CD248 Antibody

What is CD248 and why is it a significant target for antibody research?

CD248, also known as endosialin or tumor endothelial marker 1 (TEM1), is a C-type lectin-like transmembrane glycoprotein with a molecular weight of approximately 80.9 kilodaltons in its unmodified form, though the heavily glycosylated mature protein approaches 175 kDa . The protein contains a C-type lectin domain, a sushi domain, three epidermal growth factor repeats, a mucin domain, a transmembrane domain, and a short cytoplasmic tail .

• Activated pericytes and myofibroblasts in liver fibrosis

• Tumor-associated stroma and vasculature

• Cancer-associated fibroblasts (CAFs) in various cancers including non-small cell lung cancer

The significance of CD248 as a research target lies in its restricted expression pattern and role in pathological processes. It serves as:

• A biomarker for activated stromal cells in the tumor microenvironment

• A potential therapeutic target for fibrotic diseases and cancer

• A tool for understanding stromal-tumor interactions

Researchers targeting CD248 with antibodies can investigate mechanisms of fibrosis, tumor progression, and develop potential therapeutic interventions, making it a valuable target for both basic and translational research.

What are the different applications of CD248 antibodies in research?

CD248 antibodies are versatile tools employed in multiple research applications, each offering unique insights into CD248 biology:

When selecting an application:

• Consider the specific research question (protein quantification, localization, or cell identification)

• Tissue or cell type (cancer biopsies, fibrotic tissue, cell cultures)

• Available equipment and expertise

• Need for quantitative vs. qualitative data

Most commercial CD248 antibodies have been validated for specific applications, and researchers should review validation data before proceeding with experiments .

How does CD248 expression differ between normal tissues and pathological conditions?

CD248 expression exhibits a highly regulated pattern that changes dramatically between normal physiology and pathological states:

Normal Tissues:

• Primarily expressed during embryonic development

• Limited expression in adult tissues with detection only in:

  • Endometrial stroma

  • Bone marrow fibroblasts

  • Corpus luteum

  • Scattered fibroblasts in normal tissues

• Minimal or absent expression in quiescent vasculature

• Low expression in quiescent hepatic stellate cells (HSCs)

Pathological Conditions:

• Liver Fibrosis/Cirrhosis:

  • Significantly upregulated in patients with liver cirrhosis (p < 0.001)

  • Expression correlates positively with fibrosis severity

  • Predominantly expressed on α-SMA+ myofibroblasts

  • Increased in TGF-β activated HSCs and freshly isolated HSCs from CCl4-induced fibrotic mice

• Cancer:

  • Highly expressed in tumor stroma (84% of invasive breast cancers show CD248+ pericytes, 67% show CD248+ CAFs)

  • Upregulated in stromal compartments of breast, colon, and ovarian cancers

  • Expression largely restricted to perivascular-like cells and CAFs with negligible expression on tumor cells

  • Single-cell sequencing reveals highest expression in CAF clusters that co-cluster with pericytes

This distinct expression pattern makes CD248 an excellent biomarker for activated stroma in pathological conditions and provides a rationale for targeted therapies that selectively affect disease-associated tissues while sparing normal tissues.

How are CD248 antibodies being utilized to develop therapeutic strategies for liver fibrosis?

CD248 antibodies have emerged as promising tools for treating liver fibrosis through several innovative approaches:

Antibody-Drug Conjugates (ADCs):
Researchers have developed CD248-specific ADCs to selectively target and eliminate activated hepatic stellate cells (HSCs) that drive fibrosis progression. One notable example is IgG78-DM1, which consists of:

• A CD248-specific antibody (IgG78)

• The microtubule inhibitor mertansine (DM1)

• A non-cleavable SMCC linker

This ADC demonstrated:

• Selective binding to CD248+ activated HSCs

• Effective killing of CD248+ HSCs in vitro

• Significant reduction of liver fibrosis in CCl4-induced mice

• Acceptable biosafety and reproductive safety profiles in vivo

Mechanism of Action:
The therapeutic efficacy derives from:

• Selective targeting of CD248+ myofibroblasts (activated HSCs)

• Internalization of the ADC complex

• Release of the cytotoxic payload (DM1)

• Elimination of fibrosis-driving cells

Translational Potential:
This approach offers several advantages over conventional anti-fibrotic strategies:

• Cell-specific targeting (minimizing off-target effects)

• Elimination rather than inhibition of myofibroblasts

• Addresses the cellular source of fibrotic matrix production

The success of CD248-targeting ADCs in liver fibrosis models suggests this approach could be extended to other fibrotic diseases where CD248+ myofibroblasts contribute to pathology. Additionally, the biosafety data provided in these studies accelerates the path to potential clinical translation .

