Novel technologies for applying immune checkpoint blockers.

Cancer cells develop several ways to subdue the immune system among others via upregulation of inhibitory immune checkpoint (ICP) proteins. These ICPs paralyze immune effector cells and thereby enable unfettered tumor growth. Monoclonal antibodies (mAbs) that block ICPs can prevent immune exhaustion. Due to their outstanding effects, mAbs revolutionized the field of cancer immunotherapy. However, current ICP therapy regimens suffer from issues related to systemic administration of mAbs, including the onset of immune related adverse events, poor pharmacokinetics, limited tumor accessibility and immunogenicity. These drawbacks and new insights on spatiality prompted the exploration of novel administration routes for mAbs for instance peritumoral delivery. Moreover, novel ICP drug classes that are adept to novel delivery technologies were developed to circumvent the drawbacks of mAbs. We therefore review the state-of-the-art and novel delivery strategies of ICP drugs.

In vitro modelling of local gene therapy with IL-15/IL-15Rα and a PD-L1 antagonist in melanoma reveals an interplay between NK cells and CD4+ T cells.

Blockade of the immune checkpoint axis consisting of programmed death-1 (PD-1) and its ligand PD-L1 alleviates the functional inhibition of tumor-infiltrating lymphoid cells yet weakly induces their expansion. Exogenous cytokines could further expand lymphoid cells and thus synergize with αPD-L1 therapy. However, systemic delivery of most cytokines causes severe toxicity due to unspecific expansion of immune cells in the periphery. Here, we modelled local delivery of cytokines and αPD-L1 therapeutics to immune cell-containing in vitro melanoma tumors. Three-dimensional tumor models consisting of 624-MEL cells were co-cultured with human peripheral blood lymphoid cells (PBLs) in presence of the cytokines IL-2, IL-7, IL-15, IL-21 and IFN-γ. To model local gene therapy, melanoma tumors were modified with lentiviral vectors encoding IL-15 fused to IL-15Rα (IL-15/IL-15Rα) and K2-Fc, a fusion of a human PD-L1 specific single domain antibody to immunoglobulin (Ig)G1 Fc. To evaluate the interplay between PBL fractions, NK cells, CD4+ T cells or CD8+ T cells were depleted. Tumor cell killing was followed up using real time imaging and immune cell expansion and activation was evaluated with flow cytometry. Among the tested cytokines, IL-15 was the most potent cytokine in stimulating tumor cell killing and expanding both natural killer (NK) cells and CD8+ T cells. Gene-based delivery of IL-15/IL-15Rα to tumor cells, shows expansion of NK cells, activation of NK cells, CD4+ and CD8+ T cells, and killing of tumor spheroids. Both NK cells and CD8+ T cells are necessary for tumor cell killing and CD4+ T-cell activation was reduced without NK cells. Co-delivery of K2-Fc improved tumor cell killing coinciding with increased activation of NK cells, which was independent of bystander T cells. CD4+ or CD8+ T cells were not affected by the co-delivery of K2-Fc even though NK-cell activation impacted CD4+ T-cell activation. This study demonstrates that gene-based delivery of IL-15/IL-15Rα to tumor cells effectively mediates anti-tumor activity and sensitizes the tumor microenvironment for therapy with αPD-L1 therapeutics mainly by impacting NK cells. These findings warrant further investigation of gene-based IL-15 and K2-Fc delivery in vivo.

Development and evaluation of nanobody tracers for noninvasive nuclear imaging of the immune-checkpoint TIGIT.

Introduction: T cell Ig and ITIM domain receptor (TIGIT) is a next-generation immune checkpoint predominantly expressed on activated T cells and NK cells, exhibiting an unfavorable prognostic association with various malignancies. Despite the emergence of multiple TIGIT-blocking agents entering clinical trials, only a fraction of patients responded positively to anti-TIGIT therapy. Consequently, an urgent demand arises for noninvasive techniques to quantify and monitor TIGIT expression, facilitating patient stratification and enhancing therapeutic outcomes. Small antigen binding moieties such as nanobodies, are promising candidates for such tracer development. Methods: We generated a panel of anti-human or anti-mouse TIGIT nanobodies from immunized llamas. In addition, we designed a single-chain variable fragment derived from the clinically tested monoclonal antibody Vibostolimab targeting TIGIT, and assessed its performance alongside the nanobodies. In vitro characterization studies were performed, including binding ability and affinity to cell expressed or recombinant TIGIT. After Technetium-99m labeling, the nanobodies and the single-chain variable fragment were evaluated in vivo for their ability to detect TIGIT expression using SPECT/CT imaging, followed by ex vivo biodistribution analysis. Results: Nine nanobodies were selected for binding to recombinant and cell expressed TIGIT with low sub-nanomolar affinities and are thermostable. A six-fold higher uptake in TIGIT-overexpressing tumor was demonstrated one hour post- injection with Technetium-99m labeled nanobodies compared to an irrelevant control nanobody. Though the single-chain variable fragment exhibited superior binding to TIGIT-expressing peripheral blood mononuclear cells in vitro, its in vivo behavior yielded lower tumor-to-background ratios at one hour post- injection, indicating that nanobodies are better suited for in vivo imaging than the single-chain variable fragment. Despite the good affinity, high specificity and on-target uptake in mice in this setting, imaging of TIGIT expression on tumor- infiltrating lymphocytes within MC38 tumors remained elusive. This is likely due to the low expression levels of TIGIT in this model. Discussion: The excellent affinity, high specificity and rapid on-target uptake in mice bearing TIGIT- overexpressing tumors showed the promising diagnostic potential of nanobodies to noninvasively image high TIGIT expression within the tumor. These findings hold promise for clinical translation to aid patient selection and improve therapy response.

