A novel efficient bispecific antibody format, combining a conventional antigen-binding fragment with a single domain antibody, avoids potential heavy-light chain mis-pairing

Due to the technical innovations in generating bispecific antibodies (BsAbs) in recent years, BsAbs have become important reagents for diagnostic and therapeutic applications. However, the difficulty of producing a heterodimer consisting of two different arms with high yield and purity constituted a major limitation for their application in academic and clinical settings. Here, we describe a novel Fc-containing BsAb format (Fab × sdAb-Fc) composed of a conventional antigen-binding fragment (Fab), and a single domain antibody (sdAb), which avoids heavy-light chain mis-pairing during antibody assembly. In this study, the Fab x sdAb-Fc BsAbs were efficiently produced by three widely used heavy-heavy chain heterodimerization methods: Knobs-into-holes (KIH), Charge-pairs (CP) and controlled Fab-arm exchange (cFAE), respectively. The novel Fab x sdAb-Fc format provided a rapid and efficient strategy to generate BsAb with high purity and a unique possibility to further purify desired BsAbs from undesired antibodies based on molecular weight (MW). Compared to conventional BsAb formats, the advantages of Fab x sdAb-Fc format may thus provide a straightforward opportunity to apply bispecific antibody principles to research and development of novel targets and pathways in diseases such as cancer and autoimmunity

De-escalation of axillary treatment in the event of a positive sentinel lymph node biopsy in cT1-2 N0 breast cancer treated with mastectomy:nationwide registry study (BOOG 2013-07)

Background: Trials have demonstrated the safety of omitting completion axillary lymph node dissection in patients with cT1-2 N0 breast cancer operated with breast-conserving surgery who have limited metastatic burden in the sentinel lymph node. The aim of this registry study was to provide insight into the oncological safety of omitting completion axillary treatment in patients operated with mastectomy who have limited-volume sentinel lymph node metastasis. Methods: Women diagnosed in 2013-2014 with unilateral cT1-2 N0 breast cancer treated with mastectomy, with one to three sentinel lymph node metastases (pN1mi-pN1a), were identified from the Netherlands Cancer Registry, and classified by axillary treatment: no completion axillary treatment, completion axillary lymph node dissection, regional radiotherapy, or completion axillary lymph node dissection followed by regional radiotherapy. The primary endpoint was 5-year regional recurrence rate. Secondary endpoints included recurrence-free interval and overall survival, among others. Results: In total, 1090 patients were included (no completion axillary treatment, 219 (20.1%); completion axillary lymph node dissection, 437 (40.1%); regional radiotherapy, 327 (30.0%); completion axillary lymph node dissection and regional radiotherapy, 107 (9.8%)). Patients in the group without completion axillary treatment had more favourable tumour characteristics and were older. The overall 5-year regional recurrence rate was 1.3%, and did not differ significantly between the groups. The recurrence-free interval was also comparable among groups. The group of patients who did not undergo completion axillary treatment had statistically significantly worse 5-year overall survival, owing to a higher percentage of non-cancer deaths. Conclusion:In this registry study of patients with cT1-2 N0 breast cancer treated with mastectomy, with low-volume sentinel lymph node metastasis, the 5-year regional recurrence rate was low and comparable between patients with and without completion axillary treatment.</p

De-escalation of axillary treatment in the event of a positive sentinel lymph node biopsy in cT1-2 N0 breast cancer treated with mastectomy:nationwide registry study (BOOG 2013-07)

