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Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the two most widely overexpressed LSCs biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells (HSPCs) increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced Dual CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-Induced Killer (CIK) cells, co-expressing a first-generation low affinity anti-CD123 IL3-zetakine and an anti-CD33 as costimulatory receptor (CCR) without activation signaling domains, demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on HSPCs and endothelial cells. The proposed optimized Dual CAR CIK strategy could offer the opportunity to unleash the potential of specifically target CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

Introductory paragraphWhile CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells1,2. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity3–7; however, a truly safe and effective logic-gated CAR has remained elusive8. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3ζ domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumorsin vivowhile bypassing upstream signaling proteins such as CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineerLogic-gatedIntracellularNetworK(LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity9and fibrosis10. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering.

Chimeric antigen receptors (CAR)-modified T cells are an emerging therapeutic tool for chronic lymphocytic leukemia (CLL). However, in patients with CLL, well-known T-cell defects and the inhibitory properties of the tumor microenvironment (TME) hinder the efficacy of CAR T cells. We explored a novel approach combining CARs with lenalidomide, an immunomodulatory drug that tempers the immunosuppressive activity of the CLL TME. T cells from patients with CLL were engineered to express a CAR specific for CD23, a promising target antigen. Lenalidomide maintained the in vitro effector functions of CD23.CAR+ T cells effector functions in terms of antigen-specific cytotoxicity, cytokine release and proliferation. Overall, lenalidomide preserved functional CAR T-CLL cell immune synapses. In a Rag2−/−γc−/−-based xenograft model of CLL, we demonstrated that, when combined with low-dose lenalidomide, CD23.CAR+ T cells efficiently migrated to leukemic sites and delayed disease progression when compared to CD23.CAR+ T cells given with rhIL-2. These observations underline the therapeutic potential of this novel CAR-based combination strategy in CLL.

Epigenetic dysregulation is frequently observed in the disease pathology of pediatric cancers, including neuroblastoma, the most common extracranial solid tumor in pediatric patients. Neuroblastoma tumors co-opt developmentally linked adrenergic or mesenchymal super-enhancer landscapes that rewire their transcriptional programs. Here, we describe that the lineage commitment to a mesenchymal epigenetic state is an important mechanism of resistance to anti-GD2 therapy through loss of GD2 antigen, a ganglioside glycolipid expressed on the cell surface. Low GD2 expression was significantly correlated with the mesenchymal state in a large panel of neuroblastoma cell lines and a forced adrenergic-to-mesenchymal transition conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2 expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Genome-wide CRISPR/Cas9 screening to identify regulators of GD2 in neuroblastoma revealed that the ablation of the polycomb repressive complex 2 (PRC2) significantly upregulates GD2 expression in GD2-low cells. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells into an adrenergic-like state, re-expressed ST8SIA1, and restored surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential PRC2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma. Citation Format: Nathaniel W. Mabe, Min Huang, Daniel A. Schaefer, Guillermo N. Dalton, Giulia Digiovanni, Gabriela Alexe, Anna C. Geraghty, Delan Khalid, Marius M. Mader, Michal Sheffer, Miles H. Linde, Nghi Ly, Maria Caterina Rotiroti, Benjamin A. H. Smith, Marius Wernig, Carolyn R. Bertozzi, Michelle Monje, Constantine Mitsiades, Ravindra Majeti, Ansuman T. Satpathy, Kimberly Stegmaier, Robbie G. Majzner. Lineage plasticity dictates responsiveness to anti-GD2 therapy in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR003.

Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19− or CD19lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial (NCT03233854) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19−/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22−/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency., Bispecific CAR T cells targeting CD19 and CD22 exhibit clinical activity and low toxicity in patients with large B cell lymphoma and B cell acute lymphoblastic leukemia, with relapses associated with loss of CD19 but not CD22.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity invitro and invivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Acute Myeloid Leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients. Adoptive cell therapy by chimeric antigen receptor (CAR) T cells demonstrated a high therapeutic potential in B-acute lymphoblastic leukemia, but translation in AML is limited by the absence of an ideal tumor-specific antigen. CD123 (known as IL-3 receptor alfa) and CD33 satisfy several features of ideal target antigens as they are expressed in almost all AML patients (mainly overexpressed in NPM1 and FLT-3 mutated AML), and conserved at disease relapse . Despite that, CD33 expression on hemopoietic stem/progenitor cells (HSPC) induced prolonged myelotoxicity after anti-CD33 CAR-T cell therapy and CD123 expression on endothelium was responsible for severe capillary leak syndrome after anti-CD123 CAR therapy. With the aim to improve selectivity for CD123+/CD33+ AML cells while minimizing toxicity against healthy cells, we probe a dual targeting model by Cytokine Induced Killer (CIK) cells co-expressing a first generation low affinity anti-CD123 IL-3 zetakine (IL3z.CAR) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR). This trans-signaling strategy could allow: 1) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; 2) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; 3) full CAR-CIK activation only against double expressing CD123 + / CD33 + leukemic cells. Fresh and frozen peripheral blood mononuclear cells were transduced with retroviral vectors during the CIK cell differentiation process . The functional activity of CAR-CIK cells was assessed in vitro by means of short- and long- term cytotoxicity and cytokine production assays upon challenge with different CD123/CD33 positive AML cell lines (THP-1 and KG-1) or with a CD123 positive human endothelial cell line (TIME). To assess the myelotoxicity against HSPC, CD34+ cells were sorted after 24h co-culture with CAR-CIK cells and mixed with methylcellulose-based semisolid medium to evaluate Colony Forming Units (CFU) after 14 days through an automated imaging and counting system for hematopoietic colonies (Stemvision). The in vivo anti-leukemic activity was evaluated by monitoring luciferase-expressing KG1 AML cell line growth over time in NSG mice treated with 3 infusions of all different CAR-CIK conditions. Dual CAR-CIK cells (IL-3z.CAR/CD33.CCR) display a potent and specific in vitro anti-leukemic efficacy against all the AML cell lines tested, compared to single targeting IL-3z.CAR and non-transduced CIK cells. To further minimize toxicities against healthy cells exploiting differences in CD123 antigen density between leukemic and healthy cells, we generated a low affinity dual CAR decreasing the IL3z.CAR binding affinity to CD123, by site-directed mutagenesis. Low affinity dual CAR-CIK cells show irrelevant cytotoxicity against the TIME endothelial cell line, comparable to non-transduced CIK cells, while preserving the efficacy against THP1 and KG1 AML cell lines. As a confirm of the reduced reactivity against endothelial cells, low affinity dual CAR-CIK cells produce the same levels of IL-2 and IFNγ either alone or in the presence of TIME cell line. Low affinity dual CAR-CIK cells also preserve the in vitro CFU-E, CFU-GM and CFU-GEMM colony forming capacity as compared to wild type (wt) dual CAR-CIK and wt IL-3z.CAR-CIK cells, highlighting the possibility to find a therapeutic window to minimize toxicity on healthy cells. The anti-leukemic activity of low affinity dual CAR-CIK cells has been confirmed also in vivo, with a significant suppression of leukemic growth against KG1 AML cell line. Mice treated with 3 infusions of low affinity dual CAR-CIK cells exhibited a significant better anti-leukemic profile, when compared to IL-3-single targeting CAR-CIK cells in terms of tumor growth and overall survival. These preclinical data demonstrate a powerful antitumor efficacy mediated by low affinity dual targeting IL-3z.CAR/CD33.CCR CIK cells against AML targets without any relevant in vitro toxicity on HSPC and endothelial cells, offering a proof-of-concept strategy to increase selectivity for CD123+/CD33+ AML cells whilst reducing the risk of "on-target off-tumor toxicity". Disclosures Perriello: Novartis: Other: Advisory Board. Martelli: Abbvie, Amgen, Celgene, Janssen, Novartis, Pfizer, Jazz Pharmaceuticals: Honoraria. Falini: Rasna Therapeutics: Honoraria. Biondi: Novartis: Honoraria; Bluebird: Other: Advisory Board; Incyte: Consultancy, Other: Advisory Board; Amgen: Honoraria; Colmmune: Honoraria.

