CHR4 Antibody

What are the primary roles of CCR4 in immune function?

CCR4 is a chemokine receptor highly expressed on certain T-cell populations, particularly cutaneous T-cell lymphoma (CTCL) cells and T-regulatory cells (Tregs). Its high-level expression on CTCL cells is associated with their skin-homing capacity, allowing these cells to migrate to tissues producing CCL17 and CCL22 chemokines. Additionally, CCR4 plays a crucial role in enabling Tregs to migrate toward tumor sites where CCL17 and CCL22 are secreted, facilitating tumor evasion from immune surveillance .

This dual expression pattern on both malignant T-cells and immunosuppressive Tregs makes CCR4 a particularly attractive target for antibody-based immunotherapy, not only for CTCL but potentially for other cancer types where CCR4-positive Tregs contribute to immune evasion .

How does CHD4 influence B cell development and function?

CHD4 is a chromatin remodeling protein that plays critical roles throughout B cell development and function. Research has demonstrated that CHD4 is constitutively expressed in B cells and remains relatively stable during activation with various stimuli .

CHD4 contains a C-terminus ATPase module that uses energy from ATP hydrolysis to remodel nucleosomes, thereby affecting gene accessibility and expression. In B cell development, CHD4 is absolutely essential for the pro-B to pre-B cell transition in the bone marrow, as demonstrated in Mb1-cre/wt Chd4fl/fl mice which show severe loss of pre-B cells and increased frequency of pro-B cells .

Beyond development, CHD4 plays a crucial role in class switch recombination (CSR), a process by which B cells switch antibody isotype production. Depletion of CHD4 in mature B cells significantly reduces CSR to IgG1 and IgG3, without affecting AID expression or B cell proliferation and survival .

What is the basic structure of CXCR4-targeting antibodies with elongated CDRs?

CXCR4-targeting antibodies with elongated complementarity determining regions (CDRs) represent an innovative approach to antibody engineering. These antibodies are designed using the bovine antibody (BLV1H12) scaffold, which naturally contains an ultralong heavy chain CDR3 (CDRH3) .

The engineered antibodies are created by substituting the extended CDRH3 of BLV1H12 with modified CXCR4-binding peptides that adopt a β-hairpin conformation. These peptides are specifically designed to target the ligand binding pocket of the CXCR4 receptor. The basic structure involves:

• A bovine antibody framework with human IgG1 Fc domain

• Modified CXCR4-binding peptides (derived from CVX15) inserted into CDRH3

• Various β-turn linkers that connect the β-strands without affecting CXCR4 interaction

This design approach allows the antibodies to specifically bind CXCR4 with nanomolar affinity while maintaining the favorable pharmacological properties of an antibody molecule.

What methods are most effective for detecting CCR4 expression in clinical samples?

To detect CCR4 expression in clinical samples, researchers typically employ multiple complementary approaches:

Flow Cytometry: This is the most widely used method for detecting CCR4 on cell surfaces. Anti-CCR4 monoclonal antibodies such as mAb1567 (R&D Systems) and 1G1 (BD Pharmingen) are commercially available and can be used to identify CCR4-expressing cells. Flow cytometry allows for quantitative assessment of CCR4 expression and can identify specific cell populations in heterogeneous samples .

Western Blotting: For detecting CCR4 protein levels in cellular extracts, western blotting provides a reliable method. As demonstrated in studies with B cells, protein expression can be monitored before and after various stimulations to assess changes in expression levels .

Immunohistochemistry: For tissue samples, particularly skin biopsies from CTCL patients, immunohistochemical staining with anti-CCR4 antibodies allows visualization of CCR4 expression patterns in the tissue context.

ELISA: For binding studies evaluating antibody specificity, ELISA using plate-bound antibodies and various CCR4 constructs (including NT domain alone) can effectively distinguish binding characteristics .

When assessing CCR4 expression, it's important to include appropriate controls, such as CCR4-negative cell lines and isotype controls, to confirm specificity.

How can researchers evaluate the functional activity of anti-CXCR4 antibodies?

