RHAMM/CD168-R3 Peptide Vaccination of HLA-A2+ Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS) and Multiple Myeloma (MM).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2781-2781 ◽  
Author(s):  
Jochen Greiner ◽  
Krzysztof Giannopoulos ◽  
Li Li ◽  
Peter Liebisch ◽  
Christiane Wendl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Hematological Malignancies ◽  
Peptide Vaccination ◽  
Acute Myeloid

Abstract The receptor for hyaluronic acid mediated motility (RHAMM/CD168) has been described as a leukemia-associated antigen (LAA) eliciting both humoral and cellular immune responses in patients with hematological malignancies. RHAMM/CD168 is expressed in more than 80% of patients with acute myeloid leukemia (AML) or multiple myeloma (MM). Recently, we characterized the RHAMM/CD168-derived peptide R3 (ILSLELMKL) as a CD8+ T cell epitope. R3-primed CD8+ T lymphocytes were able to lyse autologous RHAMM/CD168+ AML blasts in a MHC class I-restricted and epitope-specific manner. Therefore, we initiated a phase I/II R3 peptide vaccination trial for patients with AML, MDS or MM overexpressing RHAMM/CD168. Patients were included with positive RHAMM/CD168 expression but with a limited tumor load. 300 mcg RHAMM R3 peptide emulsified with the incomplete Freund’s adjuvant (ISA-51, Montanide; day 3) and GM-CSF (Leukine, days 1–5) was administrated four times subcutaneously at a biweekly interval. The primary aim of the study is safety and feasibility of this peptide vaccination, secondary aims the evaluation of a specific T cell immune response to RHAMM/CD168 R3 peptide and the assessment of the influence of the R3 peptide vaccination on the remission status. Since January 2005, ten patients were enrolled in this study. The first eight patients (2 AML, 3 MDS, 3 MM) have completed the course of four vaccinations and four patients have been evaluated. The only side effects observed under R3-peptide vaccination were erythema and induration of the skin at the site of injection (CTC I°). In 2/4 patients, we found in the peripheral blood a significant increase of specific CD8+ T cells (from 0.01% to 0.8%) recognizing the R3 peptide in ELISPOT analysis and tetramer staining, one patient showed already initially a high number of HLA-A2/R3 tetramer+CCR7-CD27-CD45RA+ effector T cells and maintained this level of T cell response. Clinical responses have been assessed by the examination of peripheral blood and bone-marrow samples before and after vaccination. Patients showed a reduction of the tumor-specific expressed antigen RHAMM/CD168 in real-time RT-PCR analysis after vaccination. 2/4 patients with myeloid disorders (1 AML, 1 MDS RAEB1) showed a reduction of CD33+ cells in FACS analysis of the bone-marrow after 4 vaccinations from 10 and 7 % to 1–2 and <1%, respectively. One patient with MM showed a reduction of plasma cells in bone-marrow and a stable quantity of light chains in peripheral blood, one patient with AML showed a progressive disease. In summary, RHAMM/CD168 is a promising target antigen for immunotherapies in patients with hematological malignancies.

Characterization of Immune Reconstitution Following High Dose Chemotherapy in Acute Myeloid Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3888-3888
Author(s):  
Christian F. Meyer ◽  
Christopher Thoburn ◽  
Ferdynand Kos ◽  
Christopher Gocke ◽  
Hyam I. Levitsky ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Immune Reconstitution ◽  
Consolidation Therapy ◽  
Time Points ◽  
Acute Myeloid

