CPK26 Antibody

What is CD26 and why are antibodies against it important in research?

CD26 is a 110 kDa type II transmembrane glycoprotein also known as dipeptidyl peptidase IV (DPPIV/DPP IV) or adenosine deaminase binding protein (ADABP). It is expressed by T lymphocytes (increasing upon activation), B cells, NK cells, and macrophages, serving various physiological functions . CD26 has been implicated in tumorigenesis across several cancer types, including malignant lymphoma and multiple myeloma (MM) .

Antibodies against CD26 are critically important in research for several reasons. First, they allow for identification and characterization of CD26 expression patterns across different cell types and disease states. Second, humanized anti-CD26 monoclonal antibodies (huCD26mAb) have demonstrated therapeutic potential by inhibiting human osteoclast differentiation and targeting CD26-positive MM cells through antibody-dependent cytotoxicity (ADCC) . Finally, these antibodies provide valuable research tools for investigating CD26's role in normal physiology and pathological conditions.

How does CD26 expression change in multiple myeloma microenvironments?

CD26 exhibits context-dependent expression patterns in multiple myeloma that researchers must understand for effective experimental design. While CD26 expression is typically low or absent on MM cell lines cultured in isolation, it becomes intensely and uniformly expressed when these cells are co-cultured with osteoclasts (OCs) . This microenvironment-dependent expression pattern has significant implications for therapeutic targeting.

In bone marrow tissues from MM patients, CD26 expression is detectable on plasma cells, confirming its clinical relevance . The augmented expression of CD26 in MM cells co-cultured with osteoclasts creates an opportunity for enhanced efficacy of anti-CD26 therapeutic antibodies through increased ADCC activity. This pattern exemplifies how the tumor microenvironment can dramatically alter target expression and potentially influence therapeutic outcomes in experimental models.

What are the key differences between research-grade and therapeutic CD26 antibodies?

Research-grade CD26 antibodies, such as the APC Mouse Anti-Human CD26 (Clone M-A261), are optimized for flow cytometry, immunohistochemistry, and other detection methods . These antibodies typically have validated specificity against the target but may not be optimized for therapeutic functions like ADCC or complement-dependent cytotoxicity (CDC).

In contrast, therapeutic antibodies like the humanized IgG1 monoclonal antibody targeting CD26 (huCD26mAb) are specifically engineered to induce effector functions. The huCD26mAb has been demonstrated to enhance ADCC against CD26+ MM cells but does not significantly trigger CDC . Furthermore, therapeutic antibodies undergo extensive optimization for pharmacokinetics, tissue distribution, and safety profiles not typically required for research applications. Researchers should carefully select the appropriate antibody type based on their experimental goals and required functional characteristics.

How should researchers design experiments to evaluate CD26 antibody efficacy in multiple myeloma models?

When designing experiments to evaluate CD26 antibody efficacy in multiple myeloma models, researchers should consider the following methodological approach:

• Cell culture systems: Establish both monoculture and co-culture systems of MM cell lines with osteoclasts since CD26 expression is significantly upregulated in MM cells when co-cultured with osteoclasts . This ensures testing under conditions that recapitulate the physiological microenvironment.

• Functional assays: Include multiple functional readouts:

  • ADCC assays using appropriate effector cells (e.g., peripheral blood mononuclear cells or NK cells)

  • CDC assays with complement factors

  • Cell viability and proliferation assays

  • Side population (SP) fraction analysis to evaluate effects on potential cancer stem cells

• Combination studies: Test CD26 antibodies alone and in combination with standard MM therapies such as proteasome inhibitors and immunomodulatory drugs, as these combinations have shown synergistic enhancement of ADCC activity against CD26+ MM cells .

• In vivo validation: Establish xenograft models in immunocompromised mice to assess effects on tumor burden and osteoclast formation, measuring both tumor volume and analyzing bone structure through appropriate imaging techniques .

What controls are essential when working with CD26 antibodies in experimental systems?

When designing experiments with CD26 antibodies, implement these essential controls:

• Isotype controls: Include matching isotype control antibodies to distinguish between specific CD26-mediated effects and non-specific Fc-mediated effects. For mouse monoclonal antibodies, use matching mouse IgG isotypes; for humanized antibodies, use matching human IgG isotypes .

• Expression controls: Validate CD26 expression levels on target cells using flow cytometry or Western blotting before conducting functional assays. This is particularly critical when working with MM cell lines, where CD26 expression varies depending on culture conditions .

• Negative cell lines: Include CD26-negative cell lines to confirm antibody specificity and rule out off-target effects.