Targeted α-Therapy Using 225Ac Radiolabeled Single-Domain Antibodies Induces Antigen-Specific Immune Responses and Instills Immunomodulation Both Systemically and at the Tumor Microenvironment.

Targeted radionuclide therapy (TRT) using targeting moieties labeled with α-particle-emitting radionuclides (α-TRT) is an intensely investigated treatment approach as the short range of α-particles allows effective treatment of local lesions and micrometastases. However, profound assessment of the immunomodulatory effect of α-TRT is lacking in literature. Methods: Using flow cytometry of tumors, splenocyte restimulation, and multiplex analysis of blood serum, we studied immunologic responses ensuing from TRT with an antihuman CD20 single-domain antibody radiolabeled with 225Ac in a human CD20 and ovalbumin expressing B16-melanoma model. Results: Tumor growth was delayed with α-TRT and increased blood levels of various cytokines such as interferon-γ, C-C motif chemokine ligand 5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-1. Peripheral antitumoral T-cell responses were detected on α-TRT. At the tumor site, α-TRT modulated the cold tumor microenvironment (TME) to a more hospitable and hot habitat for antitumoral immune cells, characterized by a decrease in protumoral alternatively activated macrophages and an increase in antitumoral macrophages and dendritic cells. We also showed that α-TRT increased the percentage of programmed death-ligand 1 (PD-L1)-positive (PD-L1pos) immune cells in the TME. To circumvent this immunosuppressive countermeasure we applied immune checkpoint blockade of the programmed cell death protein 1-PD-L1 axis. Combination of α-TRT with PD-L1 blockade potentiated the therapeutic effect, however, the combination aggravated adverse events. A long-term toxicity study revealed severe kidney damage ensuing from α-TRT. Conclusion: These data suggest that α-TRT alters the TME and induces systemic antitumoral immune responses, which explains why immune checkpoint blockade enhances the therapeutic effect of α-TRT. However, further optimization is warranted to avoid adverse events.

Targeted Radionuclide Therapy with Low and High-Dose Lutetium-177-Labeled Single Domain Antibodies Induces Distinct Immune Signatures in a Mouse Melanoma Model.

Targeted radionuclide therapy (TRT) using probes labeled with Lutetium-177 (177Lu) represents a new and growing type of cancer therapy. We studied immunologic changes in response to TRT with 177Lu labeled anti-human CD20 camelid single domain antibodies (sdAb) in a B16-melanoma model transfected to express human CD20, the target antigen, and ovalbumin, a surrogate tumor antigen. High-dose TRT induced melanoma cell death, calreticulin exposure, and ATP-release in vitro. Melanoma-bearing mice received fractionated low and high-dose TRT via tumor targeting anti-human CD20 sdAbs, as opposed to control sdAbs. Tumor growth was delayed with both doses. Low- and high-dose TRT increased IL10 serum levels. Low-dose TRT also decreased CCL5 serum levels. At the tumor, high-dose TRT induced a type I IFN gene signature, while low-dose TRT induced a proinflammatory gene signature. Low- and high-dose TRT increased the percentage of PD-L1pos and PD-L2pos myeloid cells in tumors with a marked increase in alternatively activated macrophages after high-dose TRT. The percentage of tumor-infiltrating T cells was not changed, yet a modest increase in ovalbumin-specific CD8pos T-cells was observed after low-dose TRT. Contradictory, low and high-dose TRT decreased CD4pos Th1 cells in addition to double negative T cells. In conclusion, these data suggest that low and high-dose TRT induce distinct immunologic changes, which might serve as an anchoring point for combination therapy.

TNF-α-Secreting Lung Tumor-Infiltrated Monocytes Play a Pivotal Role During Anti-PD-L1 Immunotherapy.