Researchers can employ several functional assays to evaluate the activity of anti-CXCR4 antibodies:

Binding Affinity Determination:

• Flow cytometry using CXCR4-expressing cells (e.g., Jurkat cells)

• Tag-lite homogeneous time-resolved fluorescence (HTRF) competitive binding assays, which can determine precise Kd values by measuring competition between the antibody and fluorescently labeled SDF-1

Inhibition of Receptor Signaling:

• Calcium flux assays: CXCR4-expressing cells (e.g., Ramos cells) are loaded with calcium indicators like Fluo-4, and the ability of antibodies to block SDF-1-induced calcium release is measured

• Phosphorylation of downstream signaling molecules can be assessed by western blotting

Functional Inhibition:

• Chemotaxis assays: The ability of antibodies to block SDF-1-induced migration of CXCR4-expressing cells through transwell membranes provides a physiologically relevant measure of functional inhibition

• Cell viability assays in CXCR4-dependent cell lines

Specificity Controls:

• Testing antibody binding on CXCR4-transfected versus non-transfected cells

• Comparing activity against related receptors (e.g., CCR5, which is structurally similar to CXCR4)

These complementary approaches provide comprehensive characterization of anti-CXCR4 antibody function, from binding to downstream biological effects.

What techniques can be used to engineer CCR4-targeting antibodies with improved therapeutic properties?

Engineering CCR4-targeting antibodies with enhanced therapeutic properties involves several sophisticated techniques:

Humanization Process:

• Identification of murine antibody framework regions (FR) and complementarity-determining regions (CDRs)

• Grafting of murine CDRs onto human antibody framework

• Back-mutation of key framework residues to preserve binding affinity

• Expression in mammalian cell systems (typically HEK293 cells)

Affinity Maturation:

• Creation of antibody libraries with mutations in CDRs

• Phage display selection with decreasing antigen concentrations

• Screening of high-affinity variants using binding assays

• Combination of beneficial mutations to generate improved variants

Fc Engineering:

• Modification of the Fc region to enhance effector functions

• Introduction of specific glycosylation patterns to improve ADCC

• Mutation of key residues to enhance CDC activity

• Engineering for improved half-life through enhanced FcRn binding

Expression and Purification Optimization:

• Codon optimization for mammalian expression

• Design of stable cell lines with high antibody production

• Development of efficient purification protocols, typically using protein A/G chromatography

• Quality control testing for purity, stability, and activity

For CCR4-targeting antibodies specifically, engineering should focus on preserving recognition of both the N-terminal and extracellular domains of CCR4, as this dual recognition provides optimal targeting specificity and functional activity.

How does CHD4 mechanistically influence the class switch recombination process in B cells?

CHD4's role in class switch recombination (CSR) involves sophisticated molecular interactions at the chromatin level. While the complete mechanistic details are still being elucidated, several key insights have emerged from recent research:

Chromatin Remodeling Activity: CHD4, as a component of the nucleosome remodeling and deacetylase (NuRD) complex, uses ATP hydrolysis to remodel nucleosomes at switch regions, potentially making these regions accessible to AID (activation-induced cytidine deaminase) and other CSR factors .

Impact on B Cell Transcriptional Program: CHD4 has been suggested to negatively regulate expression of certain B cell genes like Cd79a, indicating its role in fine-tuning the B cell transcriptional landscape during activation and differentiation .

Developmental Stage-Specific Functions: The requirement for CHD4 differs across B cell developmental stages. While it is absolutely essential for pro-B to pre-B transition, in mature B cells its role becomes more specialized for processes like CSR, suggesting context-dependent functions .

Research using both shRNA-mediated knockdown and conditional knockout approaches has demonstrated that CHD4 depletion leads to approximately 75% reduction in CSR to IgG1, without affecting cell proliferation or survival, indicating a specific role in the CSR process rather than general B cell activation .

What are the structural considerations for targeting specific epitopes on CCR4 with therapeutic antibodies?

Developing therapeutic antibodies against CCR4 requires careful consideration of the receptor's complex structure and epitope accessibility:

CCR4 Structural Elements:

• N-terminal domain (NT, ~30-50 amino acids)

• Three extracellular domain loops (ECLs, each ~10-30 amino acids)

• Seven transmembrane helices (mostly inaccessible to antibodies)

• Intracellular domains (not accessible to antibodies)

Epitope Selection Considerations:

• Domain-Spanning Epitopes: Antibodies like mAb1567 that recognize both the N-terminal and extracellular domains of CCR4 show superior binding characteristics compared to those targeting single domains .

