Antibodies that block the interaction between programmed death ligand 1 (PD-L1) and PD-1 have shown impressive antitumor activity. Patients with tumors expressing PD-L1 are most likely to respond to ...this treatment. The aim of our study was to develop a noninvasive imaging technique to determine tumor PD-L1 expression in vivo. This could allow selection of patients that are most likely to benefit from anti-PD-1/PD-L1 treatment and to monitor PD-L1 expression during therapy. The monoclonal antibody PD-L1.3.1 was radiolabeled with Indium-111 ((111)In) and characterized using PD-L1-expressing MDA-MB-231 cells. Subsequently, the optimal antibody dose and time point for imaging was determined in mice with MDA-MB-231 xenografts. Finally, SPECT/CT imaging was performed in xenograft models with different PD-L1 expression levels and tumor sections were analyzed for PD-L1 expression using IHC. The optimal antibody dose of (111)In-PD-L1.3.1 (Kd = 1 nmol/L) for SPECT/CT imaging was ≤1 μg. Highest tumor-to-normal tissue contrast was obtained at days 3 and 7 after injection. (111)In-PD-L1.3.1 SPECT/CT showed efficient accumulation in high PD-L1-expressing tumors (MDA-MB-231 and SK-Br-3), whereas no specific uptake was observed in tumors with low or no detectable levels of PD-L1 (SUM149, BT474, and MCF-7). SPECT/CT and autoradiography showed a very heterogeneous distribution of (111)In-PD-L1.3.1 within the tumor. In conclusion, this is the first study showing the feasibility of noninvasive in vivo imaging of PD-L1 expression in tumors. (111)In-PD-L1.3.1 showed efficient and specific uptake in PD-L1 expressing xenografts. This technique may enable patient selection for PD-1 and PD-L1-targeted therapy.
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.
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
111
In-PD-L1xEGFR showed a significantly higher tumor uptake compared to
111
In-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.
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.
Anti-mouse-PD-L1 and isotype control antibodies were labelled with indium-111 (
InIn-DTPA-anti-mPD-L1 and
InIn-DTPA-IgG2a, respectively). We evaluated the effect of inflammatory responses on the pharmacokinetics of
InIn-DTPA-anti-mPD-L1 in RenCa tumor-bearing BALB/c mice in three conditions: lipopolysaccharide (LPS), local
, and heat-killed
. After intravenous injection of 30 or 100 µg of
InIn-DTPA-anti-mPD-L1 or
InIn-DTPA-IgG2a, blood samples were collected 1, 4, and 24 h p.i. followed by microSPECT/CT and
biodistribution analyses. PD-L1 expression, neutrophil, and macrophage infiltration in relevant tissues were evaluated immunohistochemically.
In 30 µg of
InIn-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
infections showed high PD-L1
neutrophil influx resulting in significantly increased
InIn-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
(Hk-
) challenge did not affect pharmacokinetics. Increasing
InIn-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
InIn-DTPA-IgG2a pharmacokinetics.
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.
BackgroundCD3 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.MethodsHere, 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 111In to investigate their pharmacokinetics, target binding, and biodistribution through SPECT/CT imaging and ex vivo biodistribution analyses. Co-injection of 111In- and 125I-labeled bsAb was performed to investigate the in vivo internalization by comparing tissue concentrations of cellular residing 111In versus effluxing 125I. Antitumor therapy effects were evaluated by monitoring tumor growth and immunohistochemistry.ResultsSPECT/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.ConclusionsWe 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.
Immuno-positron emission tomography (immunoPET) with 89Zr-labeled antibodies has shown great potential in cancer imaging. It can provide important information about the pharmacokinetics and ...tumor-targeting properties of monoclonal antibodies and may help in anticipating on toxicity. Furthermore, it allows accurate dose planning for individualized radioimmunotherapy and may aid in patient selection and early-response monitoring for targeted therapies. The most commonly used chelator for 89Zr is desferrioxamine (DFO). Preclinical studies have shown that DFO is not an ideal chelator because the 89Zr–DFO complex is partly unstable in vivo, which results in the release of 89Zr from the chelator and the subsequent accumulation of 89Zr in bone. This bone accumulation interferes with accurate interpretation and quantification of bone uptake on PET images. Therefore, there is a need for novel chelators that allow more stable complexation of 89Zr. In this Review, we will describe the most recent developments in 89Zr radiochemistry, including novel chelators and site-specific conjugation methods.
Platinum-based chemotherapeutics exhibit excellent antitumor properties. However, these drugs cause severe side effects including toxicity, drug resistance, and lack of tumor selectivity. ...Tumor-targeted drug delivery has demonstrated great potential to overcome these drawbacks. Herein, we aimed to design radioactive bisphosphonate-functionalized platinum (
Pt-BP) complexes to confirm preferential accumulation of these Pt-based drugs in metabolically active bone. In vitro NMR studies revealed that release of Pt from Pt BP complexes increased with decreasing pH. Upon systemic administration to mice, Pt-BP exhibited a 4.5-fold higher affinity to bone compared to platinum complexes lacking the bone-seeking bisphosphonate moiety. These Pt-BP complexes formed less Pt-DNA adducts compared to bisphosphonate-free platinum complexes, indicating that in vivo release of Pt from Pt-BP complexes proceeded relatively slow. Subsequently, radioactive
Pt-BP complexes were synthesized using
Pt(NO
)
(en) as precursor and injected intravenously into mice. Specific accumulation of
Pt-BP was observed at skeletal sites with high metabolic activity using micro-SPECT/CT imaging. Furthermore, laser ablation-ICP-MS imaging of proximal tibia sections confirmed that
Pt BP co-localized with calcium in the trabeculae of mice tibia.
In this study, we compared the tumor-targeting properties, therapeutic efficacy, and tolerability of the humanized anti-CAIX antibody (hG250) labeled with either the α-emitter actinium-225 (225Ac) or ...the β--emitter lutetium-177 (177Lu) in mice. BALB/c nude mice were grafted with human renal cell carcinoma SK-RC-52 cells and intravenously injected with 30 µg 225Ac Ac-DOTA-hG250 (225Ac-hG250) or 30 µg 177Lu Lu-DOTA-hG250 (177Lu-hG250), followed by ex vivo biodistribution studies. Therapeutic efficacy was evaluated in mice receiving 5, 15, and 25 kBq of 225Ac-hG250; 13 MBq of 177Lu-hG250; or no treatment. Tolerability was evaluated in non-tumor-bearing animals. High tumor uptake of both radioimmunoconjugates was observed and increased up to day 7 (212.8 ± 50.2 %IA/g vs. 101.0 ± 18.4 %IA/g for 225Ac-hG250 and 177Lu-hG250, respectively). Survival was significantly prolonged in mice treated with 15 kBq 225Ac-hG250, 25 kBq 225Ac-hG250, and 13 MBq 177Lu-hG250 compared to untreated control (p < 0.05). Non-tumor-bearing mice that received single-dose treatment with 15 or 25 kBq 225Ac-hG250 showed weight loss at the end of the experiment (day 126), and immunohistochemical analysis suggested radiation-induced nephrotoxicity. These results demonstrate the therapeutic potential of CAIX-targeted α-therapy in renal cell carcinoma. Future studies are required to find an optimal balance between therapeutic efficacy and toxicity.
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