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.

Bispecific antibody CD73xEGFR more selectively inhibits the CD73/adenosine immune checkpoint on cancer cells and concurrently counteracts pro-oncogenic activities of CD73 and EGFR.

BACKGROUND: CD73 is an ecto-enzyme that is involved in the conversion of pro-inflammatory extracellular ATP (eATP) excreted by cancer cells under stress to anti-inflammatory adenosine (ADO). A broad variety of solid cancer types was shown to exploit CD73 overexpression as a suppressive immune checkpoint. Consequently, CD73-antagonistic antibodies, most notably oleclumab, are currently evaluated in several multicenter trials for clinical applicability. However, the efficacy of conventional monospecific CD73-inhibiting antibodies may be limited due to on-target/off-tumor binding to CD73 on normal cells. Therefore, a novel approach that more selectively directs CD73 immune checkpoint inhibition towards cancer cells is warranted. METHODS: To address this issue, we constructed a novel tetravalent bispecific antibody (bsAb), designated bsAb CD73xEGFR. Subsequently, the anticancer activities of bsAb CD73xEGFR were evaluated using in vitro and in vivo tumor models. RESULTS: In vitro treatment of various carcinoma cell types with bsAb CD73xEGFR potently inhibited the enzyme activity of CD73 (~71%) in an EGFR-directed manner. In this process, bsAb CD73xEGFR induced rapid internalization of antigen/antibody complexes, which resulted in a prolonged concurrent displacement of both CD73 and EGFR from the cancer cell surface. In addition, bsAb CD73xEGFR sensitized cancer to the cytotoxic activity of various chemotherapeutic agents and potently inhibited the proliferative/migratory capacity (~40%) of cancer cells. Unexpectedly, we uncovered that treatment of carcinoma cells with oleclumab appeared to enhance several pro-oncogenic features, including upregulation and phosphorylation of EGFR, tumor cell proliferation (~20%), and resistance towards cytotoxic agents and ionizing radiation (~39%). Importantly, in a tumor model using immunocompetent BALB/c mice inoculated with syngeneic CD73pos/EGFRpos CT26 cancer cells, treatment with bsAb CD73xEGFR outperformed oleclumab (65% vs 31% tumor volume reduction). Compared with oleclumab, treatment with bsAb CD73xEGFR enhanced the intratumoral presence of CD8pos T cells and M1 macrophages. CONCLUSIONS: BsAb CD73xEGFR outperforms oleclumab as it inhibits the CD73/ADO immune checkpoint in an EGFR-directed manner and concurrently counteracts several oncogenic activities of EGFR and CD73. Therefore, bsAb CD73xEGFR may be of significant clinical potential for various forms of difficult-to-treat solid cancer types.

Applications of Anti-Cytomegalovirus T Cells for Cancer (Immuno)Therapy.

Infection with cytomegalovirus (CMV) is highly prevalent in the general population and largely controlled by CD8pos T cells. Intriguingly, anti-CMV T cells accumulate over time to extraordinarily high numbers, are frequently present as tumor-resident 'bystander' T cells, and remain functional in cancer patients. Consequently, various strategies for redirecting anti-CMV CD8pos T cells to eliminate cancer cells are currently being developed. Here, we provide an overview of these strategies including immunogenic CMV peptide-loading onto endogenous HLA complexes on cancer cells and the use of tumor-directed fusion proteins containing a preassembled CMV peptide/HLA-I complex. Additionally, we discuss conveying the advantageous characteristics of anti-CMV T cells in adoptive cell therapy. Utilization of anti-CMV CD8pos T cells to generate CAR T cells promotes their in vivo persistence and expansion due to appropriate co-stimulation through the endogenous (CMV-)TCR signaling complex. Designing TCR-engineered T cells is more challenging, as the artificial and endogenous TCR compete for expression. Moreover, the use of expanded/reactivated anti-CMV T cells to target CMV peptide-expressing glioblastomas is discussed. This review highlights the most important findings and compares the benefits, disadvantages, and challenges of each strategy. Finally, we discuss how anti-CMV T cell therapies can be further improved to enhance treatment efficacy.

A Novel Bispecific Antibody for EpCAM-Directed Inhibition of the CD73/Adenosine Immune Checkpoint in Ovarian Cancer.

