Search

BackgroundGrowth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member physiologically expressed mainly in placenta and linked to feto-maternal tolerance. Under pathophysiologic conditions, prevention of excessive immune cell infiltration during tissue damage and cachexia induction have been ascribed to GDF-15. Recent research has though indicated a prominant role in modulation of the tumor microenvironment and the immune synapse, too1 2 indicating that GDF-15 may be a major tumor-derived immunosuppressant. Importantly, several cancer entities secrete high levels of GDF-15, correlating with poor prognosis and reduced overall survival [Front Immunol 2020 May 19;11:951]. To block this effect the GDF-15 neutralizing antibody CTL-002 was generated. In preclinical models CTL-002 demonstrated potent effector T cell shifting into tumor tissue by neutralizing GDF-15.MethodsThis is a phase 1, first-in-human (FIH), two-part, open-label clinical trial of intravenous (IV) administration of CTL-002 given as monotherapy and in combination with an anti-PD-1 antibody in subjects with advanced-stage, relapsed/refractory solid tumors who relapsed or were refractory to a prior anti-PD-1/PD-L1 therapy. Eligible subjects have exhausted all available approved standard treatments, including prior anti-PD1/-PD-L1 treatment, and present with a biopsy-accessible tumor for serial biopsy taking. The trial is termed GDFATHER, for ”GDF-15 Antibody-mediaTed Effector cell Relocation”.Main endpoints are safety of CTL-002 monotherapy and CTL-002 combination with an anti-PD-1 antibody, pharmacokinetics, pharmacodynamics (e.g. degree of GDF-15 neutralization achieved and change in immune-cell number and composition in the tumor tissue) as well as preliminary clinical efficacy (tumor mass reduction; anticachexia effect)In part A of the trial (dose escalation) up to 24 subjects will receive escalating doses of CTL-002 IV (0.3 – 20 mg/kg) in a ”mono-followed-by-combination”-design with CTL-002 given as monotherapy and followed by combination with an anti-PD-1 checkpoint inhibitor. In part B (expansion) up to 5 cohorts with up to 25 subjects per cohort with defined tumor entities expected to be GDF-15 dependent will be treated to determine the recommended phase 2 dose (RP2D) and further evaluate safety and preliminary efficacy of CTL-002 monotherapy and the combination.The study was initiated in December 2020 and enrolled the first patient on Dec 09, 2020. Cohort 4 is ongoing at time of submission (07/2021) and so far no DLT has occurred. Updated safety, biomarker and response assessments will be reported at the meeting. The ClinicalTrials.gov Identifier is NCT04725474. For more information please contact info@catalym.com.Trial RegistrationNCT04725474ReferencesWischhusen J, Wistuba-Hamprecht K, Harter PN, Cheng P, Martens A, Gogolla F, Nonomura Y, Romer P, Koch SD, Haake M, Schuberth-Wagner C, Rudiger M, Leo E, Mittelbronn M, Levesque MP, Hackl H, Dummer R, Weide B. Identifying GDF-15 as potential novel immunotherapeutic target linked to immune cell exclusion in tumors and resistance to anti-PD-1 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27–28 and Jun 22–24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl): Abstract nr 2161.Hurt E, Thomas S, Mulgrew K, Blackmore S, Moynihan J, Cusdin F, Dodd R, Cariuk P, Sigurdardottir A, Brannigan E, Dobson C, Kumar R, Cobbold M. AZD8853: A novel antibody targeting GDF15 for immunotherapy refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10–15 and May 17–21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl): Abstract nr 1828.Ethics ApprovalAll participants gave informed consent prior to participation. EC approval by Gobierno de Navarra, Departamento de Salud, EC_2020/30, Dated: Oct 13, 2020 in Pamplona, Spain. Respective additional national lead EC approvals for Germany (Ethikkommission der Universität Würzburg, 203–20ff of Oct 26, 2020) and Switzerland (Kantonale Ethikkommission Zürich, 2020–02308 of Nov 24, 2020).