What roles do CD248-expressing cancer-associated fibroblasts (CAFs) play in tumor progression?

CD248-expressing CAFs serve as critical mediators in the tumor microenvironment, influencing cancer progression through multiple mechanisms:

Epithelial-Mesenchymal Transition (EMT) Promotion:
In non-small cell lung cancer (NSCLC), CD248+ CAFs promote EMT, a key process in metastasis, through:

• Regulation of macrophage polarization toward the M2 phenotype

• Indirect promotion of cancer cell invasiveness

• Creation of a pro-metastatic microenvironment

Tumor Stroma Remodeling:
CD248+ CAFs contribute to matrix remodeling by:

• Interacting with extracellular matrix components like fibronectin and collagen I/IV

• Enhancing cellular migration and adhesion properties

• Supporting tumor progression and invasion

Heterogeneity and Functional Specialization:
Single-cell sequencing has revealed that CD248 expression varies among CAF populations:

• Five distinct CAF clusters exist in lung tumors

• CD248 expression is elevated in most CAF clusters compared to normal fibroblasts

• The CAF cluster with highest CD248 expression co-clusters with pericytes

Experimental Evidence:
Research using genetic models has demonstrated that:

• CD248 knockout in fibroblasts reduces M2 macrophage polarization

• This leads to decreased EMT in cancer cells both in vitro and in vivo

• CD248+ CAFs influence the immune microenvironment to favor tumor progression

These findings highlight CD248+ CAFs as potential therapeutic targets, with strategies aimed at either eliminating these cells or reprogramming them to adopt anti-tumorigenic functions. Antibodies targeting CD248 could disrupt the pro-tumorigenic functions of CAFs, potentially limiting cancer progression and metastasis.

How does CD248 antibody staining correlate with clinical outcomes in cancer research?

CD248 antibody staining has emerged as a valuable prognostic tool in cancer research, with several studies establishing correlations between CD248 expression patterns and clinical outcomes:

Expression Patterns and Clinical Significance:

Stromal Heterogeneity and Prognosis:
Single-cell sequencing studies have revealed distinct CD248-expressing stromal populations with different prognostic implications:

• CAF clusters with high CD248 expression show distinct gene signatures

• The perivascular-associated CAF cluster with highest CD248 expression may indicate more aggressive disease

• Quantitative analysis of CD248+ cell density provides additional prognostic information beyond traditional histopathological parameters

Therapeutic Response Prediction:
CD248 antibody staining can potentially predict response to targeted therapies:

• Tumors with high CD248 expression in the stroma may be more responsive to anti-stromal therapies

• CD248 expression pattern helps identify patients who might benefit from therapies targeting the tumor microenvironment

• Serial biopsies with CD248 staining can monitor stromal changes during treatment

Methodological Considerations:
When using CD248 antibody staining for prognostic purposes, researchers should:

• Use validated antibodies with demonstrated specificity

• Employ standardized staining protocols and scoring systems

• Consider co-staining with other markers (α-SMA, pericyte markers) to identify specific stromal cell populations

• Correlate findings with established clinical parameters and outcomes

These correlative studies underscore the potential of CD248 not only as a research tool but also as a clinically relevant biomarker that could inform personalized treatment strategies targeting the tumor microenvironment.

What are the optimal conditions for using CD248 antibodies in immunohistochemistry and immunocytochemistry?

