CD33 Antibody

What is CD33 and why is it a significant target in myeloid malignancy research?

CD33 is a 67 kDa transmembrane glycoprotein also known as SIGLEC3 (sialic acid-binding immunoglobulin-like lectin 3). It belongs to the SIGLEC family and functions as a sialic acid-binding receptor with 2-3 extracellular domains - the membrane-distal immunoglobulin V (IgV) domain and the membrane-proximal immunoglobulin C (IgC) domain - along with an intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) . CD33 is expressed on myeloid cells and myeloid progenitors, with particularly high expression on AML blasts in more than 85-90% of patients, making it an attractive therapeutic target . Notably, CD33 has endocytic properties and internalizes when bound by antibodies, which has been exploited for the delivery of cytotoxic agents to leukemic cells .

How variable is CD33 expression across AML subtypes?

CD33 expression demonstrates significant variability across different AML genetic subtypes. Comprehensive analyses of adult AML patients have revealed that:

Bone marrow blasts of AML patients express between 709 and 54,894 CD33 molecules/cell (mean 10,380 molecules/cell), compared to only 859–5137 molecules/cell (mean 2997 molecules/cell) detected in normal CD33-positive bone marrow cells . This differential expression provides a therapeutic window for targeting AML cells while potentially sparing normal hematopoietic stem cells.

How can we accurately quantify CD33 expression levels on leukemic and normal cells?

Multiple methodologies can be employed to quantify CD33 expression:

• Flow Cytometry: The gold standard for determining CD33 expression on cell surfaces. Protocols typically involve:

  • Incubating AML cells with mouse anti-human CD33 antibodies

  • Washing cells with PBS to remove unbound antibodies

  • Staining with PE-labeled goat anti-mouse IgG secondary antibodies

  • Analyzing expression through flow cytometry

• Western Blot Analysis: Used to assess total CD33 protein expression in cell lysates, particularly useful for detecting different isoforms. The protocol outlined in search result describes using PVDF membranes probed with anti-CD33 monoclonal antibodies followed by HRP-conjugated secondary antibodies.

• Immunohistochemistry: For tissue samples such as bone marrow biopsies, IHC protocols involving heat-induced epitope retrieval and DAB staining can visualize CD33 expression patterns across different tissue compartments .

When quantifying CD33, researchers should consider both the absolute number of CD33 molecules per cell and the relative intensity of expression compared to control cells.

How can researchers distinguish between different CD33 isoforms?

Distinguishing between CD33 isoforms, particularly the full-length CD33 (CD33FL) and the exon 2-skipped variant (CD33ΔE2 or CD33D2), requires isoform-specific detection methods:

• Domain-Specific Antibodies:

  • Antibodies targeting the IgV domain (e.g., clones WM53, P67.6) detect only the full-length CD33

  • Antibodies targeting the IgC domain (e.g., clone 1c7/1, HL2541, 5C11-2) can detect both CD33FL and CD33ΔE2

• Flow Cytometry with Domain-Specific Antibodies: When CD33 ΔE2-DAP12-GCaMP6m lines were tested, antibody clones WM53 and P67.6 showed no staining (1.29% ± 0.23% and 1.32% ± 0.28%, respectively), while clone 1c7/1 detected variant 2 CD33 expression (75.72% ± 6.93%) .

• PCR-Based Detection: Genotyping for the rs12459419 SNP, which leads to exon 2 skipping, can indirectly identify patients likely to express the CD33ΔE2 isoform .

• Western Blot Analysis: Can distinguish isoforms based on molecular weight differences.

Recent studies have developed novel CD33 antibodies specifically to understand the localization, biology, and therapeutic implications of CD33 isoforms .

What are the current CD33-targeted antibody therapies in development for AML?

Several antibody-based approaches targeting CD33 are being investigated:

• Antibody-Drug Conjugates:

  • Gemtuzumab ozogamicin (GO): A humanized anti-CD33 antibody conjugated to calicheamicin, reapproved in 2017 for AML treatment

  • HUM-195/rGel: An anti-CD33 immunotoxin using humanized M195 antibody conjugated to recombinant gelonin

• Bispecific T-Cell Engagers (BiTEs):

  • AMG 330: A CD33/CD3-bispecific antibody that engages T cells to eliminate CD33+ leukemic cells. It has shown potent cytolytic activity in vitro with EC50 values ranging between 0.4 pmol/L and 3 pmol/L (18–149 pg/mL)

  • Other BiTEs targeting either the IgV domain (BC133, BC269) or the IgC domain (based on the HIM3-4 antibody)

• Tetravalent Bispecific Antibodies (TandAbs):

  • CD33/CD3 TandAbs: These provide two binding sites for each antigen to maintain the avidity of a bivalent antibody and have shown efficacy in preclinical models

• Electrostatic Antibody-Protamine Nanocarriers:

  • Anti-CD33-antibody–protamine nanocarriers for siRNA delivery against mutated DNMT3A and FLT3 in leukemia

Each approach offers unique advantages in terms of mechanism of action, potency, and potential to overcome resistance mechanisms.

