C-type Lectin-like Molecule-1 Research Articles

Developing “Off-the-Shelf” CLL1 CAR-DNT Therapeutics for the R/R Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is one of the most common and highly heterogeneous hematological malignancy. The prognosis is poor especially for the elderly patients. For Relapsed/Refractory(R/R) AML patients, the one-year overall survival rate is merely 20%. CAR-T cells have shown tremendous therapeutic benefits for ALL, DLBCL and MM. One of the limitations of current CAR-T therapies is the use of autologous T cells. This can be limiting due to the cost, complicated manufacturing process, rapid disease progression, and manufacturing failures etc. Therefore, there is a need to develop the ready-to-use allogeneic cell product. Double negative T (DNT) cells are the ideal candidate for AML cell therapy as they naturally kill AML cells, and have been successfully administered to AML patients with therapeutic benefit without causing GvHD. Relying on their own natural immune activated receptors, DNT cells could lyse a wide range of tumors independent of TAA expression, which have more advantages compared to the conventional CAR-T cells. In our study, we developed the allogeneic product of CAR-DNT cells targeting C-type lectin-like molecule-1 (CLL1), which is a promising target due to its over-expression in primary AML cells and leukemia stem cells, while absence on hematopoietic stem/progenitor cells. Methods: We developed a humanized single chain variable fragment (scFv) with specific recognition of human CLL1 antigen by screening from 62 murine-derived CLL1 antibodies. Then, we designed a second-generation CLL1 CAR structure containing 4-1BB-CD3ζ and transduced it into the DNT cells by lentivirus. To clarify the GvH and HvG reactions of DNT cells, we performed the mixed lymphocyte reaction experiments in vitro and compared DNT cells with TCR and B2M double knockout T(DKO) cells and TCR knockout T(TKO) cells. We evaluated the anti-tumor function of CLL1 CAR-DNT cells through killing experiments by different effector-to-target ratios and multiple stimulations with AML cell lines (THP1 and HL60). Furthermore, we speculated that the CAR structure plays a critical role in both tumoricidal function and persistence of CAR-DNT cells. We designed nearly 20 enhanced CAR structures based on the signaling pathway of DNT cells. These newly designed structures have been tested in vitro and will be confirmed in vivo. Results: The humanized anti-CLL1 scFv candidate H1-3Q2 demonstrated high specificity toward human CLL1 antigen at the Membrane Protein Array. H1-3Q2 based CAR-T cells showed good tumor suppression in vivo. However, due to the heterogenous expression of CLL1 in AML, CLL1 CAR-T cells may not be able to eliminate AML cancer cells completely. While DNT cells could naturally lyse tumor cells through the receptors like NKG2D and DNAM-1, providing them with lots of advantages. Moreover, DNT cells have the potential to be used as allogenic cell therapy product. Co-culturing allogenic PBMCs or umbilical cord blood with DNT cells for up to 72 hours resulted in little or no killing reaction, indicated a low risk of GvHD. Conversely, the PBMCs from three different donors were used to attack allogenic DNT cells or DKO/TKO T cells in vitro. The killing effect on DKO/TKO T cells gradually increased over time, while no effect on DNT cells was observed even after 192 hours. This suggested that DNT cells are not easily cleared by host immune cells. DNT cells could efficiently lyse various hematological and solid tumor cell lines, particularly showing high sensitivity towards AML cells. The introduction of the CAR moiety further enhanced the specificity of DNT cells. The in vitro killing results demonstrated that CLL1 CAR-DNT cells exhibited stronger and faster AML resistance compared with CAR-T cells, and even eliminated tumor cells that have lost TAA. However, CLL1 CAR-DNT cells showed limited proliferation in the multiple-stimulation assays. To address this, we designed enhanced CAR structures. Screening by multiple-challenge assays, the fifth-generation CAR exhibited outstanding proliferation and superior tumor lysis ability in vitro. Conclusions: Our findings demonstrate that CAR-DNT product is an ideal candidate for allogeneic adoptive immunotherapy and exhibits superior tumoricidal function. This study underscores the potential of the “off-the-shelf” CLL1 CAR-DNT cell product for treating R/R AML.

Tandem bispecific CD123/CLL-1 CAR-T cells exhibit specific cytolytic effector functions against human acute myeloid leukaemia.

The treatment of refractory and recurrent acute myeloid leukaemia (AML) is still a challenge with poor response rates and short survival times. In an attempt to solve this problem, we constructed a tandem bispecific chimeric antigen receptor (CAR) targeting CD123 and C-type lectin-like molecule 1 (CLL-1), two different AML antigens, and verified its cytotoxic effects in vitro. We established and cultured K562 cell lines expressing both CD123 and CLL1 antigens. Single-target CAR-T cells specific to CD123 and CLL1 were engineered, alongside tandem CD123/CLL1 bispecific CAR-T cells. Flow cytometry was used to determine cell phenotypes, transfection efficiencies, cytokine release, and CAR-T-cell proliferation, and an lactate dehydrogenase assay was used to detect the cytotoxicity of CD123/CLL-1 bispecific tandem CAR-T cells in vitro. Two types of tandem CAR-T cells exhibited significant killing effects on CLL-1 + CD123+ leukaemia cell lines and primary AML tumour cells. The killing efficiency of tandem CAR-T cells in the case of single antigen expression is comparable to that of single target CAR-T cells. When faced with dual target tumour cells, dual target CAR-T cells significantly surpass single target CAR-T cells. CD123/CLL-1 CAR-T cells in tandem targeted and killed CD123- and CLL-1-positive leukaemia cell lines and released a large number of cytokines. CD123/CLL-1 CAR-T cells in tandem can simultaneously target CD123 and CLL-1 on AML cells, demonstrating a significant ability to kill single antigens and multi-target tumour cells. This suggests that CD123/CLL-1 CAR-T cells exhibit significant advantages in the expression of multiple antigens in a wide range of target cells, which may help overcome the challenges posed by tumour heterogeneity and evasion mechanisms.