Background: Growth and differentiation factor 15 (GDF-15), a divergent member of the TGF-β protein superfamily, shows low physiological baseline expression. GDF-15 is, however, strongly upregulated during pregnancy. It is further induced in stressed and damaged tissues, where it limits immune infiltration and inflammation. In solid tumors, GDF-15 was shown to be a key inhibitor of T-cell infiltration. While this might already explain the strong correlation between GDF-15 overexpression and poor survival with or without checkpoint-based immunotherapy, effects of GDF-15 on dendritic cells and monocytes may further shape the tumor microenvironment. Methods: To analyze the impact of GDF-15 in murine tumor models, GDF-15 was either deleted by CRISPR/CAS9 gene editing or neutralized by administration of a blocking antibody. Effects on tumor growth were recorded and the composition of the tumor microenvironment was characterized by flow cytometry. More detailed insight into tumor-immune interactions was obtained via the CrownBio Mouse I/O RNA-Seq Panel. Specific effects on polarization of innate and on antigen-specific priming of adaptive immune cells were confirmed in cellular assays in vitro. Results: Tumor-derived GDF-15 modulates the tumor microenvironment by inhibiting infiltration and activation of myeloid cells. GDF-15 thereby impairs the induction of antitumoral immune responses. Deletion of GDF-15 enhances infiltration of innate cells into immune-excluded tumors, supports the priming of naïve T cells by dendritic cells and generates a pro-inflammatory tumor microenvironment. In vitro, GDF-15 inhibits DC maturation and synapse formation, thus preventing successful T-cell activation. Moreover, GDF-15 interferes with M1 polarization of macrophages. Conclusion: GDF-15 secretion helps tumors to generate a microenvironment that is poorly infiltrated by immune cells. GDF-15 further polarizes myeloid cells towards a tumor-promoting, anti-inflammatory phenotype. By inducing a more pro-inflammatory tumor phenotype, anti-GDF-15 antibodies may synergize with other immunotherapeutic agents. A clinical trial combining anti-GDF-15 (CTL002) with anti-PD-1 (NCT04725474) is ongoing. Citation Format: Markus Haake, Beatrice Haack, Sabrina Genßler, Julia Weigandt, Marlene Auer, Vincent Thiemann, Wahid M. Haq, Melanie Haag, Florian Wedekink, Birgitt Fischer, Kathrin Klar, Matthias Wölfl, Christine Schuberth-Wagner, Jorg Wischhusen. Tumor-derived GDF-15 promotes immune escape of tumors by functional alteration of the myeloid compartment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6118.

Background: Growth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member physiologically expressed mainly in placenta and linked to feto-maternal tolerance. Under pathophysiologic conditions, prevention of excessive immune cell infiltration during tissue damage and cachexia induction have been ascribed to GDF-15. A recent study [Haake et al. AACR2020; Abstract #5597] elucidated a mechanism by which GDF-15 inhibits LFA-1 activation on CD8+ T cells, thus interfering with effector T cell recruitment to tissues. Importantly, several cancer entities secrete high levels of GDF-15, correlating with poor prognosis and reduced overall survival [reviewed in Front Immunol 2020 May 19;11:951]. To block this effect the GDF-15 neutralizing antibody CTL-002 was generated. In preclinical models CTL-002 demonstrated potent effector T cell shifting into tumor tissue by neutralizing GDF-15 and enhancing response to checkpoint inhibitor therapy. Methods: This is a phase 1, first-in-human (FIH), two-part, open-label clinical trial of intravenous (IV) administration of CTL-002 given as monotherapy and in combination with an anti-PD-1 antibody in subjects with advanced-stage, relapsed/refractory solid tumors who relapsed on or were refractory to a prior anti-PD-1/PD-L1 therapy. Eligible subjects have exhausted all available approved standard treatments, including prior anti-PD1/-PD-L1 treatment, and present with a biopsy-accessible tumor for serial biopsy taking. The trial is termed GDFATHER, for “GDF-15 Antibody-mediaTed Effector cell Relocation”. Main endpoints are safety of CTL-002 monotherapy and CTL-002 combination with an anti-PD-1 antibody, pharmacokinetics, pharmacodynamics (e.g., degree of GDF-15 neutralization achieved and change in immune-cell number and composition in the tumor tissue) as well as preliminary clinical efficacy (tumor mass reduction; anticachexia effect). In part A of the trial (dose escalation) up to 24 subjects will receive escalating doses of CTL-002 IV (0.3 – 20 mg/kg) in a „mono-followed-by-combination“-design with CTL-002 given as monotherapy and followed by combination with an anti-PD-1 checkpoint inhibitor. In part B (expansion) up to 5 cohorts with up to 25 subjects per cohort with defined tumor entities expected to be GDF-15 dependent will be treated to determine the recommended phase 2 dose (RP2D) and further evaluate safety and preliminary efficacy of CTL-002 monotherapy and the combination. The study was initiated in December 2020 and enrolled the first patient on Dec 09, 2020. Cohort 4 is ongoing at time of submission (07/2021) and so far no DLT has occurred. Updated safety, biomarker and response assessments will be reported. The ClinicalTrials.gov Identifier is NCT04725474. Citation Format: Ignacio Melero, Emiliano Calvo, Maria-Elisabeth Goebeler, Elena Garralda, Reinhard Dummer, María Rodríguez-Ruiz, María De Miguel, Cyrus Sayehli, Guzman Alonso Casal, Egle Ramelyte, Martin Schuler, Tanja Gromke, Miguel Sanmamed, Irene Moreno, Ralf Bargou, Maria Lostes, Julia-Tatjana Maul, Heike Richly, Petra Fettes, Kathrin Klar, Christine Schuberth-Wagner, Markus Haake, Joerg Wischhusen, Eugen Leo. A phase I, first-in-human clinical trial of the GDF-15 neutralizing antibody CTL-002 in subjects with advanced stage solid tumors (Acronym: GDFATHER) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P06-01.

BackgroundImmune checkpoint blockade (ICB) can achieve durable responses in a subgroup of patients with metastatic cancer, only. Poor immune effector cell infiltration into the tumor microenvironment is a major obstacle to successful therapy. Growth and differentiation factor 15 (GDF-15) is a divergent member of the TGF-β superfamily and has been linked to feto-maternal tolerance, anorexia but recently also to potent local immunosuppression under physiologic and pathophysiologic conditions. GDF-15 is overexpressed in a wide variety of tumors and may be key factor produced by tumors to prevent effective immune cell infiltration into the tumor and to potently block checkpoint inhibitor activity.MethodsEffects of recombinant GDF-15 and a proprietary GDF-15 neutralizing antibody (CTL-002) on immune cell trafficking and activation were analyzed by adhesion and interaction assays and in melanoma-bearing humanized mouse models. The impact of GDF-15 overexpression was tested in subcutaneously implanted, GDF-15-transgenic MC38 cells. Additionally, patient GDF-15 serum levels were correlated with immune infiltration and OS in cutaneous melanoma. Associations between GDF-15 serum levels, response to PD-1-based ICB and corresponding OS were assessed in two independent cohorts of melanoma patients.ResultsGDF-15 impairs adhesion of T and NK cells on activated endothelia. In HV18-MK bearing humanized mice, inhibition of GDF-15 strongly enhances infiltration of activated myeloid and lymphoid cells. In MC38 tumors, GDF-15 overexpression can abrogate tumor rejection upon anti-PD-1 therapy. 50% of the mice with GDF-15 overexpressing tumors were, however, rescued when anti-PD-1 was combined with anti-GDF-15 (CTL-002). Likewise, anti-GDF-15 improved responses to anti-CD40 + poly(I:C) in the same tumor model. Clinically, inverse correlations of GDF-15 levels with CD8+ T cell infiltration were shown for melanoma brain metastases. In two independent melanoma patient cohorts, low baseline serum GDF-15 levels predicted clinical response to anti-PD1 treatment and superior OS. Bivariate analysis including LDH indicates that GDF-15 is an independently predictor for poor survival in anti-PD-1 treated melanoma patients.ConclusionsTumor-derived GDF-15 blocks the infiltration of immune effector cells into tumor tissues. Neutralizing GDF-15 with CTL-002 restores the ability of immune cells to extravasate blood vessels and enter the tumor microenvironment in vivo. GDF-15 thus represents a promising target for cancer immunotherapy. Antibodies against GDF-15 may support treatments with anti-PD-1 and other immunotherapeutic agents. A clinical trial combining anti-GDF-15 (CTL002) with anti-PD-1 (NCT04725474, submitted Abstract ID 15073) is ongoing.Ethics ApprovalUse of patient samples for this study had been approved by the institutional ethics committee Tübingen (ethic vote 125/2015BO2). Use of surplus sera collected in the University of Zurich Hospital (USZ) Biobank during routine blood draws from consenting metastatic melanoma patients was performed according to IRB approval (KEK.Zh- 647/800) and followed the Declaration of Helsinki on Human Rights.ConsentAll patients had given written informed consent to have clinical data recorded by the Central Malignant Melanoma Registry (CMMR) database.