Abstract Recovery of immune function after initial treatment of acute myeloid leukemia (AML) is critical, not only for protection against infections but also for surveillance against recurrent disease. A better understanding of the nature of lymphocyte recovery following induction and consolidation therapy for AML could guide the design of immunotherapy strategies aimed at boosting the anti-leukemia activity of a reconstituted immune system. Prior studies examining thymic T cell production following bone marrow transplantation (BMT) have found varying levels of thymic output post-transplant, as measured by T cell Receptor Excision Circle (TREC) levels in the peripheral blood of adult patients. Of note, relapse of chronic myeloid leukemia (CML) following BMT is correlated with decreased levels of TREC positive T cells. In order to characterize immune reconstitution in AML, we studied 26 patients after induction or consolidation time sequential chemotherapy. Their median age was 52 (range 23–69). Thirteen patients received cytarabine, daunorubicin, and etoposide (AcDVP-16) induction therapy, 3 patients received cytarabine, daunorubicin, and cytarabine (AcDAc) consolidation therapy and 10 patients received flavopiridol, cytarabine, and mitoxantrone (FLAM) either as induction or consolidation therapy. Peripheral blood samples were collected approximately every other day for 3–5 time points after each patient’s white blood cell count exceeded 200 cells/cubic mm on three consecutive days. Among the four patients evaluated to date, flow cytometry results show that a majority of cells seen early in immune reconstitution are CD3+ lymphocytes (range 69–97%). Subset analyses on 3 of these 4 patients have shown CD4:CD8 ratios ranging from 3:1 to 4:1, while the fourth patient exhibited an inverse of this ratio at 1:5. In addition, CD25+FOXP3+ T cells represented a median of 5.1% (range 2.5–12.3%) of the CD3+ T cells. Since T cells represented the abundance of cells in the peripheral blood during early bone marrow recovery, we then assessed whether these cells represented recent thymic emigrants or naïve T cells by examining TRECs using real time PCR (RT-PCR). TRECs were present in 24 of the 26 patients with levels ranging between 100 and 100,000 copies per 100,000 cells. Furthermore, 4 control samples from normal volunteers (ages 37–43) revealed the absence of TREC positive cells. Further analyses of these time points and correlations between TREC levels and clinical responses are ongoing.

scholarly journals Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts

Blood ◽  
2009 ◽  
Vol 114 (18) ◽  
pp. 3909-3916 ◽  
Author(s):  
Rifca Le Dieu ◽  
David C. Taussig ◽  
Alan G. Ramsay ◽  
Richard Mitter ◽  
Faridah Miraki-Moud ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Absolute Number ◽  
Immune Synapses ◽  
Acute Myeloid

Abstract Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.

scholarly journals Identification and Isolation of Tumor-Specific Cytotoxic T Lymphocytes in Acute Myeloid Leukemia Patients through the Identification of T-Cells Capable to Establish Stable Interactions with Leukemic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3336-3336
Author(s):  
Estefania Garcia-Guerrero ◽  
Luis I. Sanchez-Abarca ◽  
Esther Domingo ◽  
Teresa Ramos ◽  
Jose Antonio Bejarano-García ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
T Lymphocytes ◽  
Tumor Cells ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Target Cells ◽  
Acute Myeloid

Abstract Introduction Autologous adoptive T cell therapies, based on the use of tumor infiltrating lymphocytes (TILs), have made great progress in recent years for the treatment of solid tumors, especially melanoma. However, further work is needed to isolate tumor-reactive T cells among patients diagnosed with hematologic malignancies. The dynamics of the interaction between T cells and antigen presenting cells (APC) dictate the quality of the immune responses. While stable joints between target cells and T lymphocytes lead to the induction of T cell activation and immune response, brief contacts contribute to the induction of immune-tolerance. Taking advantage of the strong interaction between target cell and activated T-cells, we show the feasibility to identify and isolate tumor-specific cytotoxic T lymphocytes (CTLs) from acute myeloid leukemia (AML) patients. Using this approach, CTLs stably bound through T cell receptor to tumor cells (doublet forming T-cells) can be identified in peripheral blood and bone marrow and subsequently selected and isolated by FACS-based cell sorting. Methods Co-cultures between PBMC from AML patients in complete remission and AML tumor cells (PKH-stained) from the same patient were performed to study the percentage of doublet-forming T cells (CD3+PKH+) (T cell bound to a tumor cell). After 15 hours of co-culture, cells were stained and sorted. Secondary co-cultures with autologous tumor cells (used in primary co-culture) were performed to study the cytotoxic activity and cytokine production of T-cells capable or not to form stable joints with the leukemic cells (doublet population vs non-doublet population). Results Doublet-forming T cells from AML patients were identified in a range of 2% to 6% (mean=3.83%, n=5). Immunophenotyping analysis showed differences between doublet-forming T cells (CD3+PKH+) and those T cells which did not form stable and strong interactions with target cells (CD3+PKH-). Doublet T cells displayed a higher percentage of CD8+ T cells and higher percentage of effector CD4+ and CD8+ T cells compared to non-doublet T cells. Next, we explored, among effector CD4+ and CD8+ cells, those with cytotoxic phenotype. As expected, a high percentage of effector CD8+ doublet T cells showed Granzyme B and perforin expression, thus corresponding with a cytotoxic immune-phenotype (n=3, mean 65.51%). Within effector CD4+ doublet T cells, a mean of 9.053 % showed expression of both Granzyme B and perforin corresponding with CD4+ CTL (n=3). Regarding CD57 and CD16 markers, a mean of 18.62% of effector CD4+ doublet T cells were positive for both markers, compared to 65.84% of effector CD8+ doublet T cells (n=3). Further, we performed secondary co-cultures to analyze the CD69 activation marker after 24h of co-culture. A high percentage of CD69+ cells was observed in co-cultures with doublet-forming T cells against target cells as compared to non-doublet T cells (n=3, p=0.0053). Finally, analysis of supernatants of co-culture of doublet T cells and non-doublet T cells with target cells revealed specific secretion of IFNγ and IL-2 (n=3, p=0.0001; p=0.0005, respectively). The cytolytic activity was evaluated comparing the viability of tumor cells cultured alone or with doublet-forming T cells or non-doublet T cells from the same patient. A significant increase of the specific lysis of AML cells was observed when doublet T cells were co-cultured as compared to non-doublet T cells (p=0.0424, n=5). This encouraged us to examine whether we were able to identify doublet-forming T cells from bone marrow of AML patients at diagnosis. Analyses of bone marrow by flow cytometry reveled a small percentage of CD3+CD34+ population corresponding with bone marrow-doublet-forming T cells (n=3, mean=2.9%). Interestingly, bone marrow-doublet-forming T cells show a higher percentage of CD4+ T cells, whereas bone marrow-non-doublet T cells show a higher percentage of CD8+ T cells. Conclusions Our data demonstrate that when T cells from AML patients are co-cultured with tumor cells, a "doublet T cell" population appears. This population consists of T cells capable to bind tumor cells. These CTLs display higher percentage of effector cells and a marked cytotoxic activity against AML blasts. In conclusion, we have developed a new procedure to identify and select specific cytotoxic T cells in both bone marrow and peripheral blood from patients diagnosed with acute myeloid leukemia. Figure. Figure. Disclosures Sanchez-Abarca: Virgen del Rocio University Hospital: Patents & Royalties. Ramos:Takeda Oncology: Research Funding.

