Jacqueline Caroline Mock

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Mock

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Jacqueline Caroline

ORCID

0000-0002-4618-1113

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Publications 1 - 10 of 13

Antibody-mediated delivery of LIGHT to the tumor boosts natural killer cells and delays tumor progression
Stringhini, Marco; Mock, Jacqueline Caroline; Fontana, Vanessa; et al. (2021)
mAbs
LIGHT is a member of the tumor necrosis factor superfamily, which has been claimed to mediate anti-tumor activity on the basis of cancer cures observed in immunocompetent mice bearing transgenic LIGHT-expressing tumors. The preclinical development of a LIGHT-based therapeutic has been hindered by the lack of functional stability exhibited by this protein. Here, we describe the cloning, expression, and characterization of five antibody-LIGHT fusion proteins, directed against the alternatively spliced extra domain A of fibronectin, a conserved tumor-associated antigen. Among the five tested formats, only the sequential fusion of the F8 antibody in single-chain diabody format, followed by the LIGHT homotrimer expressed as a single polypeptide, yielded a protein (termed "F8-LIGHT") that was not prone to aggregation. A quantitative biodistribution analysis in tumor-bearing mice, using radio-iodinated protein preparations, confirmed that F8-LIGHT was able to preferentially accumulate at the tumor site, with a tumor-to-blood ratio of ca. five to one 24 hours after intravenous administration. Tumor therapy experiments, performed in two murine tumor models (CT26 and WEHI-164), featuring different levels of lymphocyte infiltration into the neoplastic mass, revealed that F8-LIGHT could significantly reduce tumor-cell growth and was more potent than a similar fusion protein (KSF-LIGHT), directed against hen egg lysozyme and serving as negative control of irrelevant specificity in the mouse. At a mechanistic level, the activity of F8-LIGHT was mainly due to an intratumoral expansion of natural killer cells, whereas there was no evidence of expansion of CD8 + T cells, neither in the tumor, nor in draining lymph nodes.
Tumor-Targeted Interleukin 2 Boosts the Anticancer Activity of FAP-Directed Radioligand Therapeutics
Galbiati, Andrea; Dorten, Paulina; Gilardoni, Ettore; et al. (2023)
The Journal of Nuclear Medicine
We studied the antitumor efficacy of a combination of ¹⁷⁷Lu-labeled radioligand therapeutics targeting the fibroblast activation protein (FAP) (OncoFAP and BiOncoFAP) with the antibody–cytokine fusion protein L19-interleukin 2 (L19-IL2) providing targeted delivery of interleukin 2 to tumors. Methods: The biodistribution of ¹⁷⁷Lu-OncoFAP and ¹⁷⁷Lu-BiOncoFAP at different molar amounts (3 vs. 250 nmol/kg) of injected ligand was studied via SPECT/CT in mice bearing subcutaneous HT-1080.hFAP tumors, and self-absorbed tumor and organ doses were calculated. The in vivo anticancer effect of 5 MBq of the radiolabeled preparations was evaluated as monotherapy or in combination with L19-IL2 in subcutaneously implanted HT-1080.hFAP and SK-RC-52.hFAP tumors. Tumor samples from animals treated with ¹⁷⁷Lu-BiOncoFAP, L19-IL2, or both were analyzed by mass spectrometry–based proteomics to identify therapeutic signatures on cellular and stromal markers of cancer and on immunomodulatory targets. Results: ¹⁷⁷Lu-BiOncoFAP led to a significantly higher self-absorbed dose in FAP-positive tumors (0.293 ± 0.123 Gy/MBq) than did ¹⁷⁷Lu-OncoFAP (0.157 ± 0.047 Gy/MBq, P = 0.01) and demonstrated favorable tumor-to-organ ratios at high molar amounts of injected ligand. Administration of L19-IL2 or ¹⁷⁷Lu-BiOncoFAP as single agents led to cancer cures in only a limited number of treated animals. In ¹⁷⁷Lu-BiOncoFAP–plus–L19-IL2 combination therapy, complete remissions were observed in all injected mice (7/7 complete remissions for the HT-1080.hFAP model, and 4/4 complete remissions for the SK-RC-52.hFAP model), suggesting therapeutic synergy. Proteomic studies revealed a mechanism of action based on the activation of natural killer cells, with a significant enhancement of the expression of granzymes and perforin 1 in the tumor microenvironment after combination treatment. Conclusion: The combination of OncoFAP-based radioligand therapeutics with concurrent targeting of interleukin 2 shows synergistic anticancer effects in the treatment of FAP-positive tumors. This experimental finding should be corroborated by future clinical studies.
Cancer therapy in mice using a pure population of CD8+ T cell specific to the AH1 tumor rejection antigen
Stringhini, Marco; Spadafora, Ilaria; Catalano, Marco; et al. (2021)
Cancer Immunology, Immunotherapy
