CD72 Antibody

What is CD72 and what cellular functions does it regulate?

CD72 is a 40-45 kDa type II transmembrane glycoprotein that functions primarily as a negative regulator of B cell activation, which is essential for maintaining immune homeostasis . The mature human CD72 protein consists of a 95 amino acid cytoplasmic domain containing two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), a 21 amino acid transmembrane segment, and a 243 amino acid extracellular domain with a coiled-coil domain and a C-type lectin domain . CD72 is predominantly expressed on B-lineage cells from the pro-B stage through to mature B cells, but is also found on NK cells, monocytes, dendritic cells, and mast cells .

Methodologically, CD72's regulatory function can be demonstrated through comparative proliferation assays with wild-type versus CD72-deficient B cells. Such experiments reveal that CD72-/- B cells exhibit hyperproliferative responses, confirming CD72's inhibitory role in B cell activation .

What applications are CD72 antibodies commonly used for in research?

CD72 antibodies serve multiple research applications across immunology and oncology fields:

For optimal results when using anti-CD72 antibodies, researchers should validate antibody specificity using appropriate positive controls (such as B cell lines) and negative controls (non-B cells) .

How can researchers detect CD72 expression in different cellular contexts?

To reliably detect CD72 expression across different experimental systems, researchers can implement the following methodological approaches:

Flow cytometry detection protocol:

• Isolate peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation

• Block Fc receptors with appropriate blocking reagent (10 minutes at room temperature)

• Stain with anti-CD72 antibody (such as clone 10.1.D2) along with anti-CD19 to identify B cells

• Include appropriate isotype controls

• Analyze populations considering that CD72 expression may vary across B cell developmental stages

For tissue samples, immunohistochemistry protocols should focus on lymphoid tissues such as tonsil, spleen, lymph node, bone marrow, and appendix, where CD72 expression is most notable . Western blotting can be effectively performed using whole cell lysates from B cell lines like RAW264.7, with expected band size around 40 kDa .

How does CD72 function as a pattern recognition receptor for RNA-containing self-antigens?

CD72 has been identified as an inhibitory pattern recognition receptor that specifically recognizes RNA-containing self-antigens, particularly Sm/ribonucleoprotein (RNP) and ribosomes . This function is critical in preventing autoimmunity, especially systemic lupus erythematosus (SLE).

Experimental approach to study CD72's interaction with RNA-containing self-antigens:

• Binding assays: Utilize recombinant CD72 C-type lectin-like domain (CTLD) with purified Sm/RNP or ribosomes to measure direct binding through co-immunoprecipitation or surface plasmon resonance

• B cell functional studies: Compare responses of CD72+/+ versus CD72-/- B cells to:

  • NP-Sm/RNP (RNA-containing self-antigen)

  • NP-BSA (control protein antigen)

  • Imiquimod (synthetic TLR7 agonist)

  Results reveal that CD72 specifically inhibits B cell responses to RNA-containing self-antigens but not to synthetic TLR7 ligands or conventional protein antigens

• BCR endocytosis assays: Measure antigen internalization rates using flow cytometry. CD72-/- B cells show accelerated endocytosis of RNA-containing antigens compared to CD72+/+ B cells, but no difference for conventional antigens

Research data shows that CD72-deficient mice spontaneously produce anti-ribosome autoantibodies, indicating that CD72 induces B cell self-tolerance to RNA-containing self-antigens by recognizing these antigens and inhibiting RNA-dependent B cell activation .

What strategies can be employed to develop CD72-targeted CAR-T cell therapies for B-cell malignancies?

CD72 has emerged as a promising target for chimeric antigen receptor (CAR) T-cell therapy in B-cell malignancies, particularly for patients who relapse after CD19-targeted therapies. Recent research has utilized nanobody-based approaches to develop CD72-specific CAR-T cells .

Methodological framework for CD72-targeted CAR-T development:

• Target validation: Surface proteomics identified CD72 as highly expressed in KMT2A/MLL1-rearranged (MLLr) B-ALL and other B-cell malignancies. Expression persists even after CD19 loss, making it suitable for patients relapsing after CD19 CAR-T therapy

• Binding domain development: Novel approach using in vitro nanobody yeast display screening:

  • Fully synthetic yeast display library (eliminates need for llama immunization)

  • Selection against recombinant CD72 protein

  • Isolation of high-affinity binders like "NbD4"

• Framework humanization: Converting llama-derived framework regions to human sequences:

  • Clone "H24" showed enhanced potency against B-cell tumors

  • Moderate increase in binding affinity to CD72

  • No improvement with further affinity maturation (KD<1 nM)

• CAR design optimization:

  • Comparison of different costimulatory domains:

    • 4-1BB-based backbone with CD8 hinge (analogous to tisagenlecleucel)

    • IgG4 (EQ) hinge with CD28 costimulatory domain

    • CD28 hinge+transmembrane+costimulatory domain

• Toxicity assessment: Co-culture experiments with CD72(NbD4) CAR and normal donor tissues showed no significant off-tumor toxicity

This research demonstrates that CD72-targeted nanobody CAR-T cells represent a promising therapeutic approach for CD19 CAR-T failures, with framework humanization enhancing antitumor potency .

