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Cancer immunotherapy has witnessed a new renaissance with the advent of immune checkpoint inhibitors, which reactivate T cells and foster endogenous anti-tumor responses. The excellent results of immunotherapy in the field of melanoma, renal cancer, lung cancer, and other cancer types that have traditionally been known to be immunogenic, rekindled the interest of the oncology community in extending the benefits to all cancers including breast cancer (BC). In this review, we highlight the current state of using T cells as both markers for clinical practice and therapeutic options for BC.

BackgroundNatural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step for cancer progression through yet unidentified mechanisms.MethodsNK cell antibody-dependent cellular cytotoxicity (ADCC) is a major determinant of the clinical efficacy of some therapeutic antibodies including the anti-HER2 Trastuzumab. Thus, we screened sera of Trastuzumab-resistant HER2 +patients with breast cancer for molecules that could inhibit NK cell ADCC. We validated our findings in vitro using cytotoxicity assays and confocal imaging of the lytic granule machinery and in vivo using syngeneic and xenograft murine models.ResultsWe found that sera from Trastuzumab-refractory patients could inhibit healthy NK cell ADCC in vitro. These sera contained high levels of the inflammatory protein chitinase 3-like 1 (CHI3L1) compared with sera from responders and healthy controls. We demonstrate that recombinant CHI3L1 inhibits both ADCC and innate NK cell cytotoxicity. Mechanistically, CHI3L1 prevents the correct polarization of the microtubule-organizing center along with the lytic granules to the IS by hindering the receptor of advanced glycation end-products and its downstream JNK signaling. In vivo, CHI3L1 administration drastically impairs the control of NK cell-sensitive tumors, while CHI3L1 blockade synergizes with ADCC to cure mice with HER2 +xenografts.ConclusionOur work highlights a new paradigm of tumor immune escape mediated by CHI3L1 which acts on the cytotoxic machinery and prevents granule polarization. Targeting CHI3L1 could mitigate immune escape and potentiate antibody and cell-based immunotherapies.

Background Targeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). The mutational landscape, heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are equipped with synthetic agonistic receptors (SARs) that are selectively activated only in the presence of a target AML-associated antigen, and a cross-linking tandem single chain variable fragment (taFv) specific for both (SAR) T cell and tumour cell. Materials and Methods A SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific taFvs that target AML-associated antigens were designed and expressed in Expi293FTM cells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigens CD33 and CD123. Results Anti-CD33-EGFRvIII and anti-CD123 EGFRvIII taFv, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by taFv could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR-taFv combination could also mediate specific cytotoxicity against patient-derived AML blasts and leukemic stem cells whilst driving SAR T cell activation. In vivo, treatment with SAR-taFv combination could efficiently eradicate leukemia and enhance survival in an AML xenograft models. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility and modularity of said activation upon depletion of the T cell engaging molecule, both in vitro and in vivo. Conclusions Here we apply the SAR-taFv platform in efforts to deliver specific and conditional activation of SAR-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its taFv facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells. Disclosure Information M. Benmebarek: None. B. Loureiro Cadilha: None. M. Herrmann: None. S. Schmitt: None. S. Lesch: None. S. Stoiber: None. A. Darwich: None. C. Augsberger: None. B. Brauchle: None. M. Schwerdtfeger: None. A. Gottschlich: None. A. Gottschlich Rataj: None. N.C. Fenn: None. C. Klein: None. M. Subklewe: None. S. Endres: None. K. Hopfner: None. S. Kobold: None.

Background and Aims The SARS-CoV-2 pandemic has overwhelmed the treatment capacity of the health care systems during the highest viral diffusion rate. Patients reaching the emergency department had to be either hospitalized (inpatients) or discharged (outpatients). Still, the decision was taken based on the individual assessment of the actual clinical condition, without specific biomarkers to predict future improvement or deterioration, and discharged patients often returned to the hospital for aggravation of their condition. Here, we have developed a new combined approach of omics to identify factors that could distinguish coronavirus disease 19 (COVID-19) inpatients from outpatients. Methods Saliva and blood samples were collected over the course of two observational cohort studies. By using machine learning approaches, we compared salivary metabolome of 50 COVID-19 patients with that of 270 healthy individuals having previously been exposed or not to SARS-CoV-2. We then correlated the salivary metabolites that allowed separating COVID-19 inpatients from outpatients with serum biomarkers and salivary microbiota taxa differentially represented in the two groups of patients. Results We identified nine salivary metabolites that allowed assessing the need of hospitalization. When combined with serum biomarkers, just two salivary metabolites (myo-inositol and 2-pyrrolidineacetic acid) and one serum protein, chitinase 3-like-1 (CHI3L1), were sufficient to separate inpatients from outpatients completely and correlated with modulated microbiota taxa. In particular, we found Corynebacterium 1 to be overrepresented in inpatients, whereas Actinomycetaceae F0332, Candidatus Saccharimonas, and Haemophilus were all underrepresented in the hospitalized population. Conclusion This is a proof of concept that a combined omic analysis can be used to stratify patients independently from COVID-19.

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment., Keypoints Modular platform enabling controlled targeting of AML by SAR-transduced T cells in combination with tandem scFv constructs. Efficient lysis of primary AML blasts in vitro and strong antitumoral effects and T cell persistence in xenograft models.

Background Targeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). Due to the mutational landscape and heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target AML-associated antigen, and a cross-linking bispecific T cell engager (BiTE) specific for both (SAR) T cell and tumour cell. Materials and Methods A SAR composed of an extracellular EGFRvIII, trans-membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap-extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific bispecific T cell engagers (BiTE) that target AML-associated antigens were designed and expressed in Expi293FTMcells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigen CD33. Results CD33-EGFRvIII BiTE, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by BiTE could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR.BiTE combination could also mediate specific cytotoxicity against patient-derived AML blasts whilst driving SAR T cell activation. In vivo, treatment with SAR.BiTE combination could efficiently eradicate leukemia and enhance survival in an AML xenograft model. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility of said activation upon depletion of the T cell engaging molecule. Conclusions Here we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of agonistic receptor-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its BiTE facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells. Disclosure Information M. Benmebarek: None. B.L. Cadilha: None. M. Hermann: None. S. Lesch: None. C. Augsburger: None. B. Brauchle: None. S. Stoiber: None. A. Darwich: None. F. Rataj: None. C. Klein: A. Employment (full or part-time); Significant; Roche. K. Hopfner: None. M. Subklewe: None. S. Endres: None. S. Kobold: None.