TPS2658 Background: Growth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member physiologically expressed mainly in placenta and linked to feto-maternal tolerance. Under pathophysiologic conditions, prevention of excessive immune cell infiltration during tissue damage and cachexia induction have been ascribed to GDF-15. A recent study [Haake et al. AACR2020; Abstract #5597] elucidated a mechanism by which GDF-15 inhibits LFA-1 activation on CD8+ T cells, thus interfering with effector T cell recruitment to tissues. Importantly, several cancer entities secrete high levels of GDF-15, correlating with poor prognosis and reduced overall survival [reviewed in Front Immunol 2020 May 19;11:951]. To block this effect the GDF-15 neutralizing antibody CTL-002 was generated. In preclinical models CTL-002 demonstrated potent effector T cell shifting into tumor tissue by neutralizing GDF-15. Methods: This is a phase 1, first-in-human (FIH), two-part, open-label clinical trial of intravenous (IV) administration of CTL-002 given as monotherapy and in combination with an anti-PD-1 antibody in subjects with advanced-stage, relapsed/refractory solid tumors who relapsed post or were refractory to a prior anti-PD-1/PD-L1 therapy. Eligible subjects have exhausted all available approved standard treatments. Further key eligibility criteria include having received at least one prior anti-PD1/-PD-L1 treatment and having relapsed on or after it or having been refractory to it, and presenting with a biopsy-accessible tumor for serial biopsy taking. The trial is termed GDFATHER, for “GDF-15 Antibody-mediaTed Effector cell Relocation”. Main endpoints are safety of CTL-002 monotherapy and CTL-002 combination with an anti-PD-1 antibody, pharmacokinetics, pharmacodynamics (e.g. degree of GDF-15 neutralization achieved and change in immune-cell number and composition in the tumor tissue) as well as preliminary clinical efficacy (tumor mass reduction; anticachexia effect) In part A of the trial (dose escalation) up to 24 subjects will receive escalating doses of CTL-002 IV (0.3 – 20 mg/kg) in a „mono-followed-by-combination“-design with CTL-002 given as monotherapy and followed by combination with an anti-PD-1 checkpoint inhibitor. In part B (expansion) up to 5 cohorts with up to 25 subjects per cohort with defined tumor entities expected to be GDF-15 dependent will be treated to determine the recommended phase 2 dose (RP2D) and further evaluate safety and preliminary efficacy of CTL-002 monotherapy and the combination. The study was initiated in December 2020 and enrolled the first patient on Dec 09, 2020. Cohort 1 has been completed without DLT and enrollment for cohort 2 began in February 2021. Clinical trial information: NCT04725474.