Preclinical development of ARD103, a CLL-1 CAR-T produced by a 3-day DashCAR technology, for acute myeloid leukemia.

119 Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of the AML patients experienced primary refractory or relapse after current treatments, such as chemotherapy, hematopoietic stem cell transplantation, and targeted therapy. AML patients are in urgent need of novel therapeutic strategies. Leukemic stem cells (LSCs), which cause the treatment failure and leukemia relapsed. Human C-type lectin-like molecule-1 (CLL-1) is a surface antigen expressed on both AML malignant blast cells and LSCs, but not on normal hematopoietic stem cells, suggesting that CLL-1 is a compelling target with low off-tumor toxicity for novel AML treatment. Methods: ARD103 is a second-generation chimeric antigen receptor (CAR)-T cell, expressing a novel extracellular anti-CLL-1 scFv fused to a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ activation domain. ARD103 was generated using lentiviral vector in an optimized 3-day DashCAR manufacturing process and demonstrated high percentage of CAR population (>80%), high percentages of memory T cells (Tscm and Tcm are >70%) and profound effector cytokine production (IFN-γ, granzyme A, perforin and granulysin). Results: ARD103, CLL-1 CAR-T exhibited specific killing on various CLL-1-expressing AML cell lines, such as U937, THP-1, and HL60, correlated to the levels of CLL-1 expression. In the AML U937-Luc xenograft mouse model, the CLL-1 CAR-T cells prolonged the survival time significantly (p < 0.0001), with superior CAR-T cell persistence, to the end point of the study at 48 days compared to the median survival time at 23 days in the untreated or control groups. Furthermore, the xenograft mice receiving as minimum as 3 x105 CAR-T cells per mouse were able to elicit effective recall responses after tumor rechallenge and survived for more than 60 days. The off-target binding interactions of ARD103 CAR-T cells using a human cell microarray showed a specific and strong interaction with CLL-1 but not with other human membrane/extracellular proteins. In the aspect of on-target off-tumor toxicity, the hematotoxicity assessment on ARD103 exerted modest (5%-23%) or no effect on colony formation inhibition of CD34+ cells derived from human normal bone marrow or peripheral blood, respectively. Furthermore, autologous ARD103 cells were successfully prepared from relapsed/refractoryAML patients and demonstrated to mediate specific cytotoxicity to primary AML blasts and LSCs derived from the same patients. Conclusions: Taken together, the ARD103 CLL-1 CAR-T candidate has demonstrated excellent anti-AML potency and efficacy with minimal safety concerns, and warrant to be further developed preclinically and clinically for the treatment of relapsed/refractory AML patients. Keywords: Acute myeloid leukemia; CLL-1; Chimeric antigen receptor-T cell; Immunotherapy.

Anti-CLL1-based CAR T-cells with 4-1-BB or CD28/CD27 stimulatory domains in treating childhood refractory/relapsed acute myeloid leukemia.

Though the efficacy of anti C-type lectin-like molecule-1 (CLL1) CAR T-cells in refractory/relapsed acute myeloid leukemia (R/R-AML) have been occasionally reported, the influence of co-stimulatory domain CAR T-cells is not investigated so far. Seven R/R-AML children treated with anti-CLL1 CAR T-cells were enrolled onto this preliminary comparison study. Among these seven patients, four received CD28/CD27-based CAR T-cells therapy, and three received 4-1BB-based CAR T-cells therapy. The overall response rates were 75% and 66.7% in CD28/CD27 and 4-1BB group respectively. All patients experienced grade 1 to 2 cytokine release syndrome, with only one patient experiencing grade 2 immune effector cell-associated neurotoxicity syndrome. The maximum CAR T-cells durations were 156 and 274 days for CD28/CD27 group and 4-1BB group respectively. The 1-yr overall survival rate was 57.1%. A preliminary similar efficacy/safety index was observed in anti-CLL1-based CAR T-cells with 4-1BB or CD28/CD27 co-stimulatory elements in treating pediatric R/R-AML.

Characteristics of anti-CLL1 based CAR-T therapy for children with relapsed or refractory acute myeloid leukemia: the multi-center efficacy and safety interim analysis.

C-type lectin-like molecule-1 (CLL1) is preferentially expressed on acute myeloid leukemia (AML) stem cells and AML blasts, which can be considered as AML-associated antigen. Anti-CLL1-based CAR-T cells exhibited effective tumor-killing capacity in vitro and in AML-bearing mouse model. In this report, eight children with relapsed or refractory AML (R/R-AML) were recruited for a phase 1/2 clinical trial of autologous anti-CLL1 CAR-T cell immunotherapy. The objectives of this clinical trial were to evaluate the safety and the preliminary efficacy of anti-CLL1 CAR-T cell treatment. Patients received one dose of autologous anti-CLL1 CAR-T cells after lymphodepletion conditioning. After CAR-T treatment, patients developed grade 1-2 cytokine release syndrome (CRS) but without any lethal events. 4 out of 8 patients achieved morphologic leukemia-free state (MLFS) and minimal residual disease (MRD) negativity, 1 patient with MLFS and MRD positivity, 1 patient achieved complete remission with incomplete hematologic recovery (CRi) but MRD positivity, 1 patient with partial remission (PR), and 1 patient remained at stable disease (SD) status but had CLL1-positive AML blast clearance. These results suggested that anti-CLL1-based CAR-T cell immunotherapy can be considered as a well-tolerated and effective option for treating children with R/R-AML.

