Search

Chimeric Antigen Receptor (CAR) Cytokine-Induced Killer (CIK) cell therapy is an emerging treatment for acute myeloid leukemia (AML) although some implementations are required. Specifically, it appears crucial to improve CAR-CIK infiltration ability into the bone marrow (BM) niche to eradicate leukemia stem cells (LSCs) at their location. CAR-CIK ex vivo manipulation influences the expression of several chemokine receptors and may dampen the capacity of infused cells to migrate to the BM. The chemokine ligand 12 (CXCL12), produced by mesenchymal stromal cells (MSCs) within the niche, and its chemokine receptor 4 (CXCR4) modulate leukocytes trafficking to the BM. In AML, CXCL12 binds CXCR4 overexpressed on blasts, enhancing their homing in the niche. CXCR4 expression is consistently downregulated during the culture of CIKs. Therefore, combining the expression of CD33.CAR and CXCR4 might promote CAR-CIK homing to the BM and subsequent leukemia eradication, specifically of LSCs. 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. Phenotype and CAR-related in vitro effector functions were compared. 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. CAR-CIKs engraftment was assessed in BM, blood, spleen, and lungs. We noticed that both CD33.CAR(2A)CXCR4-CIKs (n=22, P

Chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has been quite elusive due to tumor heterogeneity, lack of ideal target antigens and the role of the tumor microenvironment in leukemia protection and leukemia stem cells (LSCs) maintenance. Therefore, CAR design must be improved for an effective anti-AML activity. Besides selecting a good target antigen, it is crucial to improve CAR T-cells infiltration, to eradicate LSCs at their location within the bone marrow (BM) niche. The chemokine receptor 4 (CXCR4) and its ligand, CXC motif ligand 12 (CXCL12) are two fundamental factors orchestrating leukocytes trafficking to the niche. In AML, the CXCL12-CXCR4 axis is co-opted by leukemic cells. CXCL12, released by mesenchymal stromal cells (MSCs), interacts with CXCR4 overexpressed on blasts, boosting their homing in the BM, where they can find a protecting environment. We hypothesize that CXCR4 overexpression by Cytokine-Induced Killer cells (CIKs) combined with a CD33.CAR may improve their homing to the BM and subsequent leukemia eradication. We designed two bicistronic vectors: CXCR4(IRES)CD33.CAR and CD33.CAR(2A)CXCR4. CIKs were non-virally engineered with the Sleeping Beauty transposon system to express our constructs or the monocistronic CD33.CAR, used as control. We assessed the chemotactic activity of the bicistronic constructs employing the transwell migration assay, toward rhCXCL12 and human MSCs supernatants. Additionally, in vitro effector functions were compared. Considering that CXCR4 expression on CAR-CIKs is downregulated over culture, we induced it artificially. CXCR4(IRES)CD33.CAR-CIKs (n=8) expressed higher levels of CXCR4, but lower CD33.CAR expression compared with CD33.CAR-CIKs, while CD33.CAR(2A)CXCR4-CIKs (n=9) showed a significant co-expression of both proteins, as compared to control (P≤ 0.05). The analysis of T-cell markers and memory phenotype uncovered that CXCR4(IRES)CD33.CAR-CIKs (n=7) are enriched in CD8+ compartment and include a higher fraction of TEMRA contrary to CD33-CAR CIKs (P≤0.05), while CD33.CAR(2A)CXCR4-CIKs (n=8) phenotype is comparable to control. Chemotaxis assays toward rhCXCL12 confirmed that CXCR4(IRES)CD33.CAR-CIKs (n=7, P≤0.05) and CD33.CAR(2A)CXCR4-CIKs (n=7, P≤0.05) achieved a migration advantage over CD33.CAR-CIKs alone (n=11), with a mean percentage of migration of 58.5% and 68.8% respectively, compared to 40.5%. We were also able to observe a specific chemotactic response toward MSCs supernatants, as proved by the use of Plerixafor. Moreover, correlation analysis strengthened our observation, as higher CXCR4 expression resulted in increased migratory activity. Concerning CAR-related effector functions, CXCR4(IRES)CD33.CAR-CIKs and CD33.CAR(2A)CXCR4-CIKs displayed similar killing of the CD33+ KG1 cell line, compared to CD33.CAR-CIKs alone, with a mean lysis of 56.6% (E:T ratio 5:1, n=7) for CXCR4(IRES)CD33.CAR-CIKs, and of 66.9% (n=9) for CD33.CAR(2A)CXCR4-CIKs, compared to a mean lysis of 62.5% (n=12) of CD33.CAR-CIKs. The bicistronic constructs also maintained their capacity to produce IL-2 and IFN-y, and to proliferate after CD33 antigen exposure. CXCR4(IRES)CD33.CAR-CIKs displayed not significant but inferior effector responses as compared to control, due to lower CAR expression; CD33.CAR(2A)CXCR4-CIKs performed similarly to CD33.CAR CIKs alone. Compared to conventional CD33.CAR-CIKs, CXCR4-overexpressing CD33.CAR-CIKs exhibit enhanced migration capacity while retaining functional activity against CD33+ target cells. Considering these results, we believe that CD33.CAR(2A)CXCR4 is the best candidate for further in vivo homing and anti-leukemic tests.