• Functional vs. Non-Functional Epitopes: Targeting epitopes involved in ligand binding (CCL17/CCL22) can yield antibodies that block chemokine signaling, while other epitopes may be better for pure targeting without functional interference.

• Specificity Determination: Chimeric receptor studies using CCR4/CCR8 chimeras can identify which domains contribute to antibody specificity. This approach revealed that mAb1567 binding involves both NT and ECL regions of CCR4 .

• Accessibility in Native Conformation: The conformation of CCR4 on the cell surface may differ from recombinant or fixed protein, making cell-based screening methods crucial for identifying antibodies that recognize the native structure.

For optimal therapeutic antibodies, targeting epitopes that span multiple extracellular domains may provide both high specificity and functional activity, making domain-mapping studies an essential component of antibody development.

How can CDR engineering be optimized to create bi-specific antibodies targeting both CXCR4 and other disease-relevant antigens?

Creating bi-specific antibodies that target both CXCR4 and other disease-relevant antigens through CDR engineering represents an advanced application of antibody design. Based on current research, several approaches offer promising strategies:

Dual CDR Utilization Strategy:
Research has demonstrated that multiple CDRs within the same antibody can be engineered independently. The CDRH2 of the bovine antibody BLV1H12 has been successfully used for peptide grafting to create a CXCR4-binding antibody (bAb-AC4) with nanomolar affinity (Kd = 0.92 nM) . This finding suggests that CDRH2 and CDRH3 could potentially be engineered simultaneously to target different antigens.

β-Hairpin Scaffold Optimization:
The success of engineering β-hairpin structures into CDRs to target CXCR4 provides a template for targeting other GPCRs or receptors with similar ligand binding pockets. Key considerations include:

• Optimal β-turn linker selection (glycine-containing linkers showed superior flexibility)

• Proper spatial arrangement to avoid steric hindrance between the two targeting moieties

• Preservation of the critical binding residues within each engineered CDR

Expression and Stability Considerations:
CDRH2-engineered antibodies (bAb-AC4) demonstrated significantly higher expression yields (17 mg/L) compared to CDRH3-engineered variants (5 mg/L), suggesting that CDRH2 modification may be preferred when expression efficiency is critical .

Functional Validation Approach:
For bi-specific antibodies targeting CXCR4 and another target, a comprehensive functional validation strategy should include:

• Independent binding assays for each target

• Functional assays specific to each target (e.g., calcium flux inhibition for CXCR4)

• Combined effect assessment in cells expressing both targets

• In vivo distribution and efficacy studies

This approach could be particularly valuable for creating antibodies that simultaneously target CXCR4 (for blocking metastasis or targeting cancer stem cells) and tumor-specific antigens, potentially enhancing therapeutic efficacy through dual-targeting mechanisms.

What are common pitfalls when measuring antibody-dependent cellular cytotoxicity (ADCC) of anti-CCR4 antibodies?

Measuring ADCC activity of anti-CCR4 antibodies presents several technical challenges that researchers should be aware of:

Variability in Effector Cells:

• Natural killer (NK) cells from different donors can display significant variability in their killing capacity

• The activation state of NK cells affects ADCC potency

• Cryopreservation of NK cells may diminish their cytotoxic activity

Assay Format Variables:

• Effector-to-target (E:T) ratios need optimization for each system

• Incubation times can significantly impact results (typically 4-6 hours)

• The choice of readout (chromium release, calcein release, or flow cytometry-based) affects sensitivity

Fc Receptor Polymorphisms:

• FcγRIIIa (CD16) polymorphisms in effector cells affect ADCC potency

• The V158F polymorphism particularly influences binding affinity to IgG1

• Using effector cells with defined FcγR genotypes can reduce variability

To overcome these challenges, researchers should:

• Include a well-characterized reference antibody in all assays

• Use pooled NK cells from multiple donors when possible

• Validate results using multiple E:T ratios and incubation times

• Consider complementary approaches like complement-dependent cytotoxicity (CDC)

How can researchers address specificity concerns when targeting CXCR4 with engineered antibodies?

