Chimeric Antigen Receptor (CAR) Cytokine-Induced Killer cells (CIKs)-cell therapy for acute myeloid leukemia (AML) may be limited by dampened effector cells trafficking to the bone marrow (BM) niche, where leukemia stem cells (LSCs) reside. The chemokine receptor 4 (CXCR4) and its ligand, CXC motif ligand 12 (CXCL12) regulate leukocytes trafficking to the niche. In AML, CXCL12, secreted by mesenchymal stromal cells (MSCs), binds CXCR4 overexpressed on blasts, promoting their homing in the BM. CXCR4 expression on CIKs is drastically down-regulated during culture. Combining expression of CD33.CAR and CXCR4 might therefore facilitate CAR-CIK homing to the BM allowing more efficient targeting of LSCs in the leukemic niche. Two bicistronic Sleeping Beauty transposon vectors were designed: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. Migration in vitro toward rhCXCL12 or MSC supernatants was tested using a transwell migration assay. CAR-CIKs in vivo BM homing ability was evaluated in sub-lethally irradiated NSG mice. We noticed 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 overexpression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). Nevertheless, 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). Therefore, CD33.CAR(2A)CXCR4 construct was chosen to proceed with further investigations. Noticeably, CD33.CAR-CXCR4-CIKs maintained all CAR-related in vitro effector functions, eliminating CD33+ KG1 and MOLM14 target cell lines, releasing cytokines (IL-2 and IFN-γ) and proliferating in an antigen-specific fashion. Furthermore, CD33.CAR-CXCR4–CIKs displayed improved migratory response toward rhCXCL12 as compared to CD33.CAR-CIKs (n = 10, P < 0.0001). Moreover, to mimic the conditions of the BM microenvironment, we employed supernatant of MSCs derived from healthy donors (HD) (n = 6) or AML patients at diagnosis (n = 10) as a chemotactic stimulus. CD33.CAR-CXCR4-CIKs still demonstrated an increased chemotactic response compared to control, which could be inhibited by CXCR4 antagonist Plerixafor. Notably, 7 days after infusion into sub-lethally irradiated NSG mice, CD33.CAR+-CXCR4-CIKs presented a significant increase in their ability to enter BM compartment when compared to CD33.CAR+-CIKs. Contrarily, CD33.CAR+-CXCR4-CIK frequency in peripheral blood and spleen displayed only minimal differences compared to control CD33.CAR+-CIKs. Overall CD33.CAR(2A)CXCR4-CIKs display enhanced in vitro migration and superior in vivo BM homing ability while maintaining CAR functionalities. This boosted migratory potential might facilitate an improved clearing of CD33+ AML blasts and LSCs residing in the BM, which is being explored in ongoing experiments.

Biondi, M., Cerina, B., Tomasoni, C., Dotti, G., Tettamanti, S., Biondi, A., et al. (2022). CXCR4-MODIFIED CD33.CAR-CIK WITH ENHANCED BONE MARROW HOMING IN ACUTE MYELOID LEUKEMIA. Intervento presentato a: XVII Congress of the Italian Society of Experimental Hematology - March 31 – April 1-2, 2022, Roma, Italia [10.3324/haematol.2022.s1].

CXCR4-MODIFIED CD33.CAR-CIK WITH ENHANCED BONE MARROW HOMING IN ACUTE MYELOID LEUKEMIA

Biondi, M
Primo
;
Tomasoni, C;Tettamanti, S;Pievani, A;
2022

Abstract

Chimeric Antigen Receptor (CAR) Cytokine-Induced Killer cells (CIKs)-cell therapy for acute myeloid leukemia (AML) may be limited by dampened effector cells trafficking to the bone marrow (BM) niche, where leukemia stem cells (LSCs) reside. The chemokine receptor 4 (CXCR4) and its ligand, CXC motif ligand 12 (CXCL12) regulate leukocytes trafficking to the niche. In AML, CXCL12, secreted by mesenchymal stromal cells (MSCs), binds CXCR4 overexpressed on blasts, promoting their homing in the BM. CXCR4 expression on CIKs is drastically down-regulated during culture. Combining expression of CD33.CAR and CXCR4 might therefore facilitate CAR-CIK homing to the BM allowing more efficient targeting of LSCs in the leukemic niche. Two bicistronic Sleeping Beauty transposon vectors were designed: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. The monocistronic CD33.CAR was used as control. Migration in vitro toward rhCXCL12 or MSC supernatants was tested using a transwell migration assay. CAR-CIKs in vivo BM homing ability was evaluated in sub-lethally irradiated NSG mice. We noticed 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 overexpression during culture, whereas in CD33.CAR-CIKs was drastically downregulated (n=22). Nevertheless, 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). Therefore, CD33.CAR(2A)CXCR4 construct was chosen to proceed with further investigations. Noticeably, CD33.CAR-CXCR4-CIKs maintained all CAR-related in vitro effector functions, eliminating CD33+ KG1 and MOLM14 target cell lines, releasing cytokines (IL-2 and IFN-γ) and proliferating in an antigen-specific fashion. Furthermore, CD33.CAR-CXCR4–CIKs displayed improved migratory response toward rhCXCL12 as compared to CD33.CAR-CIKs (n = 10, P < 0.0001). Moreover, to mimic the conditions of the BM microenvironment, we employed supernatant of MSCs derived from healthy donors (HD) (n = 6) or AML patients at diagnosis (n = 10) as a chemotactic stimulus. CD33.CAR-CXCR4-CIKs still demonstrated an increased chemotactic response compared to control, which could be inhibited by CXCR4 antagonist Plerixafor. Notably, 7 days after infusion into sub-lethally irradiated NSG mice, CD33.CAR+-CXCR4-CIKs presented a significant increase in their ability to enter BM compartment when compared to CD33.CAR+-CIKs. Contrarily, CD33.CAR+-CXCR4-CIK frequency in peripheral blood and spleen displayed only minimal differences compared to control CD33.CAR+-CIKs. Overall CD33.CAR(2A)CXCR4-CIKs display enhanced in vitro migration and superior in vivo BM homing ability while maintaining CAR functionalities. This boosted migratory potential might facilitate an improved clearing of CD33+ AML blasts and LSCs residing in the BM, which is being explored in ongoing experiments.
Si
abstract + slide
Acute leukemias; CAR-T cell therapy;
English
XVII Congress of the Italian Society of Experimental Hematology - March 31 – April 1-2, 2022
Biondi, M., Cerina, B., Tomasoni, C., Dotti, G., Tettamanti, S., Biondi, A., et al. (2022). CXCR4-MODIFIED CD33.CAR-CIK WITH ENHANCED BONE MARROW HOMING IN ACUTE MYELOID LEUKEMIA. Intervento presentato a: XVII Congress of the Italian Society of Experimental Hematology - March 31 – April 1-2, 2022, Roma, Italia [10.3324/haematol.2022.s1].
Biondi, M; Cerina, B; Tomasoni, C; Dotti, G; Tettamanti, S; Biondi, A; Pievani, A; Serafini, M
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/381223
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