Search

Introduction: Combined radiotherapy and immune checkpoint inhibition is a potential treatment option for head and neck squamous cell carcinoma (HNSCC). Immunocompetent mouse models can help to successfully develop radio- immunotherapy combinations and to increase our understanding of the effects of radiotherapy on the tumor microenvironment for future clinical translation. Therefore, the aim of this study was to develop a homogeneous, reproducible HNSCC model originating from the Mouse Oral Cancer 1 (MOC1) HNSCC cell line, and to explore the radiotherapy-induced changes in its tumor microenvironment, using flow cytometry and PD-L1 microSPECT/CT imaging., Materials and Methods: In vivo growing tumors originating from the parental MOC1 line were used to generate single cell derived clones. These clones were screened in vitro for their ability to induce programmed cell death ligand 1 (PD-L1) and major histocompatibility complex class I (MHC-I) following IFNγ exposure. Clones with different IFNγ sensitivity were inoculated in C57BL/6 mice and assessed for tumor outgrowth. The composition of the tumor microenvironment of a stably growing (non)irradiated MOC1-derived clone was assessed by immunohistochemistry, flow cytometry and PD-L1 microSPECT/CT., Results: Low in vitro inducibility of MHC-I and PD-L1 by IFNγ was associated with increased tumor outgrowth of MOC1 clones in vivo. Flow cytometry analysis of cells derived from a stable in vivo growing MOC1 clone MOC1.3D5 low showed expression of MHC-I and PD-L1 on several cell populations within the tumor. Upon irradiation, MHC-I and PD-L1 increased on leukocytes (CD45.2 + ) and cancer associated fibroblasts (CD45.2 - /EpCAM - /CD90.1 + ). Furthermore, PD-L1 microSPECT/CT showed increased tumor uptake of radiolabeled PD-L1 antibodies with a heterogeneous spatial distribution of the radio signal, which co-localized with PD-L1 + and CD45.2 + areas., Discussion: PD-L1 and MHC-I inducibility by IFNγ in vitro is associated with tumor outgrowth of MOC1 clones in vivo. In tumors originating from a stably growing MOC1-derived clone, expression of these immune-related markers was induced by irradiation shown by flow cytometry on several cell populations within the tumor microenvironment such as immune cells and cancer associated fibroblasts. PD-L1 microSPECT/CT showed increased tumor uptake following radiotherapy, and autoradiography showed correlation of uptake with areas that are heavily infiltrated by immune cells. Knowledge of radiotherapy-induced effects on the tumor microenvironment in this model can help optimize timing and dosage for radio- immunotherapy combination strategies in future research., (© 2024. The Author(s).)

CD3 bispecific antibody (CD3 bsAb) therapy is clinically approved for refractory hematological malignancies, but responses in solid tumors have been limited so far. One of the main hurdles in solid tumors is the lack of sufficient T-cell infiltrate. Here, we show that pre-treatment vaccination, even when composed of tumor-unrelated antigens, induces CXCR3-mediated T-cell influx in immunologically 'cold' tumor models in male mice. In the absence of CD3 bsAb, the infiltrate is confined to the tumor invasive margin, whereas subsequent CD3 bsAb administration induces infiltration of activated effector CD8 T cells into the tumor cell nests. This combination therapy installs a broadly inflamed Th1-type tumor microenvironment, resulting in effective tumor eradication. Multiple vaccination formulations, including synthetic long peptides and viruses, empower CD3 bsAb therapy. Our results imply that eliciting tumor infiltration with vaccine-induced tumor-(un)related T cells can greatly improve the efficacy of CD3 bsAbs in solid tumors., (© 2024. The Author(s).)