Immunological and Clinical Responses in Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL) after RHAMM-R3 Peptide Vaccination.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1806-1806 ◽  
Author(s):  
Jochen Greiner ◽  
Anita Schmitt ◽  
Krzysztof Giannopoulos ◽  
Jinfei Chen ◽  
Marlies Goetz ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Immune Responses ◽  
Myeloid Leukemia ◽  
Hematological Malignancies ◽  
Lymphocytic Leukemia ◽  
Peptide Vaccination ◽  
Chromium Release ◽  
Clinical Responses ◽  
Acute Myeloid

Abstract We initiated a phase I/II R3 peptide vaccination for patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) overexpressing the receptor for hyaluronic acid mediated motility (RHAMM). RHAMM is a leukemia associated antigen (LAA) that is strongly expressed in several hematological malignancies and induces humoral and cellular immune responses. In this study, patients with AML, MDS, MM or CLL were included with RHAMM expression but with a limited tumor load or a minimal residual disease. To date, 25 patients were enrolled. The first 12 patients were vaccinated with 300 mcg and further patients with 1000 mcg R3 peptide emulsified with the incomplete Freund’s adjuvant. The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Only mild drug-related adverse events were observed such as erythema and induration of the skin at the site of injection. Immunological analysis were performed using enzyme linked immunospot (ELISpot) assays for Interferon gamma and Granzyme B, tetramer staining and chromium release assays. We detected specific immune responses in 70% of patients. In most patients, we found an increase of CD8+/HLA-A2/RHAMM R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Moreover, we measured IL-2 and IL-10 levels in sera before and after vaccination. While IL-10 levels remained at a rather low level, we detected an increase of IL-2 in four of ten patients who showed also clinical responses. RHAMM transcripts in bone marrow and peripheral blood were quantified by real-time RT-PCR. Responding patients showed a decrease of RHAMM after vaccination. We detected positive clinical effects in several patients with myeloid disorders showing a reduction of blasts in the bone marrow. One MDS patient did not need any longer erythrocyte transfusions. Two patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies.

