 18F-FDHT. Conclusion: Despite its lower apparent molar activity, 18F-enzalutamide shows higher tumor uptake and better metabolic stability than 18F-FDHT and thus seems to have more favorable properties for imaging of AR with PET. However, further evaluation in other oncologic animal models and patients is warranted to confirm these results.

Bispecific antibody CD73xEpCAM selectively inhibits the adenosine-mediated immunosuppressive activity of carcinoma-derived extracellular vesicles.

Tumor-derived extracellular vesicles (EVs) carry potent immunosuppressive factors that affect the antitumor activities of immune cells. A significant part of the immunoinhibitory activity of EVs is attributable to CD73, a GPI-anchored ecto-5'-nucleotidase involved in the conversion of tumor-derived proinflammatory extracellular ATP (eATP) to immunosuppressive adenosine (ADO). The CD73-antagonist antibody oleclumab inhibits cell surface-exposed CD73 and is currently undergoing clinical testing for cancer immunotherapy. However, a strategy to selectively inhibit CD73 exposed on EVs is not available. Here, we present a novel bispecific antibody (bsAb) CD73xEpCAM designed to bind with high affinity the common EV surface marker EpCAM and concurrently inhibit CD73. Unlike oleclumab, bsAb CD73xEpCAM potently inhibited the immunosuppressive activity of EVs from CD73pos/EpCAMpos carcinoma cell lines and patient-derived colorectal cancer cells. Taken together, selective blockade of EV-exposed CD73 by bsAb CD73xEpCAM may be useful as an alternate or complementary targeted approach in cancer immunotherapy.

Bispecific antibody approach for EGFR-directed blockade of the CD47-SIRPα "don't eat me" immune checkpoint promotes neutrophil-mediated trogoptosis and enhances antigen cross-presentation.

Cancer cells overexpress CD47 to subvert phagocytic elimination and evade immunogenic processing of cancer antigens. Moreover, CD47 overexpression inhibits the antibody-dependent cellular phagocytosis (ADCP) and cytotoxicity (ADCC) activities of therapeutic anticancer antibodies. Consequently, CD47-blocking antibodies have been developed to overcome the immunoevasive activities of cancer cell-expressed CD47. However, the wide-spread expression of CD47 on normal cells forms a massive "antigen sink" that potentially limits sufficient tumor accretion of these antibodies. Additionally, a generalized blockade of CD47-SIRPα interaction may ultimately lead to unintended cross-presentation of self-antigens potentially promoting autoimmunity. To address these issues, we constructed a bispecific antibody, designated bsAb CD47xEGFR-IgG1, that blocks cancer cell surface-expressed CD47 in an EGFR-directed manner. BsAb CD47xEGFR-IgG1 selectively induced phagocytic removal of EGFRpos/CD47pos cancer cells and endowed neutrophils with capacity to kill these cancer cells by trogoptosis; an alternate form of ADCC that disrupts the target cell membrane. Importantly, bsAb CD47xEGFR-IgG1 selectively enhanced phagocytosis and immunogenic processing of EGFRpos/CD47pos cancers cells ectopically expressing viral protein CMVpp65. In conclusion, bsAb CD47xEGFR-IgG1 may be useful to reduce on-target/off-tumor effects of CD47-blocking approaches, enhance cancer cell elimination by trogoptosis, and promote adaptive anticancer immune responses.

Targeted delivery of a designed sTRAIL mutant results in superior apoptotic activity towards EGFR-positive tumor cells.