There is a growing interest in the use of patient-derived T cells for the treatment of various types of malignancies. The expansion of a polyclonal and polyspecific population of tumor-reactive T cells, with a subsequent infusion into the same donor patient, has been implemented, sometimes with positive results. It is not known, however, whether a set of T cells with a single antigen specificity may be sufficient for an effective therapy. To gain more insights in this matter, we used naturally occurring T cells recognizing a retroviral peptide (AH1), which is endogenous in many tumor cell lines of BALB/c origin and which serves as potent tumor rejection antigen. We were able to isolate and expand this rare population of T cells to numbers suitable for therapy experiments in mice (i.e., up to 30 × 106 cells/mouse). After the expansion process, T cells efficiently killed antigen-positive tumor cells in vitro and demonstrated tumor growth inhibition in two syngeneic murine models of cancer. However, AH1-specific T cells failed to induce complete regressions of established tumors. The incomplete activity was associated with a failure of injected T cells to survive in vivo, as only a very limited amount of T cells was found in tumor or secondary lymphoid organs 72 h after injection. These data suggest that future therapeutic strategies based on autologous T cells may require the potentiation of tumor-homing and survival properties of cancer-specific T cells.
Small Organic Carbonic Anhydrase IX Ligands from DNA-Encoded Chemical Libraries for Tumor-Targeted Delivery of Radionuclides
Müller, Marco; Georgiev, Tony; Mock, Jacqueline Caroline; et al. (2025)
Journal of the American Chemical Society
Carbonic anhydrase IX (CAIX) is a membrane protein that is highly expressed in clear cell renal cell carcinoma (ccRCC) and in hypoxic tumors. Being virtually absent in most healthy tissues, CAIX became an attractive target for the selective delivery of diagnostic and therapeutic payloads. Here, we report the discovery and characterization of DNA-encoded chemical library (DEL)-derived CAIX ligands for radionuclide-based imaging applications. Methods: DELs were screened against CAIX and CAII to prioritize hits based on their selectivity and enrichment against CAIX. In vitro characterization of hits was performed by fluorescence polarization (FP), surface plasmon resonance (SPR), and flow cytometry. In vivo biodistribution studies of Lutetium-177 and Gallium-68-radiolabeled compounds were performed in SK-RC-52 tumor-bearing mice. Results: DEL-based CAIX ligands with different affinities and selectivities could be identified. Selectivity and high affinity toward the target correlated with higher tumor-to-organ ratios and improved tumor retention. The best candidate, named OncoCAIX, reached up to similar to 55% injected dose per gram in SK-RC-52 lesions at early time points with very low healthy organ uptake (tumor-to-kidney ratio of >23). Conclusion: OncoCAIX demonstrated rapid and selective tumor uptake, which is a key feature for the development of radionuclide-based imaging agents for early and late-stage ccRCC and hypoxic tumors.
Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA
Backhaus, Philipp; Gierse, F.; Burg, Matthias C.; et al. (2022)
European Journal of Nuclear Medicine and Molecular Imaging
Purpose The fibroblast activation protein (FAP) is an emerging target for molecular imaging and therapy in cancer. OncoFAP is a novel small organic ligand for FAP with very high affinity. In this translational study, we establish [Ga-68]Ga-OncoFAP-DOTAGA (Ga-68-OncoFAP) radiolabeling, benchmark its properties in preclinical imaging, and evaluate its application in clinical PET scanning. Methods Ga-68-OncoFAP was synthesized in a cassette-based fully automated labeling module. Lipophilicity, affinity, and serum stability of Ga-68-OncoFAP were assessed by determining logD(7.4), IC50 values, and radiochemical purity. Ga-68-OncoFAP tumor uptake and imaging properties were assessed in preclinical dynamic PET/MRI in murine subcutaneous tumor models. Finally, biodistribution and uptake in a variety of tumor types were analyzed in 12 patients based on individual clinical indications that received 163 +/- 50 MBq Ga-68-OncoFAP combined with PET/CT and PET/MRI. Results Ga-68-OncoFAP radiosynthesis was accomplished with high radiochemical yields. Affinity for FAP, lipophilicity, and stability of Ga-68-OncoFAP measured are ideally suited for PET imaging. PET and gamma counting-based biodistribution demonstrated beneficial tracer kinetics and high uptake in murine FAP-expressing tumor models with high tumor-to-blood ratios of 8.6 +/- 5.1 at 1 h and 38.1 +/- 33.1 at 3 h p.i. Clinical Ga-68-OncoFAP-PET/CT and PET/MRI demonstrated favorable biodistribution and kinetics with high and reliable uptake in primary cancers (SUVmax 12.3 +/- 2.3), lymph nodes (SUVmax 9.7 +/- 8.3), and distant metastases (SUVmax up to 20.0). Conclusion Favorable radiochemical properties, rapid clearance from organs and soft tissues, and intense tumor uptake validate Ga-68-OncoFAP as a powerful alternative to currently available FAP tracers.
An engineered 4-1BBL fusion protein with “activity on demand”
Mock, Jacqueline Caroline; Stringhini, Marco; Villa, Alessandra; et al. (2020)
Proceedings of the National Academy of Sciences of the United States of America
Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues. © 2020 National Academy of Sciences