Background: Trials have demonstrated the safety of omitting completion axillary lymph node dissection in patients with cT1-2 N0 breast cancer operated with breast-conserving surgery who have limited metastatic burden in the sentinel lymph node. The aim of this registry study was to provide insight into the oncological safety of omitting completion axillary treatment in patients operated with mastectomy who have limited-volume sentinel lymph node metastasis. Methods: Women diagnosed in 2013-2014 with unilateral cT1-2 N0 breast cancer treated with mastectomy, with one to three sentinel lymph node metastases (pN1mi-pN1a), were identified from the Netherlands Cancer Registry, and classified by axillary treatment: no completion axillary treatment, completion axillary lymph node dissection, regional radiotherapy, or completion axillary lymph node dissection followed by regional radiotherapy. The primary endpoint was 5-year regional recurrence rate. Secondary endpoints included recurrence-free interval and overall survival, among others. Results: In total, 1090 patients were included (no completion axillary treatment, 219 (20.1%); completion axillary lymph node dissection, 437 (40.1%); regional radiotherapy, 327 (30.0%); completion axillary lymph node dissection and regional radiotherapy, 107 (9.8%)). Patients in the group without completion axillary treatment had more favourable tumour characteristics and were older. The overall 5-year regional recurrence rate was 1.3%, and did not differ significantly between the groups. The recurrence-free interval was also comparable among groups. The group of patients who did not undergo completion axillary treatment had statistically significantly worse 5-year overall survival, owing to a higher percentage of non-cancer deaths. Conclusion:In this registry study of patients with cT1-2 N0 breast cancer treated with mastectomy, with low-volume sentinel lymph node metastasis, the 5-year regional recurrence rate was low and comparable between patients with and without completion axillary treatment.</p

Determinants of Ligand-Functionalized DNA Nanostructure-Cell Interactions

Synthesis of ligand-functionalized nanomaterials with control over size, shape, and ligand orientation facilitates the design of targeted nanomedicines for therapeutic purposes. DNA nanotechnology has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study, we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors, including the programmed cell death protein 1, the epidermal growth factor receptor, and the human epidermal growth factor receptor 2. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the absolute number of bound surface receptors is lower compared to soluble antibodies due to receptor accessibility by the nanostructure. We explore structural determinants of this phenomenon to improve efficiency, revealing that receptor binding is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.ISSN:0002-7863ISSN:1520-512

Development of 89 Zr-anti-CD103 PET imaging for non-invasive assessment of cancer reactive T cell infiltration

Purpose CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (89 Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses. Experimental design First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an 89 Zr-anti-murine CD103 PET tracer. Healthy, non-tumor bearing C57BL/6 mice underwent serial PET imaging after intravenous injection, followed by ex vivo biodistribution. Tracer specificity and macroscopic tissue distribution were studied using autoradiography combined with CD103 immunohistochemistry. Next, we generated and screened six unique mAbs that specifically target human CD103 positive cells. Optimal candidates were selected for 89 Zr-anti-human CD103 PET development. Nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103 expressing Chinese hamster ovary (CHO) or CHO wild-type xenografts were injected with 89 Zr-anti-human CD103 mAbs and underwent serial PET imaging, followed by ex vivo biodistribution. Results 89 Zr-anti-murine CD103 PET imaging identified CD103-positive tissues at clinically relevant target densities. For human anti-human CD103 PET development two clones were selected based on strong binding to the CD103 + CD8 + T cell subpopulation in ovarian cancer tumor digests, non-overlapping binding epitopes and differential CD103 blocking properties. In vivo, both 89 Zr-anti-human CD103 tracers showed high target-to-background ratios, high target site selectivity and a high sensitivity in human CD103 positive xenografts. Conclusion CD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration

Development of 89Zr-anti-CD103 PET imaging for non-invasive assessment of cancer reactive T cell infiltration

Purpose CD103, an integrin specifically expressed on the surface of cancer-reactive T cells, is significantly increased during successful immunotherapy across human malignancies. In this study, we describe the generation and zirconium-89 (89Zr) radiolabeling of monoclonal antibody (mAb) clones that specifically recognize human CD103 for non-invasive immune positron-emission tomography (PET) imaging of T cell infiltration as potential biomarker for effective anticancer immune responses.Experimental design First, to determine the feasibility of anti-CD103 immuno-PET to visualize CD103-positive cells at physiologically and clinically relevant target densities, we developed an 89Zr-anti-murine CD103 PET tracer. Healthy, non-tumor bearing C57BL/6 mice underwent serial PET imaging after intravenous injection, followed by ex vivo biodistribution. Tracer specificity and macroscopic tissue distribution were studied using autoradiography combined with CD103 immunohistochemistry. Next, we generated and screened six unique mAbs that specifically target human CD103 positive cells. Optimal candidates were selected for 89Zr-anti-human CD103 PET development. Nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103 expressing Chinese hamster ovary (CHO) or CHO wild-type xenografts were injected with 89Zr-anti-human CD103 mAbs and underwent serial PET imaging, followed by ex vivo biodistribution.Results 89Zr-anti-murine CD103 PET imaging identified CD103-positive tissues at clinically relevant target densities. For human anti-human CD103 PET development two clones were selected based on strong binding to the CD103+ CD8+ T cell subpopulation in ovarian cancer tumor digests, non-overlapping binding epitopes and differential CD103 blocking properties. In vivo, both 89Zr-anti-human CD103 tracers showed high target-to-background ratios, high target site selectivity and a high sensitivity in human CD103 positive xenografts.Conclusion CD103 immuno-PET tracers visualize CD103 T cells at relevant densities and are suitable for future non-invasive assessment of cancer reactive T cell infiltration

