Antitumor effects of intracranial injection of B7-H3-targeted Car-T and Car-Nk cells in a patient-derived glioblastoma xenograft model.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor for which novel therapies are needed. Recently, chimeric antigen receptor (CAR) T cell therapy has been shown to be effective against GBM, but it is a personalized medicine and requires high cost and long time for the cell production. CAR-transduced natural killer (NK) cells can be used for "off-the-shelf" cellular immunotherapy because they do not induce graft-versus-host disease. Therefore, we aimed to analyze the anti-GBM effect of CAR-T or NK cells targeting B7-H3, which is known to be highly expressed in GBM. CAR-T cells targeting B7-H3 were generated using previously reported anti-B7-H3 scFv sequences. Cord blood (CB)-derived NK cells transduced with the B7-H3 CAR were also generated. Their anti-GBM effect was analyzed in vitro. The antitumor effect of intracranial injection of the B7-H3 CAR-T or NK cells was investigated in an in vivo xenograft model with patient-derived GBM cells. Both B7-H3 CAR-T cells and CAR-NK cells exhibited marked cytotoxicity against patient-derived GBM cells in vitro. Furthermore, intracranial injection of CAR-T cells and CAR-NK cells targeting B7-H3 resulted in a significant antitumor effect against patient-derived GBM xenografts. Not only CAR-T cells but also CB-derived CAR-NK cells targeting B7-H3 may have the potential to eliminate GBM cells.

Tachi T ，Kijima N ，Kuroda H ，Ikeda S ，Murakami K ，Nakagawa T ，Yaga M ，Nakagawa K ，Utsugi R ，Hirayama R ，Okita Y ，Kagawa N ，Kishima H ，Imai C ，Hosen N ... -  《-》

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》

CD19 CAR-expressing iPSC-derived NK cells effectively enhance migration and cytotoxicity into glioblastoma by targeting to the pericytes in tumor microenvironment.

In cancer immunotherapy, chimeric antigen receptors (CARs) targeting specific antigens have become a powerful tool for cell-based therapy. CAR-natural killer (NK) cells offer selective anticancer lysis with reduced off-tumor toxicity compared to CAR-T cells, which is beneficial in the heterogeneous milieu of solid tumors. In the tumor microenvironment (TME) of glioblastoma (GBM), pericytes not only support tumor growth but also contribute to immune evasion, underscoring their potential as therapeutic targets in GBM treatment. Given this context, our study aimed to target the GBM TME, with a special focus on pericytes expressing CD19, to evaluate the potential effectiveness of CD19 CAR-iNK cells against GBM. We performed CD19 CAR transduction in induced pluripotent stem cell-derived NK (iNK) cells. To determine whether CD19 CAR targets the TME pericytes in GBM, we developed GBM-blood vessel assembloids (GBVA) by fusing GBM spheroids with blood vessel organoids. When co-cultured with GBVA, CD19 CAR-iNK cells migrated towards the pericytes surrounding the GBM. Using a microfluidic chip, we demonstrated CD19 CAR-iNK cells' targeted action and cytotoxic effects in a perfusion-like environment. GBVA xenografts recapitulated the TME including human CD19-positive pericytes, thereby enabling the application of an in vivo model for validating the efficacy of CD19 CAR-iNK cells against GBM. Compared to GBM spheroids, the presence of pericytes significantly enhanced CD19 CAR-iNK cell migration towards GBM and reduced proliferation. These results underline the efficacy of CD19 CAR-iNK cells in targeting pericytes within the GBM TME, suggesting their potential therapeutic value for GBM treatment.

Kong D ，Kwon D ，Moon B ，Kim DH ，Kim MJ ，Choi J ，Kang KS ... -  《-》

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 ... -  《-》

ErbB2/HER2-Specific NK Cells for Targeted Therapy of Glioblastoma.

Glioblastoma (GBM) is the most common and malignant intracranial tumor in adults and currently incurable. To specifically target natural killer (NK) cell activity to GBM, we employed NK-92/5.28.z cells that are continuously expanding human NK cells expressing an ErbB2-specific chimeric antigen receptor (CAR). ErbB2 expression in 56 primary tumors, four primary cell cultures, and seven established cell lines was assessed by immunohistochemistry and flow cytometry. Cell killing activity of NK-92/5.28.z cells was analyzed in in vitro cytotoxicity assays. In vivo antitumor activity was evaluated in NOD-SCID IL2Rγ(null) (NSG) mice carrying orthotopic human GBM xenografts (6 to 11 mice per group) and C57BL/6 mice carrying subcutaneous and orthotopic ErbB2-expressing murine GBM tumors (5 to 8 mice per group). Statistical tests were two-sided. We found elevated ErbB2 protein expression in 41% of primary GBM samples and in the majority of GBM cell lines investigated. In in vitro assays, NK-92/5.28.z in contrast to untargeted NK-92 cells lysed all ErbB2-positive established and primary GBM cells analyzed. Potent in vivo antitumor activity of NK-92/5.28.z was observed in orthotopic GBM xenograft models in NSG mice, leading to a marked extension of symptom-free survival upon repeated stereotactic injection of CAR NK cells into the tumor area (median survival of 200.5 days upon treatment with NK-92/5.28.z vs 73 days upon treatment with parental NK-92 cells, P < .001). In immunocompetent mice, local therapy with NK-92/5.28.z cells resulted in cures of transplanted syngeneic GBM in four of five mice carrying subcutaneous tumors and five of eight mice carrying intracranial tumors, induction of endogenous antitumor immunity, and long-term protection against tumor rechallenge at distant sites. Our data demonstrate the potential of ErbB2-specific NK-92/5.28.z cells for adoptive immunotherapy of glioblastoma, justifying evaluation of this approach for the treatment of ErbB2-positive GBM in clinical studies.

Zhang C ，Burger MC ，Jennewein L ，Genßler S ，Schönfeld K ，Zeiner P ，Hattingen E ，Harter PN ，Mittelbronn M ，Tonn T ，Steinbach JP ，Wels WS ... -  《-》