Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer.

Chimeric antigen receptor (CAR) T cells have revolutionized blood cancer immunotherapy; however, their efficacy against solid tumors has been limited. A common mechanism of tumor escape from single target therapies is downregulation or mutational loss of the nominal epitope. Targeting multiple antigens may thus improve the effectiveness of CAR immunotherapies. We generated dual CAR-T cells targeting two tumor antigens: TAG-72 (tumor-associated glycoprotein 72) and CD47. TAG-72 is a pan-adenocarcinoma oncofetal antigen, highly expressed in ovarian cancers, with increased expression linked to disease progression. CD47 is ubiquitously overexpressed in multiple tumor types, including ovarian cancer; it is a macrophage "don't eat me" signal. However, CD47 is also expressed on many normal cells. To avoid this component of the dual CAR-T cells killing healthy tissue, we designed a truncated CD47 CAR devoid of intracellular signaling domains. The CD47 CAR facilitates binding to CD47+ cells, increasing the prospect of TAG-72+ cell elimination via the TAG-72 CAR. Furthermore, we could reduce the damage to normal tissue by monomerizing the CD47 CAR. Our results indicate that the co-expression of the TAG-72 CAR and the CD47-truncated monomer CAR on T cells could be an effective, dual CAR-T cell strategy for ovarian cancer, also applicable to other adenocarcinomas.

CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature.

Chemokines and chemokine receptors have been posited to have important roles in several common malignancies, including breast and lung cancer. Here, we demonstrate that CXCR7 (RDC1, CCX-CKR2), recently deorphanized as a chemokine receptor that binds chemokines CXCL11 and CXCL12, can regulate these two common malignancies. Using a combination of overexpression and RNA interference, we establish that CXCR7 promotes growth of tumors formed from breast and lung cancer cells and enhances experimental lung metastases in immunodeficient as well as immunocompetent mouse models of cancer. These effects did not depend on expression of the related receptor CXCR4. Furthermore, immunohistochemistry of primary human tumor tissue demonstrates extensive CXCR7 expression in human breast and lung cancers, where it is highly expressed on a majority of tumor-associated blood vessels and malignant cells but not expressed on normal vasculature. In addition, a critical role for CXCR7 in vascular formation and angiogenesis during development is demonstrated by using morpholino-mediated knockdown of CXCR7 in zebrafish. Taken together, these data suggest that CXCR7 has key functions in promoting tumor development and progression.

A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development.

The chemokine stromal cell-derived factor (SDF-1; also known as chemokine ligand 12 [CXCL12]) regulates many essential biological processes, including cardiac and neuronal development, stem cell motility, neovascularization, angiogenesis, apoptosis, and tumorigenesis. It is generally believed that SDF-1 mediates these many disparate processes via a single cell surface receptor known as chemokine receptor 4 (CXCR4). This paper characterizes an alternate receptor, CXCR7, which binds with high affinity to SDF-1 and to a second chemokine, interferon-inducible T cell alpha chemoattractant (I-TAC; also known as CXCL11). Membrane-associated CXCR7 is expressed on many tumor cell lines, on activated endothelial cells, and on fetal liver cells, but on few other cell types. Unlike many other chemokine receptors, ligand activation of CXCR7 does not cause Ca2+ mobilization or cell migration. However, expression of CXCR7 provides cells with a growth and survival advantage and increased adhesion properties. Consistent with a role for CXCR7 in cell survival and adhesion, a specific, high affinity small molecule antagonist to CXCR7 impedes in vivo tumor growth in animal models, validating this new receptor as a target for development of novel cancer therapeutics.

Proteolytic activation of alternative CCR1 ligands in inflammation.

Although chemokines CCL3/MIP-1alpha and CCL5/RANTES are considered to be primary CCR1 ligands in inflammatory responses, alternative CCR1 ligands have also been described. Indeed, four such chemokines, CCL6/C10/MIP-related protein-1, CCL9/MIP-1gamma/MIP-related protein-2, CCL15/MIP-1delta/hemofiltrate CC chemokine-2/leukotactin-1, and CCL23/CKbeta8/myeloid progenitor inhibitory factor-1, are unique in possessing a separately encoded N-terminal domain of 16-20 residues and two additional precisely positioned cysteines that form a third disulfide bridge. In vitro, these four chemokines are weak CCR1 agonists, but potency can be increased up to 1000-fold by engineered or expression-associated N-terminal truncations. We examined the ability of proinflammatory proteases, human cell supernatants, or physiological fluids to perform N-terminal truncations of these chemokines and thereby activate their functions. Remarkably, most of the proteases and fluids removed the N-terminal domains from all four chemokines, but were relatively unable to cleave the truncated forms further. The truncated chemokines exhibited up to 1000-fold increases in CCR1-mediated signaling and chemotaxis assays in vitro. In addition, N-terminally truncated CCL15/MIP-1delta and CCL23/CKbeta8, but not CCL3/MIP-1alpha or CCL5/RANTES, were detected at relatively high levels in synovial fluids from rheumatoid arthritis patients. These data suggest that alternative CCR1 ligands are converted into potent chemoattractants by proteases released during inflammatory responses in vivo.