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Acute myeloid leukemia

Targeting the membrane-proximal C2-set domain of CD33 for improved CD33-directed immunotherapy


There is increasing interest in targeting CD33 in malignant and non-malignant disorders. In acute myeloid leukemia, longer survival with the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this strategy. Still, GO benefits only some patients, prompting efforts to develop more potent CD33-directed therapeutics. As one limitation, CD33 antibodies typically recognize the membrane-distal V-set domain. Using various artificial CD33 proteins, in which this domain was differentially positioned within the extracellular portion of the molecule, we tested whether targeting membrane-proximal epitopes enhances the effector functions of CD33 antibody-based therapeutics. Consistent with this idea, a CD33V-set/CD3 bispecific antibody (BsAb) and CD33V-set-directed chimeric antigen receptor (CAR)-modified T cells elicited substantially greater cytotoxicity against cells expressing a CD33 variant lacking the entire C2-set domain than cells expressing full-length CD33, whereas cytotoxic effects induced by GO were independent of the position of the V-set domain. We therefore raised murine and human antibodies against the C2-set domain of human CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain (“CD33PAN antibodies”). These antibodies internalized when bound to CD33 and, as CD33PAN/CD3 BsAb, had potent cytolytic effects against CD33+ cells. Together, our data provide the rationale for further development of CD33PAN antibody-based therapeutics.

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Fig. 1: Membrane proximity of the target epitope modulates the anti-tumor efficacy of CD33/CD3 BsAbs and CAR T cells.
Fig. 2: Biophysical characterization of the CD33PAN antibody, 1H7.
Fig. 3: A murine CD33PAN/CD3 BsAb redirects T cell-mediated cytotoxicity against AML cells in a CD33-specific and epitope-specific manner.
Fig. 4: A human CD33PAN/CD3 BsAb redirects T cell-mediated cytotoxicity against AML cells in a CD33-specific and epitope-specific manner.


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We would like to thank Dr. Christopher Mehlin, Dr. James M. Olson, Jane Carter, and other members of the Molecular Design and Therapeutics (MDT) core facility at Fred Hutchinson Cancer Research Center for help with the generation of CD33 antibodies, and Dr. Della J. Friend for conducting the SPR experiments. Research reported in this publication was supported by the Leukemia & Lymphoma Society (Translational Research Program, grant 6489-16) and the National Institutes of Health/National Cancer Institute (NIH/NCI) (R21 CA223409, R21-CA234203, R21-CA245594, P30-CA015704, and P50-CA100632 [MD Anderson Cancer Center Leukemia SPORE]). CDG is supported by a fellowship training grant from the NIH/National Heart, Lung, and Blood Institute (NHLBI; T32-HL007093), an institutional K12 grant from the NIH/NCI (K12-CA076930) an American Society of Clinical Oncology/Conquer Cancer Foundation Young Investigator Award and an Alex’s Lemonade Stand Young Investigator Grant. The Fred Hutchinson Cancer Research Center Antibody Technology Resource received support from the M.J. Murdock Charitable Trust.

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Correspondence to Roland B. Walter.

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Conflict of interest

CDG received research funding from Immunogen and Pfizer. HPK is a consultant to and has ownership interests with Rocket Pharma and Homology Medicines and is a consultant to CSL Behring and Magenta Therapeutics. CJT received research funding from AstraZeneca, Juno Therapeutics/Bristol Myers Squibb, Minerva, Nektar Therapeutics, and TCR2 Therapeutics; has ownership interests with ArsenalBio, Caribou Biosciences, Eureka Therapeutics, Myeloid Therapeutics, Precision Biosciences; is an inventor on a patent licensed to Juno Therapeutics; and is (or has been) a consultant to Amgen, ArsenalBio, Astra Zeneca, Caribou Biosciences, Century Therapeutics, Eureka Therapeutics, Myeloid Therapeutics, Nektar Therapeutics, PACT Pharma, Precision Biosciences, and T-CURX. RBW received laboratory research grants and/or clinical trial support from Amgen, Aptevo, Celgene, Immunogen, Macrogenics, Jazz, Pfizer, and Selvita; has ownership interests with Amphivena; and is (or has been) a consultant to Agios, Amphivena, Aptevo, Astellas, Bristol Myers Squibb, Celgene, Genentech, Janssen, Kite, Macrogenics, and Pfizer. The other authors declare no competing financial interests.

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Godwin, C.D., Laszlo, G.S., Fiorenza, S. et al. Targeting the membrane-proximal C2-set domain of CD33 for improved CD33-directed immunotherapy. Leukemia (2021).

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