Co-Expression of an shRNA Targeting MICA/Micb Improves the Clinical Activity of a NKG2D-Based CAR T in Patients with Relapsed / Refractory AML/MDS

While CAR T cell therapy has delivered impressive clinical efficacy in B cell malignancies, similar levels of clinical activity have not been demonstrated in acute myeloid leukemia (AML). One underlying reason for this lack of translation is the relative paucity of validated CAR T targets. Major Histocompatibility Complex Class 1 related proteins MICA and MICB along with the UL16 binding proteins 1-6 (ULBP1-6) are frequently over-expressed on AML and myelodysplastic syndrome (MDS) blasts. The Natural Killer Group 2D (NKG2D) is a single receptor able to bind MICA, MICB and ULBP1-6, thus providing a potentially powerful approach that could be exploited to target this family of targets thereby providing a novel CAR T approach for AML/MDS. Expressing the NKG2D receptor fused to the human CD3z cytoplasmic domain generates a CAR that, when expressed in primary T cells, generates a CAR T product (termed CYAD-01) that showed good anti-tumor activity in preclinical models. In clinical trials, CYAD-01 showed a good tolerability profile but with a disappointing level of clinical activity when combined with cyclophosphamide / fludarabine preconditioning (DEPLETHINK trial, NCT03466320). We hypothesised that the transient expression of MICA/MICB on the CAR T itself may inhibit the activity of the NKG2D CAR through self-targeting and consequent fratricide. We developed a single shRNA able to knockdown both MICA and MICB. Co-expression of this shRNA with the NKG2D CAR generated a second generation CAR T product termed CYAD-02 which was examined in a highly similar AML/MDS patient population and preconditioning regimen as employed in the DEPLETHINK trial. CYAD-02 was evaluated in the the first-in-human CYCLE-1 trial (NCT04167696) in patients with r/r AML/MDS. The dose-escalation phase evaluated three dose-levels (DL) (1x10 8, 3x10 8 and 1x10 9 total cells per infusion), administered as a single infusion after standard preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classic 3+3 design. As of July 2021, 11 patients have been treated with CYAD-02 at the different dose-levels (3 at each DL1 and 2 and 5 at DL3). In terms of safety and tolerability, there was no evidence of a differential safety profile of CYAD-02 within the CYCLE-1 trial as compared to that observed in the CYAD-01 clinical trial. Of note, five patients enrolled in all 3 DLs experienced Grade ≥ 3 adverse events (AE) at least possibly related to CYAD-02 (cytokine release syndrome, febrile neutropenia, white blood cell count decreased, infusion related reaction). With respect to clinical activity, seven patients in total achieved stable disease (2 at DL1, 3 at DL2 and 2 at DL3) with 4 demonstrating evidence of transient blast reduction or anti-leukemic activity (decrease of at least 50% of the bone marrow blasts). In addition, two MDS patients at DL3 achieved a marrow complete response. Overall, this suggests dose dependent response to CYAD-02. Furthermore, patients receiving CYAD-02 cells seem to display a greater duration of response and stronger blast reduction as compared to patients who received CYAD-01 in DEPLETHINK. Interestingly, peak engraftment levels were higher for CYAD-02 compared to CYAD-01 at the same dose (DEPLETHINK trial). At DL3, CYAD-02 cells could be readily detected in peripheral blood of 4/5 patients through month 2. In addition, and unlike what is reported in B cell CAR T-cells trials, there was very limited evidence of elevated homeostatic cytokines (IL-7/IL-15) following CyFlu administration with IL-15 not detected in any of the patients and IL-7 showing only a minor increase in 2 patients. The knockdown of MICA/MICB appears to have a positive contribution to the initial clinical activity of CYAD-02 as compared to that achieved with the first generation CYAD-01 CAR T, together with good safety and tolerability. The lack of homeostatic cytokines after preconditioning, likely limiting the engraftment and activity of CAR T cells, may be related to the biology of myeloid malignancies. One approach to further drive the potency of NKG2D-based CAR T cells would likely be armoring the CAR T through using the T cell as a vehicle to secrete cytokines alongside the CAR. Overall, shRNA knockdown technology provides a means to modify CAR T function and here shows that single shRNA can target two independent genes to enhance the phenotype of the CAR Ts. Disclosures Deeren: Alexion: Consultancy; BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Sanofi: Consultancy, Research Funding; Sobi: Consultancy; Takeda: Consultancy. Lin: AbbVie, Aptevo Therapeutics, Astellas Pharma, Bio-Path Holdings, Celgene, Celyad, Genentech-Roche, Gilead Sciences, Incyte, Jazz Pharmaceuticals, Novartis, Ono Pharmaceutical, Pfizer, Prescient Therapeutics, Seattle Genetics, Tolero, Trovagene: Research Funding. Alcantar-Orozco: Celyad Oncology: Current Employment. Dheur: Celyad Oncology: Current Employment. Breman: Celyad Oncology: Current Employment. Braun: Celyad Oncology: Current Employment. Lonez: Celyad Oncology: Current Employment. Gilham: Celyad Oncology: Current Employment. Flament: Celyad Oncology: Current Employment. Lehmann: Celyad Oncology: Current Employment.