Chimeric Antigen Receptor for Specific Targeting of Acute Myeloid Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4225-4225
Author(s):  
Irene Pizzitola ◽  
Fernando Anjos-Afonso ◽  
Kevin Rouault-Pierre ◽  
Francois Lassailly ◽  
Sarah Tettamanti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Cord Blood ◽  
Myeloid Leukemia ◽  
Genetically Modified ◽  
Target Cells ◽  
Cik Cells ◽  
Acute Myeloid

Abstract Abstract 4225 Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after conventional chemotherapy regimens. Therefore it is necessary to develop novel alternative approaches. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial T-cell receptors constituted by a specific antigen-binding domain, and a signaling region, that, upon antigen recognition, leads to T-cell activation, and lysis of the target cells. AML is a potential optimal target for CAR strategy because of the over-expression of a number of surface antigens like CD33, CD123. Since CD33 is also expressed on normal hematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently emerged as new potential attractive molecules based on its differential expression pattern, being still wildly overexpressed by AML population, and at the same time less expressed on HSPCs. Here we describe the in vivo efficacy and the safety of this approach based on Cytokine-Induced-Killers (CIK) cells genetically modified to express CAR molecules specific for the CD33 or CD123 antigen. Once injected into low-level AML engrafted NSG mice (median of hCD45+CD33+ 0.6% before treatment), genetically modify T cells had a potent antitumor effect. Indeed, the bone marrow of control untreated animals or mice treated with un-manipulated CIK cells, was infiltrated by leukemic cells (86% and 81% leukemic engraftment), while in 7/8 anti-CD33-CD28-OX40-ζ and 8/10 anti-CD123-CD28-OX40-ζ treated mice we couldn't detect any AML cells. Similar results have been obtained when the treatment via T cell injection start when high AML burden has been obtained (median of hCD45+CD33+ 70% before treatment). One week after the last CIK's injection the level of AML engraftment was 96%, 87%, 0.35% and 0.34% for untreated mice, mice treated with un-manipulated CIK cells and with anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells respectively. We performed secondary transplantation on the residual AML cells present in these animals and mice were treated again with transduced CIK cells. Residual AML cells were still sensitive to CARs approach, leading once again to an almost complete eradication of the disease (median level of hCD45+CD33+ engraftment was 98%, 0.02% and 0.04% respectively for untreated mice, anti-CD33-CD28-OX40-ζ and anti-CD123-CD28-OX40-ζ transduced CIK-cells). Furthermore, a fundamental issue was to determine the safety profile of such approach against normal hematopoietic precursors. In untreated mice injected with primary cord blood derived CD34+ cells the level of engraftment of hCD45 compartment was 42% whilst in mice treated with un-manipulated, anti-CD33-CD28-OX40-ζ or anti-CD123-CD28-OX40-ζ transduced CIK-cells the levels of human compartment was 40%, 11.7% and 26.3% respectively. Moreover when we consider specifically the CD34+CD38- compartment, enriched in HSC, the level of engraftment was 1.92%, 1.02%, 0.55% and 0.83%. Secondary transplantations are now ongoing to give a more complete profile about the remaining HSC repopulating capability after treatment. To more closely mimic a physiological context, similar experiments are ongoing using mice engrafted with normal adult bone marrow instead of umbilical cord blood. These experiments should offer relevant information concerning the efficacy and safety of the proposed strategy particularly in the context of minimal residual disease in high-risk transplanted AML patients. Moreover CAR approach could be potentially used to treat patients resistant to conventional chemotherapeutic approaches or for whom high dose chemotherapy treatment could not be proposed. Disclosures: No relevant conflicts of interest to declare.

Emergence of antitumor cytolytic T cells is associated with maintenance of hematologic remission in children with acute myeloid leukemia

Blood ◽  
2006 ◽  
Vol 108 (12) ◽  
pp. 3843-3850 ◽  
Author(s):  
Daniela Montagna ◽  
Rita Maccario ◽  
Franco Locatelli ◽  
Enrica Montini ◽  
Sara Pagani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Induction Chemotherapy ◽  
Myeloid Leukemia ◽  
Allogeneic Bone Marrow Transplantation ◽  
T Cell Response ◽  
Allogeneic Bone ◽  
Acute Myeloid