• Blocking controls: Where applicable, use soluble CD26 or CD26-derived peptides to competitively inhibit antibody binding and confirm specificity of observed effects.

• Compensation controls: For flow cytometry experiments, use BD® CompBeads to assess fluorescence spillover, recognizing that spillover values may differ slightly between beads and cells for certain fluorochromes .

How can researchers optimize antibody-dependent cellular cytotoxicity (ADCC) assays for CD26 antibodies?

Optimizing ADCC assays for CD26 antibodies requires attention to several methodological factors:

• Effector cell preparation: Use freshly isolated peripheral blood mononuclear cells (PBMCs) or purified NK cells as effector cells. Pre-activate NK cells with low-dose IL-2 (100 IU/ml) for 24 hours to enhance cytotoxic potential while maintaining physiological relevance.

• Effector-to-target ratios: Test multiple effector-to-target (E:T) ratios, typically ranging from 5:1 to 50:1, to determine the optimal ratio for detecting ADCC activity. The huCD26mAb has demonstrated significant ADCC effects across various E:T ratios against CD26+ MM cells .

• Target cell preparation: For MM research, use MM cell lines co-cultured with osteoclasts to upregulate CD26 expression, making them more susceptible to ADCC. This approach more accurately reflects the in vivo tumor microenvironment .

• Antibody concentration titration: Test a range of antibody concentrations (typically 0.1-10 μg/ml) to establish dose-response relationships and determine the minimum effective concentration.

• Readout methods: Employ multiple complementary readout systems, such as:

  • Chromium-51 release assays (gold standard)

  • Flow cytometry-based assays using cell death markers

  • Luminescence-based cytotoxicity assays that measure target cell viability

How can researchers address variable CD26 expression in experimental models?

Addressing variable CD26 expression in experimental models requires several methodological approaches:

• Standardized co-culture systems: Establish standardized co-culture protocols with osteoclasts, as these consistently upregulate CD26 expression on MM cells. Document the timing of maximal CD26 induction (typically 48-72 hours of co-culture) and use this timepoint consistently for experiments .

• Flow cytometric quantification: Before each experiment, quantify CD26 expression levels using standardized flow cytometry with antibodies like APC Mouse Anti-Human CD26 (Clone M-A261). Use mean fluorescence intensity (MFI) to quantify expression levels rather than simply percent positive cells .

• Cell sorting strategies: When heterogeneous expression is observed, consider fluorescence-activated cell sorting (FACS) to isolate CD26-high and CD26-low/negative populations for comparative studies.

• Inducible expression systems: For mechanistic studies, consider developing cell lines with inducible CD26 expression systems that allow controlled, consistent expression levels independent of co-culture conditions.

• Patient-derived samples: When using patient-derived MM samples, screen for CD26 expression prior to experiments and stratify samples based on expression levels to account for patient-to-patient variability.

What are common pitfalls in antibody specificity testing and how can they be avoided?

Common pitfalls in antibody specificity testing and their methodological solutions include:

• Cross-reactivity: Antibodies may bind to structurally similar proteins. To address this:

  • Test antibodies on cells overexpressing or lacking CD26 through genetic manipulation

  • Use western blots to confirm the detected protein is the expected molecular weight (110 kDa for CD26)

  • Perform epitope mapping to characterize the exact binding region

• Background binding: Non-specific binding can obscure results. Mitigate by:

  • Optimizing blocking conditions with appropriate sera or protein solutions

  • Titrating antibody concentrations to find the optimal signal-to-noise ratio

  • Including appropriate isotype controls in all experiments

• Epitope masking: Protein interactions or conformational changes may mask epitopes. Address by:

  • Testing multiple antibody clones targeting different CD26 epitopes

  • Using different sample preparation methods (native vs. denaturing conditions)

  • Evaluating antibody performance in different assay formats (flow cytometry, IHC, Western blot)

• Batch-to-batch variability: Antibody performance may vary between lots. Control by:

  • Maintaining reference samples for comparison between batches

  • Documenting lot numbers and standardizing antibody quantities by functional testing rather than concentration alone

How should researchers validate anti-CD26 antibody performance over time?

To ensure consistent anti-CD26 antibody performance over time:

• Stability testing protocol: Establish a standardized protocol for periodic antibody validation including:

  • Flow cytometric analysis against reference cell lines with known CD26 expression

  • Functional ADCC assays against standard target cells

  • Western blotting to confirm molecular weight specificity

• Storage condition optimization: Store antibodies according to manufacturer recommendations, typically at 4-8°C for working solutions and -20°C or -80°C for long-term storage. Avoid repeated freeze-thaw cycles by preparing single-use aliquots .

