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.

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.

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.

Drug Sensitivity Prediction Models Reveal a Link between DNA Repair Defects and Poor Prognosis in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is characterized by the frequent manifestation of DNA crosslink repair defects. We established novel expression-based DNA repair defect markers to determine the clinical impact of such repair defects. Using hypersensitivity to the DNA crosslinking agents, mitomycin C and olaparib, as proxies for functional DNA repair defects in a panel of 25 HNSCC cell lines, we applied machine learning to define gene expression models that predict repair defects. The expression profiles established predicted hypersensitivity to DNA-damaging agents and were associated with mutations in crosslink repair genes, as well as downregulation of DNA damage response and repair genes, in two independent datasets. The prognostic value of the repair defect prediction profiles was assessed in two retrospective cohorts with a total of 180 patients with advanced HPV-negative HNSCC, who were treated with cisplatin-based chemoradiotherapy. DNA repair defects, as predicted by the profiles, were associated with poor outcome in both patient cohorts. The poor prognosis association was particularly strong in normoxic tumor samples and was linked to an increased risk of distant metastasis. In vitro, only crosslink repair-defective HNSCC cell lines are highly migratory and invasive. This phenotype could also be induced in cells by inhibiting rad51 in repair competent and reduced by DNA-PK inhibition. In conclusion, DNA crosslink repair prediction expression profiles reveal a poor prognosis association in HNSCC. SIGNIFICANCE: This study uses innovative machine learning-based approaches to derive models that predict the effect of DNA repair defects on treatment outcome in HNSCC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/21/5597/F1.large.jpg.

The In Vitro Response of Tissue Stem Cells to Irradiation With Different Linear Energy Transfers.

PURPOSE: A reduction in the dose, irradiated volume, and sensitivity of, in particular, normal tissue stem cells is needed to advance radiation therapy. This could be obtained with the use of particles for radiation therapy. However, the radiation response of normal tissue stem cells is still an enigma. Therefore, in the present study, we developed a model to investigate the in vitro response of stem cells to particle irradiation. METHODS AND MATERIALS: We used the immortalized human salivary gland (HSG) cell line resembling salivary gland (SG) cells to translate the radiation response in 2-dimensional (2D) to 3-dimensional (3D) conditions. This response was subsequently translated to the response of SG stem cells (SGSCs). Dispersed single cells were irradiated with photons or carbon ions at different linear energy transfers (LETs; 48.76 ± 2.16, 149.9 ± 10.8, and 189 ± 15 keV/µm). Subsequently, 2D or 3D clonogenicity was determined by counting the colonies or secondary stem cell-derived spheres in Matrigel. γH2AX immunostaining was used to assess DNA double strand break repair. RESULTS: The 2D response of HSG cells showed a similar increase in dose response to increasing higher LET irradiation as other cell lines. The 3D response of HSG cells to increasing LET irradiation was reduced compared with the 2D response. Finally, the response of mouse SGSCs to photons was similar to the 3D response of HSG cells. The response to higher LET irradiation was reduced in the stem cells. CONCLUSIONS: Mouse SGSC radiosensitivity seems reduced at higher LET radiation compared with transformed HSG cells. The developed model to assess the radiation response of SGSCs offers novel possibilities to study the radiation response of normal tissue in vitro.