How can CD72 expression be modulated to affect B cell responses in autoimmune diseases?

CD72 expression levels correlate with autoimmune disease activity, particularly in immune thrombocytopenic purpura (ITP) and systemic lupus erythematosus (SLE). Modulating CD72 expression or function represents a potential therapeutic strategy for these conditions .

Methods to investigate CD72 modulation:

• Expression analysis: Flow cytometry reveals CD72 is significantly increased in B cells from newly diagnosed or persistent ITP compared to ITP in remission, suggesting CD72 expression elevation accompanies the active status of ITP

• B cell proliferation assays:

  • In vitro addition of CD72 antibody significantly decreases B cell proliferation in both ITP patients and controls

  • Mechanism appears related to IL-1 and MIF secretion in ITP patients' cell culture supernatant

• Pharmacological modulation approaches:

  • SHIP1 inhibition increases CD72 surface density, potentially enhancing its inhibitory function

  • This represents a strategy to boost CD72's natural regulatory activity

• Allelic variant studies:

  • CD72c (lupus-susceptible allele) binds to Sm/RNP less strongly than CD72a (lupus-resistant allele)

  • X-ray crystallographic analysis reveals considerable alteration in charge at the putative ligand-binding site

  • These findings provide mechanistic insight into how CD72 polymorphisms affect autoimmune susceptibility

These approaches offer complementary methods to study CD72's role in autoimmunity and identify potential therapeutic interventions.

What is the significance of soluble CD72 (sCD72) in T cell activation and autoimmunity?

Recent research has identified a soluble form of CD72 (sCD72) that, contrary to membrane-bound CD72's inhibitory role in B cells, activates T cells through binding to CD6 . This represents a novel immunoregulatory mechanism with implications for understanding autoimmune diseases.

Experimental approach to characterize sCD72-T cell interactions:

• Cell proliferation analysis:

  • CFSE-labeled CD4+ T cells were cultured with 0, 1, or 10 μg/ml of sCD72 for seven days

  • Results showed a significant dose-dependent proliferative effect of sCD72 on T cells:

    • Control: ~75% cells did not divide

    • 1 μg/ml sCD72: ~30% cells did not divide, ~45% underwent >5 division cycles

    • 10 μg/ml sCD72: ~15% cells did not divide, ~70% underwent >5 division cycles

• Receptor identification:

  • Co-immunoprecipitation experiments with anti-V5 beads (targeting tagged sCD72) followed by western blot

  • Mass spectrometry identified CD6 as the primary receptor for sCD72 on T cells

  • Validation showed CD6 co-immunoprecipitated with sCD72, confirming the interaction

• Cell viability assessment:

  • WST-1 reagent was used to measure cell viability/proliferation over 1-7 days in presence of sCD72

  • Optical density measurements at 450 nm provided quantitative assessment of cell growth

• Signaling pathway investigation:

  • B cells were stimulated with sCD72, followed by phosphorylation analysis

  • Western blotting with phospho-specific antibodies revealed activation of specific signaling pathways

The discovery that sCD72 levels are increased in autoimmune diseases like SLE and primary Sjögren's syndrome suggests it may contribute to T cell hyperactivity in these conditions, presenting a potential new therapeutic target .

What are the optimal conditions for using CD72 antibodies in flow cytometry applications?