Achieving optimal staining with CD248 antibodies requires careful attention to sample preparation, antibody selection, and staining protocols:

Sample Preparation:

For Immunohistochemistry (IHC-P):

• Fixation: 10% neutral buffered formalin (24-48 hours)

• Processing: Standard paraffin embedding

• Sectioning: 4-5 μm thick sections

• Antigen retrieval: Critical step - typically heat-mediated in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Note: Some CD248 antibody clones (e.g., VI-71) are not suitable for formalin-fixed paraffin-embedded tissue

For Immunocytochemistry (ICC):

• Fixation: Ice-cold methanol (10 minutes) often preserves CD248 epitopes better than formaldehyde for some clones

• Alternative: 4% paraformaldehyde (10-15 minutes) works well for certain antibodies

• Cells should be grown on coated coverslips for optimal adhesion

Antibody Selection and Dilution:

Detection Systems:

• For brightfield IHC: HRP/DAB systems provide good signal with minimal background

• For fluorescence: Secondary antibodies conjugated to Alexa Fluor dyes (particularly Alexa Fluor 557 or 647) have shown excellent results

Protocol Optimization:

• Always include positive controls (tissues known to express CD248, such as tumor stroma)

• Include negative controls (primary antibody omission, isotype controls)

• Consider dual staining with α-SMA to confirm myofibroblast/pericyte localization

• For quantitative analysis, standardize exposure settings and acquisition parameters

Troubleshooting Tips:

• Weak signal: Increase antibody concentration, extend incubation time, or optimize antigen retrieval

• High background: Increase blocking time, reduce antibody concentration, or include additional washing steps

• Non-specific binding: Use more stringent blocking with 5% BSA or 10% normal serum from the secondary antibody species

Following these guidelines will help ensure consistent and specific staining of CD248 in both tissue sections and cell preparations.

Multi-color flow cytometry with CD248 antibodies requires careful panel design and optimization to achieve reliable results:

Panel Design Considerations:

• Fluorochrome Selection for CD248:

  • CD248 is typically expressed at moderate levels, making it suitable for mid-brightness fluorochromes

  • Recommended fluorochromes include:

    • Alexa Fluor 647 (optimal for CD248 detection with minimal compensation requirements)

    • PE or PE-Cy7 (bright options for detecting lower expression levels)

  • Avoid using fluorochromes that require compensation with markers used to identify the same cell populations

• Complementary Markers for Stromal Cell Identification:

Staining Protocol Optimization:

• Sample Preparation:

  • Cell suspensions from tissues require gentle enzymatic digestion to preserve CD248 epitopes

  • Recommended: Collagenase D (1 mg/mL) + DNase I (0.1 mg/mL) at 37°C for 30-45 minutes

  • Filter through 70-100 μm mesh to remove aggregates

• Staining Conditions:

  • Buffer: PBS + 2% FBS + 2 mM EDTA (prevents aggregation)

  • Concentration: ≤1.0 μg per million cells in 100 μL volume

  • Incubation: 30 minutes at 4°C protected from light

  • Washing: Two gentle washes before analyzing

• Controls:

  • Fluorescence Minus One (FMO) controls are essential for accurate gating

  • Isotype controls matched to CD248 antibody concentration

  • Positive controls (cell lines with known CD248 expression)

Gating Strategy for CD248+ Cells:

• Initial exclusion gates:

  • FSC/SSC to identify cells of interest

  • Singlet selection

  • Viability dye exclusion

• Specific CD248+ population identification:

  • First gate on stromal cell markers (e.g., PDGFRβ+)

  • Then identify CD248+ subpopulations

  • Confirm with additional markers for specific stromal cell types

• For activated HSCs from liver:

  • Use CD248 in combination with Vitamin A autofluorescence to distinguish quiescent (Vitamin A+/CD248-) from activated (Vitamin A+/CD248+) HSCs

Following these best practices will enable precise identification and isolation of CD248-expressing cell populations for further functional or molecular studies.

What methods can be used to compare the efficacy of different CD248 antibodies for therapeutic applications?