Ensuring specificity of engineered anti-CXCR4 antibodies is critical, particularly given the structural similarity between CXCR4 and other chemokine receptors. Researchers can implement the following approaches to address specificity concerns:

Comprehensive Cross-Reactivity Testing:

• Test antibody binding against cells expressing related chemokine receptors, particularly CCR5, which is structurally most similar to CXCR4

• Evaluate binding to panels of cell lines expressing various GPCRs

• Perform tissue cross-reactivity studies on human tissue arrays

Epitope Mapping and Binding Site Verification:

• Create chimeric receptors exchanging domains between CXCR4 and related receptors

• Use site-directed mutagenesis to identify critical binding residues

• Employ hydrogen-deuterium exchange mass spectrometry to identify interacting regions

Competitive Binding Assays:

• Conduct competition studies with known CXCR4 ligands (SDF-1) and antagonists (AMD3100)

• Use Tag-lite HTRF assays to quantitatively measure binding competition

• Determine precise binding constants (Kd) under various conditions

Functional Specificity Validation:

• Compare inhibition of SDF-1-induced calcium flux in CXCR4+ versus CXCR4- cells

• Assess inhibition of chemotaxis in various cell types

• Evaluate potential off-target effects on related signaling pathways

Appropriate Controls:

• Include non-transfected parental cell lines as negative controls

• Use both the original scaffold antibody (without CXCR4-targeting inserts) and irrelevant antibodies as controls

• For cell-based assays, include CXCR4 knockout or knockdown conditions

By implementing these approaches, researchers can confidently establish the specificity of their engineered anti-CXCR4 antibodies and minimize concerns about off-target effects.

What technical difficulties arise when generating conditional knockout models to study CHD4 in specific B cell populations?

Generating and working with conditional knockout models to study CHD4 in specific B cell populations presents several technical challenges:

Developmental Lethality Constraints:

• Complete CHD4 knockout is embryonically lethal, necessitating conditional approaches

• The critical role of CHD4 in early B cell development can complicate studies of its function in mature B cells

• Sequential developmental requirements make interpretation of phenotypes challenging

Cre Driver Selection Challenges:

• Different Cre drivers (Mb1-cre, Cd21-cre) induce deletion at different developmental stages

• Efficiency of Cre-mediated recombination varies between drivers and even between mice

• Incomplete deletion can result in outgrowth of cells retaining CHD4 expression

Verification of Deletion Efficiency:

• PCR genotyping may not accurately reflect protein levels due to protein stability

• Western blotting is necessary to confirm protein depletion

• Potential compensation by related proteins (CHD3, CHD5) requires careful analysis

Phenotypic Analysis Complexities:

• Early developmental blocks (e.g., at pre-B cell stage with Mb1-cre) limit analysis of later stages

• Distinguishing direct effects from indirect consequences of developmental abnormalities

• Potential confounding from altered cellular composition in primary and secondary lymphoid organs

Experimental Approaches to Overcome These Challenges:

• Use of multiple Cre drivers with different temporal expression patterns (e.g., Mb1-cre for early deletion, Cd21-cre for mature B cells)

• Complementary in vitro approaches using shRNA knockdown or CRISPR in mature B cells

• Inducible systems (e.g., tamoxifen-inducible Cre) to control the timing of deletion

• Single-cell analyses to account for heterogeneity in deletion efficiency

• Careful analysis of littermate controls and consistent monitoring of deletion efficiency

These approaches can help researchers navigate the complexities of studying CHD4 function across different B cell developmental stages and functional contexts.

What emerging approaches might enhance the efficacy of anti-CCR4 antibodies in treating cutaneous T-cell lymphomas?

Several innovative approaches are being explored to enhance the efficacy of anti-CCR4 antibodies for CTCL treatment:

Antibody-Drug Conjugates (ADCs):
Conjugating potent cytotoxic payloads to anti-CCR4 antibodies could enhance their direct killing capacity while maintaining the targeting specificity. This approach might be particularly effective against tumors with heterogeneous CCR4 expression due to bystander effects .

Bispecific Antibody Formats:
Developing bispecific antibodies that simultaneously target CCR4 and another CTCL-associated antigen (such as CD25 or CD30) could improve specificity and reduce the potential for escape variants. Alternatively, bispecifics targeting CCR4 and engaging T cells (like CD3) could enhance cytotoxic T cell responses against tumor cells .

Combination with Immunomodulatory Agents:
Anti-CCR4 antibodies' ability to deplete immunosuppressive Tregs makes them attractive candidates for combination with other immunotherapies such as checkpoint inhibitors. By simultaneously targeting the tumor and alleviating immunosuppression, such combinations might produce synergistic effects .