PD-1/PD-L1-inhibiting antibodies have shown disappointing efficacy in patients with refractory ovarian cancer (OC). Apparently, OC cells exploit nonoverlapping immunosuppressive mechanisms to evade the immune system. In this respect, the CD73-adenosine inhibitory immune checkpoint is of particular interest, as it rapidly converts pro-inflammatory ATP released from cancer cells to immunosuppressive adenosine (ADO). Moreover, cancer-cell-produced ADO is known to form a highly immunosuppressive extra-tumoral 'halo' that chronically inhibits the anticancer activity of various immune effector cells. Thus far, conventional CD73-blocking antibodies such as oleclumab show limited clinical efficacy, probably due to the fact that it indiscriminately binds to and blocks CD73 on a massive surplus of normal cells. To address this issue, we constructed a novel bispecific antibody (bsAb) CD73xEpCAM that inhibits CD73 expressed on the OC cell surface in an EpCAM-directed manner. Importantly, bsAb CD73xEpCAM showed potent capacity to inhibit the CD73 enzyme activity in an EpCAM-directed manner and restore the cytotoxic activity of ADO-suppressed anticancer T cells. Additionally, treatment with bsAb CD73xEpCAM potently inhibited the proliferative capacity of OC cells and enhanced their sensitivity to cisplatin, doxorubicin, 5FU, and ionizing radiation. BsAb CD73xEpCAM may be useful in the development of tumor-directed immunotherapeutic approaches to overcome the CD73-mediated immunosuppression in patients with refractory OC.

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.

Impact of cumulative complications on 1-year treatment-related healthcare costs in patients with colorectal peritoneal metastases undergoing cytoreductive surgery with hyperthermic intraperitoneal chemotherapy.

BACKGROUND: The aim of this study was to evaluate the impact of all minor and major complications on treatment-related healthcare costs in patients who undergo cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for the treatment of colorectal peritoneal metastases (PMs). METHOD: Patients with histologically proven colorectal PMs who underwent CRS + HIPEC from March 2006 to October 2019 in a tertiary referral centre were retrospectively identified from a prospectively maintained database. Patients were divided into six subgroups according to the severity of the complications, which were scored using the comprehensive complication index (CCI) (CCI 0-9.9, CCI 10-19.9, CCI 20-29.9, CCI 30-39.9, CCI 40-49.9, and CCI 50 or higher). Treatment-related healthcare costs up to 1 year after CRS + HIPEC were obtained from the financial department. Differences in costs and survival outcomes were compared using the chi-squared test and Kruskal-Wallis H test. RESULTS: A total of 142 patients were included (CCI 0-9.9, 53 patients; CCI 10-19.9, 0 patients; CCI 20-29.9, 45 patients; CCI 30-39.9, 14 patients; CCI 40-49, 9 patients; and CCI 50 or higher, 21 patients). Median (interquartile range) treatment-related healthcare costs increased significantly and exponentially for the CCI 30-39, CCI 40-49, and CCI 50 or higher groups (€48 993 (€44 262-€84 805); €57 167 (€43 047-€67 591); and €82 219 (€55 487-€145 314) respectively) compared with those for the CCI 0-9.9 and CCI 20-29.9 groups (€33 856 (€24 433-€40 779) and €40 621 (€31 501-€58 761) respectively, P < 0.010). CONCLUSION: Treatment-related healthcare costs increase exponentially as more complications develop among patients who undergo CRS + HIPEC for the treatment of colorectal PMs. Anastomotic leakages after CRS + HIPEC lead to an increase of 295 per cent of treatment-related healthcare costs.

Neoantigen-based cancer vaccination using chimeric RNA-loaded dendritic cell-derived extracellular vesicles.

Cancer vaccines critically rely on the availability of targetable immunogenic cancer-specific neoepitopes. However, mutation-based immunogenic neoantigens are rare or even non-existent in subgroups of cancer types. To address this issue, we exploited a cancer-specific aberrant transcription-induced chimeric RNA, designated A-Pas chiRNA, as a possible source of clinically relevant and targetable neoantigens. A-Pas chiRNA encodes a recently discovered cancer-specific chimeric protein that comprises full-length astrotactin-2 (ASTN2) C-terminally fused in-frame to the antisense sequence of the 18th intron of pregnancy-associated plasma protein-A (PAPPA). We used extracellular vesicles (EVs) from A-Pas chiRNA-transfected dendritic cells (DCs) to produce the cell-free anticancer vaccine DEXA-P . Treatment of immunocompetent cancer-bearing mice with DEXA-P inhibited tumour growth and prolonged animal survival. In summary, we demonstrate for the first time that cancer-specific transcription-induced chimeric RNAs can be exploited to produce a cell-free cancer vaccine that induces potent CD8+ T cell-mediated anticancer immunity. Our novel approach may be particularly useful for developing cancer vaccines to treat malignancies with low mutational burden or without mutation-based antigens. Moreover, this cell-free anticancer vaccine approach may offer several practical advantages over cell-based vaccines, such as ease of scalability and genetic modifiability as well as enhanced shelf life.