Evaluating CD248 antibodies for therapeutic potential requires systematic assessment of multiple parameters:

1. Binding Characteristics Assessment:

2. Functional Assays:

• Cell-Based Assays:

  • Cell viability assays to assess direct cytotoxicity against CD248+ cells

  • Migration/invasion assays to evaluate effects on CD248-mediated motility

  • Adhesion assays to assess disruption of CD248-ECM interactions

  • Co-culture systems to evaluate effects on tumor-stroma interactions

• Mechanism-Specific Assays:

  • For antibody-drug conjugates (ADCs): Internalization rate using pH-sensitive fluorophores

  • For blocking antibodies: Inhibition of CD248-ligand binding (fibronectin, collagen I/IV)

  • For immune-engaging antibodies: Antibody-dependent cellular cytotoxicity (ADCC) assays

3. In Vivo Efficacy Models:

• Liver Fibrosis Models:

  • CCl4-induced fibrosis in mice (as used for IgG78-DM1)

  • Bile duct ligation model for cholestatic fibrosis

  • Endpoints: Histological assessment of fibrosis (Sirius red staining), hydroxyproline content, α-SMA expression

• Cancer Models:

  • Subcutaneous xenografts of tumors with CD248+ stroma

  • Orthotopic models that better recapitulate the tumor microenvironment

  • Genetically engineered mouse models with fibroblast-specific CD248 knockout

4. Comparative Analysis Framework:

For direct comparison between different CD248 antibodies:

• Use standardized assays with the same concentrations across antibodies

• Include both in vitro and in vivo assessments

• Compare multiple parameters:

  • Target engagement in vivo (by tissue analysis)

  • Pharmacokinetic profiles

  • Efficacy in disease models

  • Safety profiles

5. Advanced Analysis Techniques:

• Multiplex immunohistochemistry to assess effects on the tumor microenvironment

• Single-cell RNA sequencing to evaluate transcriptional changes in CD248+ cells

• Spatial transcriptomics to analyze antibody effects on stromal-parenchymal interactions

• Intravital imaging to visualize antibody localization and effects in real-time

This comprehensive evaluation framework allows researchers to systematically compare different CD248 antibodies and select optimal candidates for further therapeutic development based on multiple efficacy and safety parameters.

How can researchers optimize CD248 antibody-drug conjugates for targeting activated stromal cells?

Optimizing CD248 antibody-drug conjugates (ADCs) for targeting activated stromal cells requires careful consideration of multiple components and their interactions:

1. Antibody Selection and Engineering:

• Isotype Selection:

  • IgG1 for potential immune effector engagement

  • IgG4 for minimal immune system interaction when using pure ADC mechanism

• Binding Properties:

  • Optimize affinity (KD < 10 nM typically ideal)

  • Select antibodies targeting epitopes that facilitate internalization

  • Consider humanization/fully human antibodies to reduce immunogenicity

• Engineering Strategies:

  • Site-specific conjugation using engineered cysteines or non-natural amino acids

  • Fc engineering to optimize half-life and reduce effector functions if needed

2. Linker-Drug Optimization:

• Drug Selection:

  • Microtubule inhibitors (DM1, MMAE) - standard for most ADCs

  • DNA damaging agents (PBD dimers) - highly potent but may increase toxicity

  • Novel payloads (topoisomerase inhibitors) - may offer improved therapeutic window

• Drug-to-Antibody Ratio (DAR):

  • Optimize between 2-4 for balanced efficacy/PK

  • Higher DAR increases potency but may reduce serum half-life and increase off-target toxicity

3. Preclinical Optimization Strategies:

• In Vitro Screening:

  • Internalization assays using pH-sensitive dyes

  • Cytotoxicity against primary activated HSCs, CAFs, and pericytes

  • Specificity testing against CD248- cells

• Pharmacokinetic Optimization:

  • Assess plasma stability of different linker chemistries

  • Measure ADC half-life in relevant animal models

  • Determine tumor/tissue penetration using imaging studies

• Efficacy Models for Stromal Targeting:

  • Liver fibrosis models (as used for IgG78-DM1)

  • Cancer models with significant stromal component

  • Evaluate both target cell depletion and disease modification

4. Safety Considerations for CD248-Targeted ADCs:

• On-Target, Off-Tumor Effects:

  • Comprehensive tissue cross-reactivity studies

  • Evaluate effects on normal CD248-expressing tissues (endometrium, bone marrow)

  • Consider conditional activation strategies if needed

• Reproductive Safety:

  • Evaluate effects on fertility and embryo-fetal development as done for IgG78-DM1

  • Consider development stage-specific administration for clinical translation

5. Translational Considerations:

• Patient Selection Biomarkers:

  • Develop IHC or other assays to quantify CD248 expression

  • Establish minimum threshold for likely response

  • Consider spatial distribution of CD248+ cells

• Combination Strategies:

  • With anti-fibrotic agents for liver disease

  • With immune checkpoint inhibitors for cancer

  • With standard-of-care therapies for specific indications

By systematically optimizing each component of the CD248-targeted ADC, researchers can develop therapeutics with improved efficacy and safety profiles for treating fibrotic diseases and cancers characterized by CD248+ activated stromal cells.

