The Hematologist

September-October 2019, Volume 16, Issue 5

Compelling Evidence for Targeted Immunotherapy As Treatment for AML

Amanda B. Kagan, MD Hematology Fellow
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins Hospital, Baltimore, MD
Amy DeZern, MD, MHS Associate Professor of Oncology and Medicine
Johns Hopkins University School of Medicine, Baltimore, MD

Published on: July 24, 2019

Borot F, Wang H, Ma Y, et al. Gene-edited stem cells enable CD33-directed immune therapy for myeloid malignancies. Proc Natl Acad Sci U S A. 2019;116:11978-11987.

Despite numerous advances in the treatment of acute myeloid leukemia (AML) in recent years, including the development of multiple molecularly targeted agents, relapse remains a major problem, even after allogeneic stem cell transplantation. While immunologic therapies, particularly the bispecific T-cell engager blinatumomab, antibody-drug conjugate (ADC) inotuzumab, and CAR T-cells (CAR-T), have been remarkably successful in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL),1-3 similar approaches have yet to be successfully implemented in AML. A major roadblock limiting the success of immunotherapies in AML is the paucity of AML-specific antigens; in their absence, immunotherapies target antigens on both AML and normal hematopoietic progenitors, leading to unacceptable toxicity.

In this study, Dr. Florence Borot and colleagues present a novel mechanism using CD33-directed immunotherapy in AML that overcomes the hindrance posed by the lack of AML-specific antigens. Their approach combines targeting myeloid lineage–specific antigen (LSA), CD33, a transmembrane protein expressed on more than 90 percent of AML blasts, with the transplantation of genetically engineered stem cells lacking CD33. CD33 knockout mice develop normally without apparent hematologic defects.4 This group shows that hematopoietic stem/progenitor cells (HSPCs) that have undergone genetic ablation of CD33 do function normally, providing further evidence that CD33 may be dispensable. They provide additional proof that ablating a myeloid LSA such as CD33 in HSPCs enables the use of anti-CD33 CAR-T or antibody therapy, with concomitant engraftment and growth of CD33-deleted hematopoietic cells transplanted into the same host.

The group used CRISPR/ CRISPR-associated protein 9 (Cas9) to ablate expression of CD33. The Cas9 endonuclease can locate, bind, and cleave double-stranded DNA targets complementary to guide CRISPR RNAs (crRNAs).5 In this study, guides were designed to target exon 3 of CD33, since this sequence is common to all CD33 transcripts and has minimal similarity with the family of pseudogenes of which CD33 is a member. There was high deletion efficiency; on average, CD33 expression was reduced from 85 percent to 10 percent of CD34+ cells. They then tested the ability of CD33 gene-edited stem cells (CD33del) to engraft and contribute to hematopoiesis. Mice were injected with CD33del HPSCs, and peripheral blood analysis post-transplantation revealed the presence of mature cells of myeloid and lymphoid lineages, all CD33-negative. Importantly, there was no functional difference in the CD34+CD33del and CD34+CD33WT myeloid subsets, and whole-genome sequencing of HSPCs edited with Cas9/RNA complexes did not identify any insertions or deletions at off-target loci.

The CAR-T designed in this study contains a single-chain variable region of anti-CD33, along with a costimulatory, transmembrane, and intracellular domain. The CAR cDNA was cloned into a lentiviral vector. Peripheral blood from healthy donors was transduced with lentiviral particles containing the CAR construct. This demonstrated robust expression of the CAR as well as binding with CD33. CAR-T cytotoxicity was proportional to the expression level of CD33 on target cells, with the highest cytotoxicity seen in CD33+ myeloid leukemia cells. Irradiated mice were injected simultaneously with both CD33+ HL-60 leukemia cells and CD34+cd33del cells, to mimic relapsed AML in the model. One week later, the mice were treated with allogeneic CD33-targeted CAR-T, control T cells, gemtuzumab ozogamicin (GO), or a combination of CAR-T and GO. No leukemia cells were found in bone marrow aspirates at 3.5 weeks or eight weeks in the CAR-T group, GO group, or combined CAR-T/GO group. Furthermore, engraftment of CD34+CD33del cells was demonstrated in the therapy model by the presence of CD45+, CD33 negative cells in the bone marrow aspirate of all groups.

Although other groups have used similar approaches to enable CAR-T cell immunotherapy in AML,6,7 this study’s approach is novel in several ways. Dr. Miriam Y. King and colleagues6 first injected mice with CD33 gene-edited CD34+ cells to allow for complete engraftment before leukemia introduction and treatment. In contrast, Dr. Borot and colleagues co-injected leukemia cells and gene-edited stem cells, followed by CAR-Ts or ADCs. This likely more closely mimics clinical AML relapse. Another unique feature of this study was the stringent selection of CD33 guide RNAs with high on-target and low off-target activity, allowing for increased safety and feasibility of using this approach in human studies. Lastly, the use of an allogeneic donor for both gene-edited HPSCs and CAR-Ts, as opposed to autologous CAR-Ts, may have greater clinical applicability.

In summary, AML, especially in the relapsed setting, is a disease with a largely unmet need in the area of immunotherapy. This group illustrates a novel approach of utilizing CD33-targeted CAR-Ts or ADCs coupled with transplantation of engineered stem cells to protect normal hematopoiesis. They show that this approach, in an animal model, can lead to a full remission as well as full hematopoietic reconstitution, due to CD33’s expression by both normal and malignant cells and functional redundancy. This represents an exciting potential avenue for pursuing new targets, both in other hematologic malignancies and in solid malignancies that share similar properties.


  1. Kantarjian H, Stein A, Gökbuget N, et al. Blinatumomab versus chemotherapy for advanced acute lymphoblastic leukemia. N Engl J Med. 2017;376:836-847.
  2. Kantarjian HM, DeAngelo DJ, Stelljes M, et al. Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia. N Engl J Med. 2016;375:740-753.
  3. Park JH, Rivière I, Gonen M, et al. Long-term follow-up of CD19 CAR therapy in acute lymphoblastic leukemia. N Engl J Med. 2018;378:449-459.
  4. Brinkman-Van der Linden EC, Angata T, Reynolds SA, et al. CD33/Singlec-3 binding specificity, expression pattern, and consequences of gene deletion in mice. Mol Cell Biol. 2003;23:4199-4206.
  5. Murugan K, Babu K, Sundaresan R, et al. The revolution continues: Newly discovered systems expand the CRISPR-Cas toolkit. Mol Cell. 2017;68:15-25.
  6. Kim MY, Yu KR, Kenderian SS, et al. Genetic inactivation of CD33 in hematopoietic stem cells to enable CAR T cell immunotherapy for acute myeloid leukemia. Cell. 2018;173:1439-1453.e19.
  7. Humbert O, Laszlo GS, Sichel S, et al. Engineering resistance to CD33-targeted immunotherapy in normal hematopoiesis by CRISPR/Cas9-deletion of CD33 exon 2. Leukemia. 2019;33:762-808.

Conflict of Interests

Dr. Kagan and Dr. DeZern indicated no relevant conflicts of interest. back to top