La terapia con cellule CAR-CIK per la leucemia mieloide acuta (LMA) è limitata dalla ridotta infiltrazione delle cellule ingegnerizzate all’interno della nicchia midollare, rifugio delle cellule staminali leucemiche (LSC). Queste ultime, over-esprimendo il recettore CXCR4, sfruttano l’interazione con la chemochina CXCL12 rilasciata dalle cellule mesenchimali stromali (MSC) per annidarsi nel microambiente. L’espressione di CXCR4 si riduce drasticamente sulle cellule CIK durante la coltura. Conseguentemente, combinare il CD33.CAR con l’over-espressione di CXCR4 potrebbe promuovere la migrazione delle CAR-CIK nel midollo, contribuendo all’eradicazione della malattia. Abbiamo disegnato il vettore bicistronico non virale CD33.CAR(2A)CXCR4 utilizzando il sistema di trasposoni Sleeping Beauty. Abbiamo osservato che le CD33.CAR(2A)CXCR4-CIK (n=22) presentano un’elevata espressione del CAR e mantengono l’espressione di CXCR4 durante la coltura, mentre le CD33.CAR-CIK lo down-regolano progressivamente (n=22, P

Regional delivery of chimeric antigen receptor (CAR) T cells in glioblastoma represents a rational therapeutic approach as an alternative to intravenous administration to avoid the blood-brain barrier impediment. Here, we developed a fibrin gel that accommodates CAR-T cell loading and promotes their gradual release. Using a model of subtotal glioblastoma resection, we demonstrated that the fibrin-based gel delivery of CAR-T cells within the surgical cavity enables superior antitumor activity compared to CAR-T cells directly inoculated into the tumor resection cavity.

CAR T therapy targeting solid tumors is restrained by limited infiltration and persistence of those cells in the tumor microenvironment (TME). Here, we developed approaches to enhance the activity of CAR T cells using an orthotopic model of locally advanced breast cancer. CAR T cells generated from Th/Tc17 cells given with the STING agonists DMXAA or cGAMP greatly enhanced tumor control, which was associated with enhanced CAR T cell persistence in the TME. Using single-cell RNA sequencing, we demonstrate that DMXAA promoted CAR T cell trafficking and persistence, supported by the generation of a chemokine milieu that promoted CAR T cell recruitment and modulation of the immunosuppressive TME through alterations in the balance of immune-stimulatory and suppressive myeloid cells. However, sustained tumor regression was accomplished only with the addition of anti-PD-1 and anti-GR-1 mAb to Th/Tc17 CAR T cell therapy given with STING agonists. This study provides new approaches to enhance adoptive T cell therapy in solid tumors.

Background Chimeric antigen receptor (CAR) T cells have shown considerable promise as a personalized cellular immunotherapy against B cell malignancies. However, the complex and lengthy manufacturing processes involved in generating CAR T cell products ex vivo result in substantial production time delays and high costs. Furthermore, ex vivo expansion of T cells promotes cell differentiation that reduces their in vivo replicative capacity and longevity. Methods Here, to overcome these limitations, CAR-T cells are engineered directly in vivo by administering a lentivirus expressing a mutant Sindbis envelope, coupled with a bispecific antibody binder that redirects the virus to CD3 + human T cells. Results This redirected lentiviral system offers exceptional specificity and efficiency; a single dose of the virus delivered to immunodeficient mice engrafted with human peripheral blood mononuclear cells generates CD19-specific CAR-T cells that markedly control the growth of an aggressive pre-established xenograft B cell tumor. Conclusions These findings underscore in vivo engineering of CAR-T cells as a promising approach for personalized cancer immunotherapy.

