FLT3 Antibody, FITC conjugated

What is FLT3 and why is it targeted in hematological research?

FLT3 (FMS-related tyrosine kinase receptor 3) is a class III receptor tyrosine kinase predominantly expressed on leukemic cells of the myeloid and lymphoid lineage. It has emerged as a significant therapeutic target in hematologic malignancies due to its frequent overexpression or constitutive activation in acute myeloid leukemia (AML) . FLT3 is often affected by internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, which contribute to leukemogenesis and are associated with poor clinical outcomes . The receptor's relatively specific expression pattern on leukemic cells makes it an attractive target for antibody-based therapies, allowing for selective targeting of malignant cells while potentially sparing normal tissues .

How does FITC conjugation enhance FLT3 antibody applications?

FITC (fluorescein isothiocyanate) conjugation provides a direct fluorescent labeling approach that enables visualization and quantification of FLT3 expression through flow cytometry, fluorescence microscopy, and other fluorescence-based detection methods. The conjugation process involves covalent binding of the fluorescent dye to the antibody without significantly altering binding properties when optimally performed. When working with FITC-conjugated FLT3 antibodies, researchers should be aware that:

• Optimal excitation occurs at approximately 495 nm with emission at 519 nm

• FITC conjugation allows direct detection without secondary antibodies

• The conjugation ratio (number of FITC molecules per antibody) affects both signal intensity and potential interference with antigen binding

What expression levels of FLT3 can be expected on different cell populations?

Based on research findings, FLT3 expression varies significantly across different cell types:

When designing experiments, it's critical to account for these expression differences when setting detection thresholds and interpreting results .

What are the optimal protocols for detecting FLT3 using FITC-conjugated antibodies?

For reliable FLT3 detection using FITC-conjugated antibodies, follow these methodological guidelines:

• Sample preparation:

  • Use freshly isolated cells when possible

  • For peripheral blood or bone marrow, perform red blood cell lysis using ammonium chloride-based buffers

  • Maintain cells at 4°C throughout processing to prevent receptor internalization

• Staining protocol:

  • Use 5-10 μL of antibody per 10⁶ cells (adjust based on titration experiments)

  • Incubate for 30 minutes at 4°C in the dark

  • Wash cells twice with phosphate-buffered saline containing 2% fetal bovine serum

  • Fix cells with 1-2% paraformaldehyde if analysis will be delayed

• Controls and validation:

  • Include unstained and isotype controls

  • For multicolor panels, include fluorescence minus one (FMO) controls

  • Validate specificity using FLT3-blocking experiments with excess unconjugated antibody

How can researchers distinguish between wild-type FLT3 and mutated forms using antibody-based approaches?

Most commercially available FLT3 antibodies, including FITC-conjugated versions, bind to epitopes present in both wild-type and mutated FLT3, making direct distinction challenging. Alternative approaches include:

• Complementary molecular testing (PCR) to identify FLT3-ITD or FLT3-TKD mutations

• Analysis of downstream signaling molecules specific to constitutively activated FLT3

• Differential binding patterns of epitope-specific antibodies

For example, antibodies like 4G8 (binding to domain 4) and BV10 (binding to domain 2) have different binding characteristics that can be leveraged for comparative analysis . Research has shown that antibodies binding to membrane-proximal domains may show different internalization kinetics between wild-type and mutated FLT3 .

How do bispecific FLT3 antibodies function in targeting leukemic cells?

Bispecific FLT3 antibodies represent an advanced approach for targeted leukemia therapy. These antibodies simultaneously bind to:

• FLT3 on leukemic cells (targeting component)

• Immune effector molecules like CD3 on T-cells (effector component)

The mechanism involves:

• Formation of an immunological synapse between leukemic cells and effector T-cells

• T-cell activation independent of antigen recognition or MHC restriction

• Induction of cytotoxic activity against FLT3-positive cells

For example, the 4G8 X UCHT1 Fabsc-antibody format shows superior properties compared to traditional bispecific single chain (bssc) formats, including:

• Higher affinity to the target antigen FLT3

• Better production yield by transfected cells

• Reduced aggregation tendency

• Effective T-cell activation and efficient killing of leukemic blasts in primary PBMC cultures of AML patients

What advantages do antibody-drug conjugates (ADCs) offer for FLT3 targeting?

FLT3-targeting ADCs provide several advantages over conventional therapeutic approaches:

• Enhanced potency through delivery of cytotoxic payloads directly to FLT3-expressing cells

• Activity independent of FLT3 mutation status, overcoming resistance mechanisms

• Reduced off-target effects compared to traditional chemotherapy

Recent development of the 20D9-ADC utilizing P5 conjugation technology has demonstrated:

• Potent cytotoxicity against cells expressing both wild-type FLT3 and FLT3-ITD

• Significant tumor reduction and durable complete remission in AML xenograft models

• Minimal hematotoxicity at therapeutically relevant concentrations

• Strong synergistic effects when combined with tyrosine kinase inhibitors like midostaurin

These properties make ADCs particularly promising for patients with poor prognosis despite treatment with FLT3 inhibitors .