AbstractAlthough the graft-versus-leukemia effect of allogeneic bone marrow transplantation (BMT) is of paramount importance in the maintenance of disease remission, the role played by the autologous T-cell response in antitumor immune surveillance is less defined. We evaluated the emergence of antileukemia cytotoxic T-lymphocyte precursors (CTLp's) and the correlation of this phenomenon with maintenance of hematologic remission in 16 children with acute myeloid leukemia (AML), treated with either chemotherapy alone (5 patients) or with autologous BMT (A-BMT, 11 patients). Antileukemia CTLp's were detectable in 8 patients in remission after induction chemotherapy; none of them subsequently had a relapse. Of the 8 patients who did not show detectable CTLp frequency while in remission after induction chemotherapy, 7 subsequently experienced leukemia relapse. In patients undergoing A-BMT, molecular fingerprinting of the TCR-Vβ repertoire, performed on antileukemia lines, demonstrated that selected antileukemia T-cell clonotypes, detectable in bone marrow before transplantation, survived ex vivo pharmacologic purging and were found in the recipient after A-BMT. These data provide evidence for an active role of autologous T cells in the maintenance of hematologic remission and also suggest that quantification of antileukemia CTLp frequency may be a useful tool to identify patients at high risk for relapse, thus potentially benefiting from an allogeneic antitumor effect.

scholarly journals Combining the Expression of CD33.CAR and CXCR4 to Increase CAR-CIK Cell Homing to Bone Marrow Niche and Leukemic Stem Cell Eradication in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2791-2791
Author(s):  
Marta Biondi ◽  
Beatrice Cerina ◽  
Chiara Tomasoni ◽  
Gianpietro Dotti ◽  
Sarah Tettamanti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Sleeping Beauty ◽  
Advisory Board ◽  
Over Expression ◽  
Car T ◽  
Acute Myeloid

Abstract Chimeric Antigen Receptor (CAR) T cell therapy is a promising treatment for acute myeloid leukemia (AML), but a limited efficacy was reported from ongoing clinical trials. The capacity of engineered T cells to infiltrate into the bone marrow (BM) niche, where leukemic stem cells (LSC) reside, strongly impacts the success of the treatment. Ex vivo manipulation of CAR T cells affects the expression of several chemokine receptors and may alter the capacity of infused cells to migrate to BM. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) regulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 over-expressed on blasts, promoting their homing in the niche. CXCR4 expression is drastically downregulated during the culture of cytokine induced killer (CIK) cells, an interesting effector T cell population with acquired NK-like cytotoxicity along with minimal alloreactivity. Therefore, combining the expression of CD33.CAR with the over-expression of CXCR4 might facilitate CAR-CIKs homing to the BM and subsequent leukemia eradication. We designed two bicistronic Sleeping Beauty transposon vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. We observed that both CD33.CAR(2A)CXCR4-CIKs (n=22, P<0.0001) and CXCR4(IRES)CD33.CAR-CIKs (n=9, P<0.0001) maintained CXCR4 over-expression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). However, CD33.CAR expression was lower in CXCR4(IRES)CD33.CAR-CIKs (n=8, P<0.0001) compared to CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=11) exhibited a significant co-expression of both proteins against control (P=0.001). CXCR4(IRES)CD33.CAR-CIKs and CD33.CAR(2A)CXCR4-CIKs maintained all CAR-associated in vitro effector functions, eliminating CD33+ KG1 target cell line, releasing cytokines (IL-2 and IFN-γ) and proliferating in an antigen-specific manner. However, CXCR4(IRES)CD33.CAR-CIKs exhibited lower effector responses against control, due to lower CAR expression. Chemotaxis assays toward recombinant CXCL12 confirmed both CXCR4(IRES)CD33.CAR-CIKs (n=7, P=0.01) and CD33.CAR(2A)CXCR4-CIKs (n=8, P=0.0006) displayed a migration advantage over CD33.CAR-CIKs (n=12) with a mean percentage of migration of 58.5% and 67.2% respectively, compared to 40.1%. Interestingly, CD33.CAR(2A)CXCR4-CIKs (n=2) showed an increased specific chemotactic response toward healthy (n=3) and AML-derived MSC (n=2) supernatants, which could be inhibited by the use of the CXCR4 antagonist Plerixafor. Moreover, when infused intravenously into NSG mice, significantly higher proportions of CD33.CAR(2A)CXCR4-CIKs were recovered in the femur BM compared to controls (P=0.0068). In conclusion, CD33.CAR(2A)CXCR4-CIKs, reaching the medullary niche more effectively, have the potential to more efficiently target the residing LSC responsible for the high relapse rates in AML. Disclosures Dotti: Tessa Therapeutics: Consultancy; Bellicum Pharmaceuticals: Consultancy; Catamaran: Consultancy. Biondi: Bluebird: Other: Advisory Board; Amgen: Honoraria; Incyte: Consultancy, Other: Advisory Board; Novartis: Honoraria; Colmmune: Honoraria.

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