Background: CD3 bispecific antibodies (CD3-bsAbs) require binding of both a tumor-associated surface antigen and CD3 for their immunotherapeutic effect. Their efficacy is, therefore, influenced by the tumor uptake and the extracellular dose. To optimize their currently limited efficacy in solid tumors, increased understanding of their pharmacokinetics and in vivo internalization is needed., Methods: Here, were studied the pharmacokinetics and in vivo internalization of CD3xTRP1, a fully murine Fc-inert bsAb, in endogenous TRP1-expressing immunocompetent male C57BL/6J mice bearing TRP1-positive and negative tumors over time. Matching bsAbs lacking TRP1-binding or CD3-binding capacity served as controls. BsAbs were radiolabeled with 111 In to investigate their pharmacokinetics, target binding, and biodistribution through SPECT/CT imaging and ex vivo biodistribution analyses. Co-injection of 111 In- and 125 I-labeled bsAb was performed to investigate the in vivo internalization by comparing tissue concentrations of cellular residing 111 In versus effluxing 125 I. Antitumor therapy effects were evaluated by monitoring tumor growth and immunohistochemistry., Results: SPECT/CT and biodistribution analyses showed that CD3xTRP1 specifically targeted TRP1-positive tumors and CD3-rich lymphoid organ and uptake peaked 24 hours pi (KPC3-TRP1: 37.7%ID/g±5.3%ID/g, spleen: 29.0%ID/g±3.9%ID/g). Studies with control bsAbs demonstrated that uptake of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues was primarily receptor-mediated. Together with CD3xTRP1 in the circulation being mainly unattached, this indicates that CD3 + T cells are generally not traffickers of CD3-bsAbs to the tumor. Additionally, target-mediated clearance by TRP1-expressing melanocytes was not observed. We further demonstrated rapid internalization of CD3xTRP1 in KPC3-TRP1 tumors (24 hours pi: 54.9%±2.3% internalized) and CD3-rich tissues (spleen, 24 hours pi: 79.7%±0.9% internalized). Therapeutic effects by CD3xTRP1 were observed for TRP1-positive tumors and consisted of high tumor influx of CD8 + T cells and neutrophils, which corresponded with increased necrosis and growth delay., Conclusions: We show that CD3xTRP1 efficiently targets TRP1-positive tumors and CD3-rich tissues primarily through receptor-mediated targeting. We further demonstrate rapid receptor-mediated internalization of CD3xTRP1 in TRP1-positive tumors and CD3-rich tissues. Even though this significantly decreases the therapeutical available dose, CD3xTRP1 still induced effective antitumor T-cell responses and inhibited tumor growth. Together, our data on the pharmacokinetics and mechanism of action of CD3xTRP1 pave the way for further optimization of CD3-bsAb therapies., Competing Interests: Competing interests: TVH received a research grant from Genmab. Bispecific antibodies were provided by Genmab. TVH and JM are authors on a patent involving the combination of CD3-bsAb therapy in combination with vaccination. The other authors declare that they have no competing interests., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Background: Programmed death-ligand 1 (PD-L1) regulates immune homeostasis by promoting T-cell exhaustion. It is involved in chronic infections and tumor progression. Nuclear imaging using radiolabeled anti-PD-L1 antibodies can monitor PD-L1 tissue expression and antibody distribution. However, physiological PD-L1 can cause rapid antibody clearance from blood at imaging doses. Therefore, we hypothesized that inflammatory responses, which can induce PD-L1 expression, affect anti-PD-L1 antibody distribution. Here, we investigated the effects of three different infectious stimuli on the pharmacokinetics and tumor targeting of radiolabeled anti-PD-L1 antibodies in tumor-bearing mice., Materials/methods: Anti-mouse-PD-L1 and isotype control antibodies were labelled with indium-111 ([ 111 In]In-DTPA-anti-mPD-L1 and [ 111 In]In-DTPA-IgG2a, respectively). We evaluated the effect of inflammatory responses on the pharmacokinetics of [ 111 In]In-DTPA-anti-mPD-L1 in RenCa tumor-bearing BALB/c mice in three conditions: lipopolysaccharide (LPS), local Staphylococcus aureus , and heat-killed Candida albicans . After intravenous injection of 30 or 100 µg of [ 111 In]In-DTPA-anti-mPD-L1 or [ 111 In]In-DTPA-IgG2a, blood samples were collected 1, 4, and 24 h p.i. followed by microSPECT/CT and ex vivo biodistribution analyses. PD-L1 expression, neutrophil, and macrophage infiltration in relevant tissues were evaluated immunohistochemically., Results: In 30 µg of [ 111 In]In-DTPA-anti-mPD-L1 injected tumor-bearing mice the LPS-challenge significantly increased lymphoid organ uptake compared with vehicle controls (spleen: 49.9 ± 4.4%ID/g versus 21.2 ± 6.9%ID/g, p < 0.001), resulting in lower blood levels (3.6 ± 1.6%ID/g versus 11.5 ± 7.2%ID/g; p < 0.01) and reduced tumor targeting (8.1 ± 4.5%ID/g versus 25.2 ± 5.2%ID/g, p < 0.001). Local S. aureus infections showed high PD-L1 + neutrophil influx resulting in significantly increased [ 111 In]In-DTPA-anti-mPD-L1 uptake in affected muscles (8.6 ± 2.6%ID/g versus 1.7 ± 0.8%ID/g, p < 0.001). Heat-killed Candida albicans (Hk- C. albicans ) challenge did not affect pharmacokinetics. Increasing [ 111 In]In-DTPA-anti-mPD-L1 dose to 100 µg normalized blood clearance and tumor uptake in LPS-challenged mice, although lymphoid organ uptake remained higher. Infectious stimuli did not affect [ 111 In]In-DTPA-IgG2a pharmacokinetics., Conclusions: This study shows that anti-PD-L1 antibody pharmacokinetics and tumor targeting can be significantly altered by severe inflammatory responses, which can be compensated for by increasing the tracer dose. This has implications for developing clinical PD-L1 imaging protocols in onco-immunology. We further demonstrate that radiolabeled anti-PD-L1 antibodies can be used to evaluate PD-L1 expression changes in a range of infectious diseases. This supports the exploration of using these techniques to assess hosts' responses to infectious stimuli., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sandker, Adema, Molkenboer-Kuenen, Wierstra, Bussink, Heskamp and Aarntzen.)

With the advent of novel immunotherapies, interest in ex vivo autologous cell labeling for in vivo cell tracking has revived. However, current clinically available labeling strategies have several drawbacks, such as release of radiolabel over time and cytotoxicity. Poly(lactic- co -glycolic acid) nanoparticles (PLGA NPs) are clinically used biodegradable carriers of contrast agents, with high loading capacity for multimodal imaging agents. Here we show the development of PLGA-based NPs for ex vivo cell labeling and in vivo cell tracking with SPECT. We used primary amine-modified PLGA polymers (PLGA-NH 2 ) to construct NPs similar to unmodified PLGA NPs. PLGA-NH 2 NPs were efficiently radiolabeled without chelator and retained the radionuclide for 2 weeks. Monocyte-derived dendritic cells labeled with [ 111 In]In-PLGA-NH 2 showed higher specific activity than those labeled with [ 111 In]In-oxine, with no negative effect on cell viability. SPECT/CT imaging showed that radiolabeled THP-1 cells accumulated at the Staphylococcus aureus infection site in mice. In conclusion, PLGA-NH 2 NPs are able to retain 111 In, independent of chelator presence. Furthermore, [ 111 In]In-PLGA-NH 2 allows cell labeling with high specific activity and no loss of activity over prolonged time intervals. Finally, in vivo tracking of ex vivo labeled THP-1 cells was demonstrated in an infection model using SPECT/CT imaging.