Previously, we have shown that epidermal growth factor receptor (EGFR)-selective delivery of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), by genetic fusion to antibody fragment scFv425, enhances the tumor-selective pro-apoptotic activity of sTRAIL. Insight into the respective contribution of the agonistic receptors TRAIL-R1 and TRAIL-R2 to TRAIL-induced apoptosis may provide a rational approach to further optimize TRAIL-based therapy. Recently, this issue has been investigated using sTRAIL mutants designed to selectively bind to either receptor. However, the relative contribution of the respective TRAIL receptors, in particular TRAIL-R1, in TRAIL signaling is still unresolved. Here, we fused scFv425 to designed sTRAIL mutant sTRAILmR1-5, reported to selectively activate TRAIL-R1, and investigated the therapeutic apoptotic activity of this novel fusion protein. EGFR-specific binding of scFv425:sTRAILmR1-5 potently induced apoptosis, which was superior to the apoptotic activity of scFv425:sTRAIL-wt and a nontargeted MOCK-scFv:sTRAILmR1-5. During cotreatment with cisplatin or the histone deacetylase inhibitor valproic acid, scFv425:sTRAILmR1-5 retained its superior pro-apoptotic activity compared to scFv425:sTRAIL-wt. However, in catching-type Enzyme-Linked ImmunoSorbent Assays with TRAIL-R1:Fc and TRAIL-R2:Fc, scFv425:sTRAILmR1-5 was found to not only bind to TRAIL-R1 but also to TRAIL-R2. Binding to TRAIL-R2 also had functional consequences because the apoptotic activity of scFv425:sTRAILmR1-5 was strongly inhibited by a TRAIL-R2 blocking monoclonal antibody. Moreover, scFv425:sTRAILmR1-5 retained apoptotic activity upon selective knockdown of TRAIL-R1 using small inhibitory RNA. Collectively, these data indicate that both agonistic TRAIL receptors are functionally involved in TRAIL signaling by scFv425:sTRAILmR1-5 in solid tumor cells. Moreover, the superior target cell-restricted apoptotic activity of scFv425:sTRAILmR1-5 indicates its therapeutic potential for EGFR-positive solid tumors.

Bispecific Antibody Approach for Improved Melanoma-Selective PD-L1 Immune Checkpoint Blockade.

Reactivation of functionally-impaired anticancer T cells by programmed cell death protein 1 (PD-1) and programmed cell death receptor ligand-1 (PD-L1)-blocking antibodies shows prominent therapeutic benefit in advanced melanoma and patients with non-small cell lung cancer. However, current PD-L1-blocking antibodies lack intrinsic tumor selectivity. Therefore, efficacy may be reduced resulting from on-target and off-tumor binding to PD-L1-expressing normal cells. This may lead to indiscriminate activation of antigen-experienced T cells, including those implicated in autoimmune-related adverse events. To direct PD-L1 blockade to chondroitin sulfate proteoglycan 4 (CSPG4)-expressing cancers and to reactivate anticancer T cells more selectively, we constructed bispecific antibody PD-L1xCSPG4. CSPG4 is an established target antigen that is selectively overexpressed on malignant melanoma and various other difficult-to-treat cancers. PD-L1xCSPG4 showed enhanced capacity for CSPG4-directed blockade of PD-L1 on cancer cells. Importantly, treatment of mixed cultures containing primary patient-derived CSPG4-expressing melanoma cells and autologous tumor-infiltrating lymphocytes with PD-L1xCSPG4 significantly enhanced activation status, IFN-γ production, and cytolytic activity of anticancer T cells. In conclusion, tumor-directed blockade of PD-L1 by PD-L1xCSPG4 may improve efficacy and safety of PD-1/PD-L1 checkpoint blockade for treatment of melanoma and other CSPG4-overexpressing malignancies.

Quantitative proteomics analysis identifies MUC1 as an effect sensor of EGFR inhibition.

Tumor responses to cancer therapeutics are generally monitored every 2-3 months based on changes in tumor size. Dynamic biomarkers that reflect effective engagement of targeted therapeutics to the targeted pathway, so-called "effect sensors", would fulfill a need for non-invasive, drug-specific indicators of early treatment effect. Using a proteomics approach to identify effect sensors, we demonstrated MUC1 upregulation in response to epidermal growth factor receptor (EGFR)-targeting treatments in breast and lung cancer models. To achieve this, using semi-quantitative mass spectrometry, we found MUC1 to be significantly and durably upregulated in response to erlotinib, an EGFR-targeting treatment. MUC1 upregulation was regulated transcriptionally, involving PI3K-signaling and STAT3. We validated these results in erlotinib-sensitive human breast and non-small lung cancer cell lines. Importantly, erlotinib treatment of mice bearing SUM149 xenografts resulted in increased MUC1 shedding into plasma. Analysis of MUC1 using serial blood sampling may therefore be a new, relatively non-invasive tool to monitor early and drug-specific effects of EGFR-targeting therapeutics.

A novel bispecific antibody for EGFR-directed blockade of the PD-1/PD-L1 immune checkpoint.