622MO An asymmetrical CLL1/CD3 bispecific antibody, ABL602, exhibits CLL1 binding-dependent CD3 binding/activation and antitumor activity in acute myeloid leukemia (AML) mouse model and leukemia blasts from AML patients

C-type lectin-like molecule 1 (CLL1) is highly expressed in acute myeloid leukemia blast cells but not in normal hematopoietic stem cells (HSCs). This limited expression of CLL1 makes it as a promising target for the AML therapy compared with other targets such as CD33 and CD123, which are also expressed in HSCs. Previously we have shown potent CLL1-specific antitumor activity using two AML cell line/models. In this study, we determined whether binding affinity of ABL602 to CD3 can be augmented upon binding to CLL1 and expanded our study to various AML cell lines and blasts from AML patients. To determine CLL1 binding-dependent CD3 binding of ABL602, T cell binding was measured by flow cytometry in the presence of CLL1-spiked beads. ABL602-mediated antitumor activity and T cell activation were tested in CLL1 expressing AML cell lines and AML blasts from patients. In vivo antitumor activity was determined in humanized mice bearing AML tumors. ABL602 (2+1), with its CD3 binding arm sterically hindered by CLL1 binding arm, binds to CD3 with a lower affinity compared with 1+1 format in the absence of CLL1-expressing cells. By incubating T cells with beads spiked with various amounts of CLL1 in the presence of ABL602, we proved that ABL602’s binding affinity to CD3 was increased in a CLL1-dependent manner, reaching comparable level to that of 1+1. ABL602 induced superior tumor cell killing than 1+1 reference antibody JNJ-67571244. Moreover, ABL602 showed a dose-dependent anti-tumor activity with tumor regression in OCIAML2 xenograft and HL-60 orthotopic model. In AML patient blasts, CLL1 was highly expressed and ABL602 exhibited a strong T cell activation and tumor killing activity on CLL1+ AML blasts. This study supports the target-specific T cell activation and tumor killing activity of 2+1 heterodimeric structure of ABL602 on both AML cell lines and patient-originated AML blasts and warrants further pre-clinical development.

A Novel Asymmetrical Anti-CLL-1×CD3 Bispecific Antibody, ABL602, Induces Potent CLL1-Specific Antitumor Activity with Minimized Sensitization of Pro-Inflammatory Cytokines

Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Expression of C-type lectin-like molecule-1 (CLL-1; also known as CLEC12A, c-type lectin domain family 12 member A) is mainly restricted to LSCs but absent in normal hematopoietic stem cells (HSCs), suggesting CLL-1 as an excellent therapeutic target for AML. This unique expression pattern paves the way to develop therapies that potentially eliminate CLL1-positive LSC while preserving CLL1-negative HSC.To re-direct T cells to AML cells, we generated IgG-based asymmetric (2+1, ABL602) bispecific antibody (BsAb) targeting CLL-1 and CD3. As a 2+1 format BsAb, ABL602 has bivalent binding to CLL-1 for target arm and monovalent binding to CD3. ABL602 exhibited higher binding activity to CLL-1-expressing AML cell lines and greater tumor-killing efficacy than 1+1 format BsAb and benchmark antibody MCLA-117 (Merus; CLEC12AxCD3 bispecific antibody). ABL602 induced potent cytotoxic activities on CLL1-expressing AML cell lines (EC 50 of 0.04~3.05pM and 0.97~16.64pM for U937 and HL-60, respectively) with concomitant T cell activation (EC 50 of 0.10~3.54pM and 0.94~4.92pM for U937 and HL-60, respectively) and cytokine/granzyme B release. Despite strong tumor-killing activity, ABL602 did not kill CLL1-negative cancer cell lines, suggesting that ABL602 induces CLL-1-dependent cytotoxicity. Moreover, ABL602 did not or minimally induce TNF-α and IL-6 in PBMC in the absence of AML cell lines, while MCLA-117 triggered high level of expression of those cytokines. In established orthotopic AML mouse model using HL-60 Luc, ABL602 demonstrated statistically significant anti-tumor activity in a dose-dependent manner. Proportions of bone marrow CD33 + AML blasts diminished in a dose-dependent manner, while CD3 + T cells more infiltrated to the bone marrow.Overall, our results indicate that ABL602, appropriately engineered 2+1 asymmetric BsAb, promotes T-cell activity specifically against CLL1-expressing AML cells and is a promising treatment strategy for AML patients by achieving the desired balance between antitumor activity and safety. DisclosuresNo relevant conflicts of interest to declare.

Tc Buster Transposon Engineered CLL-1 CAR-NK Cells Efficiently Target Acute Myeloid Leukemia

