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

Abstract

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 CD33^V-set/CD3 bispecific antibody (BsAb) and CD33^V-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 ("CD33^PAN antibodies"). These antibodies internalized when bound to CD33 and, as CD33^PAN/CD3 BsAb, had potent cytolytic effects against CD33⁺ cells. Together, our data provide the rationale for further development of CD33^PAN antibody-based therapeutics.

Figure 1. Membrane proximity of the target epitope modulates the anti-tumor efficacy of CD33/CD3 BsAbs and CAR T cells.

(A) Schematic of full-length CD33 (CD33^FL) and artificial CD33 molecules with deletion of exons 3 and 4, resulting in membrane proximal relocation of the V-set domain (CD33^ΔE3-4), or insertion of of either 2 C2-set domains of CD22 (CD33^FL + CD22 2D) or 4 C2-set domains of CD22 (CD33^FL + CD22 4D). (B-D) AML cell lines with CRISPR/Cas9-mediated deletion of the endogenous CD33 locus were engineered to overexpress either CD33^FL or CD33^ΔE3-4 via lentiviral gene transfer. Relative expression of the target proteins was flow cytometrically assessed via V-set domain CD33 antibody, P67.6, with representative histograms shown in the far-right panel. Cells were then treated with a V-set domain-targeting CD33/CD33 BsAb at a concentration of 1000 pg/mL and at the effector:target (E:T) cell ratios shown (left panel). Myeloid cells were also treated with gemtuzumab ozogamicin (GO) at the concentrations shown (middle panel). (E) The AML cell line ML-1 with CRISPR/Cas9-mediated deletion of the endogenous CD33 locus was engineered to overexpress CD33^FL, CD33^FL + CD22 2D or CD33^FL + CD22 4D via lentiviral gene transfer. Relative expression of the CD33 constructs was flow cytometrically assessed using the V-set domain CD33 antibody, P67.6 (right panel). Cells were then treated with a V-set domain-targeting CD33/CD33 BsAb at the concentrations shown at an E:T cell ratio of 1:1. (F) V-set domain-directed CAR T cells were assessed for cytotoxicity in a chromium⁵¹ release assay against the CD33^FL and CD33^ΔE3-4 expressing K562 sublines from (D). Mean+SEM from a minimum of three separate experiments is shown. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2. Biophysical characterization of the CD33^PAN antibody, 1H7.

(A) Murine CD33^PAN antibodies (clones 1H7, 9G2, 6H9) were tested flow cytometrically against parental ML-1 cells as well as ML-1 cells with CRISPR/Cas9-mediated deletion of CD33 (“CD33 KO”), as well as CD33^neg REH sublines engineered to express CD33^FL or CD33yyyyyyy^ΔE2, as indicated. A control without primary antibody was included. (B) Binding of recombinant 1H7 and anti-V-set domain antibody P67.6 was compared flow cytometrically against parental CD33^neg REH cells and subclones engineered via lentiviral gene transfer to overexpress full-length CD33 (CD33^FL), a CD33 variant with deletion of exon 2 resulting in loss of the V-set domain (CD33^ΔE2), and an artificial CD33 molecule with deletion of exons 3 and 4 resulting in loss of the C2-set domain (CD33^ΔE3-4). (C) Surface plasmon resonance assessment of purified extracellular domains from CD33^FL or CD33^ΔE2 binding to captured 1H7. Black lines are data and gray lines are the model fits. (D) Binding of 1H7 and P67.6 to parental ML-1 and TF-1 cells and cells with CRISPR/Cas9-mediated knockout of the CD33 locus (CD33 KO) was assessed flow cytometrically. Secondary antibody only negative control is shown. (E) Binding of 1H7 and P67.6 to parental AML cell lines was assessed flow cytometrically. Secondary antibody only negative control is shown. (F) Internalization of 1H7 and P67.6 s. AML cell lines were incubated with CD33 antibody at 37°C for the time indicated. Fluorescently labeled secondary antibody was then added to quantify remaining CD33 antibody on the cell surface. Results are presented as a percentage of the fluorescence signal present at time 0. Mean±SEM of 3 separate experiments is shown. (G) Modulation of CD33 expression by continuous exposure to anti-CD33 antibodies 1H7 and P67.6. ML-1 and HL-60 cells were exposed continuously to 1H7 or P67.6 for 24 hours. Primary antibody was then added again in excess to bind all available CD33 molecules, and fluorescently labeled secondary antibody was added for flow cytometric quantification. Results are presented as percentage change in fluorescence compared to control cells untreated with antibody. Mean±SEM of 3 separate experiments are shown. For all AML cell line experiments, CD33 rs12459419 genotype is shown in parentheses.

Figure 3. A murine CD33^PAN/CD3 BsAb redirects T cell-mediated cytotoxicity against AML cells in a CD33- and epitope-specific manner.

(A) Parental AML cell lines were treated with healthy donor T cells at the effector:target (E:T) cell ratios shown and 1H7/CD3 BsAb. (B) Parental ML-1 cells and a subline with CRISPR/Cas9-mediated knockout (KO) of CD33 were treated with 1H7 scFv-scFv BsAb at 500 pg/mL and healthy donor T cells at an E:T of 1:1. (C) Parental REH cell or a subline engineered to overexpress CD33^ΔE2 (left panel), and parental ML-1 cells, ML-1 CD33 KO cells, and CD33 KO cells with overexpression of CD33^ΔE2 (CD33 KO + CD33^ΔE2) were treated with 1H7/CD3 IgG-scFv BsAb at a concentration of 500 pg/mL and healthy donor T cells at an E:T of 1:1. In cell line experiments, dead leukemic cells were enumerated after 48 hours via flow cytometry, and change in dead cells compared to no BsAb treatment is shown. CD33 rs12459419 genotype is shown in parentheses. Mean±SEM of 3 separate experiments are shown. *p<0.05; **p<0.01. (D) A panel of 11 primary AML patient samples was treated with the 1H7/CD3 BsAb and healthy donor T-cells at the E:T ratios shown. Mean±SEM across the 11 patient samples is shown. Cytotoxicity was determined enumerating both dead cells and total cell number as described.^,

Figure 4. A human CD33^PAN/CD3 BsAb redirects T cell-mediated cytotoxicity against AML cells in a CD33- and epitope-specific manner.

(A) Human CD33^PAN antibody clones (clones 1A9, 1H10, 1B9, 1E6, and 1D2) were tested flow cytometrically against parental ML-1 cells as well as ML-1 cells with CRISPR/Cas9-mediated deletion of CD33 (“CD33 KO”), as well as CD33^neg REH sublines engineered to express CD33^FL or CD33^ΔE2, as indicated. A control without primary antibody was included. (B) Parental AML cell lines were treated with healthy donor T cells at the effector:target (E:T) cell ratios shown and various doses of 1E6/CD3 BsAb. (C) Parental ML-1 cells and a subline with CRISPR/Cas9-mediated knockout (KO) of CD33 were treated with 1E6 BsAb at the indicated concentrations and healthy donor T cells at an E:T of 1:1. (D) Parental CD33^neg REH cells or sublines engineered to overexpress CD33^FL or CD33^ΔE2 were treated with anti-V-set CD33/CD3 BsAb or 1E6 BsAb at a dose of 1000 pg/mL and an E:T of 3:1. In cell line experiments, dead leukemic cells were enumerated after 48 hours via flow cytometry, and change in dead cells compared to no BsAb treatment is shown. CD33 rs12459419 genotype is shown in parentheses. Mean±SEM of three separate experiments are shown. ***p<0.001; ****p<0.0001. (E) A panel of 11 primary AML patient samples was treated with the 1E6/CD3 BsAb and healthy donor T-cells at the E:T ratios shown. Mean cytotoxicity±SEM across the 11 patient samples is shown. Cytotoxicity was determined enumerating both dead cells and total cell number as described.^,