Generation of a novel fully human non-superagonistic anti-CD28 antibody with efficient and safe T-cell co-stimulation properties
Elsayed, Abdullah; Pellegrino, Christian; Plüss, Louis; et al. (2023)
mAbs
Antibody-based therapeutics represent an important class of biopharmaceuticals in cancer immunotherapy. CD3 bispecific T-cell engagers activate cytotoxic T-cells and have shown remarkable clinical outcomes against several hematological malignancies. The absence of a costimulatory signal through CD28 typically leads to insufficient T-cell activation and early exhaustion. The combination of CD3 and CD28 targeting products offers an attractive strategy to boost T-cell activity. However, the development of CD28-targeting therapies ceased after TeGenero's Phase 1 trial in 2006 evaluating a superagonistic anti-CD28 antibody (TGN1412) resulted in severe life-threatening side effects. Here, we describe the generation of a novel fully human anti-CD28 antibody termed "E1P2" using phage display technology. E1P2 bound to human and mouse CD28 as shown by flow cytometry on primary human and mouse T-cells. Epitope mapping revealed a conformational binding epitope for E1P2 close to the apex of CD28, similar to its natural ligand and unlike the lateral epitope of TGN1412. E1P2, in contrast to TGN1412, showed no signs of in vitro superagonistic properties on human peripheral blood mononuclear cells (PBMCs) using different healthy donors. Importantly, an in vivo safety study in humanized NSG mice using E1P2, in direct comparison and contrast to TGN1412, did not cause cytokine release syndrome. In an in vitro activity assay using human PBMCs, the combination of E1P2 with CD3 bispecific antibodies enhanced tumor cell killing and T-cell proliferation. Collectively, these data demonstrate the therapeutic potential of E1P2 to improve the activity of T-cell receptor/CD3 activating constructs in targeted immunotherapeutic approaches against cancer or infectious diseases.
A single-chain variable fragment-based bispecific T-cell activating antibody against CD117 enables T-cell mediated lysis of acute myeloid leukemia and hematopoietic stem and progenitor cells
Volta, Laura; Myburgh, Renier; Hofstetter, Mara; et al. (2024)
HemaSphere
Acute myeloid leukemia (AML) derives from hematopoietic stem and progenitor cells (HSPCs). To date, no AML-exclusive, non-HSPC-expressed cell-surface target molecules for AML selective immunotherapy have been identified. Therefore, to still apply surface-directed immunotherapy in this disease setting, time-limited combined immune-targeting of AML cells and healthy HSPCs, followed by hematopoietic stem cell transplantation (HSCT), might be a viable therapeutic approach. To explore this, we generated a recombinant single-chain variable fragment-based bispecific T-cell engaging and activating antibody directed against CD3 on T-cells and CD117, the surface receptor for stem cell factor, expressed by both AML cells and healthy HSPCs. Bispecific CD117xCD3 targeting induced lysis of CD117-positive healthy human HSPCs, AML cell lines and patient-derived AML blasts in the presence of T-cells at subnanomolar concentrations in vitro. Furthermore, in immunocompromised mice, engrafted with human CD117-expressing leukemia cells and human T-cells, the bispecific molecule efficiently prevented leukemia growth in vivo. Additionally, in immunodeficient mice transplanted with healthy human HSPCs, the molecule decreased the number of CD117-positive cells in vivo. Therefore, bispecific CD117xCD3 targeting might be developed clinically in order to reduce CD117-expressing leukemia cells and HSPCs prior to HSCT.
TNF superfamily members as payloads for antibody-cytokine anticancer therapeutics
Mock, Jacqueline Caroline (2021)
A novel strategy to generate immunocytokines with activity-on-demand using small molecule inhibitors
Rotta, Giulia; Gilardoni, Ettore; Ravazza, Domenico; et al. (2024)
EMBO Molecular Medicine
Cytokine-based therapeutics have been shown to mediate objective responses in certain tumor entities but suffer from insufficient selectivity, causing limiting toxicity which prevents dose escalation to therapeutically active regimens. The antibody-based delivery of cytokines significantly increases the therapeutic index of the corresponding payload but still suffers from side effects associated with peak concentrations of the product in blood upon intravenous administration. Here we devise a general strategy (named “Intra-Cork”) to mask systemic cytokine activity without impacting anti-cancer efficacy. Our technology features the use of antibody-cytokine fusions, capable of selective localization at the neoplastic site, in combination with pathway-selective inhibitors of the cytokine signaling, which rapidly clear from the body. This strategy, exemplified with a tumor-targeted IL12 in combination with a JAK2 inhibitor, allowed to abrogate cytokine-driven toxicity without affecting therapeutic activity in a preclinical model of cancer. This approach is readily applicable in clinical practice.

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Jacqueline Caroline Mock

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