INFLUENCE OF HOMEOSTATIC CYTOKINES – IL-7 AND IL-15 ON T-REGULATORY CELLS IN VITRO

Cytokines IL-7 and IL-15 are the most important humoral factors providing T-conventional cell pool reconstitution during homeostatic proliferation caused by lymphopenia. However, whether these cytokines can provide homeostatic maintenance and proliferation of T-regulatory (Treg) cells is largely unknown. Considering the association between homeostatic proliferation and the development of autoimmunity, we decided to investigate the ability of these factors to cause differentiation of Treg-cells into Th17-lymphocytes. Therefore, the purpose of this study was to investigate the influence of humoral factors of homeostatic proliferation (IL-7 and IL-15) on Treg-cells in vitro. The study used peripheral blood sampled from 22 healthy donors. PBMC fraction was isolated by Ficoll density gradient centrifugation. Proliferation was induced by IL-7, IL-15, and by a combination of IL-2 with anti-CD3-antibodies. The proliferation intensity of Tregs was evaluated by flow cytometry using CFSE in PBMC cultures by phenotype CD3+CD4+CD25+FoxP3+ and in the previously purified population of CD3+CD4+CD25+CD127lo-cells. In this case Treg-cells were obtained by immunomagnetic separation from PBMCs using a MACS Treg Isolation Kit. Also, the RORyt expression in CD3+CD4+CD25+FoxP3+-cells was evaluated during cultivation. Here, we have shown that IL-7 and IL-15 could support Treg-cells by number and phenotype. Also, we revealed that these factors provide FoxP3 expression in Treg-cells; meanwhile, stimulation with IL-2 + anti-CD3 can also cause induction of FoxP3 expression de novo in conventional CD4+ cells. Also, we have shown that IL-7 and IL-15 can cause lower-intensity proliferation of Treg-cells in comparison with IL-2 + anti-CD3. Herewith homeostatic cytokines didn’t have the ability to induce RORyt expression in both T-regulatory cells and CD4+ conventional T-lymphocytes. Thus, it has been shown that IL-7 and IL-15 can potentially participate in maintaining the total pool of Treg-cells during lymphopenia, when IL-2 deficiency occurs, without causing the induction of RORyt expression. However, how homeostatic cytokines affect the functional activity of Treg-cells remains unclear and requires further investigation.

Stromal cell-derived factor: pathological and clinical potentialities

Chemotactic cytokines are a family of signaling proteins secreted by various human tissues. Their functioning is based on the ability to induce directed chemotaxis in nearby susceptible cells. This paper reviews recent published data on one of the most important homeostatic cytokines, stromal cell-derived factor-1 (SDF-1). The leading role of this factor in the pathogenesis of various conditions, i.e., toxic, ischemic, and necrotic tissue damage, carcinogenesis (including the mechanisms of invasion and metastasis) is demonstrated. SDF-1 acts as a powerful inductor of angiogenesis and stimulates the proliferation of endothelial cells in reproductive organs. This chemokine is one of the candidate factors involved in the regulation of proliferation and differentiation of human endometrium, i.e., the implementation of epithelial-stromal interaction of tissue elements in uterine mucosa. Clear understanding of the regulatory mechanisms of SDF-1-mediated signaling during trophoblast functioning and placental angiogenesis may help develop novel therapeutic approaches to pregnancy-related disorders.KEYWORDS: stromal cell-derived factor, chemokines, cytokines, pathogenesis, pregnancy, homeostasis.FOR CITATION: Prokhorova O.V., Olina A.A., Tolibova G.Kh., Tral’ T.G. Stromal cell-derived factor: pathological and clinical potentialities. Russian Journal of Woman and Child Health. 2020;3(3):198–204. DOI: 10.32364/2618-8430-2020-3-3-198-204.

Evaluation of Bone Marrow CD8+ tissue-Resident Memory T Cells in Multiple Myeloma