e14532 Background: Growth and differentiation factor 15 (GDF-15) is a divergent member of the TGF-β superfamily with low to absent expression in healthy tissue. GDF-15 has been linked to feto-maternal immune tolerance, to prevention of excessive immune cell infiltration during tissue damage, and to anorexia. Various major tumor types secrete high levels of GDF-15. In cancer patients, elevated GDF-15 serum levels correlate with poor prognosis and reduced overall survival (OS). Methods: Impact of a proprietary GDF-15 neutralizing antibody (CTL-002) regarding T cell trafficking was analyzed by whole blood adhesion assays, a HV18-MK melanoma-bearing humanized mouse model and a GDF-15-transgenic MC38 model. Additionally, patient GDF-15 serum levels were correlated with clinical response and overall survival in oropharyngeal squamous cell carcinoma (OPSCC) and melanoma brain metastases. Results: In whole blood cell adhesion assays GDF-15 impairs adhesion of T and NK cells to activated endothelial cells. Neutralization of GDF-15 by CTL-002 rescued T cell adhesion. In HV18-MK-bearing humanized mice CTL-002 induced a strong increase in TIL numbers. Subset analysis revealed an overproportional enrichment of T cells, in particular CD8+ T cells. As immune cell exclusion is detrimental for checkpoint inhibitor (CPI) therapy, a GDF-15-transgenic MC38 model was tested for anti-PD-1 therapy efficacy. In GDF-15 overexpressing MC38 tumors response to anti PD-1 therapy was reduced by 90% compared to wtMC38 tumors. Combining aPD-1 with CTL-002 resulted in 50% of the mice rejecting their GDF-15 overexpressing tumors. Clinically, inverse correlations of GDF-15 levels with CD8+ T cell infiltration were shown for HPV+ OPSCC and for melanoma brain metastases. GDF-15 serum levels were significantly higher in HPV- than in HPV+ OPSCC patient (p < 0.0001). Low GDF-15 levels corresponded to longer OS in both HPV- and HPV+ OPSCC. In two independent melanoma patient cohorts treated with nivolumab or pembrolizumab low baseline serum GDF-15 levels were predictive for clinical response to anti-PD1 treatment and superior OS. Bivariate analysis including LDH indicates that GDF-15 independently predicts poor survival in aPD-1 treated melanoma patients. Conclusions: Taken together our in vitro and in vivo data show that elevated GDF-15 levels block T-cell infiltration into tumor tissues. Neutralizing GDF-15 with CTL-002 restores the ability of T cells to extravasate blood vessels and enter tumor tissue both in vitro and in vivo. In melanoma, patients with higher GDF-15 levels have significantly shorter survival and are less likely to respond to anti-PD1 therapy. GDF-15 may thus serve as a new predictive biomarker for anti-PD1 response, but most importantly also represents a novel target for cancer immunotherapy to improve tumor immune cell infiltration and response to anti-PD1 therapy.

Background: Growth and differentiation factor 15 (GDF-15) is a remote member of the TGF-β protein family with low to absent expression in healthy tissue. During pregnancy GDF-15 is secreted in high amounts by placenta contributing to feto-maternal tolerance. Similarly, GDF-15 expression is upregulated following tissue injury serving as a key inhibitor of excessive and disruptive immune infiltration. Importantly, various major tumor types secrete high levels of GDF-15 correlating with poor prognosis and reduced overall survival. Methods: In a flow-adhesion assay different immune cell subsets pre-treated +/- GDF-15 were perfused over an activated layer of endothelial cells or recombinant adhesion molecules. Adhesion and transmigration processes were monitored by live imaging microscopy. Impact of a proprietary GDF-15 neutralizing antibody (CTL-002) regarding T cell trafficking were analyzed. In a humanized (CD34+-HPSC engrafted) PDX mouse model inoculated with the PD-L1hi and GDF-15 secreting human melanoma tumor cell line HV18-MK T cell infiltration was analysed +/- CTL-002 by FACS. Results: Adhesion of T cells to the endothelial cell layer was significantly impaired by addition of GDF-15. Among T-cell subsets CD8+ T-cells were most affected while adhesion of other immune cells was not reduced. Inhibitory effects of GDF-15 on CD8+ T-cell adhesion