Development of [⁸⁹Zr]Zr-hCD103.Fab01A and [⁶⁸Ga]Ga-hCD103.Fab01A for PET imaging to noninvasively assess cancer reactive T cell infiltration:Fab-based CD103 immunoPET

Background: CD103 is an integrin specifically expressed on the surface of cancer-reactive T cells. The number of CD103+ T cells significantly increases during successful immunotherapy and might therefore be an attractive biomarker for noninvasive PET imaging of immunotherapy response. Since the long half-life of antibodies preclude repeat imaging of CD103+ T cell dynamics early in therapy, we therefore here explored PET imaging with CD103 Fab fragments radiolabeled with a longer (89Zr) and shorter-lived radionuclide (68Ga). Methods: Antihuman CD103 Fab fragment Fab01A was radiolabeled with 89Zr or 68Ga, generating [89Zr]Zr-hCD103.Fab01A and [68Ga]Ga-hCD103.Fab01A, respectively. In vivo evaluation of these tracers was performed in male nude mice (BALB/cOlaHsd-Foxn1nu) with established CD103-expressing CHO (CHO.CD103) or CHO-wildtype (CHO.K1) xenografts, followed by serial PET imaging and ex vivo bio-distribution. Results: [89Zr]Zr-hCD103.Fab01A showed high tracer uptake in CD103+ xenografts as early as 3 h post-injection. However, the background signal remained high in the 3- and 6-h scans. The background was relatively low at 24 h after injection with sufficient tumor uptake. [68Ga]Ga-hCD103.Fab01Ashowed acceptable uptake and signal-to-noise ratio in CD103+ xenografts after 3 h, which decreased at subsequent time points. Conclusion: [89Zr]Zr-hCD103.Fab01A demonstrated a relatively low background and high xenograft uptake in scans as early as 6 h post-injection and could be explored for repeat imaging during immunotherapy in clinical trials. 18F or 64Cu could be explored as alternative to 68Ga in optimizing half-life and radiation burden of the tracer.</p

Development and internal validation of prediction models for colorectal cancer survivors to estimate the 1-year risk of low health-related quality of life in multiple domains

Background: Many colorectal cancer (CRC) survivors experience persisting health problems post-treatment that compromise their health-related quality of life (HRQoL). Prediction models are useful tools for identifying survivors at risk of low HRQoL in the future and for taking preventive action. Therefore, we developed prediction models for CRC survivors to estimate the 1-year risk of low HRQoL in multiple domains. Methods: In 1458 CRC survivors, seven HRQoL domains (EORTC QLQ-C30: Global QoL; cognitive, emotional, physical, role, social functioning; fatigue) were measured prospectively at study baseline and 1 year later. For each HRQoL domain, scores at 1-year follow-up were dichotomized into low versus normal/high. Separate multivariable logistic prediction models including biopsychosocial predictors measured at baseline were developed for the seven HRQoL domains, and internally validated using bootstrapping. Results: Average time since diagnosis was 5 years at study baseline. Prediction models included both non-modifiable predictors (age, sex, socio-economic status, time since diagnosis, tumor stage, chemotherapy, radiotherapy, stoma, micturition, chemotherapy-related, stoma-related and gastrointestinal complaints, comorbidities, social inhibition/negative affectivity, and working status) and modifiable predictors (body mass index, physical activity, smoking, meat consumption, anxiety/depression, pain, and baseline fatigue and HRQoL scores). Internally validated models showed good calibration and discrimination (AUCs: 0.83-0.93). Conclusions: The prediction models performed well for estimating 1-year risk of low HRQoL in seven domains. External validation is needed before models can be applied in practice.</p