Chimeric antigen receptor (CAR)-redirected T lymphocytes are a promising immunotherapeutic approach and object of pre-clinical evaluation for the treatment of acute myeloid leukemia (AML). We developed a CAR against CD123, overexpressed on AML blasts and leukemic stem cells. However, potential recognition of low CD123-positive healthy tissues, through the on-target, off-tumor effect, limits safe clinical employment of CAR-redirected T cells. Therefore, we evaluated the effect of context-dependent variables capable of modulating CAR T cell functional profiles, such as CAR binding affinity, CAR expression, and target antigen density. Computational structural biology tools allowed for the design of rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity without affecting the overall CAR design. We defined both lytic and activation antigen thresholds, with early cytotoxic activity unaffected by either CAR expression or CAR affinity tuning but later effector functions impaired by low CAR expression. Moreover, the anti-CD123 CAR safety profile was confirmed by lowering CAR binding affinity, corroborating CD123 is a good therapeutic target antigen. Overall, full dissection of these variables offers suitable anti-CD123 CAR design optimization for the treatment of AML.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric high-risk patients, thus demanding advanced and personalized therapies. In this regard, different targeted immunotherapeutic approaches are available, ranging from naked monoclonal antibodies (mAb) to conjugated and multifunctional mAbs (i.e., BiTEs and DARTs). Recently, researchers have focused their attention on novel techniques of genetic manipulation specifically to redirect cytotoxic T cells endowed with chimeric antigen receptors (CARs) toward selected tumor associated antigens. So far, CAR T cells targeting the CD19 antigen expressed by B-cell origin hematological cancers have gained impressive clinical results, leading to the possibility of translating the CAR platform to treat other hematological malignancies such as AML. However, one of the main concerns in the field of AML CAR immunotherapy is the identification of an ideal target cell surface antigen, being highly expressed on tumor cells but minimally present on healthy tissues, together with the design of an anti-AML CAR appropriately balancing efficacy and safety profiles. The current review focuses mainly on AML target antigens and the related immunotherapeutic approaches developed so far, deeply dissecting methods of CAR T cell safety improvements, when designing novel CARs approaching human studies.

Background Chimeric Antigen Receptor (CAR)-T cell therapy has been successfully clinically deployed in the context of B-cell malignancies, paving the way for further development also in Acute Myeloid Leukemia (AML), a still unmet clinical need in the field of oncohematology. Among the potential AML targetable antigens, CD33 is so far one of the main validated molecule. Objectives The aim of the present study was to optimize a non-viral gene transfer method to engineer Cytokine-Induced Killer (CIK) cells with a CD33.CAR by using a novel version of the Sleeping Beauty (SB) transposon system, named "SB100X-pT4". Further, a preclinical assessment of SB-modified CD33.CAR-CIK cells was performed in chemoresistant AML Patient-Derived Xenografts (PDX), in order to address the unmet need of targeting drug-resistant AML cells. Methods Donor derived-CIK cells were stably transduced with a CD33.CAR by exploiting the novel hyperactive SB100X transposase and the pT4 transposon (SB100X-pT4). The novel SB system has been in vitro compared to the previous established SB11-pT. In vitro anti-AML activity of CD33.CAR-CIK cells was assessed by flow cytometry-based cytotoxicity (AnnV-7AAD), proliferation (CFSE) and cytokine production (intracellular IFNg and IL2 detection) assays. In vivo efficacy was evaluated in NSG mice transplanted with MA9-NRas AML cell line or PDX samples. A xenograft chemotherapy model mimicking induction therapy ("5+3" Ara-C and doxorubicin) was exploited to examine the potential benefit of CD33.CAR-CIK cells on chemoresistant/residual AML cells. Results By significantly reducing the amount of DNA transposase, the novel SB100X-pT4 combination resulted in higher CAR levels than the SB11-pT. SB100X-pT4-modified CD33.CAR CIK cells showed efficient expansion after 3 weeks (median fold increase of 38.89, n=4). Both transpositions conferred to CD33.CAR-CIK cells a specific killing (up to 70%) against CD33+ AML target cell lines and primary AML cells. The anti-AML proliferative response of SB-modified CD33.CAR-CIK cells was also considerable (up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% for IFN-γ and up to 25% for IL-2). To evaluate the effect of SB100X-pT4-modified CD33.CAR-CIK cells particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an "early treatment" in mice transplanted with the MA9-NRas cell line, which retains a high frequency of LICs. At sacrifice, CD33.CAR-CIK cell-treated mice showed a significant bone marrow (BM) engraftment reduction (median 27.80 for the untreated group and 22.60 for the unmanipulated CIK cells vs 6.45 for CD33.CAR-CIK cell, n=4 NSG mice per group, p= 0.02). PDX of two different AML samples at the onset were established to be used as models mimicking different disease conditions. In an "early treatment" model using secondary