were comparable to potent blockade of LFA-1 by TS1/18 antibody and could be rescued by the anti-GDF-15 antibody CTL-002. In vivo, neutralization of GDF-15 in HV18-MK melanoma-bearing humanized mice by CTL-002 resulted in a strong increase of tumor infiltrating leukocyte numbers. Subset analysis revealed an overproportional enrichment of T-cells, especially CD8+-T cells. Conclusion: Our in vitro and in vivo data show that elevated GDF-15 levels block T-cell infiltration into the tumor tissue. Neutralizing GDF-15 with a proprietary antibody (CTL-002) restored the ability of T cells (especially CD8+-T-cells) to extravasate blood vessels and enter tumor tissue both in vitro and in vivo. As it is known that presence of tumor-infiltrating lymphocytes correlates with better patient outcomes in a multitude of cancers and is a predictor of response to checkpoint inhibitors, high levels of GDF-15 in the tumor may contribute to failure of immunotherapies and poor overall survival. Neutralization of GDF-15 could be a new and promising approach to increase response rates of immunotherapies and increase overall survival of cancer patients. Citation Format: Markus Haake, Neha Vashist, Sabrina Genssler, Kristin H. Eichler, Birgitt Fischer, Jessica Kammer, Paula S. Romer, Manfred Rudiger, Eugen Leo, Falk Nimmerjahn, Christine Schuberth-Wagner, Jorg Wischhusen. Tumor-derived GDF-15 suppresses T-lymphocyte recruitment to the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5597.

Introduction: Growth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member mainly expressed in placenta and prostate of healthy individuals. GDF-15 has been linked to feto-maternal tolerance, prevention of excessive immune cell infiltration during tissue damage and to anorexia. In cancer patients, GDF-15 serum levels are frequently elevated and associated with poor prognosis, via so far mostly unknown mechanism(s). A recent study elucidated a mechanism by which GDF-15 inhibits LFA-1 activation on CD8+ T cells, thus interfering with T cell recruitment to tissues [Haake et al. AACR 2020; submitted]. To further delineate the role of GDF-15 in cancer in this study GDF-15 serum and tissue levels were analyzed and correlated with tumoral immune-cell infiltration and clinical anti-PD1 response. Methods: In-silico, TCGA-derived mRNA levels of GDF-15 were compared in cancer vs. normal tissue. Two independent melanoma patient cohorts (88 and 34 patients) treated with nivolumab or pembrolizumab were analyzed regarding baseline GDF-15 serum levels, correlation with clinical response and overall survival. Melanoma brain metastases from 80 patients were collected to assess and compare intratumoral GDF-15 levels vs. CD3+, CD8+ and Foxp3+ cell numbers by immunohistochemistry (IHC). Results: TCGA-based analyses demonstrated significantly elevated GDF-15 mRNA levels in tumor vs. surrounding normal tissue in various major cancer types such as e.g. colorectal, prostate, head & neck and melanoma. In the two independent, anti-PD1 treated melanoma patient cohorts baseline serum GDF-15 levels were predictive for superior overall survival and clinical response to anti-PD1 treatment (p Conclusion: GDF-15 is elevated in serum and tumor tissue of various major cancer types. In melanoma, patients with higher GDF-15 levels have significantly shorter survival and are less likely to respond to anti-PD1 therapy. In addition, intratumoral GDF-15 levels in melanoma brain metastasis correlate inversely with CD3+ and CD8+ T cell infiltration. Consequently, GDF-15 may serve as a predictive biomarker for anti-PD1 response and potentially represent a novel target in the immunotherapy of cancer to improve tumor immune cell infiltration and anti-PD1 response. Citation Format: Jorg Wischhusen, Kilian Wistuba-Hamprecht, Patrick N. Harter, Phil Cheng, Alexander Martens, Falk Gogolla, Yumi Nonomura, Paula Romer, Sven D. Koch, Markus Haake, Christine Schuberth-Wagner, Manfred Rudiger, Eugen Leo, Michael Mittelbronn, Mitchell P. Levesque, Hubert Hackl, Reinhard Dummer, Benjamin Weide. Identifying GDF-15 as potential novel immunotherapeutic target linked to immune cell exclusion in tumors and resistance to anti-PD-1 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2161.