Introduction:DNA transposons are efficient, integrating, non-viral vector systems which have been applied to clinical scale CAR-T cell production, aiming to reduce cost and deliver larger genetic cargos relative to viral transduction. CAR-NK cells are a promising cell therapy platform, with a potential for ‘off-the-shelf’, allogeneic application. Inefficient delivery of plasmid DNA and substantial associated toxicity have limited the utility of DNA transposon systems in primary NK cell engineering. Here we describe an efficient process for the production of transposon engineered primary CAR-NK cells, and the in vitro activity of a CAR-NK cell product targeting human C-type lectin-like molecule-1 (CLL-1/ C-type lectin domain family 12 member A, CLEC12A) for acute myeloid leukemia (AML) produced using the Tc Buster (TcB) transposon system. Since CLL-1 is frequently expressed by leukemic stem cells (LSC) but absent on hematopoietic stem cells, it represents a rational antigenic target to enhance the innate activity of allogeneic NK cell therapies in AML.Methods:NK cells were isolated from 30ml of healthy donor peripheral blood by negative immunomagnetic selection (NK Cell Isolation Kit, Miltenyi Biotec), then activated and expanded using a good manufacturing practice (GMP) grade, 100 Gy irradiated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) in the presence of IL-2 and IL-21. On day 4, NK cells were electroporated with a nanoplasmid TcB transposon carrying a second generation CLL-1 CAR (CD28/CD3ζ or 41BB/CD3ζ) and hyperactive TcB transposase mRNA (Maxcyte ATx). Following transposition, the NK cells were stimulated with the feeder line for a second time. Purified CAR-NK cell populations were produced by immunomagnetic selection of CAR expressing cells using anti-biotin beads (Miltenyi Biotec) and biotinylated CLL-1 protein (ACRO Biosystems), which was also used to detect CAR expression by flow cytometry. For CRISPR/Cas9 gene knockout (KO), pooled sgRNAs targeting 3 sites within the target gene were complexed with Cas9 protein prior to co-electroporation with the TcB payload. CLL-1 CAR-NK cell function was evaluated in co-culture with AML cell lines or biobanked AML patient samples and analyzed by flow cytometry.Results:This approach (Fig 1A) produced efficient transposition with retained clinical-scale expansion capacity of primary CAR-NK cells with mean CAR expression of 37% (n=3, range 26-46%) at day 21, without selection (Fig 1B). By extrapolation, a mean 4,275-fold expansion was observed by day 21, increasing to 14,023-fold by day 25, sufficient to support many clinical doses of 1x10 7 cells/Kg (Fig 1C). Mean NK cell purity of the final product was 98%. A further increase in the proportion of CAR-NK cells was achieved by immunomagnetic selection, with mean CAR expression increasing to 89.5% (range 84-97%, n=3). A further feeder cell stimulation of these CLL-1 CAR selected cells produced a similar expansion trajectory, 3 days delayed relative to unselected cells (Fig 1C). CLL-1 CAR-NK cell function was confirmed by an enhanced ability to target CLL-1 positive AML cell lines and primary AML blasts relative to control electroporated NK cells (Fig 1 D,E). Increased elimination of a primary AML population enriched for LSC (CD34+, CD38-, CLL-1+) by CLL-1 CAR-NK cells was confirmed (mean 96% vs. 32%, p=0.0002, n=3 AML, n=2 NK donors) (Fig 1E). Preliminary data shows simultaneous KO of the NK cell checkpoint cytokine-inducible SH2 containing protein (CISH) using CRISPR/Cas9 while maintaining efficient transposition (Fig 1F).Conclusions:We describe a novel, non-viral approach to CAR-NK cell production using the TcB DNA transposon system, supported by a clinically validated, GMP grade, irradiated, EBV-LCL with clinical scale expansion capability. Purified CAR-NK cell populations were achieved by immunomagnetic sorting without requiring a selection marker or cytotoxic exposure. Preliminary data supports the ability to simultaneously perform CRISPR/Cas9 gene editing - in this case applied to KO of the CISH gene, an NK cell checkpoint with multiple established benefits, including enhanced in vivo NK cell persistence and improved metabolic health. Manufactured using this process, we also present the first reported pre-clinical activity of a CLL-1 CAR-NK cell therapy in AML, demonstrating enhanced targeting of populations enriched for LSC. [Display omitted] DisclosuresGurney: ONK Therapeutics: Research Funding. O'Reilly: ONK Therapeutics: Research Funding. Corcoran: ONK Therapeutics: Current Employment. Brophy: ONK Therapeutics: Current Employment; Autolus: Ended employment in the past 24 months. Hardwicke: ONK Therapeutics: Current Employment; Novartis: Ended employment in the past 24 months. Hermanson: Bio-Techne: Current Employment. Szegezdi: ONK Therapeutics: Research Funding. O'Dwyer: Janssen: Consultancy; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

Site-specific antibody-drug conjugates with variable drug-to-antibody-ratios for AML therapy

Random conjugations of chemotherapeutics to monoclonal antibodies result in heterogeneous antibody-drug conjugates (ADCs) with suboptimal pharmacological properties. We recently developed a new technology for facile generation of homogeneous ADCs by harnessing human CD38 catalytic domain and its dinucleotide-derived covalent inhibitor, termed ADP-ribosyl cyclase-enabled ADCs (ARC-ADCs). Herein we advance this technology by designing and synthesizing ARC-ADCs with customizable drug-to-antibody ratios (DARs). Through varying numbers and locations of CD38 fused to an antibody targeting human C-type lectin-like molecule-1 (hCLL-1), ARC-ADCs featuring DARs of 2 and 4 were rapidly generated via a single step with cytotoxic monomethyl auristatin F (MMAF) as payloads. In contrast to anti-hCLL-1 ARC-ADC carrying 2 drug molecules, anti-hCLL-1 ARC-ADC with a DAR of 4 shows highly potent activity in killing hCLL-1-positive acute myeloid leukemia (AML) cells both in vitro and in vivo. This work provides novel ADC candidates for combating AML and supports ARC-ADC as a general and versatile approach for producing site-specific ADCs with defined DARs.

Plasma-Derived Exosomes Carrying Leukemia-Associated Antigens Associate with Leukemia Relapse in AML Patients after Chemotherapy