For researchers using CD72 antibodies in flow cytometry, the following protocol optimizations are recommended:

Optimized flow cytometry protocol:

• Sample preparation:

  • Use freshly isolated PBMCs or cultured B cells

  • For whole blood: lyse red blood cells with commercial lysing solution before staining

  • Cell concentration: 1×10^6 cells per 100 μl staining buffer

• Blocking and staining:

  • Block Fc receptors with 2% normal serum from the species of the secondary antibody (if using indirect staining)

  • For direct staining, use PE, FITC, or Alexa Fluor 700-conjugated anti-CD72 antibodies

  • Recommended working dilution: 5-10 μg/ml (optimal concentration should be determined experimentally)

  • Incubation: 30 minutes at 4°C in the dark

• Multi-parameter considerations:

  • Combine with anti-CD19 (PE or FITC-conjugated) to identify B cell populations

  • Additional markers: anti-CD20, anti-CD27 (memory B cells), anti-CD38 (plasma cells)

  • Include appropriate isotype controls (e.g., mouse IgG1 kappa for clone 10.1.D2)

• Data analysis:

  • Gate on lymphocytes based on FSC/SSC properties

  • Further gate on CD19+ B cells

  • Analyze CD72 expression with appropriate controls for accurate quantification

This protocol enables reliable detection of CD72 on B cells across different developmental stages and activation states.

How can researchers effectively investigate CD72 interactions with RNA-containing self-antigens?

To study the specific interaction between CD72 and RNA-containing self-antigens, researchers should employ these methodological approaches:

Binding and functional analysis protocol:

• Purification of CD72 CTLD domain:

  • Express recombinant CTLD of CD72 in prokaryotic or eukaryotic expression systems

  • Purify using affinity chromatography with appropriate tags

• Preparation of RNA-containing self-antigens:

  • Isolate Sm/RNP complexes from cell lysates using anti-Sm antibodies

  • Purify ribosomes using sucrose gradient ultracentrifugation

  • Create control proteins lacking RNA components through RNase treatment

• Direct binding assays:

  • ELISA-based binding assays with immobilized RNA-containing antigens

  • Surface plasmon resonance to measure binding kinetics and affinity

  • Co-immunoprecipitation assays with tagged CD72 proteins

• Functional B cell assays:

  • Prepare NP-conjugated antigens (NP-Sm/RNP, NP-ribosomes, NP-BSA)

  • Compare calcium mobilization, proliferation, and BCR signaling in CD72+/+ versus CD72-/- B cells

  • Analyze BCR endocytosis rates using flow cytometry

• Structural analysis:

  • X-ray crystallography to determine structural differences between CD72 alleles

  • Mapping of the RNA-binding sites within the CTLD through mutagenesis studies

This comprehensive approach allows researchers to characterize both the physical interaction between CD72 and RNA-containing self-antigens and the functional consequences of this interaction in B cells.

What are the key considerations when developing and validating nanobody-based CD72 CARs?

Developing nanobody-based chimeric antigen receptor (CAR) T cells targeting CD72 requires careful consideration of several factors:

Development and validation framework:

• Nanobody selection process:

  • Utilize synthetic yeast display libraries for in vitro selection

  • Screen against purified CD72 protein and CD72-expressing cells

  • Select candidates based on binding affinity, specificity, and stability

• Framework optimization:

  • Convert llama-derived frameworks to humanized versions to reduce immunogenicity

  • Test multiple humanized variants (e.g., H6, H15, H20, H23, H24)

  • Evaluate binding affinity changes through surface plasmon resonance

• CAR construct design:

  • Test different hinge regions: CD8 hinge vs. mutated IgG4 (EQ) hinge

  • Compare costimulatory domains: 4-1BB vs. CD28

  • Evaluate transmembrane domains: CD8 vs. CD28

• Efficacy testing pipeline:

  Assay Type	Methodology	Output Measurements
  In vitro cytotoxicity	24-hour co-culture with target cells	% target cell killing, EC50 values
  Proliferation	Cell counting, flow cytometry	Expansion rate, divisions
  Cytokine production	ELISA, flow cytometry	IL-2, IFN-γ, TNF-α levels
  In vivo models	Xenograft models in mice	Tumor growth, survival, CAR-T persistence
  Patient-derived samples	Co-culture with primary patient samples	Efficacy against CD19 CAR-T failures

• Safety assessment:

  • Screen for off-target binding using protein arrays

  • Co-culture with normal tissue panels

  • Monitor for antigen downregulation in relapse samples (partially reversible in CD72)

By following this framework, researchers can develop optimized CD72-targeted CAR-T cells with enhanced efficacy and safety profiles for clinical translation.

How can CD72 antibodies be utilized to study and potentially treat B-cell malignancies?