How does CD33 expression heterogeneity impact therapeutic response to antibody-based treatments?

CD33 expression heterogeneity significantly impacts therapeutic efficacy:

Understanding this heterogeneity is crucial for patient selection and predicting therapeutic response.

How does the CD33ΔE2 isoform differ from full-length CD33 in terms of cellular localization and function?

The CD33ΔE2 isoform presents significant differences from full-length CD33:

These findings highlight the importance of understanding isoform-specific biology when developing CD33-targeted therapeutics.

What strategies can overcome resistance to CD33-targeted therapies in AML?

Several strategies have been developed to address resistance mechanisms:

• Targeting Alternative CD33 Domains:

  • Developing antibodies against the IgC domain to target both CD33FL and CD33ΔE2 isoforms

  • Using dual-targeting approaches that recognize multiple CD33 epitopes

• Combination with Epigenetic Modifiers:

  • Panobinostat (a histone deacetylase inhibitor) and azacitidine (a DNA methyltransferase inhibitor) increased CD33 expression in some cell lines and augmented AMG 330-induced cytotoxicity

  • These epigenetic modifiers can potentially resensitize resistant cells by upregulating CD33 expression

• Addressing ABC Transporter Activity:

  • Unlike traditional antibody-drug conjugates, BiTE antibodies like AMG 330 showed activity independent of adenosine triphosphate-binding cassette (ABC) transporter proteins like P-glycoprotein or breast cancer resistance protein

  • This independence from efflux mechanisms represents an important advance over previous CD33-targeted agents

• Novel Delivery Systems:

  • Electrostatic anti-CD33 antibody-protamine nanocarriers for siRNA delivery represent an alternative approach that may overcome certain resistance mechanisms

• T-Cell Engagement Strategies:

  • AMG 330 efficiently recruited and expanded residual CD3+/CD45RA−/CCR7+ memory T cells within patient samples

  • Even at low effector to target ratios, the recruited T cells lysed autologous blasts completely in the majority of samples in a time-dependent manner

These approaches may be particularly valuable for patients with low CD33 expression or CD33 isoform variations.

What are the optimal experimental conditions for evaluating anti-CD33 antibody efficacy in vitro?

For robust evaluation of anti-CD33 antibodies, consider these methodological approaches:

• Cell Line Selection:

  • Use multiple CD33+ AML cell lines with varying CD33 expression levels (e.g., MOLM-13, HL-60, U937)

  • Include CD33- cell lines as negative controls

  • For BiTE antibodies, ensure lines with different CD33 expression levels are tested to establish dose-response relationships

• Primary Cell Experiments:

  • Use MS-5 feeder cell-based long-term cultures that can support primary AML blast growth for up to 36 days

  • Include samples representing different genetic backgrounds and CD33 expression levels

  • For T-cell engaging antibodies, assess both allogeneic and autologous T-cell responses

• Assay Parameters:

  • Cytotoxicity Assays: Test multiple effector-to-target ratios (typically ranging from 1:1 to 10:1)

  • Dose Range: Use wide concentration ranges (e.g., for AMG 330, from sub-picomolar to nanomolar)

  • Time Course: Measure responses at multiple time points (24, 48, 72 hours) as complete lysis may require up to 40 hours

• Functional Readouts:

  • T-Cell Activation: Measure CD69 and CD25 expression

  • Cytokine Release: Assess IFN-γ, TNF, IL-2, IL-10, and IL-6 levels

  • Cytotoxicity: Use flow cytometry-based or luminescence-based cell death assays

• Controls and Variables to Consider:

  • Test antibody specificity using CD33 knockout cells

  • Include soluble CD33 at physiologically relevant concentrations to assess potential interference

  • Evaluate the impact of CD33 polymorphisms and isoforms

What are the key considerations for translating CD33 antibody research from in vitro to in vivo models?

Successful translation to in vivo models requires:

• Animal Model Selection:

  • Xenograft Models: Both cell line-derived (e.g., MOLM-13, HL-60) and patient-derived xenografts in immunodeficient mice have been used

  • Humanized Immune System Models: For T-cell engaging antibodies, models with reconstituted human immune components are preferable

• Dosing Strategies:

  • For AMG 330, daily intravenous administration at doses as low as 0.002 mg/kg significantly prolonged survival in mouse models

  • Consider pharmacokinetic data from similar antibody constructs; for example, HUM-195/rGel showed highest blood levels of 200-300 ng/mL with a half-life of ~20 hours

• Efficacy Endpoints:

  • Tumor Growth Inhibition: Monitor both prophylactic and established tumor models

  • Survival Analysis: Kaplan-Meier survival curves with appropriate statistical analysis

  • Pharmacodynamic Markers: Monitor changes in CD33+ cells in peripheral blood and bone marrow

• Safety Considerations:

  • Monitor for cytokine release syndrome, a common side effect of T-cell engaging antibodies

  • Assess potential off-target effects on normal CD33+ myeloid cells

  • In the phase 1 study of HUM-195/rGel, the dose-limiting toxicity was infusion-related allergic reaction including hypoxia and hypotension

• Translation to Non-Human Primates:

  • AMG 330 binds with low nanomolar affinity to CD33 and CD3ϵ of both human and cynomolgus monkey origin

  • Ex vivo studies in cynomolgus monkey bone marrow aspirates can assess autologous depletion of CD33-positive cells

How should researchers interpret CD33 expression data in the context of potential therapeutic response?

Interpretation of CD33 expression data requires consideration of several factors:

• Expression Level Thresholds:

  • No universal cut-off has been established for "high" versus "low" CD33 expression

  • Consider quartile-based analyses as used in clinical studies: patients with CD33 expression in the highest quartile may show differential response compared to those in the lowest quartile

• Genetic Context:

  • Integrate CD33 expression data with genetic information such as:

    • NPM1 mutation status (associated with higher CD33)

    • FLT3-ITD status (associated with higher CD33)

    • Core binding factor translocations (associated with lower CD33)

• Leukemic Stem Cell (LSC) Expression:

  • Consider CD33 expression specifically on the CD34+/CD38- LSC population

  • Higher CD33 on LSCs compared to normal HSCs suggests potential for selective targeting and long-term disease control

• Isoform Analysis:

  • Determine rs12459419 genotype, which predicts CD33ΔE2 expression

  • Consider both surface and intracellular expression patterns of CD33 isoforms

• Dynamic Changes:

  • CD33 expression may change with disease progression or treatment

  • Consider repeat assessment at relapse or after exposure to modifying agents

A comprehensive interpretation would integrate these factors to assess the likelihood of response to CD33-targeted therapies and help guide clinical decision-making.

What are the most significant challenges in developing CD33 antibodies that address inter-patient variability?

Developing universally effective CD33 antibodies faces several challenges:

• Genetic Heterogeneity:

  • The rs12459419 SNP drives exon 2 skipping, creating the CD33ΔE2 isoform that lacks the IgV domain targeted by most current antibodies

  • Patients heterozygous for this variant show diminished response to gemtuzumab ozogamicin

• Expression Level Variability:

  • AML patients show a wide range of CD33 expression (709-54,894 molecules/cell)

  • Lower expression levels may reduce efficacy of CD33-targeted therapies

  • Strategies to upregulate CD33 expression (e.g., with epigenetic modifiers) may not work uniformly across patients

• Technical Challenges in Dual-Domain Targeting:

  • Developing antibodies that effectively target both IgV and IgC domains simultaneously

  • Ensuring adequate accessibility of the IgC domain, which may be partially obscured in the full-length protein

• Balancing Efficacy and Safety:

  • Higher-affinity antibodies may provide better efficacy but potentially greater toxicity to normal myeloid cells

  • The phase 1 study of HUM-195/rGel determined that 28 mg/m² was the maximally tolerated dose due to infusion-related reactions

• Soluble CD33 Interference:

  • Soluble CD33 in patient circulation may interfere with antibody binding

  • Although some in vitro evidence suggests that soluble CD33 may not affect the activity of CD33-targeted immunotherapy, this remains a consideration

Addressing these challenges requires multi-domain targeting approaches, personalized dosing strategies, and combination therapies tailored to individual patient characteristics.