Introduction: Exosomes are the smallest (30-150nm) type of extracellular vesicles that are produced by normal and cancerous cells, including leukemia blasts. Exosomes accumulating in the plasma of patients with cancer carry molecular cargo resembling that of parental tumor cells. Patients newly diagnosed with AML prior to any therapy have significantly higher plasma levels of exosomes (in µg protein/mL plasma) compared to levels in the plasma of normal controls. We previously showed that exosomes in AML patients carry leukemia-associated antigens (LAAs) and hypothesized that exosomes could define disease progression and outcome. Here, we evaluated the molecular cargo of exosomes at AML diagnosis, monitored changes in the exosome cargo during and post therapy, and correlated these changes with the blast content of LAAs detected in leukemia blasts using multi-parameter flow cytometry.Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML and following therapy. Exosomes were isolated from plasma by exclusion chromatography and characterized for protein levels, number and size by qNano, and for morphology by transmission electron microscopy. Exosome cargos, including the presence and level of LAA, were evaluated by Western blots using exosomes isolated from serial plasma specimens obtained during and post chemotherapy. The data were correlated for expression of LAAs.Results: Exosomes isolated from the plasma of 15 patients newly diagnosed with AML carried CD34, CD117, C-type lectin-like molecule-1 (CLL-1), and interleukin-3 receptor subunit alpha (CD123). These antigens were carried by exosomes of all patients, but the antigen levels or profiles were distinct for each patient. The exosome antigen profile detected at AML diagnosis was comparable to that detected in each patient's leukemic blasts by multi-parameter flow cytometry. In AML patients with persistent leukemia following therapy, at the time when blasts were detectable in the bone marrow but not in the peripheral blood, circulating exosomes carried the LAAs. In all patients who achieved complete remission (n=7), at the time when no blasts were detected by morphologic and flow cytometric evaluation of the bone marrow, circulating exosomes carried LAAs with the same profile as that detected at AML diagnosis. All complete remission patients with LAA+ exosomes in the plasma eventually relapsed.Conclusions: We demonstrated, for the first time, that exosomes in patients newly diagnosed with AML have the LAA profiles that are identical to the profiles detected on leukemic blasts by conventional multi-parameter flow cytometry. The persistence of LAA+ exosomes in patients' plasma post-chemotherapy at the time of complete remission suggests the presence of residual leukemia and may be predictive of leukemia relapse. DisclosuresNo relevant conflicts of interest to declare.

Cytotoxic effect of CLL‑1 CAR‑T cell immunotherapy with PD‑1 silencing on relapsed/refractory acute myeloid leukemia.

The activation of chimeric antigen receptor (CAR)-T cells can lead to persistently high levels of programmed cell death 1 (PD-1) antigen and eventually causes the exhaustion of T cells. The effectiveness of CAR-T cells targeting C-type lectin-like molecule-1 (CLL-1) combined with PD-1 silencing therapy for acute myeloid leukemia (AML) was evaluated in the present study. CLL-1 levels in primary AML bone marrow samples was examined using flow cytometric analysis. We designed a CLL-1 CAR-T, containing CLL-1-specific single-chain variable fragment, CD28, OX40, CD8 hinge and TM and CD3-ζ signaling domains. CLL-1 CAR-T with PD-1 silencing was constructed. It was confirmed that CLL-1 is expressed on the surface of AML cells. CLL-1 CAR-T showed specific lysing activity against CLL-1+ AML cells. PD-1 silencing enhanced the killing ability of CLL-1 CAR-T. Furthermore, it was found that CAR-T derived from healthy donor T cells was more effective in killing THP-1 cells (a human acute monocytic leukemia cell line) than those from patient-derived T cells. These results indicated that CLL-1 CAR-T and PD-1 knockdown CLL-1 CAR-T could be used as a potential immunotherapy to treat relapsed or refractory AML.

Ligand Density Controls C-Type Lectin-Like Molecule-1 Receptor-Specific Uptake of Polymer Nanoparticles.

The newest generation of drug delivery systems (DDSs) exploits ligands to mediate specific targeting of cells and/or tissues. However, studies investigating the link between ligand density and nanoparticle (NP) uptake are limited to a small number of ligand-receptor systems. C-type lectin-like molecule-1 (CLL1) is uniquely expressed on myeloid cells, which enables the development of receptors specifically targeting treat various diseases. This study aims to investigate how NPs with different CLL1 targeting peptide density impact cellular uptake. To this end, poly(styrene-alt-maleic anhydride)-b-poly(styrene) NPs are functionalized with cyclized CLL1 binding peptides (cCBP) ranging from 240 ± 12 to 31000 ± 940 peptides per NP. Unexpectedly, the percentage of cells with internalized NPs is decreased for all cCBP-NP designs regardless of ligand density compared to unmodified NPs. Internalization through CLL1 receptor-mediated processes is further investigated without confounding the effects of NP size and surface charge. Interestingly, high density cCBP-NPs (>7000 cCBP per NP) uptake is dominated by CLL1 receptor-mediated processes while low density cCBP-NPs (≈200 cCBP per NP) and untargeted NP occurred through non-specific clathrin and caveolin-mediated endocytosis. Altogether, these studies show that ligand density and uptake mechanism should be carefully investigated for specific ligand-receptor systems for the design of targeted DDSs to achieve effective drug delivery.

Leukemic Stem Cells Persistence Measured By Multiparametric Flow Cytometry Is a Biomarker of Poor Prognosis in Adult Patients with Acute Myeloid Leukemia