transplanted PDX, a setting which presumably reflects the typical LIC properties, a clear engraftment reduction in the treated cohort was observed, nearly undetectable in 2/5 mice, as compared to the untreated mice (up to 70% in BM). A significant leukemia reduction was also measured in the peripheral blood and spleen of treated mice, showing CD33.CAR-CIK cell potential of reducing AML dissemination in the periphery. When ex vivo re-exposed to CD33.CAR-CIK cells residual AML cells were still sensitive to the treatment, indicating that no resistance mechanisms occurred. CD33.CAR-CIK cells were also effective in a second model by which the treatment started when AML engraftment was clearly manifested in the BM (> 1%). Finally, when starting CD33.CAR-CIK cell treatment after disease recurrence post induction therapy, a significant disease reduction was observed in the CD33.CAR-CIK-treated group, reaching undetectable levels in half of the mice, as compared to chemotherapy-only treated mice (up to 60% of engraftment in BM)(Figure 1). Conclusions The employment of a non-viral SB-based CD33.CAR-gene transfer approach, which is overall associated to less cumbersome protocols and reduces the cost of goods, offers a unique alternative to current viral-based strategies to be explored in the setting of resistant forms of AML. Disclosures No relevant conflicts of interest to declare.

Background Acute Myeloid Leukemia (AML) is still associated with high relapse rates when treated with conventional chemotherapeutic and hematopoietic transplantation regimens. Thus, new treatment options are urgently needed. Immunotherapy adopting T cells engineered to express tumor-directed Chimeric Antigen Receptors (CARs) has shown striking results particularly in the context of B-cell malignancies, sparking a keen interest in extending this approach also to other hematological malignancies such as AML. Among the surface molecules identified, the CD33 molecule represents so far one of the main validated target in AML and, being broadly expressed on AML blasts, represents a suitable antigen to be targeted with CAR-T cells. Objectives The aim of the present study is to preclinically evaluate the efficacy and safety profiles of CD33.CAR redirected Cytokine Induced Killer (CIK) cells alone and in combination with standard chemotherapeutic agents. Methods Donor derived- and autologous-CIK cells were stably or transiently transduced with a third generation anti-CD33.CAR by Sleeping Beauty transposon- or mRNA-mediated engineering. In vitro anti-AML activity has been assessed by means of Flow cytometry-based cytotoxicity (AnnV-7AAD staining), proliferation (Ki67 staining and CFSE dilution) and cytokine production (intracellular IFNg and IL2 detection) assays, upon challenge with AML samples. In vivo efficacy has been evaluated in NSG mice transplanted with MA9-NRas AML cell line or primary AML samples. Moreover, an already established xenograft chemotherapy model has been exploited to examine the potential benefit of combining CD33.CAR-CIK cells with standard AML induction therapy (Ara-C and doxorubicin). Results CD33.CAR stably expressing CIK cells were able to induce a potent anti-leukemic activity in vitro, in terms of specific killing either in short term (>70% at 4h, E:T ratio 5:1) and long term cytotoxic assays (>90% at 1 week, E:T ratio 1:10), with statistically significant differences as compared to the unmanipulated condition. Moreover, CD33.CAR-CIK cells were able to retain a significant cytotoxic activity when re-challenged with the CD33+ target following a previous stimulation (up to 65%). The proliferative response to AML target cells was also considerable and CAR-specific (up to 60% of Ki67+CAR-CIK cells and up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% of IFN-γ producing CAR-CIK cells and up to 25% of IL-2 producing CAR-CIK cells). CIK cells transiently expressing the CD33.CAR were also effective towards the AML target. In vivo results showed that CD33.CAR-CIK cells were able to control the disease in MA9 grafted mice in all the districts analyzed (peripheral blood, bone marrow, spleen, liver and kidney), as compared to untreated mice. To evaluate the effect of CD33.CAR-CIK cell immunotherapy particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an early treatment approach, treating mice 5 days after i.v. injection of a secondary transplanted PDX sample. We observed a clear engraftment reduction in the treated cohort, nearly undetectable in 2 out 5 mice, while a high leukemic burden has been detected in untreated mice (up to 70% of engraftment in bone marrow). Furthermore, by exploiting CD33.CAR-CIK cell treatment in mice experiencing disease recurrence after the "5+3" chemotherapy-induction protocol, preliminary data showed that CD33.CAR-CIK cells were also capable to target chemotherapy resistant/residual AML cells. Conclusions Considering our in vivo preliminary results, we aim to further evaluate CD33.CAR-CIK cell immunotherapy efficacy, particularly against chemotherapy resistant/residual AML cells. Concerning the safety aspect, since the CD33 targeting raises concerns for a potential myelotoxicity, we will assess the potential long-term off-target effects of CD33.CAR-CIK cells (comparing stably with transiently expressed CD33.CARs) on normal hematopoietic stem/myeloid progenitor cells. Disclosures No relevant conflicts of interest to declare.