Multi-antigen immunotherapy approaches against Staphylococcus aureus are expected to have the best chance of clinical success when used in combinatorial therapy, potentially incorporating opsonic killing of bacteria and toxin neutralization. We recently reported the development of a murine monoclonal antibody specific for the immunodominant staphylococcal antigen A (IsaA), which showed highly efficient staphylococcal killing in experimental infection models of S. aureus. If IsaA-specific antibodies are to be used as a component of combination therapy in humans, the binding specificity and biological activity of the humanized variant must be preserved. Here, we describe the functional characterization of a humanized monoclonal IgG1 variant designated, hUK-66. The humanized antibody showed comparable binding kinetics to those of its murine parent, and recognized the target antigen IsaA on the surface of clinically relevant S. aureus lineages. Furthermore, hUK-66 enhances the killing of S. aureus in whole blood (a physiological environment) samples from healthy subjects and patients prone to staphylococcal infections such as diabetes and dialysis patients, and patients with generalized artery occlusive disease indicating no interference with already present natural antibodies. Taken together, these data indicate that hUK-66 mediates bacterial killing even in high risk patients and thus, could play a role for immunotherapy strategies to combat severe S. aureus infections.

Environmental pollutants can influence the expression of immunoregulatory molecules and, in this way, promote allergies. The local synthesis of proinflammatory chemokines is an important aspect in the development of allergic airway inflammation. We have characterized the influence of pyrene, a polycyclic aromatic hydrocarbon (PAH) contained, for example, in diesel exhaust particles (DEP), on transcription and secretion of the chemokines interleukin-8 (IL-8) and eotaxin. Reporter genes under control of the respective promoters were tested in the human cell lines A549 and HeLa, mRNA production was assayed in A549 cells and protein production was measured by ELISA in cell supernatants from primary human fibroblasts. Pyrene content of cell supernatants was measured by analytical HPLC. Promoter activity, mRNA production and protein expression of IL-8 were increased by pyrene. The activating effect in reporter gene studies was abolished by mutating either an NF-κB or an AP-1 binding site in the IL-8 promoter. In contrast, pyrene showed no effect on transcription from the eotaxin promoter, despite the important role of this chemokine in asthma. Our data show that pyrene has specific effects on chemokine synthesis, which are not restricted to mediators primarily associated with atopic diseases. Pyrene also affected cells not derived from lung tissue, which suggests a broader immunoregulatory influence for this pollutant.

The transcription factor Stat6 (signal transducer and activator of transcription 6) is activated following stimulation with interleukin (IL)-4 or IL-13. Stat6 binds via a single SH2 domain first to tyrosine-phosphorylated motifs in the IL-4Ralpha chain, and then to another Stat6 molecule, which results in the formation of active dimers. We show here that a peptide derived from the Stat6-binding region of IL-4Ralpha (Stat6BP) is an effective inhibitor when it is delivered into cells by coupling with a membrane-permeable peptide. Stat6BP completely inhibited IL-4 dependent phosphorylation of Stat6, as well as basal and IL-4 stimulated transcription from a reporter gene construct with a Stat6-dependent promoter, while IL-3 and IL-4 dependent phosphorylation of Stat5 was not affected. The inhibitory effect of Stat6BP was transient, but could be prolonged by treating the cells with the phosphatase inhibitor pervanadate.

In lymphocytes, glucocorticoids (GC)- and interleukin-4-signaling pathways are known to interact, as evidenced by inhibition of IL-4-mediated proliferation by dexamethasone or suppression of GC-induced apoptosis by IL-4. In this study, we characterized the molecular basis for this reciprocal interference. We report that, in murine CTLL-2 cells, IL-4 inhibits GC-induced MMTV (mouse mammary tumor virus) promoter transactivation, and that GC suppress IL-4-induced transactivation of a STAT6 (signal transducers and activators of transcription 6)-responsive promoter without affecting IL-4-stimulated STAT6 DNA-binding. Moreover, we evidenced a physical association between GC receptor and STAT6, which proved to be functionally relevant, since STAT6 overexpression increased the IL-4 inhibitory effect on GC-induced MMTV transactivation.