How does the Fabsc antibody format compare to traditional bssc formats for FLT3 targeting?

The Fabsc (Fab-single chain) format represents an optimization over the bispecific single chain (bssc) format with several measurable advantages:

The Fabsc format's superior properties are attributed to its more physiologic antibody structure that resembles normal antibody architecture more closely than the bssc format .

What factors contribute to variability in FLT3 detection across patient samples?

Several factors affect FLT3 detection variability in clinical specimens:

• Heterogeneous FLT3 expression levels:

  • AML patients show FLT3 expression ranging from 400 to 3,300 molecules per cell

  • Some samples may have expression below detection thresholds

• Technical considerations:

  • Sample handling and processing time affect receptor integrity

  • Antibody clone selection impacts epitope accessibility

  • Flow cytometer settings and compensation parameters influence detection sensitivity

• Biological factors:

  • FLT3 mutation status affects receptor turnover and surface expression

  • Presence of FLT3 ligand in samples can interfere with antibody binding

  • Prior treatment with FLT3 inhibitors may alter receptor expression and conformation

How can non-specific binding of FLT3 antibodies be identified and minimized?

Non-specific binding can lead to false positive results and misinterpretation of data. To address this issue:

• Identification methods:

  • Use isotype-matched control antibodies conjugated to the same fluorochrome

  • Perform blocking experiments with excess unconjugated antibody

  • Test antibody binding on known FLT3-negative cell lines

• Minimization strategies:

  • Optimize antibody concentrations through titration experiments

  • Include Fc receptor blocking reagents in staining buffer

  • Implement stringent gating strategies based on fluorescence minus one (FMO) controls

  • Use alternative clones targeting different epitopes for confirmation

Research has identified that monocytes expressing trace amounts of FLT3 (300-600 molecules per cell) can cause off-target activation of PBMC cultures, which can be specifically blocked by depletion of CD14+ cells or excess parental FLT3 antibody .

What controls are essential when evaluating the therapeutic potential of FLT3 antibodies?

When assessing FLT3 antibodies for therapeutic applications, these controls are critical:

• Target specificity controls:

  • Antibodies targeting unrelated antigens (e.g., 9.2.27 antibody targeting CSPG4)

  • Excess unconjugated FLT3 antibodies to block specific binding

• Functional validation controls:

  • Testing on FLT3-negative cells to confirm specificity

  • Comparison with established therapeutic antibodies

  • Assessment in the presence and absence of target cells to determine off-target activation

• Patient-derived controls:

  • Include FLT3-negative patient samples

  • Test on normal hematopoietic cells to assess potential toxicity

  • Evaluate on samples with varying FLT3 expression levels

Studies have demonstrated that rigorous controls can identify important characteristics, such as the specific blockade of PBMC activation after depletion of monocytes with magnetic beads carrying CD14 and CD33, confirming the specificity of therapeutic antibodies .

How can FLT3 antibodies be combined with other therapeutics for enhanced efficacy?

Combination strategies involving FLT3 antibodies show promising results:

• FLT3 antibodies with tyrosine kinase inhibitors:

  • The combination of 20D9-ADC with midostaurin demonstrated strong synergy in vitro and in vivo

  • This approach led to reduction of aggressive AML cells below detection limits

  • Combining targeting mechanisms may overcome resistance to single-agent therapies

• Bispecific antibodies with immune checkpoint inhibitors:

  • Potential to overcome immune suppression in the tumor microenvironment

  • May enhance T-cell mediated killing of leukemic cells

• ADCs with conventional chemotherapy:

  • Sequential or concurrent administration strategies

  • Potential for dose reduction of conventional agents

These approaches represent the frontier of FLT3-directed immunotherapy research .

What are the latest innovations in FLT3 antibody engineering?

Recent technological advances in FLT3 antibody development include:

• Novel conjugation technologies:

  • P5 conjugation technology for next-generation ADCs as demonstrated with 20D9-ADC

  • Site-specific conjugation methods for improved stability and homogeneity

• Advanced antibody formats:

  • Fabsc format that more closely resembles normal antibody structure

  • FLT3 X CD3 bispecific antibodies for T-cell engagement

  • Fc-attenuated designs to minimize Fc-receptor mediated effects

• Emerging combination approaches:

  • Dual-targeting antibodies combining FLT3 with complementary targets

  • Antibodies designed for enhanced tissue penetration

  • Engineered antibodies with optimized pharmacokinetic properties

These innovations aim to overcome limitations of current therapies and improve outcomes for patients with FLT3-positive malignancies.