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3436 Targeting the Membrane-Proximal Domain of CD33 to Maximize the Efficacy of Natural Killer Cell-Based Immunotherapies

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies other than CAR-T Cells: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Myeloid Malignancies
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Sheryl Y. T. Lim, DPhil1*, Frances M. Cole1*, George S. Laszlo, PhD2*, Margaret C. Lunn-Halbert1*, Jenny Huo1*, Junyang Li1*, Allie R. Kehret1* and Roland B. Walter, MD, PhD, MS3,4,5

1Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA
21100 Fairview Avenue North, Fred Hutchinson Cancer Center, Seattle, WA
3Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, WA
4Fred Hutchinson Cancer Center, Seattle, WA
5Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA

Background: Improved survival of some patients with AML with the antibody-drug conjugate gemtuzumab ozogamicin (GO) validates CD33 as a therapeutic target, but GO is often ineffective. As one limitation, hP67.6 (used in GO) and almost all other CD33 antibodies recognize the membrane-distal V-set domain. This may be problematic given existence of a CD33 variant lacking this domain. Moreover, we have shown that CD33/CD3 bispecific antibodies (BiAbs) and chimeric antigen receptor (CAR)-modified T cells binding CD33 closer to the cell membrane exert greater T-cell mediated cytotoxicity than those binding distally. Here, we investigated whether this principle applies to CD33-targeted therapies harnessing natural killer (NK) cells.

Methods: We compared NK cell-mediated cytotoxicity against human leukemia cells expressing full-length CD33 (CD33FL) vs. isogenic cells expressing similar levels of a CD33 molecule lacking the membrane proximal C2-set domain (CD33ΔE3-4), bringing the V-set domain into immediate cell membrane proximity. We studied 3 types of therapeutics: 1) unconjugated V-set-directed CD33 IgG1 antibodies; 2) CD33Vset/CD16a BiAbs; and 3) CD33V-set-directed CAR-NK cells. NK cell-mediated cytotoxicity was determined flow cytometrically in co-culture assays by quantifying cell numbers and proportion of non-viable target leukemia cells.

Results: First, we investigated antibody-dependent cell-mediated cytotoxicity (ADCC) of CD33V-set antibodies with NK-92 cells transduced with high-affinity CD16a (NK-92CD16a) and primary human NK cells as effectors. Three different CD33 antibodies (including lintuzumab) elicited dose-dependent cytotoxicity against human leukemia cells overexpressing CD33FL or CD33∆E3-4 but not against cells lacking CD33. Importantly, the CD33V-set antibodies exhibited greater ADCC against cells expressing CD33∆E3-4 than cells expressing CD33FL. We then tested the effect of membrane proximity on the efficacy of CD33Vset/CD16a BiAbs in IgG4-scFv format. Like CD33V-set antibodies, the CD33Vset/CD16a BiAb (using sequences from lintuzumab) induced greater cytotoxicity against cells expressing CD33∆E3-4 than cells expressing CD33FL. In a third series of experiments, we investigated the effect of membrane proximity on the efficacy of human NK cells (KHYG-1 cells) transduced with CD33V-set-directed CARs. Both the lintuzumab- and hP67.6-based CAR-NK cells demonstrated dose-dependent, CD33-specific cytotoxicity. Both CAR-NK cell products exhibited greater cytotoxicity against cells expressing CD33∆E3-4 than cells expressing CD33FL. Consistent with this notion of greater activation upon membrane proximal engagement, CD33V-set CAR-NK cells showed higher intracellular TNFα and IFNγ levels after co-culture with CD33∆E3-4-expressing cells compared to CD33FL-expressing cells.

Our observations provided the rationale to explore CD33C2-set-directed therapeutics. We previously generated a panel of murine and human antibodies that bind the C2-set domain regardless of the presence or absence of the V-set domain (“CD33PAN antibodies). In the presence of NK-92CD16a cells, all 4 CD33PAN antibodies we tested induced CD33-specific ADCC against human AML cell lines. Finally, we generated CD33C2-set-directed CAR-NK cells using the sequences from 5 CD33PAN antibodies. Compared to non-targeting CAR-NK cells, CD33PAN CAR-NK cells induced substantially greater cytotoxicity against AML cell lines as well as primary human AML cells with a range of CD33 molecules expressed on leukemic cells.

Conclusions: Our data indicate decreasing the distance between CD33 binding epitope and leukemia cell membrane enhances the efficacy of CD33-directed NK cell therapies. These findings are the first to show that membrane proximity may matter for ADCC efficacy even for a relatively small target antigen like CD33, with a change in epitope position by ~4 nm sufficing to yield differences. Our studies are also the first to show an advantage of targeting CD33 membrane-proximally to enhance CAR-NK efficacy. Together, our findings support the further development of CD33C2-set-directed NK cell-based therapies for AML and other CD33-expressing neoplasms.

Disclosures: Walter: Pfizer: Research Funding; VOR: Research Funding; Jazz: Research Funding; Aptevo: Research Funding; Janssen: Research Funding; Kura: Research Funding; ImmunoGen: Research Funding; Wugen, Inc.: Consultancy; Kite: Research Funding; Celgene/Bristol Myers Squibb: Research Funding.

*signifies non-member of ASH