PD-L1-blocking antibodies produce significant clinical benefit in selected cancer patients by reactivating functionally-impaired antigen-experienced anticancer T cells. However, the efficacy of current PD-L1-blocking antibodies is potentially reduced by 'on-target/off-tumor' binding to PD-L1 widely expressed on normal cells. This lack of tumor selectivity may induce a generalized activation of all antigen-experienced T cells which may explain the frequent occurrence of autoimmune-related adverse events during and after treatment. To address these issues, we constructed a bispecific antibody (bsAb), designated PD-L1xEGFR, to direct PD-L1-blockade to EGFR-expressing cancer cells and to more selectively reactivate anticancer T cells. Indeed, the IC50 of PD-L1xEGFR for blocking PD-L1 on EGFR+ cancer cells was ∼140 fold lower compared to that of the analogous PD-L1-blocking bsAb PD-L1xMock with irrelevant target antigen specificity. Importantly, activation status, IFN-γ production, and oncolytic activity of anti-CD3xanti-EpCAM-redirected T cells was enhanced when cocultured with EGFR-expressing carcinoma cells. Similarly, the capacity of PD-L1xEGFR to promote proliferation and IFN-γ production by CMVpp65-directed CD8+ effector T cells was enhanced when cocultured with EGFR-expressing CMVpp65-transfected cancer cells. In contrast, the clinically-used PD-L1-blocking antibody MEDI4736 (durvalumab) promoted T cell activation indiscriminate of EGFR expression on cancer cells. Additionally, in mice xenografted with EGFR-expressing cancer cells 111In-PD-L1xEGFR showed a significantly higher tumor uptake compared to 111In-PD-L1xMock. In conclusion, PD-L1xEGFR blocks the PD-1/PD-L1 immune checkpoint in an EGFR-directed manner, thereby promoting the selective reactivation of anticancer T cells. This novel targeted approach may be useful to enhance efficacy and safety of PD-1/PD-L1 checkpoint blockade in EGFR-overexpressing malignancies.

CD20-selective inhibition of CD47-SIRPα "don't eat me" signaling with a bispecific antibody-derivative enhances the anticancer activity of daratumumab, alemtuzumab and obinutuzumab.

Here, we report on a novel bispecific antibody-derivative, designated RTX-CD47, with unique capacity for CD20-directed inhibition of CD47-SIRPα "don't eat me" signaling. RTX-CD47 comprises a CD20-targeting scFv antibody fragment derived from rituximab fused in tandem to a CD47-blocking scFv. Single agent treatment with RTX-CD47 triggered significant phagocytic removal of CD20pos/CD47pos malignant B-cells, but not of CD20neg/CD47pos cells, and required no pro-phagocytic FcR-mediated signaling. Importantly, treatment with RTX-CD47 synergistically enhanced the phagocytic elimination of primary malignant B cells by autologous phagocytic effector cells as induced by therapeutic anticancer antibodies daratumumab (anti-CD38), alemtuzumab (anti-CD52) and obinutuzumab (anti-CD20). In conclusion, RTX-CD47 blocks CD47 "don't eat me" signaling by cancer cells in a CD20-directed manner with essentially no activity towards CD20neg/CD47pos cells and enhances the activity of therapeutic anticancer antibodies directed to B-cell malignancies.

Target cell-restricted apoptosis induction of acute leukemic T cells by a recombinant tumor necrosis factor-related apoptosis-inducing ligand fusion protein with specificity for human CD7.

Current treatment of human T-cell leukemia and lymphoma is predominantly limited to conventional cytotoxic therapy and is associated with limited therapeutic response and significant morbidity. Therefore, more potent and leukemia-specific therapies with favorable toxicity profiles are urgently needed. Here, we report on the construction of a novel therapeutic fusion protein, scFvCD7:sTRAIL, designed to induce target antigen-restricted apoptosis in human T-cell tumors. ScFvCD7:sTRAIL consists of the death-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) genetically linked to an scFv antibody fragment specific for the T-cell surface antigen CD7. Treatment with scFvCD7:sTRAIL induced potent CD7-restricted apoptosis in a series of malignant T-cell lines, whereas normal resting leukocytes, activated T cells, and vascular endothelial cells (human umbilical vein endothelial cells) showed no detectable apoptosis. The apoptosis-inducing activity of scFvCD7:sTRAIL was stronger than that of the immunotoxin scFvCD7:ETA. In mixed culture experiments with CD7-positive and CD7-negative tumor cells, scFvCD7:sTRAIL induced very potent bystander apoptosis of CD7-negative tumor cells. In vitro treatment of blood cells freshly derived from T-acute lymphoblastic leukemia patients resulted in marked apoptosis of the malignant T cells that was strongly augmented by vincristin. In conclusion, scFvCD7:sTRAIL is a novel recombinant protein causing restricted apoptosis in human leukemic T cells with low toxicity for normal human blood and endothelial cells.