Vα24-invariant natural killer T (NKT) cells have shown potent anti-tumor properties in murine tumor models and have been linked to favorable outcomes in patients with cancer. However, low numbers of these cells in humans have hindered their clinical applications. Here we report interim results from all three patients enrolled on dose level 1 in a phase 1 dose-escalation trial of autologous NKT cells engineered to co-express a GD2-specific chimeric antigen receptor (CAR) with interleukin-15 in children with relapsed or resistant neuroblastoma (NCT03294954). Primary and secondary objectives were to assess safety and anti-tumor responses, respectively, with immune response evaluation as an additional objective. We ex vivo expanded highly pure NKT cells (mean ± s.d., 94.7 ± 3.8%) and treated patients with 3 × 106 CAR-NKT cells per square meter of body surface area after lymphodepleting conditioning with cyclophosphamide/fludarabine (Cy/Flu). Cy/Flu conditioning was the probable cause for grade 3–4 hematologic adverse events, as they occurred before CAR-NKT cell infusion, and no dose-limiting toxicities were observed. CAR-NKT cells expanded in vivo, localized to tumors and, in one patient, induced an objective response with regression of bone metastatic lesions. These initial results suggest that CAR-NKT cells can be expanded to clinical scale and safely applied to treat patients with cancer.

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15.

Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure, increases blood perfusion, releases antigens, and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells can potentially increase the therapeutic index of these cells in solid tumors. It is found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma mice engrafted with the human melanoma WM115 cell line have superior antitumor activity after photothermal ablation of the tumor. The findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within solid tumors.

Tumors are inherently heterogeneous in antigen expression, and escape from immune surveillance due to antigen loss remains one of the limitations of targeted immunotherapy. Despite the clinical use of adoptive therapy with chimeric antigen receptor (CAR)–redirected T cells in lymphoblastic leukemia, treatment failure due to epitope loss occurs. Targeting multiple tumor-associated antigens (TAAs) may thus improve the outcome of CAR-T cell therapies. CARs developed to simultaneously target multiple targets are limited by the large size of each single-chain variable fragment and compromised protein folding when several single chains are linearly assembled. Here, we describe single-domain antibody mimics that function within CAR parameters but form a very compact structure. We show that antibody mimics targeting EGFR and HER2 of the ErbB receptor tyrosine kinase family can be assembled into receptor molecules, which we call antibody mimic receptors (amR). These amR can redirect T cells to recognize two different epitopes of the same antigen or two different TAAs in vitro and in vivo

Tumor-associated adipocytes promote tumor growth by providing energy and causing chronic inflammation. Here, we have exploited the lipid metabolism to engineer adipocytes that serve as a depot to deliver cancer therapeutics at the tumor site. Rumenic acid (RA), as an anticancer fatty acid, and a doxorubicin prodrug (pDox) with a reactive oxygen species (ROS)-cleavable linker, are encapsulated in adipocytes to deliver therapeutics in a tumor-specific bioresponsive manner. After intratumoral or postsurgical administration, lipolysis releases the RA and pDox that is activated by intracellular ROS-responsive conversion, subsequently promoting antitumor efficacy. Furthermore, downregulation of PD-L1 expression is observed in tumor cells, favoring the emergence of CD4+ and CD8+ T cell-mediated immune responses. © 2019 Elsevier Inc.Adipocytes were engineered for local delivery of anticancer therapeutics, including fatty acid and small-molecule drug. The lipolysis triggered by tumor cells promoted the release of these anticancer therapeutics. In addition, the immunogenic tumor phenotype induced by anticancer fatty acid combined with tumor-responsive doxorubicin prodrug enhanced cancer therapy. © 2019 Elsevier Inc.The tumor microenvironment composed of nonmalignant cells often promotes tumor growth by providing growth factors and preventing the infiltration of tumor-killing immune cells. It could be valuable to leverage the therapeutic potential of the nonmalignant cells within the tumor microenvironment for anticancer treatment. In this work, the adipocytes have been engineered with the encapsulation of an anticancer fatty acid and a bioresponsive doxorubicin prodrug for chemotherapy and simultaneously inducing an immunogenic tumor phenotype. These Trojan horse-like injectable engineered adipocytes can serve as a drug-delivery depot for sustained drug release with suppressed primary tumor growth and postsurgical tumor recurrence. This adipocyte-mediated drug-delivery strategy expands the scope of cell therapy and could be extended for treating other diseases associated with lipid metabolism pathways.

Cancer recurrence after surgical resection remains a significant cause of treatment failure. Here, we have developed an in situ formed immunotherapeutic bioresponsive gel that controls both local tumour recurrence after surgery and development of distant tumours. Briefly, calcium carbonate nanoparticles pre-loaded with the anti-CD47 antibody are encapsulated in the fibrin gel and scavenge H+ in the surgical wound, allowing polarization of tumour-associated macrophages to the M1-like phenotype. The released anti-CD47 antibody blocks the ‘don’t eat me’ signal in cancer cells, thereby increasing phagocytosis of cancer cells by macrophages. Macrophages can promote effective antigen presentation and initiate T cell mediated immune responses that control tumour growth. Our findings indicate that the immunotherapeutic fibrin gel ‘awakens’ the host innate and adaptive immune systems to inhibit both local tumour recurrence post surgery and potential metastatic spread.