CD72 represents a promising target for B-cell malignancies, particularly in cases where standard therapies fail or resistance develops. Researchers can utilize CD72 antibodies in several translational applications:

Research approaches for B-cell malignancy applications:

• Malignancy profiling:

  • Flow cytometry with anti-CD72 antibodies to assess expression levels across different B-cell malignancy subtypes

  • Surface proteomics revealed CD72 as an optimal target for poor-prognosis KMT2A/MLL1-rearranged (MLLr) B-ALL

  • Immunohistochemistry of tissue biopsies to evaluate CD72 expression in diffuse large B-cell lymphoma

• Therapeutic development strategies:

  • Antibody-drug conjugates (ADCs) targeting CD72

  • Bispecific antibodies linking CD72+ malignant B cells to effector cells

  • CAR-T cells with CD72-specific nanobody binding domains

  • Combined targeting of CD19 and CD72 to prevent antigen escape

• Resistance mechanism studies:

  • Monitor CD72 expression in tumors relapsing after CD19-directed therapies

  • Research shows CD72 expression persists after CD19 loss

  • In vivo relapse after CD72 CAR-T treatment was accompanied by CD72 antigen downregulation, which was partially reversible

• Biomarker applications:

  • CD72 expression profiling as a prognostic biomarker

  • Monitoring of soluble CD72 levels in patient serum

  • Correlation with disease burden and treatment response

These approaches provide a comprehensive framework for translating CD72 research into potential clinical applications for B-cell malignancies.

What is the role of CD72 in autoimmune diseases and how can it be targeted therapeutically?

CD72 plays a critical role in maintaining B cell tolerance to self-antigens, and dysregulation of CD72 function is implicated in several autoimmune diseases. Understanding these mechanisms can guide therapeutic development:

CD72 in autoimmunity - research and therapeutic approaches:

• Disease-specific expression patterns:

  • Flow cytometry analysis shows CD72 is significantly increased in B cells from newly diagnosed or persistent immune thrombocytopenic purpura (ITP) compared to ITP in remission

  • CD72-/- mice spontaneously produce anti-ribosome and anti-Sm/RNP autoantibodies, mimicking SLE features

• Functional mechanism studies:

  • CD72 specifically recognizes RNA-containing self-antigens (Sm/RNP, ribosomes)

  • It inhibits BCR signaling induced by these self-antigens through recruitment of SHP-1

  • CD72 allelic variants (CD72c vs. CD72a) show differential binding to self-antigens, explaining genetic susceptibility to lupus

• Therapeutic targeting strategies:

  • Agonistic approaches: Enhance CD72's natural inhibitory function

    • Develop CD72-specific agonistic antibodies

    • Small molecule enhancers of CD72-SHP-1 interaction

  • Modulation approaches: Increase CD72 expression

    • SHIP1 inhibition increases CD72 surface density

    • Cytokine-based therapies to upregulate CD72 expression

• Monitoring protocol:

  • Measure soluble CD72 levels in patient serum (increased in SLE and Sjögren's syndrome)

  • Track CD72 expression on B cells as a biomarker of disease activity

  • Correlate with autoantibody titers and clinical manifestations

These approaches provide a foundation for developing CD72-targeted therapies for autoimmune diseases, particularly those characterized by B cell hyperactivity and production of autoantibodies against RNA-containing self-antigens.

What experimental systems best model CD72 function in health and disease?

To effectively study CD72 biology and its role in various diseases, researchers should consider these experimental models:

Recommended experimental systems:

• Cell line models:

  Cell Type	Applications	Advantages
  Raji, Daudi (human B cell lines)	Basic CD72 signaling, antibody validation	Well-characterized, easy to manipulate
  SEM (MLLr B-ALL line)	CAR-T efficacy testing	High CD72 expression, clinically relevant
  JeKo-1 (mantle cell lymphoma)	CD72 targeting in NHL	Models CD72+ B-cell lymphoma
  RAW264.7 (mouse macrophage)	Western blot positive control	Reliable CD72 expression

• Primary cell systems:

  • Human PBMCs with focus on CD19+ B cells

  • Purified primary B cells from peripheral blood or tonsil

  • Bone marrow-derived B cell precursors

  • Patient-derived B cells from autoimmune disease subjects

• Mouse models:

  • CD72-/- knockout mice to study autoimmunity development

  • CD72c congenic mice (lupus-susceptible allele)

  • Humanized CD72 mouse models

  • Xenograft models for CD72 CAR-T efficacy assessment

• In vitro functional assays:

  • BCR signaling analysis with phospho-flow cytometry

  • B cell proliferation assays with CFSE or BrdU

  • Calcium mobilization assays

  • Co-immunoprecipitation studies of CD72 interacting partners

• Disease-specific models:

  • Pristane-induced lupus in CD72+/+ vs. CD72-/- mice

  • ITP mouse models with anti-platelet antibody production

  • Patient-derived xenografts of B-cell malignancies