Chimeric Antigen Receptors (CARs)-redirected T lymphocytes are a promising novel immunotherapeutic approach, nowadays object of accurate preclinical evaluation also for the treatment of Acute Myeloid Leukemia (AML). In this context, we recently developed a CAR against CD123 (IL-3 receptor α subunit), a tumor associated antigen over-expressed on AML blasts and leukemic stem cells. However, the potential recognition by anti-CD123 CAR-T cells of low CD123 positive healthy tissues, i.e. endothelium and monocytes, through the known "on-target-off-organ" effect, warrants careful preclinical investigation for a safe employment of this approach in the clinic. Therefore, in search for an optimization of this strategy, we evaluated the effect of several variables implicated in the CAR design, known to modulate CAR T-cell functional profiles in a context-dependent manner, such as CAR binding affinity, CAR expression and target antigen density. Computational structural biology tools have been exploited to design rational mutations in the anti-CD123 CAR antigen binding domain that altered CAR expression and CAR binding affinity. To this aim, Cytokine-Induced Killer (CIK) effector cells have been genetically modified with four Chimeric Affinity Mutants (CAMs), represented by CAM-H1 (High Affinity 1), CAM-H2, which maintain the same binding affinity of the wild type (wt) anti-CD123 CAR (10-9 M), CAM-M (Medium affinity) and CAM-L (Low affinity), displaying a 10- and 100-fold affinity reduction, respectively. The functional characterization of CAM-CIK cells revealed both their specificity and effectiveness in early cytotoxic responses against both the high CD123+ THP-1 AML cell line and primary AML cells, reaching more than 70% of killing activity as the wt CAR-CIK cells. However, in this context, among all the variables analyzed, a CAR expression level up to ≈40% proved to be necessary for inducing effective later effector functions towards a high CD123+target, such as proliferation and cytokine production. To mimic the CD123 expression on leukemic and healthy tissues, a panel of cell lines with different surface levels of CD123 was introduced. In particular, a number of ≈1600 CD123 molecules was sufficient to induce a good cytotoxic response by all the CARs tested, with the CAM-L being the less powerful against both the AML U937 cell line and healthy TIME endothelial cell line, sharing the same target antigen density, and allowing for the identification of ≈1600 CD123 molecules as the "lytic threshold" in our setting. At the same time, this antigen density was not enough to determine a good proliferative capability for all the CAR-CIK cells tested, which instead occurred with leukemic cells expressing ≈5000 or more CD123 molecules. Cytokine release induced by all CAM-CIK cells was instead directly proportional to target antigen density, starting from ≈1600 molecules/cell, indicating that both these "activation thresholds" depend on different target antigen densities for being respectively triggered. As a matter of fact, a proportional CAR down-modulation in response to increasing target antigen densities was observed, implying this mechanism as one of the principal determinant for CAR-CIK cell related functional outcomes, independently of CAR binding affinity. Furthermore, with the aim of evaluating the response of CAM-CIK cells against the endothelium in a more physiological context, we set up a co-culture of Matrigel-embedded endothelial cells and CIK cells. The same and limited impairment of vessel formation was observed, with no differences encountered between un-manipulated and CAR-redirected CIK cells, implying that anti-CD123 CAR CIK cells are not activated more than the un-manipulated counterpart towards this target, independently of CAR binding affinity. In conclusion, we were able to define both "lytic" and "activation" antigen thresholds (Figure 1), showing that whereas the early T-cell cytotoxic activity is not affected either by CAR expression or CAR affinity tuning, later effector functions are impaired by low CAR expression. Moreover, a promising balance in the efficacy and safety profiles of CAR-CIK cells was observed in the lowest affinity mutant in response to targets with low CD123 densities. Overall, the full dissection of all these variables offers additional knowledge for the proper design of a suitable anti-CD123 CAR for the treatment of AML. Disclosures Biondi: Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