A versatile pretargeting approach for tumour-selective delivery and activation of TNF superfamily members.

TNFR superfamily (TNFRSF) members have important immunoregulatory functions and are of clear interest for cancer immunotherapy. Various TNFRSF agonists have been clinically evaluated, but have met with limited efficacy and/or toxicity. Recent insights indicate that 'first-generation' TNFRSF agonists lack efficacy as they do not effectively cross-link their corresponding receptor. Reversely, ubiquitous TNFRSF receptor(s) cross-linking by CD40 and Fas agonistic antibodies resulted in dose-limiting liver toxicity. To overcome these issues, we developed a novel pretargeting strategy exploiting recombinant fusion proteins in which a soluble form of TRAIL, FasL or CD40L is genetically fused to a high-affinity anti-fluorescein scFv antibody fragment (scFvFITC). Fusion proteins scFvFITC:sTRAIL and scFvFITC:sFasL induced potent target antigen-restricted apoptosis in a panel of cancer lines and in primary patient-derived cancer cells, but only when pretargeted with a relevant FITC-labelled antitumour antibody. In a similar pretargeting setting, fusion protein scFvFITC:sCD40L promoted tumour-directed maturation of immature monocyte-derived dendritic cells (iDCs). This novel tumour-selective pretargeting approach may be used to improve efficacy and/or reduce possible off-target toxicity of TNFSF ligands for cancer immunotherapy.

Programmed Death Ligand 1 (PD-L1)-targeted TRAIL combines PD-L1-mediated checkpoint inhibition with TRAIL-mediated apoptosis induction.

Antibodies that block PD-L1/PD-1 immune checkpoints restore the activity of functionally-impaired antitumor T cells. These antibodies show unprecedented clinical benefit in various advanced cancers, particularly in melanoma. However, only a subset of cancer patients responds to current PD-L1/PD-1-blocking strategies, highlighting the need for further advancements in PD-L1/PD-1-based immunotherapy. Here, we report on a novel approach designed to combine PD-L1 checkpoint inhibition with the tumor-selective induction of apoptosis by TNF-related Apoptosis Inducing Ligand (TRAIL). In brief, a new bi-functional fusion protein, designated anti-PD-L1:TRAIL, was constructed comprising a PD-L1-blocking antibody fragment genetically fused to the extracellular domain of the pro-apoptotic tumoricidal protein TRAIL. Treatment of PD-L1-expressing cancer cells with anti-PD-L1:TRAIL induced PD-L1-directed TRAIL-mediated cancer cell death. Treatment of T cells with anti-PD-L1:TRAIL augmented T cell activation, as evidenced by increased proliferation, secretion of IFNγ and enhanced killing of cancer cell lines and primary patient-derived cancer cells in mixed T cell/cancer cell culture experiments. Of note, elevated levels of IFNγ further upregulated PD-L1 on cancer cells and simultaneously sensitized cancer cells to TRAIL-mediated apoptosis by anti-PD-L1:TRAIL. Additionally, anti-PD-L1:TRAIL converted immunosuppressive PD-L1-expressing myeloid cells into pro-apoptotic effector cells that triggered TRAIL-mediated cancer cell death. In conclusion, combining PD-L1 checkpoint inhibition with TRAIL-mediated induction of apoptosis using anti-PD-L1:TRAIL yields promising multi-fold and mutually reinforcing anticancer activity that may be exploited to enhance the efficacy of therapeutic PD-L1/PD-1 checkpoint inhibition.

CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαß+ CD8αß+ T cells that can be targeted for cancer immunotherapy.

CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in high-grade serous epithelial ovarian cancer (HGSC). However, whether these cells are induced as part of an ongoing anti-HGSC immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown. In this study, we first confirmed that CD103+ TIL from HGSC were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαß+ CD8αß+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFßR1-dependent manner. Finally, CD103+ TIL isolated from primary HGSC showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27. Taken together, our data indicate CD103+ TIL in HGSC are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition.