CML33 Antibody

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

CD33, also known as Siglec-3 (Sialic acid-binding immunoglobulin-like lectin 3), is a 67-kDa glycoprotein expressed predominantly on cells of myelomonocytic lineage. It consists of an N-terminal Ig-like V-type domain, one Ig-like C2-type domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs) . CD33 has gained significant attention in hematological research because its expression is restricted to myeloid cells, making it an ideal target for acute myeloid leukemia (AML) therapy. The protein binds sialic acid with preference for alpha 2,3-linkage over alpha 2,6-linkage and can mediate inhibitory signals through recruitment of phosphatases SHP-1 and SHP-2 to its ITIMs .

How do CD33 antibodies function in research and therapeutic applications?

CD33 antibodies function through multiple mechanisms depending on their design and application. In research settings, they serve as valuable tools for cell identification, isolation, and characterization of myeloid populations. Therapeutically, anti-CD33 antibodies can work through:

• Direct binding and signaling - Some antibodies like clones P67.6 and 1c7/1 have been validated as agonistic antibodies that can activate the CD33 pathway .

• Antibody-dependent cellular cytotoxicity (ADCC) - Fc-engineered antibodies like BI 836858 significantly induce both autologous and allogeneic natural killer (NK) cell degranulation and NK-cell-mediated ADCC against AML cells .

• Targeted drug delivery - When conjugated with cytotoxic agents (antibody-drug conjugates or ADCs), CD33 antibodies can deliver potent payloads specifically to CD33-expressing cells, as demonstrated by the FDA-approved Mylotarg (gemtuzumab ozogamicin) .

Importantly, methodological considerations include antibody clone selection, as different epitope binding can significantly affect function and specificity for CD33 variants .

Which CD33 antibody clones are most appropriate for different experimental applications?

The selection of CD33 antibody clones should be based on the specific experimental requirements and the CD33 domain of interest:

• Clone 1c7/1: Binds an epitope in the constant C2-set Ig-like domain, allowing detection of both full-length CD33M and the exon 2-deleted variant (CD33 ΔE2) . This makes it ideal for studies examining both CD33 isoforms.

• Clones WM53 and P67.6: Bind epitopes proximal to the sialic acid binding domain in the V-set Ig-like domain, making them specific for full-length CD33M only . In flow cytometry experiments, these antibodies showed high specificity with 93.72% ± 1.55% and 92.42% ± 1.89% positive cells respectively for full-length CD33, while showing no significant binding (1.29% ± 0.23% and 1.32% ± 0.28%) to the CD33 ΔE2 variant .

For functional studies, both P67.6 and 1c7/1 have been validated as agonistic antibodies capable of activating CD33 signaling and modulating TREM2 signaling at the level of SYK phosphorylation .

What are the optimal protocols for using CD33 antibodies in flow cytometry applications?

For optimal flow cytometry results with CD33 antibodies, researchers should consider:

• Sample preparation: Use fresh samples when possible. For cell lines like U937 human histiocytic lymphoma (CD33-positive) and MOLT-4 human acute lymphoblastic leukemia (CD33-negative), immersion fixation has produced reliable results .

• Antibody concentration: Titration is essential, but starting concentrations of 2-8 μg/mL have been effective in published protocols. For example, 8 μg/mL for 3 hours at room temperature has been successful for immunocytochemistry applications .

• Controls: Always include appropriate isotype controls and known positive (e.g., U937) and negative (e.g., MOLT-4) cell lines to validate staining specificity .

• Detection system: For indirect staining, secondary antibodies such as NorthernLights™ 557-conjugated Anti-Mouse IgG have shown good results when counterstained with DAPI .

• CD33 variant consideration: When studying CD33 variants, use multiple antibody clones (e.g., 1c7/1 for all variants and WM53 or P67.6 for full-length specific detection) to distinguish between isoforms .

How can CD33 antibodies be optimized for antibody-drug conjugate (ADC) development?

