Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia.

The absence of cancer-restricted surface markers is a major impediment to antigen-specific immunotherapy using chimeric antigen receptor (CAR) T cells. For example, targeting the canonical myeloid marker CD33 in acute myeloid leukemia (AML) results in toxicity from destruction of normal myeloid cells. We hypothesized that a leukemia-specific antigen could be created by deleting CD33 from normal hematopoietic stem and progenitor cells (HSPCs), thereby generating a hematopoietic system resistant to CD33-targeted therapy and enabling specific targeting of AML with CAR T cells. We generated CD33-deficient human HSPCs and demonstrated normal engraftment and differentiation in immunodeficient mice. Autologous CD33 KO HSPC transplantation in rhesus macaques demonstrated long-term multilineage engraftment of gene-edited cells with normal myeloid function. CD33-deficient cells were impervious to CD33-targeting CAR T cells, allowing for efficient elimination of leukemia without myelotoxicity. These studies illuminate a novel approach to antigen-specific immunotherapy by genetically engineering the host to avoid on-target, off-tumor toxicity.

Kim MY ，Yu KR ，Kenderian SS ，Ruella M ，Chen S ，Shin TH ，Aljanahi AA ，Schreeder D ，Klichinsky M ，Shestova O ，Kozlowski MS ，Cummins KD ，Shan X ，Shestov M ，Bagg A ，Morrissette JJD ，Sekhri P ，Lazzarotto CR ，Calvo KR ，Kuhns DB ，Donahue RE ，Behbehani GK ，Tsai SQ ，Dunbar CE ，Gill S ... -  《-》

CD33-directed immunotherapy with third-generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and CD33-edited acute myeloid leukemia and hematopoietic stem and progenitor cells.

Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.

Liu Y ，Wang S ，Schubert ML ，Lauk A ，Yao H ，Blank MF ，Cui C ，Janssen M ，Schmidt C ，Göllner S ，Kleist C ，Zhou F ，Rahfeld JU ，Sauer T ，Schmitt M ，Müller-Tidow C ... -  《-》

In Vitro Pre-Clinical Validation of Suicide Gene Modified Anti-CD33 Redirected Chimeric Antigen Receptor T-Cells for Acute Myeloid Leukemia.

Minagawa K ，Jamil MO ，Al-Obaidi M ，Pereboeva L ，Salzman D ，Erba HP ，Lamb LS ，Bhatia R ，Mineishi S ，Di Stasi A ... -  《PLoS One》

A dual-targeting approach with anti-IL10R CAR-T cells engineered to release anti-CD33 bispecific antibody in enhancing killing effect on acute myeloid leukemia cells.

Immunotherapies, including chimeric antigen receptor (CAR) T cells and bispecific antibodies (BsAbs), encounter several challenges in the management of acute myeloid leukemia (AML), including limited persistence of these treatments, antigen loss and resistance of leukemia stem cells (LSCs) to therapy. Here, we proposed a novel dual-targeting approach utilizing engineered anti-IL10R CAR-T cells to secrete bispecific antibodies targeting CD33. This innovative strategy, rooted in our previous research which established a connection between IL-10 and the stemness of AML cells, designed to improve targeting efficiency and eradicate both LSCs and AML blasts. We first demonstrated the superior efficacy of this synergistic approach in eliminating AML cell lines and primary cells expressing different levels of the target antigens, even in cases of low CD33 or IL10R expression. Furthermore, the IL10R CAR-T cells that secret anti-CD33 bsAbs (CAR.BsAb-T), exhibited an enhanced activation and induction of cytotoxicity not only in IL10R CAR-T cells but also in bystander T cells, thereby more effectively targeting CD33-positive tumor cells. Our in vivo experiments provided additional evidence that CAR.BsAb-T cells could efficiently redirect T cells, reduce tumor burden, and demonstrate no significant toxicity. Additionally, delivering bsAbs locally to the tumor sites through this strategy helps mitigate the pharmacokinetic challenges typically associated with the rapid clearance of prototypical bsAbs. Overall, the engineering of a single-vector targeting IL10R CAR, which subsequently secretes CD33-targeted bsAb, addresses the issue of immune escape due to the heterogeneous expression of IL10R and CD33, and represents a promising progress in AML therapy aimed at improving treatment outcomes.

Yan Z ，Gu R ，Ma H ，Chen N ，Zhang T ，Xu Y ，Qiu S ，Xing H ，Tang K ，Tian Z ，Rao Q ，Wang M ，Wang J ... -  《-》

Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo.

Pizzitola I ，Anjos-Afonso F ，Rouault-Pierre K ，Lassailly F ，Tettamanti S ，Spinelli O ，Biondi A ，Biagi E ，Bonnet D ... -  《-》