Immune checkpoint blockade (ICB) of the PD-1 pathway revolutionized the survival forecast for advanced non-small cell lung cancer (NSCLC). Yet, the majority of PD-L1+ NSCLC patients are refractory to anti-PD-L1 therapy. Recent observations indicate a pivotal role for the PD-L1+ tumor-infiltrating myeloid cells in therapy failure. As the latter comprise a heterogenous population in the lung tumor microenvironment, we applied an orthotopic Lewis Lung Carcinoma (LLC) model to evaluate 11 different tumor-residing myeloid subsets in response to anti-PD-L1 therapy. While we observed significantly reduced fractions of tumor-infiltrating MHC-IIlow macrophages and monocytes, serological levels of TNF-α restored in lung tumor-bearing mice. Notably, we demonstrated in vivo and in vitro that anti-PD-L1 therapy mediated a monocyte-specific production of, and response to TNF-α, further accompanied by their significant upregulation of CD80, VISTA, LAG-3, SIRP-α and TIM-3. Nevertheless, co-blockade of PD-L1 and TNF-α did not reduce LLC tumor growth. A phenomenon that was partly explained by the observation that monocytes and TNF-α play a Janus-faced role in anti-PD-L1 therapy-mediated CTL stimulation. This was endorsed by the observation that monocytes appeared crucial to effectively boost T cell-mediated LLC killing in vitro upon combined PD-L1 with LAG-3 or SIRP-α blockade. Hence, this study enlightens the biomarker potential of lung tumor-infiltrated monocytes to define more effective ICB combination strategies.

Inhibiting Histone and DNA Methylation Improves Cancer Vaccination in an Experimental Model of Melanoma.

Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.

Emerging applications of nanobodies in cancer therapy.

Cancer is a heterogeneous disease, requiring treatment tailored to the unique phenotype of the patient's tumor. Monoclonal antibodies (mAbs) and variants thereof have enabled targeted therapies to selectively target cancer cells. Cancer cell-specific mAbs have been used for image-guided surgery and targeted delivery of radionuclides or toxic agents, improving classical treatment strategies. Cancer cell-specific mAbs can further inhibit tumor cell growth or can stimulate immune-mediated destruction of cancer cells, a feature that has also been achieved through mAb-mediated manipulation of immune cells and pathways. Drawbacks of mAbs and their variants, together with the discovery of camelid heavy chain-only antibodies and the many advantageous features of their variable domains, referred to as VHHs, single domain antibodies or nanobodies (Nbs), resulted in the exploration of Nbs as an alternative targeting moiety. We therefore review the state-of-the-art as well as novel exploitation strategies of Nbs for targeted cancer therapy.

Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment.

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1ß-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming.

Site-Specific Radiolabeling of a Human PD-L1 Nanobody via Maleimide-Cysteine Chemistry.

Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) and its ligand PD-L1 have proven to be efficient cancer therapies in a subset of patients. From all the patients with various cancer types, only 20% have a positive response. Being able to distinguish patients that do express PD-1/PD-L1 from patients that do not allows patients to benefit from a more personalized and efficient treatment of tumor lesion(s). Expression of PD-1 and PD-L1 is typically assessed via immunohistochemical detection in a tumor biopsy. However, this method does not take in account the expression heterogeneity within the lesion, nor the possible metastasis. To visualize whole-body PD-L1 expression by PET imaging, we developed a nanobody-based radio-immunotracer targeting PD-L1 site-specifically labeled with gallium-68. The cysteine-tagged nanobody was site-specifically conjugated with a maleimide (mal)-NOTA chelator and radiolabeling was tested at different nanobody concentrations and temperatures. Affinity and specificity of the tracer, referred to as [68Ga]Ga-NOTA-mal-hPD-L1 Nb, were assayed by surface plasmon resonance and on PD-L1POS or PD-L1NEG 624-MEL cells. Xenografted athymic nude mice bearing 624-MEL PD-L1POS or PD-L1NEG tumors were injected with the tracer and ex vivo biodistribution was performed 1 h 20 min post-injection. Ideal 68Ga-labeling conditions were found at 50 °C for 15 min. [68Ga]Ga-NOTA-mal-hPD-L1 Nb was obtained in 80 ± 5% DC-RCY with a RCP > 99%, and was stable in injection buffer and human serum up to 3 h (>99% RCP). The in vitro characterization showed that the NOTA-functionalized Nb retained its affinity and specificity. Ex vivo biodistribution revealed a tracer uptake of 1.86 ± 0.67% IA/g in the positive tumors compared with 0.42 ± 0.04% IA/g in the negative tumors. Low background uptake was measured in the other organs and tissues, except for the kidneys and bladder, due to the expected excretion route of Nbs. The data obtained show that the site-specific 68Ga-labeled NOTA-mal-hPD-L1 Nb is a promising PET radio-immunotracer due to its ease of production, stability and specificity for PD-L1.