Optimization of CD33-targeted ADCs requires careful consideration of three key components:

• Antibody selection: The antibody should have high specificity and affinity for CD33, with minimal cross-reactivity. Full-length antibodies are typically preferred for ADCs to maintain longer half-life, though fragment-based approaches have also been explored .

• Linker chemistry: The linker between antibody and payload must balance stability in circulation with efficient release in target cells. Current research has focused on developing novel linker technologies that improve the therapeutic index of CD33-targeted ADCs compared to earlier generation ADCs like Mylotarg .

• Payload selection: Cytotoxic payloads must be potent enough to kill target cells at low concentrations. Research has shown that optimization of all ADC components (antibody, linker, and payload) has resulted in next-generation CD33-targeted ADCs with improved safety profiles and therapeutic indices in preclinical models of AML .

What strategies can enhance the efficacy of CD33 antibody therapies in combination treatment approaches?

Several strategies have demonstrated promise for enhancing CD33 antibody efficacy in combination approaches:

• Combination with hypomethylating agents: Decitabine (DAC) has shown synergistic effects with CD33 antibodies. For example, BI 836858-mediated ADCC was significantly higher in AML samples collected 28 days post-DAC treatment compared to pre-DAC samples . The mechanism involves DAC-induced upregulation of NKG2D ligands on AML cells, enhancing their susceptibility to antibody-mediated killing .

• Fc engineering: Modification of the Fc region of CD33 antibodies can enhance effector functions. BI 836858, a fully human Fc-engineered anti-CD33 antibody, demonstrated significant induction of both autologous and allogeneic NK cell degranulation and ADCC against AML cells .

• Optimizing treatment schedules: Serial testing of BI 836858-mediated ADCC in AML patients receiving a 10-day course of DAC showed that efficacy was highest at day 28 post-DAC, suggesting that optimal timing of combination therapy is crucial .

• Rational selection of combination partners: In vitro studies have shown that combination of CD33 antibodies with agents that enhance effector cell function or increase target antigen expression can significantly improve efficacy without increasing toxicity .

How do CD33 genetic variants affect antibody binding and functional studies?

CD33 genetic variants significantly impact antibody binding and downstream applications in several important ways:

• Exon 2 deletion variant (CD33 ΔE2): The Alzheimer's disease-protective SNP rs3865444(A) is co-inherited with rs12459419(T), which mediates exon 2 splicing, resulting in a CD33 variant lacking part of the IgV sialic acid-binding domain . This has profound implications for antibody selection:

  • Antibodies targeting epitopes in the V-set domain (like WM53 and P67.6) cannot detect the CD33 ΔE2 variant

  • Antibodies binding the C2-set domain (like 1c7/1) can detect both full-length CD33M and the ΔE2 variant

• Functional differences: The CD33 ΔE2 variant shows altered functional properties. In induced pluripotent stem cell-derived microglia, agonistic CD33 antibodies directly counteracted TREM2-triggered phosphorylation of SYK and decreased phagocytic uptake of S. aureus BioParticles in wild-type CD33M expressing cells, but had no effect in CD33 ΔE2 expressing cells .

• Experimental validation: When developing CD33-targeted therapies or research tools, validation with both variants is crucial. The human cell-based CD33 reporter system developed in study confirmed that only clones 1c7/1 and P67.6 acted as agonistic CD33-specific antibodies, and their effects were dependent on the CD33 variant expressed.

What are the key considerations for developing CD33 antibodies that target specific isoforms?

Developing isoform-specific CD33 antibodies requires several methodological considerations:

• Epitope mapping: Careful identification of epitopes unique to specific CD33 isoforms is essential. For example, antibodies targeting the V-set domain will be specific to full-length CD33, while those targeting the C2-set domain may recognize multiple isoforms .

• Validation across expression systems: Testing antibody binding and function in multiple systems expressing different CD33 isoforms is critical. Flow cytometry analysis using reporter cell lines expressing either full-length CD33M-DAP12 or variant CD33 ΔE2-DAP12 has been used to validate isoform specificity .