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.

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.

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 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.

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.

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.

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.

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.

Antigen-specific active immunotherapy for ovarian cancer.

BACKGROUND: This is the second update of the review first published in the Cochrane Library (2010, Issue 2) and later updated (2014, Issue 9).Despite advances in chemotherapy, the prognosis of ovarian cancer remains poor. Antigen-specific active immunotherapy aims to induce tumour antigen-specific anti-tumour immune responses as an alternative treatment for ovarian cancer. OBJECTIVES: Primary objective• To assess the clinical efficacy of antigen-specific active immunotherapy for the treatment of ovarian cancer as evaluated by tumour response measured by Response Evaluation Criteria In Solid Tumors (RECIST) and/or cancer antigen (CA)-125 levels, response to post-immunotherapy treatment, and survival differences◦ In addition, we recorded the numbers of observed antigen-specific humoral and cellular responsesSecondary objective• To establish which combinations of immunotherapeutic strategies with tumour antigens provide the best immunological and clinical results SEARCH METHODS: For the previous version of this review, we performed a systematic search of the Cochrane Central Register of Controlled Trials (CENTRAL; 2009, Issue 3), in the Cochrane Library, the Cochrane Gynaecological Cancer Group Specialised Register, MEDLINE and Embase databases, and clinicaltrials.gov (1966 to July 2009). We also conducted handsearches of the proceedings of relevant annual meetings (1996 to July 2009).For the first update of this review, we extended the searches to October 2013, and for this update, we extended the searches to July 2017. SELECTION CRITERIA: We searched for randomised controlled trials (RCTs), as well as non-randomised studies (NRSs), that included participants with epithelial ovarian cancer, irrespective of disease stage, who were treated with antigen-specific active immunotherapy, irrespective of type of vaccine, antigen used, adjuvant used, route of vaccination, treatment schedule, and reported clinical or immunological outcomes. DATA COLLECTION AND ANALYSIS: Two reviews authors independently extracted the data. We evaluated the risk of bias for RCTs according to standard methodological procedures expected by Cochrane, and for NRSs by using a selection of quality domains deemed best applicable to the NRS. MAIN RESULTS: We included 67 studies (representing 3632 women with epithelial ovarian cancer). The most striking observations of this review address the lack of uniformity in conduct and reporting of early-phase immunotherapy studies. Response definitions show substantial variation between trials, which makes comparison of trial results unreliable. Information on adverse events is frequently limited. Furthermore, reports of both RCTs and NRSs frequently lack the relevant information necessary for risk of bias assessment. Therefore, we cannot rule out serious biases in most of the included trials. However, selection, attrition, and selective reporting biases are likely to have affected the studies included in this review. GRADE ratings were high only for survival; for other primary outcomes, GRADE ratings were very low.The largest body of evidence is currently available for CA-125-targeted antibody therapy (17 studies, 2347 participants; very low-certainty evidence). Non-randomised studies of CA-125-targeted antibody therapy suggest improved survival among humoral and/or cellular responders, with only moderate adverse events. However, four large randomised placebo-controlled trials did not show any clinical benefit, despite induction of immune responses in approximately 60% of participants. Time to relapse with CA-125 monoclonal antibody versus placebo, respectively, ranged from 10.3 to 18.9 months versus 10.3 to 13 months (six RCTs, 1882 participants; high-certainty evidence). Only one RCT provided data on overall survival, reporting rates of 80% in both treatment and placebo groups (three RCTs, 1062 participants; high-certainty evidence). Other small studies targeting many different tumour antigens have presented promising immunological results. As these strategies have not yet been tested in RCTs, no reliable inferences about clinical efficacy can be made. Given the promising immunological results and the limited side effects and toxicity reported, exploration of clinical efficacy in large well-designed RCTs may be worthwhile. AUTHORS' CONCLUSIONS: We conclude that despite promising immunological responses, no clinically effective antigen-specific active immunotherapy is yet available for ovarian cancer. Results should be interpreted cautiously, as review authors found a significant dearth of relevant information for assessment of risk of bias in both RCTs and NRSs.

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.