Introduction: Despite the recent advances in chemotherapy regimens, relapse still substantially affects prognosis of intensively treated adult acute myeloid leukemia (AML) patients. There is growing evidence that a residual populations of leukemic cells may survive chemotherapy and outgrow, eventually causing relapse. These chemo-resistant cells are particularly abundant in the fraction of leukemic stem cells (LSC), which are endowed with pronounced self-renewal properties allowing to initiate and maintain leukemic clone. These cells can be detected, by high sensitivity multiparametric flow-cytometry (MFC), in the CD34+/CD38- fraction of the leukemic populations and can be distinguished from normal hematopoietic stem cells by the expression of specific markers. In recent clinical trials, LSC have been demonstrated to represent a biomarker of poor prognosis when detected at diagnosis but also during treatment course. Moreover, the combined estimate of measurable residual disease (MRD) and LSC refines the prognostic assessment as determined by the sole application of MRD detection. Aim: We analyzed a series of patients (pts) treated in the context of GIMEMA trials, in whom the LSC frequency was assessed by MFC at diagnosis. Pts with measurable levels of LSC were tested again after the consolidation cycle. At the same timepoint "standard" MRD was also determined. The purpose of the study was to demonstrate a correlation between LSC burden at baseline and prognosis in terms of overall (OS) and disease-free survival (DFS). Furthermore, we wanted to investigate the relationship between LSC and "standard" MRD persistence (>0.035%) after consolidation, and possible correlation with outcome. Methods: LSC were evaluated by MFC as described elsewhere (Terwijn, PLoS 2014). LSC were quantified exploiting the expression of the C-type lectin-like molecule-1 (CLL1) and applying a sequential gating strategy that contained the CD34+/CD38- population. Pts were defined as LSC negative (LSCneg) in case of zero LSC count, LSClow or LSChigh when LSC were >0<0,03% or >0.03%, respectively. After consolidation, any level >0 was considered as a LSC persistence. Methods of analysis and thresholds were set according to previous publications (Zeijlemaker, Leukemia 2019). Results: We analyzed 130 pts with de novo AML, in whom LSC determination was available at the baseline. Fifty-nine (45,4%) pts were LSCneg, 49 (37,7%) LSClow, 22 (16,9%) LSChigh. We did not observe any correlation between baseline LSC level and genetic/cytogenetic risk at diagnosis. There was not a significant difference in terms of OS duration according to the 3 LSC levels, however, pts who were LSChigh had the shortest OS (36-month estimate OS of 71.5% vs. 65.4 % vs 52.4 % for the LSCneg, LSClow and LSChigh categories respectively; p=0.21). A statistically significant difference, regardless of the belonging to the LSClow or LSChigh category was observed when we focus on the subgroup of 30 pts with intermediate-risk AML, with a 36-month estimate OS of 76% vs. 77.8% vs 25% for the LSCneg, LSClowand LSChigh categories respectively (p=0.023) (Figure 1A). In 19 patients, LSC persistence was assessed at the post-consolidation time-point. Nine LSChigh pts who failed to eradicate residual LSC at this timepoint had a worse outcome as compared to those belonging to the same category but achieving a LSC clearance or those who were LSClow (36-month OS of 62.5% vs. 59.2% vs. 66.7% vs. 25% for the LSClow converted into LSCneg, LSClow not converted into LSCneg, LSChigh converted into LSCneg and LSChigh not converted into LSCneg categories, respectively; P=0.062) (Figure 1B). In 27 pts LSC and "standard" MRD determination was available. LSC persistence determined a worse 3-years OS both in MRD negative (66.7% vs 85.7%, p=0.44) and MRD positive pts (<20% vs 75.0%, p=0.041). Conclusions: In line with the experience of other European groups, we demonstrated that MFC monitoring of LSC is feasible and provides prognostic information when performed at diagnosis and during treatment course. MFC assessment of LSC also offers the opportunity to monitor pts who lack aberrant phenotypes suitable for "standard" MRD investigation. When the 2 approaches - standard "MRD" and LSC assessment - are combined together, the prognosis prediction of AML can be further refined. Finally, LSC assessment can potentially represent an effective tool to monitor the effect of LSC targeting agents. Disclosures Buccisano: Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Venditti:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy.

Optimizing C-Type Lectin-like Molecule 1 (CLL-1) Directed CAR T Cell Therapy of Acute Myeloid Leukemia

Introduction Acute Myeloid Leukemia (AML) is a heterogeneous disease with continuous clonal evolution and frequent treatment-resistant relapses, often attributed to chemoresistant leukemic stem cells (LSCs). Since LSCs can be phenotypically and metabolically distinct from AML blast cells, any curative AML therapy has to target both LSC and AML blasts. Transcriptomic and proteomic analyses show that the CLL-1 antigen may fulfil these requirements and we previously showed (Tashiro et al) that T cells expressing a C-type lectin 1 (CLL-1)-specific CAR effectively recognized and eliminated both AML blasts and primitive AML colony-forming cells.We have now modified the structure of the CLL-1 CAR to optimize performance for clinical application in patients with AML. Material and Methods We utilized a CLL-1-specific single-chain fragment variable (scFV) to create a panel of CLL-1 CARs with modifications to the spacers, transmembrane and signaling endodomains (CD28z-CD8, CD28z-sh, CD28z-CH3, 4-1BBz-sh, 4-1BBz-CH3).To determine the functionally optimal construct, we compared the phenotype and cytolytic ability of T cells transduced with each construct. We measured 72 hr cytotoxicity against the target cell lines THP-1, HL60, Molm13 and KG1a expressing different levels of the target antigen.To measure exhaustion, we set up sequential co-culture assays, collecting the T cells every 3 days, counting them and then replating with fresh tumor.Finally, we tested the cytolytic activity of the constructs against FFLuc-modified HL 60 and THP- 1 AML cell lines in NOD.SCID IL-2Rg−/− (NSG) mice.The animals were engrafted with tumor cells by iv injection, and CLL-1 CAR T cells were injected 3 days later. IL-15 was co-transduced with CLL-1 CAR to sustain a positive signal 3. Results T cells expressing 4-1BBz-containing constructs expanded less than CD28z-containing constructs. T cells expressing CD28z constructs retained a mix of naive, minimally differentiated and central memory T cells similar to 4-1BBz constructs.CD28z with a short hinge and CD8 endodomains had the greatest anti-tumor activity in vitro against tumor cell lines.In sequential killing assays against HL60 and THP-1, both the above CD28z constructs had significantly prolonged anti-tumor activity compared to 4-1BBz, with sustained CAR T cell survival.In the in vivo xenograft NSG HL-60 mouse model CD28z constructs improved survival and co-expression of this optimized CLL-1 CAR with transgenic IL-15 (signal 3) further improved T-cell persistence and survival in THP-1 model. Conclusion The CD28z CAR with a short hinge and/or CD8 endodomains appears superior to other vector constructs against CLL-1 targets in terms of proliferation, functional persistence and in vitro and in vivo anti-tumor activity. Co-expression with transgenic IL-15 further improves these functional characteristics.