In the last years, adoptive cellular immunotherapy employing T lymphocytes genetically modified with Chimeric Antigen Receptors (CARs) has demonstrated impressive clinical results, particularly in the treatment of acute/chronic lymphoblastic leukemia and B-cell lymphoma, paving the way towards the possibility to translate this approach also to other hematological malignancies, such as Acute Myeloid Leukemia (AML). In the AML context, the CD123 antigen (IL-3 receptor alpha subunit) represents a good target antigen, being a poor prognosis over-expressed marker on AML cells and leukemic stem cells (LSCs), a rare population that plays a key role in perpetuating leukemia. However, CD123 is also expressed on the surface of healthy cells such as monocytes and endothelial cells, although at lower levels as compared to leukemic cells. The potential recognition of low antigen positive healthy tissues by CAR-redirected T cells, through the so called "on-target-off-organ" effect, limits a safe clinical employment of this immunotherapeutic approach. CARs are artificial receptors generated by joining the cytoplasmic TCR (T Cell Receptor) signaling modules to the heavy and light chain variable regions of a monoclonal antibody, whose affinity toward a target antigen is a variable capable of influencing the CAR-mediated functional responses. Therefore, in our study we investigated how the CAR affinity variable in the context of CD123 targeting, together with the CAR and CD123 target antigen density, could impact anti-CD123. CAR-redirected effector cells efficacy against leukemic cells and safety towards the healthy cells. To this aim, Cytokine-Induced Killer (CIK) effector cells have been genetically modified with four Chimeric Affinity Mutants (CAMs), CAM-1, CAM-2, CAM-3 and CAM-4, identified by means of a computational docking technique. In vitro cytotoxic assays, cytokine production and proliferation experiments have been performed in order to evaluate both the efficacy and safety profile of the CAR-redirected CIK cells, using un-manipulated CIK cells (NO DNA) and wild-type anti-CD123.CAR condition as controls. The functional characterization of all the CAMs revealed both the specificity and the effectiveness of CIK-CAR+ cells against the CD123+ THP-1 cell line and primary AML cells. However we observed that, at least in the context of CD123 targeting, a good CAR expression level is necessary for inducing effective later functions, such as proliferation and cytokine production, towards a high CD123+ target. When introducing leukemic cell lines with different CD123 density on their surface we observed different effector properties minimally influenced by the CAR affinity. In particular, in terms of killing activity, we noticed that a number of ≈1600 CD123 molecules is sufficient to induce a good cytotoxic response of all the CARs tested, with the CAM-2 (2-magnitude log lower in affinity) being less powerful. At the same time, this antigen density is not enough to determine a good proliferative capability which instead occurs with leukemic target cells expressing 5000 or more CD123 molecules. Considering the cytokine production (IL-2 and IFN-gamma), we observed that all CIK-CAR+ cells showed a cytokine release that is directly proportional to the target antigen density, with CAM-2 showing a reduced response towards low-CD123 expressing leukemic targets. When analyzing the safety profile of the CAMs against low-CD123+ endothelial target cells (≈1600 molecule/cell), we observed a lower functional activity of the CAMs as compared to the leukemic cell lines expressing the same level of CD123 molecules on their surface, with the low-affinity CAM-2 showing a major sparing capability in terms of killing activity (being the only one not statistically different from NO DNA). In conclusion, exploiting our model of affinity mutants we were able to in vitro characterize the role of the CAR density balanced with the affinity of the anti-CD123.CAR towards AML cells expressing different CD123 levels and CD123-low expressing normal tissues. In particular, the results obtained with CAM-2 suggested a potential threshold of affinity below which, even if the safety profile is preserved, the anti-leukemic efficacy would be impaired. Disclosures No relevant conflicts of interest to declare.