Developing a membrane-proximal CD33-targeting CAR T cell.

CD33 is a tractable target in acute myeloid leukemia (AML) for chimeric antigen receptor (CAR) T cell therapy, but clinical success is lacking. We developed 3P14HLh28Z, a novel CD33-directed CD28/CD3Z-based CAR T cell derived from a high-affinity binder obtained through membrane-proximal fragment immunization in humanized mice. We found that immunization exclusively with the membrane-proximal domain of CD33 is necessary for identification of membrane-proximal binders in humanized mice. Compared with clinically validated lintuzumab-based CAR T cells targeting distal CD33 epitopes, 3P14HLh28Z showed enhanced in vitro functionality as well as superior tumor control and increased overall survival in both low antigen density and clinically relevant patient-derived xenograft models. Increased activation and enhanced polyfunctionality led to enhanced efficacy. Showing for the first time that a membrane-proximal CAR is superior to a membrane-distal one in the setting of CD33 targeting, our results demonstrate the rationale for targeting membrane-proximal epitopes with high-affinity binders. We also demonstrate the importance of optimizing CAR T cells for functionality in settings of both low antigen density and clinically relevant patient-derived models.

Freeman R ，Shahid S ，Khan AG ，Mathew SC ，Souness S ，Burns ER ，Um JS ，Tanaka K ，Cai W ，Yoo S ，Dunbar A ，Park Y ，McAvoy D ，Hosszu KK ，Levine RL ，Boelens JJ ，Lorenz IC ，Brentjens RJ ，Daniyan AF ... -  《Journal for ImmunoTherapy of Cancer》

Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design.

Successful development of chimeric antigen receptor (CAR) T cell immunotherapy for children and adults with relapsed/refractory acute myeloid leukemia (AML) is highly desired given their poor clinical prognosis and frequent inability to achieve cure with conventional chemotherapy. Initial experiences with CD19 CAR T cell immunotherapy for patients with B-cell malignancies highlighted the critical impact of intracellular costimulatory domain selection (CD28 vs 4-1BB (CD137)) on CAR T cell expansion and in vivo persistence that may impact clinical outcomes. However, the impact of costimulatory domains on the efficacy of myeloid antigen-directed CAR T cell immunotherapy remains unknown. In this preclinical study, we developed six CAR constructs targeting CD33, a highly expressed and validated AML target, comprised of one of three single-chain variable fragments with CD3ζ and either CD28 or 4-1BB costimulatory domains. We systematically compared the preclinical in vitro and in vivo efficacy of T cells lentivirally transduced with CD33 CAR constructs (CD33CARTs) against human AML. We observed potent in vitro cytokine production and cytotoxicity of CD33CARTs incubated with human CD33+ AML cell lines, as well as robust in vivo antileukemia activity in cell line and childhood AML patient-derived xenograft (PDX) models. Gemtuzumab-based CD33CARTs were unexpectedly toxic in vivo in animal models despite observed in vitro anti-leukemia activity. CD28-based CD33CARTs consistently induced more robust inhibition of leukemia proliferation in AML cell line and PDX models than did 4-1BB-based CD33CARTs. A 'best-in-class' lintuzumab-CD28/CD3ζ CAR construct was thus selected for clinical translation. CD33 is a critical antigen for potential immunotherapeutic targeting in patients with AML. Based on this rigorous preclinical evaluation, our validated clinical grade lintuzumab-CD28/CD3ζ CD33CART immunotherapy is now under evaluation in a first-in-child/first-in-human phase 1 clinical trial for children and adolescents/young adults with relapsed/refractory AML. clinicaltrials.gov; NCT03971799.

Qin H ，Yang L ，Chukinas JA ，Shah N ，Tarun S ，Pouzolles M ，Chien CD ，Niswander LM ，Welch AR ，Taylor N ，Tasian SK ，Fry TJ ... -  《Journal for ImmunoTherapy of Cancer》

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 ... -  《-》

CD33-Specific Chimeric Antigen Receptor T Cells with Different Co-Stimulators Showed Potent Anti-Leukemia Efficacy and Different Phenotype.

Li S ，Tao Z ，Xu Y ，Liu J ，An N ，Wang Y ，Xing H ，Tian Z ，Tang K ，Liao X ，Rao Q ，Wang M ，Wang J ... -  《-》

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》