• Positive control maintenance: Maintain frozen aliquots of standardized positive control cells (e.g., activated T cells or CD26-expressing MM cell lines) to use as reference standards across experiments .

• Documentation system: Implement a detailed documentation system recording:

  • Antibody source, lot number, and receipt date

  • Testing dates and performance metrics

  • Any observed changes in performance

  • Experimental conditions of each validation test

• Performance trending: Track antibody performance metrics over time to identify gradual degradation before it significantly impacts experimental results.

How can CD26 antibodies be combined with other therapeutic agents for synergistic effects?

CD26 antibodies can be effectively combined with other therapeutic agents through several methodological approaches:

• Combination with proteasome inhibitors: The combination of huCD26mAb with proteasome inhibitors (PIs) synergistically enhances ADCC activity against CD26+ MM cells compared to either agent alone . Researchers should:

  • Establish dose matrices testing various concentrations of both agents

  • Calculate combination indices to quantify synergy vs. additivity

  • Investigate the molecular mechanisms of synergy, potentially related to PI-induced stress response pathways

• Combination with immunomodulatory drugs (IMiDs): Similar synergistic enhancement of ADCC activity occurs when combining huCD26mAb with IMiDs . When designing these experiments:

  • Pre-treat effector cells with IMiDs to maximize NK cell activation

  • Evaluate changes in immune checkpoint expression on effector cells

  • Monitor cytokine production to understand immunomodulatory effects

• Sequential vs. simultaneous administration: Test both sequential and simultaneous administration protocols:

  • Sequential: Pre-treatment with PIs or IMiDs before huCD26mAb administration

  • Simultaneous: Concurrent administration of both agents

  • Compare these approaches for differential effects on efficacy and toxicity

• Targeting cancer stem cells: huCD26mAb reduces the side population (SP) fraction in CD26+ MM cells through ADCC . This suggests potential for targeting cancer stem cells, which could be enhanced by combining with:

  • Other stem cell-targeted therapies

  • Epigenetic modifiers that may affect stem cell phenotypes

  • Standard chemotherapeutic agents that primarily target bulk tumor cells

What novel approaches are emerging for designing CD26 antibodies with enhanced specificity?

Emerging approaches for designing CD26 antibodies with enhanced specificity include:

• Biophysics-informed modeling: This approach uses data from phage display experiments to identify different binding modes associated with particular ligands. The model:

  • Successfully disentangles binding modes even when associated with chemically similar ligands

  • Enables computational design of antibodies with customized specificity profiles

  • Allows generation of antibodies with either specific high affinity for particular targets or cross-specificity for multiple targets

• High-throughput sequencing and analysis: This methodology involves:

  • Generating antibody libraries based on a single naïve human VH domain

  • Systematically varying complementary determining regions (CDRs)

  • Using high-throughput sequencing to track antibody enrichment

  • Applying computational analysis to identify key residues determining specificity

• Specificity matrix prediction: This approach:

  • Characterizes antibody binding specificity to generate a specificity matrix

  • Uses this matrix to predict antibody reactivity against novel targets

  • Enables rational design of antibodies with predetermined specificity profiles

• Targeted modification of CDR regions: For CD26 antibodies specifically:

  • Identify key residues in CDRs that contact unique epitopes on CD26

  • Engineer modifications to enhance interactions with distinctive CD26 domains

  • Test modified antibodies against closely related proteins to confirm specificity

How can researchers evaluate CD26 antibody efficacy against cancer stem cell populations?

Evaluating CD26 antibody efficacy against cancer stem cell populations requires specialized methodological approaches:

• Side population (SP) assays: The huCD26mAb has been shown to reduce the SP fraction in CD26+ MM cells through ADCC . Researchers should:

  • Use Hoechst 33342 dye exclusion assays to identify SP cells

  • Quantify changes in SP percentage following antibody treatment

  • Confirm SP phenotype using functional assays (e.g., clonogenic potential)

• Limiting dilution assays: To assess impact on self-renewal capacity:

  • Treat MM cells with CD26 antibodies at varying concentrations

  • Perform serial dilution and replating of surviving cells

  • Quantify colony-forming efficiency as a measure of stem cell function

  • Compare antibody-treated cells with control-treated populations

• In vivo serial transplantation: For rigorous stem cell assessment:

  • Treat MM cells with CD26 antibodies in vitro

  • Transplant treated cells into primary recipient mice at limiting dilutions

  • Assess tumor development and perform secondary transplantation

  • Calculate stem cell frequency using extreme limiting dilution analysis (ELDA)

• Molecular profiling: To understand mechanisms of action:

  • Isolate SP and non-SP fractions after antibody treatment

  • Perform transcriptomic and proteomic analyses

  • Identify stem cell-associated pathways affected by antibody treatment

  • Validate key findings using functional assays

• Combinatorial approaches: As huCD26mAb in combination with novel agents synergistically enhances ADCC activity , test combinations of:

  • CD26 antibodies with standard anti-MM therapies

  • CD26 antibodies with stem cell pathway inhibitors

  • Triple combinations targeting different aspects of stem cell biology

What are emerging applications of CD26 antibodies beyond multiple myeloma?

