Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-␥, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs. (Blood. 2011;118(26):6772-6782)

CD28 superagonists (CD28SAs) are potent T-cell-activating monoclonal antibodies (mAbs).

Following inconspicuous preclinical testing, the superagonistic anti-CD28 mAb TGN1412 was applied to six study participants who all developed a devastating cytokine storm. We verified that TGN1412 treatment of fresh PBMCs induced only moderate responses, whereas restoration of tissue-like conditions by high-density preculture (HDC) allowed vigorous cytokine production. TGN1412 treatment of T cells isolated from HDC-PBMCs induced moderate cytokine responses, which upon additional anti-IgG crosslinking were significantly boosted. Moreover, coincubation of TGN1412-treated T cells with B cells expressing the intermediate affinity Fcγ receptor IIB (CD32B), or coincubation with CD32B+ transfectants, resulted in robust T cell activation. This was surprising because TGN1412 was expressed as an Ig of the subclass 4 (IgG4), which was shown before to exhibit only minor affinity to FcγRs. Transcriptome analysis of TGN1412-treated T cells revealed that similar gene signatures were induced irrespective of whether T cells derived from fresh or HDC-PBMCs were studied. Collectively, these data indicate that HDC-PBMCs and HDC-PBMC–derived T cells mount rapid TGN1412 responses, which are massively boosted by FcγR crosslinking, in particular by CD32-expressing B cells. These results qualify HDC-PBMCs as a valuable in vitro test system for the analysis of complex mAb functions.

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<0.0001 and p=0.0382, respectively). In melanoma biopsies an inverse correlation of GDF-15 levels (histoscore) with CD3+ (R=-0.26; p=0.016) and CD8+ T cells (R=-0.21; p=0.05), but no correlation with Foxp3+ T cells was shown. 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.

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.