CD16-IL15-CLEC12A Trispecific Killer Engager (TriKE) Drives NK Cell Expansion, Activation, and Antigen Specific Killing of Cancer Stem Cells in Acute Myeloid Leukemia

Our group developed a 161533 trispecific killer engager (TriKE) molecule to target acute myeloid leukemia (AML) cells using Natural Killer (NK) cells. This molecule contains an anti-CD16 camelid nanobody to activate NK cells, an anti-CD33 single chain variable fragment (scFv) to engage cancer targets, and an IL-15 molecule that drives NK cell priming, expansion and survival. Using an earlier version of this molecule, we have shown that the CD33 TriKE is effective at activating NK cells against AML targets in vitro and in vivo. This preclinical data has lead to the establishment of a clinical trial in refractory AML patients at the University of Minnesota, set to open Q3 2018. While these previous studies have validated the use of TriKEs as an effective strategy of harnessing NK cells in cancer immunotherapy, CD33 has limitations as a target antigen.The high mortality and poor five-year survival rates (26%) for AML patients can be attributed to chemotherapy resistance and disease relapse. A majority of chemotherapy resistant leukemia stem cells (LSCs), that are hypothesized to facilitate relapse, do not express CD33. In addition, all hematopoietic stem cells and normal myeloid cells express CD33, thus targeting this antigen can lead to severe defects in hematopoiesis and on-target/off-tumor toxicity. To address these limitations, we developed a TriKE that targets CLEC12A or C-type lectin-like molecule 1 (CLL-1). CLEC12A is highly expressed on AML cells and over 70% of CD33 negative cells express CLEC12A. It has been attributed as a stem cell marker in AML, being selectively overexpressed in LSCs. CLEC12A is expressed by CD34+/CD38- LSCs but not normal CD34+/CD38- hematopoietic stem cells in regenerating bone marrow, thus minimizing off-target effects.The CLEC12A TriKE was developed in a mammalian cell system to ensure that appropriate post-translational modifications are present. We confirmed that the TriKE binds specifically to HL-60 and THP-1 target cells that express CLEC12A compared to Raji cells that do not express CLEC12A. Treatment of peripheral blood mononuclear cells (PBMCs) with the CLEC12A TriKE drives a significant increase in NK cell specific proliferation over 7 days as measured by CellTrace dilution compared to treatment with a CLEC12A scFv or IL-15 alone (69.7 ± 6.7% vs 11.9 ± 2.5% vs 38.4 ± 7.3%) (Figure 1A). To measure NK cell killing, we conducted an IncuCyte Zoom assay. Here, HL-60 target cells were labeled with a caspase 3/7 reagent where a color change indicates target cell death. The CLEC12A TriKE was able to induce more target cell killing than CLEC12A scFv or IL-15 as measured by number of live target cells at the end of the 48 hour assay (53.9 ± 1.9% vs 103.3 ± 3.4% vs 71.1 ± 1.4%). The CLEC12A TriKE induces an increase in NK cell degranulation, measured by CD107a expression against HL-60 AML tumor targets in a 4 hour functional assay compared to treatment with CLEC12A scFv or IL-15 alone (62.3 ± 1.1% vs 19.4 ± 3.8% vs 27.5 ± 4.9%). In this assay, there is also an increase in cytokine production, measured by IFNg and TNFa respectively (16.7 ± 4.2% vs 2.3 ± 1.5% vs 4.7 ± 1.9% and 18.0 ± 5.1% vs 2.5 ± 1.7% vs 4.6 ± 2.5%) (Figure 1B). We observe a similar enhanced functional response with THP-1 AML tumor targets. In these functional assays, treatment with the CLEC12A TriKE produced less background activation compared to the CD33 TriKE, indicating less off-target effects on PBMCs. To confirm the clinical relevance of this molecule, we tested the efficacy of the CLEC12A TriKE against primary AML targets. AML blasts were identified as SSC low, CD45 intermediate and CD34 high cells. Out of the 9 AML samples tested, 7 expressed high levels of CD33 (70.4 ± 6.3%) and CLEC12A (78.1 ± 5.2%). In functional assays with these samples, the CLEC12A TriKE was able to induce greater CD107a and IFNg expression, and enhanced killing of tumor targets as measured by a live/dead stain compared to CLEC12A scFv or IL-15 (Figure 1C). In these assays, the efficacy of the CLEC12A TriKE was comparable to the CD33 TriKE. Our data demonstrates that the CLEC12A TriKE drives NK cell specific proliferation, degranulation, cytokine secretion, and killing of tumor targets in vitro. Apart from AML, CLEC12A is expressed on cancer cells and LSCs in patients with myelodysplastic syndromes (MDS). These findings highlight the clinical potential of the CLEC12A TriKE individually or in combination with the CD33 TriKE for the treatment of MDS and AML. [Display omitted] DisclosuresVallera:GT Biopharma: Consultancy, Research Funding. Felices:GT Biopharma: Research Funding.

CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is one of the most common types of adult acute leukemia. Standard chemotherapies can induce complete remission in selected patients; however, a majority of patients eventually relapse and succumb to the disease. Thus, the development of novel therapeutics for AML is urgently needed. Human C-type lectin-like molecule-1 (CLL-1) is a type II transmembrane glycoprotein, and its expression is restricted to myeloid cells and the majority of AML blasts. Moreover, CLL-1 is expressed in leukemia stem cells (LSCs), but absent in hematopoietic stem cells (HSCs), which may provide a potential therapeutic target for AML treatment.MethodsWe tested the expression of CLL-1 antigen on peripheral blood cells and bone marrow cells in healthy donor and AML patients. Then, we developed a chimeric antigen receptor (CAR) containing a CLL1-specific single-chain variable fragment, in combination with CD28, 4-1BB costimulatory domains, and CD3-ζ signaling domain. We further investigate the function of CLL-1 CAR-T cells.ResultsThe CLL-1 CAR-T cells specifically lysed CLL-1+ cell lines as well as primary AML patient samples in vitro. Strong anti-leukemic activity was observed in vivo by using a xenograft model of disseminated AML. Importantly, CLL-1+ myeloid progenitor cells and mature myeloid cells were specifically eliminated by CLL-1 CAR-T cells, while normal HSCs were not targeted due to the lack of CLL-1 expression.ConclusionsCLL-1 CAR-T represents a promising immunotherapy for the treatment of AML.