Background: CD8+ T cell responses are an essential component of the adaptive immune system. After resolution of infection a small population of memory cells is formed. In relation to circulatory patterns, different subsets of memory CD8+ T cells can be identified: the central memory (CM) and the effector memory T cells (EM) (Martin MD, et al., Front Immunol. 2018). In addition, it has been described a subset of resident memory T cells (TRM) permanently living in peripheral tissues, including the bone marrow (BM) (Di Rosa F., et al., Nat Rev Immunol. 2016). It is conceivable that these cells can contribute to the defence toward haematological tumours infiltrating the BM. Therefore, we performed a study to evaluate the frequency and the phenotype of BM CD8+ TRM in patients with multiple myeloma (MM). Moreover, to evaluate the contribution that the microenvironment can have on the homeostatic and functional maintenance of these cells, we performed in vitro experiments of BM-derived mononucleate cells of MM patients cultured in the presence of different homeostatic cytokines. Patients and Methods: we prospectively analysed 21 patients, 16 with a new diagnosis of IgA, IgG and light chain multiple myeloma (MM) and 5 with IgA and IgG smoldering myeloma (SM). At the time of the bone marrow assessment, we collected a sample for the flow cytometry analysis and in vitro cell culture. The ex vivo evaluation of CD8+ TRM frequency and phenotype in BM samples was performed using anti-human mAbs to CD3, CD103, CD69, CD45, CD8, CD45RA and CCR7 (CD197). The sequential gating strategy was: gate on lymphocytes population with CD45 vs. SSC, 7AAD negative cells, exclusion of doublets with FSC-H vs. FSC-A, CD8+CD3+ and evaluation of percentage of CD103+CD69+ cells. Was also established the subsets using CCR7 and CD45RA. Moreover, to evaluate the role of the microenvironment on maintenance of these cells, we performed in vitro experiments of BM-derived mononucleate cells of MM patients cultured in the presence of homeostatic cytokines in maintaining these cells for a long time. BM derived mononucleate cells from patients were then cultured in vitro in complete RPMI with 10% of human serum for 4 days with IL15 (25 ng/ml), IL7 (25 ng/ml) and TGF-β (2 ng/ml), in different combination and in RPMI alone. After culture, we analyzed the frequency of CD8+ TRM and the proliferating fraction with intracellular staining with anti human Ki67 APC. Non parametric Mann-Whitney and Kruskall-wallis tests were performed to determine statistical differences in the distribution of the results using GraphPad Prism 7.00. Values of * p<0.05 were considered significant. Results: the ex vivo average frequency of CD8+ TRM in 16 MM patients was of 0.48% and the phenotype was represented mainly by TEM (72,9%) followed by TEMRA (12.3%) and (7,6%) of naïve cells and (7,2%) of TCM (Fig. A). The comparison with the ex vivo frequency of CD8 TRM in SM patients did not show any significant difference between two groups (data not showed). To evaluate factors capable of maintaining or to induce the expansion of these cells in vitro, we maintained BM-derived mononucleate cells from MM patients for 4 days in presence of homeostatic cytokines, IL-15, IL-7 plus IL-15 and IL-7 together with IL-15 and TGF-β. The result showed an increase of the percentage of CD8+ TRM in all conditions tested, especially in presence of all cytokines (Fig. B), with a percentage of CD8 TRM of 2,74%. Regarding the phenotype distribution, we observed an expansion of CM compared to the other subsets (Fig. C). We also analysed the percentage of CD8+ TRM proliferating through the identification of Ki67 positive cells. Data highlight that IL-15 gives the strongest proliferative input, but also other cytokines contribute to the homeostatic maintenance of these cells (Fig. D). Conclusions: we evaluated the frequency and the phenotype of CD8 TRM in BM of MM patients compared to SM patients with the conclusion that these cells do not differ significantly in percentage and phenotypic distribution in both conditions. In MM patients, the increase of CD8+ TRM cells with a CM phenotype after in vitro culture with the three cytokines could have an anti-tumor role in the control of MM. Further studies are needed to investigate the cytotoxic capacity of these cells against myeloma cells, in order to study their functional role, also in the perspective of a possible use in future therapeutic programs. Disclosures No relevant conflicts of interest to declare.

Effect of Proinflammatory and Homeostatic Cytokines on the Functionality of Human Double-Negative Regulatory T Cells

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is often the only curative treatment option for patients with hematologic malignancies. Its therapeutic effect is mediated by donor T cells, recognizing and eliminating residual malignant cells of the patient. However, the main adverse effect of allo-HSCT is also mediated by these T cells: the so-called graft-versus-host disease (GvHD). A promising approach to treat GvHD is the use of immunosuppressive T cell populations to inhibit uncontrolled T cell activation. A novel regulatory T cell population described in the setting of allo-HSCT is the subset of TCRαβ+ CD4- CD8- double-negative (DN) T cells. In murine models infusion and/or activation of DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allo-HSCT. Of interest, clinical studies in patients who underwent allo-HSCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD. We have recently demonstrated that human DN T cells like their murine counterparts strongly inhibit proliferation of alloreactive CD4+ and CD8+ T-cells. Here we asked whether proinflammatory and homeostatic cytokines associated with incidence and severity of GvHD after allo-HSCT affect the suppressive activity of DN T cells. We found that the proinflammatory cytokines IL-6, IL-18, TNF and IFN-g do not affect the suppressive activity of human DN T cells nor render alloreactive T cells insensitive to inhibition. In contrast, the homeostatic cytokine IL-15 significantly diminished the immune regulatory function of DN T cells. Further analyses demonstrated that both conventional CD4+ T cells and DN T cells express the functional IL-15 receptor which elicits downstream receptor signaling. Of importance, IL-15 highly induced Akt/mTOR signaling cascade in DN T cells, a pathway reported to reverse suppressive activity of regulatory cells. Together, our findings indicate that the homeostatic cytokine IL-15 but not proinflammatory mediators impair DN T cell-mediated suppression of allogeneic T cell responses. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for using them as a cellular-based therapy to limit alloreactive immune Responses. Disclosures No relevant conflicts of interest to declare.