• Functional characterization: Beyond binding, functional effects of antibodies may differ between isoforms. For instance, agonistic antibodies like P67.6 and 1c7/1 activate signaling in cells expressing full-length CD33 but not in those expressing the ΔE2 variant .

• Cross-validation with genetic approaches: Complementing antibody studies with genetic manipulation (knockout or variant-specific expression) provides more robust validation of isoform-specific effects.

• Clinical relevance assessment: For therapeutic development, understanding the expression patterns of different CD33 isoforms in disease states is crucial. The prevalence of CD33 variants in patient populations may impact the efficacy of isoform-specific targeting strategies.

How can researchers effectively measure CD33 antibody-mediated ADCC in primary AML samples?

Measuring CD33 antibody-mediated ADCC in primary AML samples requires careful experimental design:

• Sample collection and processing: Primary AML blasts should be isolated from bone marrow aspirates or peripheral blood using density gradient centrifugation and cryopreserved in liquid nitrogen until use. Viability assessment post-thawing is essential, with samples showing >70% viability being optimal .

• Effector cell preparation: For autologous ADCC, NK cells should be isolated from the same patient sample. For allogeneic ADCC, healthy donor NK cells can be isolated using negative selection kits to avoid activation. NK cells should be assessed for purity by CD56+CD3- phenotype via flow cytometry .

• ADCC assay setup:

  • Target cells (AML blasts) should be labeled with a suitable marker (e.g., CFSE)

  • Cells should be opsonized with the CD33 antibody of interest (e.g., BI 836858 at 10 μg/mL)

  • Effector and target cells should be co-cultured at various E:T ratios (typically 1:1 to 10:1)

  • After 4-6 hours, samples can be analyzed for target cell lysis

• Readout methods: Multiple approaches can be used to quantify ADCC:

  • Flow cytometry-based methods measuring target cell viability

  • Chromium-51 release assays

  • Lactate dehydrogenase (LDH) release assays

• Controls: Include non-opsonized targets, isotype antibody controls, and known CD33-negative cells as controls. For studies on treatment effects (e.g., decitabine), include matched pre-treatment and post-treatment samples from the same patient .

Using this approach, researchers have demonstrated significantly higher ADCC in samples collected 28 days post-decitabine treatment compared to pre-treatment samples, providing evidence for enhanced CD33 antibody efficacy following hypomethylating agent therapy .

What reporter systems can verify the functionality of CD33 antibodies in research applications?

Several reporter systems have been developed to verify CD33 antibody functionality:

• DAP12-GCaMP6m reporter system: This system utilizes the calcium-sensitive fluorescent protein GCaMP6m coupled to DAP12, a signaling adaptor protein. When CD33 is activated by agonistic antibodies:

  • The reporter cells exhibit increased fluorescence intensity

  • The system can distinguish between antibodies that activate CD33 signaling (P67.6 and 1c7/1) and those that merely bind without activation

  • The system allows quantitative measurement of activation kinetics

• SYK phosphorylation assays: Since CD33 modulates SYK phosphorylation through its ITIMs, measuring changes in phospho-SYK levels by western blot or flow cytometry after antibody treatment provides a direct readout of CD33 activity. In iPSC-derived microglia, treatment with agonistic CD33 antibodies (P67.6 and 1c7/1) counteracted TREM2-triggered SYK phosphorylation .

• Functional phagocytosis assays: CD33 activation inhibits phagocytosis, so measuring the uptake of fluorescently labeled particles (e.g., pHrodo-labeled S. aureus BioParticles) in the presence of CD33 antibodies can verify their functional impact. Agonistic CD33 antibodies decreased phagocytic uptake in wild-type CD33M expressing cells but not in CD33 ΔE2 or CD33-/- cells .

• NK cell degranulation assays: For antibodies designed to induce ADCC, measuring NK cell degranulation (CD107a surface expression) upon co-culture with antibody-opsonized target cells provides functional verification .

These reporter systems not only validate antibody functionality but also help distinguish between different mechanisms of action and can identify antibodies with therapeutic potential.