Development of A Chimeric Antigen Receptor Targeting C-Type Lectin-Like Molecule-1 for Human Acute Myeloid Leukemia

The treatment of patients with acute myeloid leukemia (AML) with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR)-engineered T-cells (CAR-T-cells), a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A) is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs), and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.

Low CLL-1 Expression Is a Novel Adverse Predictor in 123 Patients with De Novo CD34+ Acute Myeloid Leukemia.

Recent reports state that C-type lectin-like molecule-1 (CLL-1) in acute myeloid leukemia (AML) is expressed primarily on myeloid cells, but there is still no investigation about its prognostic significance on leukemic blast compartment. Hence, this study aimed to evaluate the prognostic value of CLL-1 in 123 patients with de novo CD34+ Non-M3 AML. Multiparameter flow cytometry was used to assess the expression of CLL-1 on immature compartment in AML and control groups. We found that CLL-1 expression level on blast compartment was closely linked to clinical characteristics, treatment response, and survival outcome of patients. Decreased expression of CLL-1 was observed on immature compartment from AML patients as compared with controls (62.6% vs. 86.5%, P < 0.05). Logistic model exhibited that CLL-1low independently predicted low complete remission rate with an odds ratio of 4.57 (2.53-6.61, P < 0.05). Additionally, CLL-1 expression level at diagnosis was inversely correlated to the residual blast cells (residual leukemia cell) after induction chemotherapy (r = -0.423, P < 0.05). Furthermore, multivariate Cox regression model demonstrated that CLL-1low was still an independent adverse predictor (P < 0.05 for event-free survival, P < 0.05 for overall survival). Notably, CLL-1low was able to discriminate poor survival patients from intermediate- and favorable-risk groups. Taken together, CLL-1 is a novel prognostic predictor that could be exploited to supplement the current AML prognostic risk stratification system, and potentially optimize the clinical management of AML.

Treatment of Acute Myeloid Leukemia with T Cells Expressing Chimeric Antigen Receptors Directed to C-type Lectin-like Molecule 1

The successful immunotherapy of acute myeloid leukemia (AML) has been hampered because most potential antigenic targets are shared with normal hematopoietic stem cells (HSCs), increasing the risk of sustained and severe hematopoietic toxicity following treatment. C-type lectin-like molecule 1(CLL-1) is a membrane glycoprotein expressed by >80% of AML but is absent on normal HSCs. Here we describe the development and evaluation of CLL-1-specific chimeric antigen receptor Tcells (CLL-1.CAR-Ts) and we demonstrate their specificactivity against CLL-1+ AML cell lines as well as primary AML patient samples invitro. CLL-1.CAR-Ts selectively reduced leukemic colony formation in primary AML patient peripheral blood mononuclear cells compared to control Tcells. In a human xenograft mouse model, CLL-1.CAR-Ts mediated anti-leukemic activity against disseminated AML and significantly extended survival. By contrast, the colony formationof normal progenitor cells remained intact following CLL-1.CAR-T treatment. Although CLL-1.CAR-Ts are cytotoxic to mature normal myeloid cells, the selective sparing of normal hematopoietic progenitor cells should allow full myeloid recovery once CLL-1.CAR-T activity terminates. To enable elective ablation of the CAR-T, we therefore introduced the inducible caspase-9 suicide gene system and we show that exposure to the activating drug rapidly induced a controlled decrease of unwanted CLL-1.CAR-T activity against mature normal myeloid cells.

Evidence for the Role of Biologically Active Exosomes in Acute Myeloid Leukemia Relapse

Introduction: Despite of the notable achievements of multi-modality treatments in patients with acute myeloid leukemia (AML), leukemic relapse remains a formidable clinical challenge. Studies in AML patients have shown that tumor-induced impairments of immune cells contribute to leukemia progression. One recently identified immunosuppressive mechanism operating in AML is the release by leukemia blasts of virus-size (30-100nm) membrane-bound vesicles called exosomes. We have reported that plasma of patients with newly-diagnosed AML prior to any therapy contains higher levels of exosomal proteins than that of normal donors. AML exosomes carry an immunosuppressive cargo, including membrane-associated TGF-β1, MICA/MICB and markers of myeloid blasts. We hypothesize that persistence of these exosomes may contribute to the high relapse rates seen in AML.Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML prior to any treatment, from AML patients who achieved CR following initial therapy and from age-matched healthy volunteers (NC). Exosome fractions were isolated from plasma by using mini-size exclusion chromatography with Sepharose 2A. Exosome protein levels, numbers and size of exosomes (qNano) and their morphology by transmission electron microscopy were determined. Exosomes were characterized by western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44 and CD96. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels (mean fluorescence intensity) of NKG2D on NK cells.Results: Exosome fractions isolated from AML patients' plasma at diagnosis (n=16) had higher mean protein content than fractions obtained from NC plasma (96 vs 37 µg protein/mL plasma, p=0.03). In exosome fractions isolated from plasma of AML patients in CR (n=13) protein levels remained elevated (mean value=120 µg protein/mL plasma) at the time when leukemic blasts were undetectable in the bone marrow by conventional hematopathological methods. AML exosomes in patients in CR carried blast markers, CD123, CLL-1, CD44, CD96 and were enriched in TGF-β1. Co-incubation of these exosomes with activated NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Ten/13 AML patients in CR whose plasma exosome levels remained elevated eventually relapsed.Conclusion: The persistently elevated levels of biologically-active TGF-β1+ exosomes carrying leukemia blast markers in plasma of AML patients in CR impair anti-leukemia immune responses and might contribute to leukemia relapse. DisclosuresNo relevant conflicts of interest to declare.