Directing B7-H3 chimeric antigen receptor T cell homing through IL-8 induces potent antitumor activity against pediatric sarcoma.

Advances in pediatric oncology have occurred for some cancers; however, new therapies for sarcoma have been inadequate. Cellular immunotherapy using chimeric antigen receptor (CAR) T cells has shown dramatic benefits in leukemia, lymphoma, and multiple myeloma but has been far less successful in pediatric solid tumors such as rhabdomyosarcoma (RMS) and osteosarcoma (OS). Balancing issues of "on-target, off-tumor toxicity", investigators have identified B7-H3 as a broadly expressed tumor antigen with otherwise restricted expression on normal tissues. We hypothesized that rapid homing via a chemokine receptor and CAR engagement through B7-H3 would enhance CAR T cell efficacy in solid tumors. We generated B7-H3 CAR T cells that also express the Interleukin-8 (IL-8) receptor, CXCR2. Cytokine production, flow cytometry, Seahorse assays and RNA sequencing were used to compare the B7-H3 CXCR2 (BC2) CAR T cells with B7-H3 CAR T cells. We developed an IL-8 overexpressing human RMS mouse model to test homing and cytotoxicity in vivo. We demonstrate that IL-8 is expressed by RMS and OS and expression significantly increases after radiation. Overexpression of an IL-8 receptor, CXCR2, on B7-H3 CAR T cells enhances homing into IL-8 expressing tumors, augments T cell metabolism and leads to significant tumor regression. These findings warrant further investigation into the use of BC2 CAR T cells as a treatment for patients with RMS, OS and other B7-H3-expressing, IL-8 producing solid tumors.

Lake JA ，Woods E ，Hoffmeyer E ，Schaller KL ，Cruz-Cruz J ，Fernandez J ，Tufa D ，Kooiman B ，Hall SC ，Jones D ，Hayashi M ，Verneris MR ... -  《Journal for ImmunoTherapy of Cancer》

Redirecting B7-H3.CAR T Cells to Chemokines Expressed in Osteosarcoma Enhances Homing and Antitumor Activity in Preclinical Models.

Clinical efficacy of chimeric antigen receptor (CAR) T cells against pediatric osteosarcoma (OS) has been limited. One strategy to improve efficacy may be to drive chemokine-mediated homing of CAR T cells to tumors. We sought to determine the primary chemokines secreted by OS and evaluate the efficacy of B7-H3.CAR T cells expressing the cognate receptors. We developed a pipeline to identify chemokines secreted by OS by correlating RNA-seq data with chemokine protein detected in media from fresh surgical specimens. We identified CXCR2 and CXCR6 as promising receptors for enhancing CAR T-cell homing against OS. We evaluated the homing kinetics and efficiency of CXCR2- and CXCR6.T cells and homing, cytokine production, and antitumor activity of CXCR2- and CXCR6.B7-H3.CAR T cells in vitro and in vivo. T cells transgenically expressing CXCR2 or CXCR6 exhibited ligand-specific enhanced migration over T cells modified with nonfunctional control receptors. Differential homing kinetics were observed, with CXCR2.T-cell homing quickly and plateauing early, whereas CXCR6.T cells took longer to home but achieved a similar plateau. When expressed in B7-H3.CAR T cells, CXCR2- and CXCR6 modification conferred enhanced homing toward OS in vitro and in vivo. CXCR2- and CXCR6-B7-H3.CAR-treated mice experienced prolonged survival in a metastatic model compared with B7-H3.CAR T-cell-treated mice. Our patient-based pipeline identified targets for chemokine receptor modification of CAR T cells targeting OS. CXCR2 and CXCR6 expression enhanced the homing and anti-OS activity of B7-H3.CAR T cells. These findings support clinical evaluation of CXCR-modified CAR T cells to improve adoptive cell therapy for patients with OS.

Talbot LJ ，Chabot A ，Ross AB ，Beckett A ，Nguyen P ，Fleming A ，Chockley PJ ，Shepphard H ，Wang J ，Gottschalk S ，DeRenzo C ... -  《-》

CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors.

Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system. We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models. B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3. B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.

Majzner RG ，Theruvath JL ，Nellan A ，Heitzeneder S ，Cui Y ，Mount CW ，Rietberg SP ，Linde MH ，Xu P ，Rota C ，Sotillo E ，Labanieh L ，Lee DW ，Orentas RJ ，Dimitrov DS ，Zhu Z ，Croix BS ，Delaidelli A ，Sekunova A ，Bonvini E ，Mitra SS ，Quezado MM ，Majeti R ，Monje M ，Sorensen PHB ，Maris JM ，Mackall CL ... -  《-》

B7-H3-Targeting Chimeric Antigen Receptors Epstein-Barr Virus-specific T Cells Provides a Tumor Agnostic Off-The-Shelf Therapy Against B7-H3-positive Solid Tumors.

Encouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues, we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3-targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment-based domains. In efforts to expand patient access to CAR T-cell therapy, we engineered our nanobody-based CAR into human Epstein-Barr virus-specific T cells (EBVST), offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3-positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T, B, and natural killer cells were unaffected by B7H3.CAR EBVSTs, monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells, which are key mediators of cytokine release syndrome (CRS), contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably, we showed that B7H3.CAR EBVSTs can target B7-H3-expressing myeloid-derived suppressor cells (MDSC), thereby mitigating MDSC-driven immune suppression. In summary, our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3-positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment, highlighting their promising clinical potential in targeting solid tumors. Clinical application of EBVSTs armored with B7-H3-targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors.

Yeo SP ，Kua L ，Tan JW ，Lim JK ，Wong FH ，Santos MD ，Poh CM ，Goh AX ，Koh XY ，Zhou X ，Rajarethinam R ，Chen Q ，Her Z ，Horak ID ，Low L ，Tan KW ... -  《-》

B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres.

The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM. We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models. B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines. We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. FUND: Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.

Nehama D ，Di Ianni N ，Musio S ，Du H ，Patané M ，Pollo B ，Finocchiaro G ，Park JJH ，Dunn DE ，Edwards DS ，Damrauer JS ，Hudson H ，Floyd SR ，Ferrone S ，Savoldo B ，Pellegatta S ，Dotti G ... -  《EBioMedicine》