While CD26 antibodies have shown promising results in multiple myeloma research, several emerging applications warrant investigation:

• Other hematological malignancies: Given that CD26 is expressed on several tumor cells including malignant lymphoma , researchers should:

  • Screen diverse lymphoma subtypes for CD26 expression

  • Evaluate correlation between CD26 expression and clinical outcomes

  • Test huCD26mAb efficacy in lymphoma models using similar ADCC approaches as with MM

• Autoimmune disorders: Since CD26 is expressed by T lymphocytes and increases upon activation , its potential role in autoimmunity could be explored by:

  • Characterizing CD26 expression on autoreactive T cell populations

  • Testing CD26 antibodies in models of T cell-mediated autoimmunity

  • Investigating whether CD26 blockade affects T cell activation and cytokine production

• Inflammatory diseases: Building on CD26's expression on immune cells:

  • Assess CD26 expression in tissue samples from inflammatory disease patients

  • Evaluate effects of CD26 antibodies on inflammatory mediator production

  • Test therapeutic potential in inflammatory disease models

• Metabolism-related applications: Given CD26/DPP-IV's role in glucose metabolism:

  • Explore potential applications in diabetes research

  • Investigate interactions between CD26 antibodies and established DPP-IV inhibitors

  • Assess effects on metabolic parameters in preclinical models

How might computational approaches improve CD26 antibody design and prediction?

Computational approaches offer significant potential for improving CD26 antibody design:

• Biophysics-informed modeling: This approach can:

  • Identify distinct binding modes associated with CD26 epitopes

  • Enable prediction of antibody variants with customized specificity profiles

  • Generate novel antibody sequences not present in initial libraries but with desired binding properties

• Structure-based design: Using available structural data:

  • Model CD26 protein structure in complex with antibody fragments

  • Identify key interaction residues at the antibody-antigen interface

  • Design modifications to enhance binding affinity and specificity

  • Predict potential cross-reactivity with related proteins

• Machine learning applications: Advanced algorithms can:

  • Analyze large datasets of antibody sequences and their binding properties

  • Identify non-obvious patterns correlating sequence features with functionality

  • Predict optimal antibody candidates for synthesis and testing

  • Continuously improve predictions through iterative testing and feedback

• High-throughput virtual screening: Computational screening approaches:

  • Evaluate thousands of potential antibody variants in silico

  • Rank candidates based on predicted binding properties

  • Identify promising candidates for experimental validation

  • Significantly reduce time and resources needed for antibody optimization

What methodological advances are needed to improve CD26 antibody therapeutic applications?

Several methodological advances could enhance CD26 antibody therapeutic applications:

• Improved co-culture systems: Since CD26 expression in MM cells is significantly upregulated when co-cultured with osteoclasts :

  • Develop standardized 3D co-culture systems that better mimic bone marrow microenvironment

  • Establish patient-derived xenograft models that maintain CD26 expression patterns

  • Create organoid models incorporating multiple cell types from the MM microenvironment

• Enhanced antibody engineering: Building on current huCD26mAb technology:

  • Develop bispecific antibodies targeting both CD26 and other MM-associated antigens

  • Engineer antibody-drug conjugates using CD26 antibodies as targeting moieties

  • Create CD26-targeted chimeric antigen receptor (CAR) T cells

• Predictive biomarkers: To identify patients most likely to benefit:

  • Establish standardized methods for CD26 detection in clinical samples

  • Correlate CD26 expression patterns with response to huCD26mAb in preclinical models

  • Identify additional biomarkers that predict sensitivity to CD26-targeted therapies

• Resistance mechanisms: To address potential limitations:

  • Investigate mechanisms of resistance to CD26 antibody therapy

  • Identify pathways that might be activated to compensate for CD26 targeting

  • Develop rational combination strategies to overcome resistance

• Delivery optimization: To enhance in vivo efficacy:

  • Explore alternative antibody formats with improved tissue penetration

  • Investigate local delivery approaches for bone marrow targeting

  • Develop controlled release formulations for sustained antibody exposure