Fc Engineering for Enhanced Effector Functions:
Further optimization of the Fc region to enhance ADCC and CDC activities could improve clinical efficacy. Specific glycoengineering approaches or amino acid substitutions in the Fc region could enhance interactions with activating FcγRs on effector cells .

Integration with CAR-T Cell Approaches:
Anti-CCR4 antibody fragments could be incorporated into chimeric antigen receptor (CAR) constructs to redirect T cell activity against CCR4-expressing malignant cells, potentially offering more durable responses than antibody therapy alone.

These emerging approaches hold promise for enhancing the therapeutic efficacy of anti-CCR4 antibodies against CTCL and potentially other CCR4-expressing malignancies.

How might synthetic biology approaches expand the applications of engineered antibodies with elongated CDRs?

Synthetic biology presents exciting opportunities to expand applications of antibodies with elongated CDRs, particularly building on the success of CXCR4-targeting antibodies:

Multi-CDR Engineering Platforms:
The demonstration that both CDRH2 and CDRH3 can independently accommodate functional peptide insertions opens possibilities for creating antibodies with multiple functionalities. Future platforms could systematically explore combinations of modified CDRs to create multifunctional antibodies with precisely engineered properties .

Incorporation of Non-Natural Amino Acids:
Expanding beyond natural amino acids by incorporating non-canonical amino acids into elongated CDRs could enhance binding affinity, stability, or introduce novel functionalities such as photo-crosslinking, click chemistry handles, or pH-sensitive elements.

Cell-Penetrating CDR Designs:
Engineering elongated CDRs to include cell-penetrating peptide sequences could create antibodies capable of crossing cellular membranes to reach intracellular targets, dramatically expanding the range of addressable targets beyond surface proteins.

Stimulus-Responsive Antibody Systems:
Incorporating environmentally-responsive elements (pH, temperature, or protease-sensitive) into elongated CDRs could create "smart" antibodies that selectively activate in disease microenvironments. For example, a tumor-activated antibody could have its targeting function masked until reaching the acidic tumor microenvironment .

In Vivo Evolution Systems:
Development of platforms for in vivo directed evolution of elongated CDRs could enable selection of optimal binding sequences in physiologically relevant contexts, potentially identifying novel binding motifs not discoverable through traditional in vitro selection methods.

These synthetic biology approaches could transform engineered antibodies with elongated CDRs from simple targeting molecules into sophisticated biological devices with programmable, multi-functional capabilities for both research and therapeutic applications.

What potential roles might CHD4-targeting approaches play in modulating B cell responses in autoimmunity and cancer?

CHD4's critical role in B cell development and function suggests several promising therapeutic applications through targeted modulation:

Autoimmune Disease Applications:
The essential role of CHD4 in class switch recombination suggests that its selective inhibition might reduce pathogenic antibody production in autoimmune diseases. Partial inhibition of CHD4 could potentially:

• Decrease production of high-affinity autoantibodies by limiting CSR

• Alter the balance of antibody isotypes produced during autoimmune responses

• Modify B cell differentiation patterns to favor regulatory over inflammatory phenotypes

B Cell Malignancy Approaches:
CHD4's differential requirements across B cell developmental stages offers potential therapeutic windows for B cell malignancies:

• Pre-B cell ALL might be particularly vulnerable to CHD4 inhibition given its essential role at this developmental stage

• Diffuse large B cell lymphomas could potentially be sensitized to conventional therapies through CHD4 modulation

• Multiple myeloma might be affected through disruption of plasma cell maintenance programs

Targeting Strategies:
Several approaches might be deployed to modulate CHD4 function:

• Small molecule inhibitors targeting its ATPase activity

• Degrader approaches (PROTACs) to induce selective CHD4 degradation

• Disruption of specific protein-protein interactions within the NuRD complex

• Targeted delivery of CHD4 inhibitors to B cells using antibody-drug conjugates

Potential Combination Approaches:
CHD4 modulation might be particularly effective when combined with:

• BTK inhibitors in B cell malignancies

• CD20-targeting antibodies to enhance B cell depletion

• Checkpoint inhibitors in B cell-influenced solid tumors

• Conventional chemotherapies to overcome resistance mechanisms

While therapeutic targeting of chromatin remodelers presents significant challenges, the highly specific role of CHD4 in B cell biology offers potential therapeutic windows where beneficial effects might be achieved with acceptable toxicity profiles.