What are the most valuable resources for researchers studying CD248 antibodies?

Researchers investigating CD248 antibodies can benefit from these specialized resources:

Key Literature Resources:

• Seminal Papers on CD248 Biology:

  • MacFadyen et al. (2005): Initial characterization of CD248 expression patterns

  • Wilhelm et al. (2016): CD248 role in liver fibrosis

  • Teicher (2019): Comprehensive review of CD248 as a therapeutic target

• Technical Resources for CD248 Antibody Applications:

  • Single-cell sequencing data repositories containing CD248 expression (e.g., Lambrechts et al.)

  • Tissue microarray datasets with CD248 staining correlations

  • Antibody validation initiatives (e.g., The Antibody Registry)

Research Tools and Repositories:

Collaborative Networks and Consortia:

• Cancer-associated fibroblast research networks

• Fibrosis research consortia

• Tumor microenvironment focused working groups

Technology Platforms:

• Multiplex imaging platforms for studying CD248 in spatial context

• Single-cell analysis platforms for examining CD248+ cell heterogeneity

• Humanized mouse models for testing CD248-targeted therapies

These resources collectively provide researchers with the tools, data, and collaborative opportunities to advance CD248 antibody research from basic characterization to therapeutic applications.

What are the emerging applications and future directions for CD248 antibody research?

CD248 antibody research is rapidly evolving with several promising directions emerging:

1. Advanced Therapeutic Modalities:

• Bispecific Antibodies:

  • CD248 x CD3 bispecifics to redirect T cells against stromal targets

  • CD248 x CD47 bispecifics to block "don't eat me" signals while targeting stroma

• Immune-Modulating Conjugates:

  • CD248-targeted TLR agonist delivery to reprogram the tumor microenvironment

  • CD248-targeted cytokine delivery to activate local anti-tumor immunity

• Engineered Cell Therapies:

  • CAR-T cells targeting CD248+ stromal cells

  • Macrophages engineered to recognize and reprogram CD248+ CAFs

2. Diagnostic and Monitoring Applications:

• Liquid Biopsy Development:

  • Detection of CD248 in circulating tumor-derived extracellular vesicles

  • CD248 as part of multi-marker panels for fibrosis/cancer monitoring

• Imaging Applications:

  • CD248-targeted PET imaging agents for non-invasive assessment of stromal activation

  • Intraoperative imaging to guide surgical resection of tumors with stromal invasion

3. Precision Medicine Approaches:

• Patient Stratification:

  • CD248 expression patterns as predictive biomarkers for response to anti-stromal therapies

  • Integration of CD248 status into comprehensive tumor microenvironment profiles

• Combinatorial Strategies:

  • Rational combinations of CD248-targeted therapies with:

    • Immune checkpoint inhibitors

    • Anti-fibrotic agents

    • Conventional chemotherapies

4. Target Biology Innovations:

• Structural Biology:

  • High-resolution structures of CD248-ligand complexes to guide rational antibody design

  • Structure-based optimization of binding epitopes for therapeutic antibodies

• Signaling Studies:

  • Further elucidation of CD248 signaling pathways to identify synergistic targets

  • Identification of context-dependent functions in different disease settings

5. Technical Innovations:

• Next-Generation Antibody Formats:

  • Nanobodies and single-domain antibodies against CD248 for improved tissue penetration

  • pH-sensitive antibodies that selectively activate in the tumor microenvironment

• Spatially Resolved Analysis:

  • Integration of CD248 antibody staining with spatial transcriptomics

  • AI-assisted image analysis for quantifying stromal patterns and CD248 expression